The enhancement of chronic inflammation via interorgan communication like liver-adipose tissue is one of the pathological bases for the development of obesity. Although small extracellular vesicles (sEVs) are suggested to play a critical role in intercellular communications and glucose intolerance in obese and diabetic conditions, the exact target tissue/cells for hepatocyte-derived sEVs (HsEVs) in vivo and their contribution to chronic inflammation are not clear.

Therefore, we have generated a new mouse model to track HsEVs by scarlet, red fluorescent protein, in vivo.

We observed high numbers of scarlet-positive (Sc+) CD45-positive cells without expressing scarlet mRNA in the pancreas, and visceral adipose tissue (eWAT), suggesting Sc+ HsEVs-recipient cells are contained in these tissues. The scRNA-seq determined that macrophages/monocytes are the major HsEVs-recipient cell types. High-fat diet (HFD) increased mRNA expression for sEV biogenesis in the liver and Sc+ HsEVs ratio in the serum in mice. Sc+ macrophages exhibited higher levels of TNFa compared to scarlet-negative macrophages, and this level in the Sc+ macrophage was further enhanced in the HFD condition.

These data suggest that the hepatocytes in obesity are involved in generating pro-inflammatory sEVs, which may contribute to macrophage activation in the eWAT.

Thyroid hormone plays a critical role in development of central nervous system, such as neurogenesis, synaptogenesis and myelination. However, little is known about the role of the thyroid hormone during the perinatal period in the development of the mammalian circadian clock located in the hypothalamic suprachiasmatic nucleus (SCN). In this study, we examined the effects of the pharmacological insufficiency of thyroid hormone in the perinatal period on the wheel-running circadian rhythm in the adulthood as well as the gene expression in the neonatal SCN of mice. The pregnant mice were given antithyroid agents from ED17 to 14th day after the delivery and their offspring was normally grown and treated as congenic hypothyroid (CH) mice. CH mice at 8 wk ages showed the shorter period in the circadian behavioral rhythm in the constant darkness compared with control mice. The period of Per2::luciferase reporter rhythm in the explant SCN culture was shorter in CH mice than that from control mice. The mRNA expression of Sox2 gene, which is known to be critical for the normal development of the SCN clock, was upregulated in CH mice. Furthermore, the daily administration of thyroxin all ameliorated the phenotype in CH mice. These results impact the important roles of thyroid hormone in the circadian clock development.

Mitochondria are involved in various cellular functions such as energy production and apoptosis, and their dysfunction has been regarded as one of the causes of metabolic disorders such as diabetes. We recently identified the mitochondrial protein p13 with a molecular weight of 13 kDa based on the decreased expression in pancreatic islets of Langerhans in type II diabetic mice, and have been conducting research using systemically deficient mice (p13-/- mice). Here, we investigated the role of p13 in white adipose tissue (WAT) pathophysiology via multiscale omics analyses in vivo and in vitro. First, we measured the weight of various tissues in the whole body and found that the weight of white adipose tissue was markedly decreased in p13-/- mice. Moreover, as a result of comprehensive histochemical analysis, selective and marked abnormalities were observed in p13-/- WAT compared to the other tissues, and especially, a marked reduction in the size of adipocytes and lipid droplets were observed. Besides, RNA-seq and qRT-PCR analyses revealed decreases in lipid synthesis genes and increases in lipid degradation genes in p13-/- WAT, suggesting the role of p13 in lipid metabolism. In contrast, there was no significant difference in adipose differentiation marker genes, and we also confirmed that p13 deficiency did not affect adipose differentiation in an adipocyte differentiation model using mouse embryonic fibroblasts (MEFs). Serum parameter analysis revealed high levels of total ketone bodies and low levels of blood glucose, lipase, triglycerides, and HDL-C. Besides, p13-/- mice had lower steady-state insulin levels and lower levels of insulin-antagonizing corticosterone. Taken together, the present results suggest that WAT is markedly decreased in p13-/- mice due to abnormal hormone homeostasis, and that endogenous p13 may play an important role in regulating hormon release and lipid metabolism.

Objective: Sphingosine 1-phosphate (S1P) is implicated in brown adipose tissue (BAT) formation and energy consumption; however, the mechanistic role of sphingolipids, including S1P, in BAT remains unclear. Here, we sought to elucidate the physiological significance of sphingolipids in BAT.

Methods: Global sphingosine kinase 1 (SphK1)-deficient mice and their wild-type littermates were housed at both room temperature and cold environment to capture the physiological phenotypes. Subsequently, qRT-PCR, immunostaining, immunoblotting, and determinations of S1P and triglyceride were performed in isolated BAT and cultured brown adipocytes.

Results: In BAT, SphK1 was localized largely in the lysosomes of brown adipocytes, and SphK1 and its product S1P levels were upregulated in cold-activated BAT. Genetic deletion of Sphk1 resulted in a reduced number of brown adipocyte lysosomes, which was accompanied by impairment of lysosomal functions, including proteolytic activity and motility. Interestingly, Sphk1^–/– mice exhibited mild hypothermia and greater triglyceride content with larger lipid droplets dominating in the brown adipocytes. Triglyceride accumulation in brown adipocytes induced by the lysosome inhibitor chloroquine was blunted in Sphk1^–/– mice compared to wild-type mice, suggesting a reduced lysosome-mediated lipolysis in Sphk1^–/– mice.

Conclusion: Our results indicate a novel role of SphK1 in lysosomal integrity, which is required for lipolysis and thermogenesis in BAT.

Multiple drug resistance, a phenomenon in which cancer cells that acquire resistance to one type of anticancer drug are also resistant to several other drugs, often quite different in both structure and mechanism of action, has been studied. Our previous studies have identified a novel oxaliplatin resistance factor by analyzing heat shock protein 70 (Hsp70) interacting proteins. Hsp70, a stress response molecule, has been reported to be involved in the resistance to several anticancer drugs, including oxaliplatin. This study analyzed Hsp70-interacting proteins and then identified novel molecules that contribute to multidrug resistance. To identify Hsp70 interactors critical for oxaliplatin, 5-fluorouracil, paclitaxel, and irinotecan resistance in human gastric cancer cells, OCUM-2M, we performed mass spectrometry-based proteomic analysis using affinity purification with anti-Hsp70 antibodies. This led to the identification of six Hsp70 interactors common to the four resistant cell lines. These six candidates were then subjected to RNAi screening to assess drug sensitivity. Two candidates contributed to cell proliferation, while the other four candidates increased sensitivity to anticancer drugs. These results suggest that Hsp70 interactors cause multiple drug resistance.

Given the involvement of H₂S, a gaseous mediator, in multiple myeloma (MM) cell proliferation and survival, we evaluated effects of benserazide and L-carbidopa, aromatic L-amino acid decarboxylase (AADC) inhibitors capable of inhibiting cystathionine-β-synthase (CBS), an H₂S-forming enzyme, on the viability of human MM-derived KMS-11 and bortezomib (BTZ)-resistant KMS-11/BTZ cells. Three different H₂S-forming enzymes, i.e. CBS, cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), were comparably expressed in KMS-11 and KMS-11/BTZ cells. Application of BTZ at 10 nM for 24 h upregulated CBS, but not CSE or 3-MST, in KMS-11 cells. Benserazide and L-carbidopa, as well as aminooxyacetic acid, a well-known CBS inhibitor, markedly reduced viability of both MM cells, an effect mimicked by D-carbidopa that inhibited CBS, but not AADC. CSE and 3-MST inhibitors had relatively weak and little anti-MM effects, respectively. The cytotoxic effect of L-carbidopa was reversed by Na₂S, an H₂S donor. Benserazide and L- and D-carbidopa reduced phosphorylation of NF-κB p65 in KMS-11 cells. Our data suggest that the CBS/H₂S/NF-κB pathway is involved in the survival of BTZ-sensitive and -resistant MM, and that D-carbidopa, an inhibitor of CBS, but not AADC, would be useful to treat BTZ-resistant MM.

Camptothecin derivatives, such as CPT-11 (irinotecan), possess potent antitumor properties but are often hampered by their hydrophobic nature, leading to severe diarrhea as a common adverse effect. To address this challenge, we designed and synthesized novel highly hydrophilic camptothecin derivatives by conjugating SN38 with branched glycerol trimer (SN38-BGL). This unique strategy aimed to enhance the therapeutic benefits while minimizing the adverse effects. In murine xenograft models of human lung cancer, SN38-BGLs exhibited comparable or slightly superior tumor-suppressing efficacy compared to CPT-11 without the onset of early or late diarrhea. Additionally, histological analysis revealed that SN38-BGL treatment resulted in longer villi in the jejunum and ileum compared to CPT-11, indicating that SN38-BGL is less harmful. Digestion by liver microsome ex vivo did not yield SN38 but a few other molecules, suggesting possible involvement of other active metabolites than SN38. Our findings suggest that SN38-BGLs represent a promising class of hydrophilic camptothecin derivatives with the potential to mitigate severe diarrhea while maintaining antitumor efficacy, offering new prospects for pharmacological interventions.

【INTRODUCTION】Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer. Since TNBC exhibits a significantly lower 5-year survival rate than other subtypes, development of novel therapeutic strategies for TNBC is urgently needed. Tumor suppressor gene Cylindromatosis (CYLD) is associated with acquisition of malignant traits in various malignant tumors as a negative regulator for intracellular signals, such as nuclear factor-κB (NF-κB) signaling. The aim of this study was to clarify the clinical and biological significance of reduced CYLD expression in TNBC and to explore novel pharmacotherapy CYLD-negative poor-prognosis TNBC patients.

【METHODS】We knocked-downed CYLD expression by CYLD-specific siRNA in TNBC cells (MDA-MB-231 cells), and determined the therapeutic effects of anti-tumor drugs by evaluating malignant characteristics, such as, migration ability and anticancer drug sensitivity.  

【RESULTS】In CYLD knock-downed TNBC cells, cell migration and drug resistance for epirubicin and 5-FU, standard drugs for TNBC, were promoted via hyper-activation of NF-kB signaling. Indeed, NF-kB inhibitor (BAY11-7085) significantly suppressed the cell migration and cell viability caused by CYLD knock-down. Furthermore, therapeutic effects of clinically approved NF-kB inhibitors, such as, denosumab and bortezomib, were evaluated in CYLD knock-downed TNBC cells. In addition to the therapeutic effect of denosumab on cell migration, interestingly, bortezomib showed significant inhibitory effect on both cell viability and cell migration.

【DISCUSSION】Loss of CYLD expression enhanced the cell migration and drug resistance through hyper-activation of NF-kB signaling. Both clinically approved NF-kB inhibitors exhibited therapeutic effects, in particular, bortezomib might be more effective for TNBC cells with loss of CYLD expression by suppressing both cell viability and cell migration.

【CONCLUSION】Pharmacotherapy targeting NF-kB signaling may have potential to be novel therapeutic strategy for poor prognosis TNBC patients with loss of CYLD expression.

Clioquinol (Quinoform) was extensively administered to treat indigestion and diarrhea in the mid-1900s. However, it was withdrawn from the market in Japan because its use was epidemiologically linked to an increase in the incidence of subacute myelo-optic neuropathy (SMON). SMON is characterized by the subacute onset of sensory and motor disturbances in the lower extremities with occasional visual impairments, which are preceded by abdominal symptoms. The underlying mechanisms of clioquinol toxicity, however, have not been elucidated in detail. We previously reported that clioquinol induced oxidation of the copper chaperone ATOX1, leading to the impairment of the functional maturation of a copper-dependent enzyme dopamine-β-hydroxylase and the inhibition of noradrenaline biosynthesis. Effects of clioquinol on expression levels of other copper-related proteins were investigated. Quantitative PCR demonstrated that clioquinol decreased mRNA levels of SCO1 and SCO2, copper chaperones for mitochondrial respiratory chain complex IV (cytochrome c oxidase), and of COX18, a membrane insertase of a complex IV component. In clioquinol-treated cells, the assembly and activity of complex IV were suppressed. Thus, clioquinol was suggested to suppress ATP synthesis through this pathway, leading to neuronal cell death.

Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthase (NOS), thus BH4 might play a key role for maintaining endothelial functions. Here we examined whether hypoxia affects intracellular BH4 levels under inflammatory cytokine-stimulated human umbilical vein endothelial cells (HUVEC). IFN-gamma and TNF-alpha (each 15 ng/mL) markedly increased BH4 levels after incubation for 24h. Hypoxia (3%, O₂) significantly caused further increase of the BH4 levels than those in normoxia. Hypoxia also caused decreasing levels of BH2, which inhibits NOS activity and causes production of superoxide anion. Contrary to the increased BH4 levels, hypoxia reduced mRNA expression of GTP cyclohydrolase 1 (GCH1), the rate limiting enzyme in BH4 biosynthesis. As to the increased intracellular BH4 levels, we considered two possibilities for the reason: (1) elevated activity of dihydrofolate reductase, which is able to reduce BH2 to BH4; (2) turn back of once released BH4 into cells by autocrine or paracrine manner, since degradation of BH4 might less occur under hypoxic condition. The decrease of GCH1 mRNA expression suggested the presence of a feedback mechanism in BH4 biosynthesis other than the feedback mediated by GTP cyclohydrolase 1 feedback regulatory protein.

Corosolic acid (CRA) is a natural compound extracted from leaves of Banaba and exerts anti-inflammatory, anti-diabetic, anti-hyperlipidemic, anti-oxidant, and anti-tumor effects. We have recently demonstrated that CRA inhibits the expression of STAT3 and ameliorates the development of pulmonary arterial hypertension (PAH). In the present study, we focused on platelet-derived growth factor receptor (PDGFR) signaling, which enhances in PAH patients, and examined the downregulating mechanism of STAT3 expression by CRA. The expression level of PDGFRβ was higher in pulmonary arterial smooth muscle cells (PASMCs) from idiopathic pulmonary arterial hypertension (IPAH) patients than in normal PASMCs. Increased expression of PDGFRβ in IPAH-PASMCs was reduced by the treatment with CRA in a time-dependent manner. The expression of two downstream pathways of PDGFRβ signaling, STAT3 and NF-κB, was also upregulated in IPAH-PASMCs, which was attenuated by CRA. In addition, the phosphorylation levels of PDGFRβ and STAT3 were reduced by CRA. The excessive proliferation and migration of IPAH-PASMCs following PDGF stimulation were inhibited by CRA. In conclusion, CRA inhibits the development of PAH via the downregulation of PDGFRβ-STAT3 and PDGFRβ-NF-κB signaling pathways.

Yes-associated protein 1 (YAP), a transcriptional co-activator, is known to regulate cell growth and organ size. We have shown previously that YAP is activated in response to acute pressure overload (PO), and that YAP cardiac-specific heterozygous knockout (YAPchKO) mice are suppressed adaptive cardiac hypertrophy with inhibition of GLUT1 upregulation during acute PO. Furthermore, we found that YAP promotes glycolysis by upregulating GLUT1 in cultured rat ventricular myocytes. Glycolysis is intimately involved in cell growth. Thus, we examined whether AAV-mediated GLUT1 overexpression rescues adaptive cardiac hypertrophy during acute PO in YAPchKO mice. Wild-type (WT) or YAPchKO mice were injected with AAV-control or AAV-GLUT. After 14 days, these mice were subjected to sham or transverse artic constriction (TAC) for 7 days. TAC induced adaptive cardiac hypertrophy in WT mice injected with both AAVs. In contrast, left ventricular (LV) dysfunction, LV dilation, and inhibition of cardiac hypertrophy were observed in YAPchKO mice injected with AAV-control after TAC. AAV-GLUT1 injection successfully rescued GLUT1 upregulation, LV function, and cardiac hypertrophy in YAPchKO mice after TAC. These results suggest that YAP-induced upregulation of GLUT1 plays a critical role in promoting adaptive cardiac hypertrophy during acute PO.

Background: The prevalence of atrial fibrillation (AF), particularly associated with obesity, is on the rise worldwide. Ca²⁺/calmodulin-dependent protein kinase (CaMKII) is activated in the advanced condition of atrial remodeling. However, it is unclear whether CaMKII is activated and affects vulnerability to AF in the early phase of obesity. In this study, we examined the involvement of CaMKII in the inducibility and duration of AF in the early stage of diet-induced obese mice.

Methods: Mice were fed a normal chow diet (NCD) or high-fat diet (HFD). Following diet-loading for 2 weeks, HFD-fed mice were administrated CaMKII inhibitor. Induction of AF was performed by transesophageal atrial burst pacing. Furthermore, we evaluated the expression of CaMKII, blood pressure, and atrial fibrosis.

Results: HFD-fed mice increased the inducibility of AF compared to NCD mice. In addition, treatment with the CaMKII inhibitor in HFD-fed mice reduced the inducibility of AF. Expression of phosphorylated CaMKII is increased in HFD-fed mice. Inhibition of CaMKII didn’t have effects on blood pressure, and fibrosis.

Conclusion: Inhibition of CaMKII reduced the inducibility of AF in the early phase of obesity without affecting atrial structural remodeling, suggesting that CaMKII is a factor that contributes to AF from the early stage of obesity.

Diabetic cardiomyopathy (DMCM) is characterized by an early left ventricular (LV) diastolic dysfunction and subsequent progression to systolic dysfunction. In this study, we hypothesized the downregulation of cardioprotective factors is involved in the pathogenesis of DMCM. We aimed to elucidate the mechanisms underlying the DMCM development. In the streptozotocin (STZ)-induced type 1 diabetic model mice 4 weeks after STZ injection (STZ-4W), diastolic function was impaired without systolic dysfunction. Counter to expectations, the serum levels of neuregulin-1 (NRG1) were significantly up-regulated in the STZ-4W group compared to the control group. NRG1 expression was increased in the ventricle, kidney, and liver in the STZ-4W group. In the ventricles, NRG1 was localized in the vascular endothelial cells, endocardium, and epicardium. To clarify the physiological role of NRG1, trastuzumab (TRZ), an antibody against NRG1 receptor ErbB2 (HER2), was administered to mice. The systolic function, T-tubule structure, and the accumulation of Ca_V1.2 at the junctional structure were significantly impaired in the TRZ-injected STZ-4W mice compared to STZ-4W mice. These results suggest that the compensatory up-regulation of NRG1-ErbB signaling contributes to maintaining the LV systolic function in the early stage of DMCM.

In biology, embryology has explained how multiple cell types arise from a fertilized egg and organs are formed. In the following chapter, we seek to explain the mechanisms of how organs from multiple cells are maintained in a homeostatic state. Understanding this state is essential for maintaining the health of us.

In general, organs consist of two major groups of cells: parenchymal cells and stromal cells. The properties of organs have been understood through the function of parenchymal cells. For example, hepatocytes are studied when investigating the function of the liver, and myocytes are studied as well. The interstitium, on the other hand, has been regarded as a scaffold for the parenchyma and as similar independent of organ origin. However, we focused on mesenchymal stromal cells (MSCs) for understanding organs.

In this study, we performed a comparative analysis of MSCs over six organs. We revealed the organ-specific character of MSCs and identified a muscle-maintaining functional molecule whose expression is reduced by aging. Its knockout mice exhibited a loss of muscle mass and muscle strength at a young age. The concept that MSCs maintain organ function, as demonstrated by this study, can be extended to other organs. In this presentation, we also would like to discuss the possibilities for future research development from a new perspective of 'understanding organs from the interstitium'.

Aims: It has been suggested that the urinary bladder has a physiological role for absorbing water, but there were few studies published. In contrast, the bladder mucosa plays the role of a barrier mechanism that does not allow urine to penetrate. In this study, we aimed to investigate mucosal permeability of rat urinary bladder by using newly established bladder “inside-out” preparation. 

Methods: Female adult Sprague-Dawley rats were used. Using isoflurane anesthesia rats were sacrificed and whole bladder was isolated. This whole bladder was used as normal (N=12) and inside-out (N=11) samples, and inside-out sample was reversed the bladder from the bladder top. For monitoring of intravesical pressure and instillation of Krebs solution, a catheter (PE-50) was inserted through the urethra and fixed with surgical suture at the bladder neck. The bladder was fixed vertically in a 30 ml organ bath with Krebs solution gassed with 5% CO₂ and 95% O₂ at 37℃. The bladder was instilled with Krebs solution at a rate of 6 ml/h. until the intravesical pressure reached 30 cmH₂O. The bladder was kept under an isovolumetric condition and allowed to stabilize for 5 min (approximately around 15-20 cmH₂O), and then high K⁺ (KCl: 50 mM) and acetylcholine (ACh: 10 μM) were added into the organ bath, and the intravesical pressure was recorded.

Results: In the normal bladder samples, intravesical pressure was remarkably increased with KCl (42.4 ± 3.3 cmH₂O) and ACh (33.3 ± 1.9 cmH₂O). In contrast in the inside-out bladder samples, the intravesical pressure was only slightly increased with KCl (1.0 ± 0.3 cmH₂O) and ACh (1.8 ± 0.2 cmH₂O). 

Conclusions: Bladder normal samples showed remarkable increased responses of the intravesical pressure to KCl and ACh, but inside-out samples did not show such responses. The present study revealed that the bladder detrusor smooth muscle potentially has water (urine) permeability, whereas bladder mucosa has robust barrier mechanisms for preventing absorb water (urine) in at least normal rat.

Malignant hyperthermia (MH) is a life-threatening genetic disorder in which general anesthetics cause massive Ca²⁺release from the sarcoplasmic reticulum in skeletal muscle. While most MH mutations have been found in the ryanodine receptor type 1 (RyR1), other genes are also associated with MH. We have recently identified several novel mutations in CACNA1S, the α1S subunit of the dihydropyridine receptor, from MH-susceptible patients. In this study, we performed a functional analysis of MH mutants using the reconstituted depolarization-induced Ca²⁺ release (DICR) platform. HEK293 cells expressing RyR1 were infected with baculovirus containing genes essential for DICR (CACNA1S, β1a, JP2, Stac3, and Kir2.1). DICR was induced by depolarization with different concentrations of potassium (K⁺) solutions. Cells expressing two CACNA1S mutants showed greater sensitivity to K⁺ than WT cells. Consistently, charge movements were shifted to more hyperpolarizing potentials. These results suggest that CACNA1S MH mutations shift the DICR to a more hyperpolarizing potential. The two mutant cells showed greater sensitivity to caffeine than WT cells. Interestingly, the enhanced caffeine sensitivity was abolished by hyperpolarizing the resting membrane potential or by removing Stac3. Possible mechanisms of MH pathogenesis are discussed.

We have been searching for mechanism to induce smooth muscle contraction migration that involved actin remodeling and associated with phosphorylation of Myosin light chain (MLC) of smooth muscle myosin. We report that Spingosylphosphorylcholine (SPC) stimulates ATPase activity of vascular smooth muscle (A7r5) cell and increased migration of A7r5 cells and enhanced appearance magnapodia consisting of actin membrane structure. After SPC stimulation, mitogen-activated protein kinases (MAPKs),inculuding p38 MAPK (p38) and p42/44 MAPK (p42/44) were found to phosphorylated. Migration of cells toward SPC was reduced in the presence of SB-203580, an inhibitor of p38, but not PD-98059, an inhibitor of p42/44, Pertussis toxin (PTX), aGi prptein inhibitor, induced an inhibitory effect on p38 phosphorylation and A7r5 cells migration. Myosin light chain (MLC) phosphorylation occurred after SPC stimulation with or without pretreatment with SB-203580 or PTX. The MLC kinase inhibitor ML-7 and the Rho kinase inhibitor Y-27632 inhibited MLC phosphorylation but only partially inhibited SPC-directed miglation. Complete inhibition was achieved with the addition of SB-203580. After SPC 1 mM stimulation, actin cytoskeleton formed thick bundles of actin filaments around the periphery of cells, and the cells were surrounded by elongated a– actin and b- actin, but magnapodia consisited exclusively of –actin. Such a remodeling of actin was reversed by addition of SB-203580 and PTX, but not ML-7 or Y-27632. Taken together, our biochemical and morphorogical data confirmed the regulation of actin remodeling and suggest that A7r5 cells migrate toward SPC, not only by an MLC phosphorylation-dependent pathway, but also by an MLC phosphorylation-independent pathway.

We have previously reported that noradrenaline (NAd) inhibited mouse colonic motility via the α1A adrenergic receptor (α1A AR)-small conductance Ca²⁺-activated K⁺ (SK) channel signal pathway in PDGFRα⁺ cells. In the present study, we studied the effects of NAd and pituitary adenylate cyclase-activating polypeptide (PACAP) on contractile and electrical activities of circular smooth muscle cells (CSMCs) in the human colon. NAd inhibited colonic contractions through binding to α1A AR. PACAP and maxadilan, a PACAP type 1 receptor (PAC1) agonist, also inhibited colonic contractions. The inhibitory effects of these drugs were sensitive to apamin, a blocker of SK channel. However, the latency of the responses to PACAP or maxadilan were significantly longer than NAd. Similar latency of the responses was also observed when we recorded apamin-sensitive membrane hyperpolarization evoked by NAd and maxadilan in colonic CSMCs intracellularly. These results suggest that PDGFRα⁺ cells integrate inhibitory inputs from NAd and PACAP, leading to the activation of G protein coupled receptor-SK channel signal pathway in the human colon. NAd and PACAP may work together with different inhibitory time course to regulate colonic contractility during sustained stress.

Purpose and Method: Nilotinib has been reported to clinically cause QTc prolongation. We observed that nilotinib at 1 µg/mL induced repolarization delay showing early afterdepolarization (EAD) with electrical alternans under electrical pacing in human-induced pluripotent stem cell-derived cardiomyocytes sheets.  To characterize the alternans, we adopted field potential (FP), Ca²⁺ imaging, and motion vector analyses.

Results: Nilotinib induced EADs in every excitation for the most part, but excitation without EAD showed up intermittently, and EADs were sometimes terminated by electrical pacing via the MEA system, which depended on the pacing cycle lengths. Alternate phenomena of FP duration (FPD) were observed with and without EAD, which also caused alternans in the conduction speed, contraction velocity and peak amplitude of Ca peak along with Ca transient duration. During electrical pacing, electrical excitations with EADs induced conduction delays upon the next one without EAD. Transient loss of EAD increased the conduction speed, contraction velocity and Ca peak amplitude of the cell sheets upon the next electrical excitation, indicating that the shorter FPD enhanced the recovery of Ca and Na channels from their inactivation state. Alternans was not observed when only action potentials without EAD or with EAD were sustained.

Conclusion: Intermittent loss of EAD would enhance electrical, calcium, and mechanical alternans of the next beat in the in vivo heart.

Introduction: While morphine has been used for treatment-resistant dyspnea in patients with end-stage heart failure, its information on in vivo cardiovascular profile remains limited. 

Methods: Morphine was intravenously administered to the halothane-anesthetized dogs (n=4) in escalating doses of 0.1 followed by 1 mg/kg/10 min with respective 20 min observation periods under the monitoring of cardiohemodynamic and electrophysiological variables.

Results: The low dose hardly altered cardiovascular variables. The high dose reduced preload and afterload to the left ventricle for 5-15 min after the start of infusion, then decreased the left ventricular contractility along with the mean blood pressure for 10-30 min, and next suppressed the heart rate for 15-30 min. Morphine slowed the atrioventricular conduction and ventricular late repolarization, and prolonged the ventricular effective refractory period without altering the intraventricular conduction or ventricular early repolarization. A reverse-frequency-dependent delay of ventricular repolarization was confirmed. 

Conclusion: Morphine could directly dilate the resistance and capacitance vessels, whereas it would attenuate the adrenergic tone followed by an increase of vagal tone. The reverse-frequency-dependent delay of ventricular repolarization by morphine along with the prolongation of late repolarization suggests hERG K⁺ channel inhibition in vivo in spite of its large IC₅₀ value for hERG K⁺ channel in vitro (>1 mM), indicating the presence of indirect mechanisms for its inhibition.

Background

Medetomidine (MED) is used for animal experiment as mixed anesthesia (Medetomidine-Midazolam-Butorphanol: MMB). On the other hand, Dexmedetomidine (DEX), its enantiomer, is used for human sedation in the intensive care situation. It is unknown whether or not they may accelerate thrombogenesis via platelets α₂-receptors stimulation and we evaluate their effect on platelet aggregation and thrombotic vessel occlusion using animal model.

Method

ICR mice (6-month-old males) were used for the experiments.

1) Blood was collected under pentobarbital anesthesia (80　mg/kg-i.p.), and collagen　(0.8 µg/mL)-induced platelet aggregation was measured under DEX or MED supplementation.

2) Mice were injected with 1 µg/kg DEX or 0.75 mg/kg MED i.p., before pentobarbital 80mg/kg anesthesia, and p­latelet aggregation was evaluated similarly as described above.

3) Vascular occlusion time was measured by ferric chloride (FeCl3)-induced thrombosis models under MMB or pentobarbital anesthesia.

Results

1) Platelet aggregation was enhanced by higher concentrations at 10 ng/mL DEX or 100 ng/mL MED supplement. The lower concentrations under 1 ng/mL DEX or 10 ng/mL MED did not affect aggregation.

2) 1 µg/kg MED i.p. enhanced platelet aggregation, while 0.75 mg/kg DEX did not.

3) Thrombotic occlusion time of mice femoral artery was shortened in MMB-anesthetized animals in comparison with pentobarbital-anesthetized ones.

Discussion

Clinical doses of DEX seem not affect thrombogenicity, while MED in MMB-animal anesthesia was revealed to enhance it.

Introduction: Peramivir, anti-influenza drug, was reported to induce QT prolongation in some phase III studies. Such the effect on cardiac repolarization may have some potential to induce lethal ventricular arrhythmia, torsade de pointes (TdP). Accordingly, we characterized the cardiac safety pharmacological profile of peramivir by assessing ventricular and atrial electrophysiological actions in addition to cardiohemodynamic effects. 

Methods: Peramivir in doses of 1 and 10 mg/kg/10 min (subtherapeutic and clinically-relevant doses, respectively) was intravenously administered to isoflurane-anesthetized dogs under the monitoring of electropharmacological variables (n=4). 

Results: Peramivir decreased total peripheral vascular resistance, whereas it increased cardiac output and kept mean blood pressure at the basal control level. Meanwhile, peramivir prolonged QT interval/QTcV and T_peak-T_end without altering J-T_peakc or intra-atrial, atrioventricular as well as intra-ventricular conduction. Peramivir also delayed ventricular repolarization and increased refractoriness at the same site, and tended to prolong terminal repolarization period. Peramivir prolonged atrial effective refractory period, of which extent was smaller than those of existing anti-atrial fibrillatory drugs; moreover, its atrial selectivity was lower among the drugs. 

Conclusion: The clinical dose exposure of peramivir can develop a substrate for inducing TdP, but may not provide its trigger, suggesting that it would have the least potential for the onset of TdP.

Developmental lymphatic vascular defects are a major cause of fetal nuchal edema, which is characterized by a subcutaneous accumulation of extracellular fluid in the fetal neck. Fetal nuchal edema is visualized as increased nuchal translucency by ultrasonography. It is found to be associated with chromosomal anomalies in about ten percent of clinical cases, whereas causative genes have not been elucidated in many clinical cases. In this study, we aimed to identify novel causative genes of fetal nuchal edema and lymphatic vascular defects through N-Ethyl-N-nitrosourea-induced mutagenesis screening in mice. This screening detected mouse embryos exhibiting both fetal nuchal edema and lymphatic vascular defect. Exome sequencing of the genomic DNA from these embryos revealed the candidate causative genes for fetal nuchal edema. Knockout of a candidate causative gene by CRISPR/Cas9 system in mice induced fetal nuchal edema and lymphatic vascular defects. Gene silencing by small interfering RNA in culture inhibited proliferation of lymphatic endothelial cells  and also reduced mRNA expression of HMGCR and FASN encoding 3-hydroxy-3-methylglutaryl-coA reductase and fatty acid synthase, respectively. In conclusion, we identified a novel gene that regulates both proliferation and lipid metabolism in lymphatic endothelial cells.

Crohn’s disease, a chronic and recurrent gastrointestinal disease, frequently causes intestinal fibrosis. Transient receptor potential melastatin 2 (TRPM2) belonging to TRP channel family is activated by reactive oxygen species. This study investigated the role of TRPM2 in acute colitis and chronic colitis-associated fibrosis progression. Acute colitis and chronic colitis-associated fibrosis were induced in TRPM2-deficient (KO) and wild-type (WT) mice through single and repeated intrarectal injections of trinitrobenzene sulfonic acid (TNBS). Bone marrow-derived macrophages (BMDMs) were created by M-CSF stimulation. In WT, a single TNBS injection induced acute colitis and upregulated inflammatory cytokines/chemokines, Th1 and Th17-related cytokines, and their transcription factors. In contrast, repeated TNBS injections induced chronic colitis-associated fibrosis and upregulation of fibrogenic factors, Th2-related cytokines, and its transcription factor. However, these increases were considerably suppressed in KO. Treating BMDMs with H₂O₂ increased inflammatory, Th1 and Th17-related cytokines expression, and JNK and ERK phosphorylation, but these responses were significantly less in KO than those in WT. These finding suggest that TRPM2 contributes to acute colitis progression via Th1/Th17-mediated immune responses. Furthermore, TRPM2 may be directly involved in colitis-associated fibrosis induction, likely due to the regulation of Th2/TGF-β1-mediated fibrogenesis in addition to a consequence of acute colitis progression.

Chronic liver disease progresses to cirrhosis and often develops into hepatocarcinoma. The final stage of the disease is fatal liver failure, and death from liver cancer is common. The development of treatments for chronic liver diseases has been a priority to prevent the progression to cirrhosis and the development of hepatocarcinoma. In fact, there are no approved drugs for nonalcoholic fatty liver disease (NAFLD) which represents the most common cause of chronic liver disease in many countries. It is now well-established that in NAFLD, mortality is highest with advanced fibrosis. Therefore, there is an urgent need to develop therapies that inhibit or ameliorate liver fibrosis. 

Liver fibrosis is common pathological condition of chronic liver diseases caused by scarring due to excessive production and accumulation of extracellular matrix. Hepatic stellate cells, which are activated by liver injury and are a major source of collagen and other extracellular matrix, have attracted attention as a therapeutic target for liver fibrosis. We conducted a drug screening using human-derived hepatic stellate cells and found that several proteasome inhibitors including bortezomib and carfilzomib suppressed the function and proliferation of hepatic stellate cells. Furthermore, a 2^nd generation proteasome inhibitor, carfilzomib also suppressed cell proliferation and decreased the production of extracellular matrix including Collagen and Timp1 in hepatic stellate cells isolated from mouse liver. Currently, CCL4-induced liver fibrosis is being studied by using a mouse model. 

Inhibiting the function and proliferation of hepatic stellate cells with proteasome inhibitors, especially carfilzomib, could potentially suppress and improve liver fibrosis and provide a new treatment widely applicable to chronic liver disease.

Objective: In recent years, most prevalent liver disease is metabolism dysfunction-associated steatotic liver disease (MASLD). MASLD-associated inflammation develops steatohepatitis, which further progresses to liver cirrhosis and liver cancer. Inflammation is regulated by the interaction between the nervous system and the immune system. We reported that a neuropeptide, calcitonin gene-related peptide CGRP acts on the CGRP receptor, receptor activity modulating protein 1 (RAMP1), to suppress inflammation. The objective of the present study examined whether RAMP1 signaling contributed to the progression of liver inflammation in MASLD.

Methods and Results: Male RAMP1 deficient (RAMP1^–/–) or wild-type (WT) mice were fed a normal diet (ND) or HFD for 12 weeks. HFD-fed RAMP1^–/– mice showed heavier body weights than HFD-fed WT mice, which was associated with high levels of liver weights, ALT, total cholesterol, and glucose. HFD-fed RAMP1^–/– mice had higher gene expression levels related to fibrosis including alpha SMA and collagen 1a1 and to inflammation including TNF and IL-1beta than HFD-fed WT mice. Flow cytometry analysis revealed that both genotypes fed with HFD decreased Kupffer cells (KCs). HFD-fed WT mice showed increased monocyte-derived KCs and monocyte-derived macrophages, while HFD-fed RAMP1^–/– mice showed no changes in monocyte-derived KCs and macrophages.  

Conclusions:  These results suggested RAMP1 signaling deficiency enhanced diet-induced liver steatosis with liver inflammation and fibrosis.

【Introduction】 Resident macrophages within gastrointestinal muscularis layer (MMφ) are located near sympathetic nerves. However, the role of adrenergic signaling in regulating MMφ activation during inflammation remains unclear. This study aims to elucidate the the role of adrenergic receptors in MMφ activation.

【Methods】 The expression of adrenergic receptors mRNA in isolated MMφ was examined using RT-PCR. The J774.1 macrophage cell line was treated with LPS for 24 hours, either in the presence of adrenergic receptor agonists or antagonists. The production of nitric oxide (NO) was evaluated using the Griess method as an indicator for MMφ activation.

【Results】 MMφ expressed mRNA for α₁, α₂, β₁, and β₂ adrenergic receptors. Stimulation of J774.1 with LPS increased NO production. This increase in NO production was significantly reduced by both dobutamine and clenbuterol. Treatment with noradrenaline significantly decreased the LPS-induced NO production. Propranolol suppressed the inhibitory effect of noradrenaline on NO production. Additionally, both atenolol and butoxamine suppressed the noradrenaline-induced inhibitory effect on NO production.

【Conclusion】 Our results suggest that noradrenaline released from sympathetic nerves might suppress the inflammatory activation of MMφ via β₁ and β₂ adrenergic receptors.

The goal of antiviral treatment for chronic hepatitis B is to prevent disease progression by suppressing hepatitis activity; 48-week treatment with peg-IFN is used as first-line antiviral treatment for persistently infected individuals. However, because treatment efficacy is heterogeneous among individuals and treatment has side effects, it is desirable to be able to identify early which individuals would benefit from treatment. In this study, time series data on three serum biomarkers, HBV DNA, HBsAg and HBcrAg, which determine treatment response, were used to stratify the treatment efficacy of patients. First, a mathematical model describing the time-series dynamics of these biomarkers was constructed and parameters were estimated for each individual using non-linear mixed effects modeling. Clustering of patients based on these parameters showed that patients with high treatment efficacy were concentrated in a specific cluster. This cluster was characterized by lower biomarker levels at baseline compared to other clusters, with HBsAg and HBV DNA declining by more than 1 log10 during the first several weeks of treatment. The degree of decline in the amount of cccDNA remaining in hepatocytes was also greater. Therefore, a machine learning model was created to predict this cluster using random forest. The results showed that using both the initial biomarker levels at the start of treatment and the cumulative levels up to several weeks after treatment, it was possible to identify a group of patients with high treatment efficacy with sufficient accuracy. Other blood markers and patient background factors were also found to be associated with treatment response. Thus, dynamical systems phenotyping based on multivariable time-series biomarkers allows patient stratification and prediction of treatment efficacy. We expect that such a method could also be used to stratify patients with other diseases.

The tumor tissue environment is considered hypoxic condition. The hypoxic condition inhibits hypoxia inducible factor (HIF) degradation and HIFs are constitutively active through prolylhydrosylase deactivation. Furthermore, in tumor cells, the activation of HIF-1a is considered as a tumor exacerbation factor that promotes proliferation, metastasis, and resistance to therapy in tumor cells. On the other hand, in immune cells, HIF-1α is involved in enhancing the inflammatory and immune response, activating T cells, and then acting as a tumor suppressor. These controversial things are not clear in vivo models. Therefore, it is needed to reveal whether upregulating HIFs level is beneficial for cancer therapy or not.

we have used Lewis lung carcinoma (LLC) syngeneic tumor mouse models and used macrophage-specific VHL knockout mice. Macrophage were collected from transplanted mice tumor and performed RNA-seq analysis. We explored genes that were highly expressed in tumor suppress macrophages and found candidate genes regulated by HIF. These candidate genes were evaluated and confirmed to be secreted from macrophages and inhibit tumor growth in vivo. These results suggest that upregulation of HIF-1 in macrophage could contribute to inhibit tumor growth.

Thrombomodulin (TM) expressed on vascular endothelial cells plays an important role in activation of the anti-coagulant protein C pathway. TM interacts with multiple ligands and regulates several endothelial cell functions. Here, we found hornerin as a candidate protein interacting to TM on endothelial cell surface by mass spectrometry-based approach. It has reported that hornerin has similar features to filaggrin and implicates in the epidermal barrier function. However, the expression and role of hornerin in endothelial cells is still elusive.

In this study, we confirmed that hornerin protein and mRNA was expressed in endothelium of aorta and cultured endothelial cells by immunofluorescence staining, western blot, and RT-PCR. Lipopolysaccharide (LPS)-mediated sterile inflammation increased serum concentration of hornerin in mice, whereas reduced hornerin from cultured cell. Of note, TM knockdown cells emerged the reduction of hornerin on endothelial cell surface. Furthermore, HRNR knockdown impaired angiogenic tube formation of endothelial cells.

Our findings demonstrated that hornerin is expressed on vascular endothelial cells via the coordinating with TM. Therefore, endothelial hornerin might be an important component in the regulatory machinery of vascular inflammation and angiogenesis.

CD44 plays important roles in the tumor progression and has various isoforms, which are generated by the alternative splicing of CD44 mRNA. The mRNA of CD44 standard (CD44s) isoform is produced by constant region exons including the first five (1 to 5) and the last five (16 to 20). The mRNAs of CD44 variant (CD44v) isoform are produced by the assembling of variant exons (v1–v10) with the constant region exons of CD44s. CD44s and CD44v receive the post-translational modifications, such as N-glycosylation and O-glycosylation. Both CD44s and CD44v (pan-CD44) can attach to hyaluronic acid, which is important for cellular adhesion, homing, and motility. CD44v isoverexpressed in tumors and promotes tumor malignant progression through the binding to growth factors, and the acquisition of invasiveness, stemness, and drug resistance. These were mediated by the unique functions of the variant’s exon-encoded region. Our group have established anti-CD44 monoclonal antibodies (mAbs) using the Cell-Based Immunization and Screening (CBIS) method. We would like to introduce the isoform-specific mAbs against CD44 and their application including flow cytometry and immunohistochemistry.

Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is known to be sensitive to chemotherapy. However, 15-20% of patients experience relapse, and survival rates after relapse can drop to 5-10%. We isolated heptelidic acid (HA) from the probiotic Aspergillus oryzae and demonstrated HA exerted antitumor functions against several cancers. In this study, we assessed the antitumor effects of HA in B-ALL. HA exhibited cytotoxicity in two B-ALL cell lines and three pediatric B-ALL patient samples, while sparing CD34+ hematopoietic stem cells (HSCs). GAPDH activity was measured, as HA can bind to GAPDH. GAPDH activity of HSCs was lower than B-ALL cells. The cytotoxic effect of HA was associated with the reduction of GAPDH activity. Oral daily administration of HA prolonged the survival of the B-ALL patient derived xenograft (PDX) mice. HA demonstrated synergistic effects when combined with chemotherapeutics, especially vincristin (VCR). The combination therapy of HA and VCR resulted in improved survival outcomes in the B-ALL PDX model compared to single-agent treatments. HA treatment caused G2/M arrest, and this was accompanied by increased cleavage of PARP. Moreover, the cytotoxicity of HA was attenuated by RIPK1 inhibition. In conclusion, HA is cytotoxic in B-ALL cells by disrupting glycolysis through the inhibition of GAPDH. The therapeutic efficacy of HA is enhanced when combined with chemotherapeutic agents. The underlying mechanisms of HA involve the induction of RIPK-1 mediated programmed cell death. This is the first study to demonstrate HA as a novel therapeutic agent for B-ALL.

Breast cancer is the most common cancer type in woman worldwide. Recurrence of breast cancer is connected to poor prognosis, therefore novel treatments to prevent the recurrence are required. PDH generates acetyl-CoA from pyruvate and drives TCA cycle. PDH complex is composed of five subunits. In our previous study, knockdown of PDH-E1β subunit in breast cancer MDA-MB-231 cells reduced tumor formation in mice. In the present study, we used spheroid as an in vitro model to clarify the mechanism behind the tumor suppression in PDH-E1β KD cells. Wild type (WT) cells formed spheroid in three days, whereas PDH-E1β KD cells required seven days to form one. To investigate the effects of PDH-E1β on extracellular matrix (ECM) degradation, we examined the expression of MMPs, which are the main enzymes to degrade ECM. Expression of MMP19 and MMP28 were increased in PDH-E1β KD cells. Further, spheroid formation was rescued and formed in three days by the addition of MMP inhibitor. We have previously shown that CREB, a transcription factor, induces MMP1. Thus, we hypothesized that CREB is activated and induces MMP19 and MMP28 in PDH-E1β KD cells. Phosphorylation of CREB was increased in PDH-E1β KD cells, and PDH-E1β KD cells transfected with CREB siRNA also formed spheroid in three days. Knockdown of CREB decreased MMP19 but not MMP28 in PDH-E1β KD cells. Altogether, it is indicated that CREB induces the transcription of MMP19, and inhibit spheroid formation.

Autoimmune hepatitis (AIH) is a refractory inflammatory disease that causes progressive liver damage. While it has been suggested that regulatory T cells (Treg) have a significant role on the pathophysiology of AIH, the precise mechanism how AIH is controlled by Treg is still elusive.

We have previously demonstrated that nardilysin (NRDC), a member of the M16 family of metalloendopeptidase, is involved in several inflammatory diseases (e.g. rheumatoid arthritis, non-alcoholic fatty liver) via the activation of TNF-α. NRDC was also reported to be involved in antigen processing, suggesting that NRDC in immune cells may play a significant role in the pathogenesis of autoimmune diseases including AIH.

In this study, T cell-specific NRDC-deficient mice (CD4-CKO) and control mice (CONT) were intravenously injected with concanavalin A (ConA), which is a well-established model of acute AIH. Examination of liver histology and serum hepatic enzymes demonstrated that liver injury in CD4-CKO was significantly milder than that in CONT. Transcriptome analysis of splenic T cells revealed that the downstream genes of Foxp3, the master transcription factor of Tregs, were significantly altered in the CD4-CKO.

Consistently, the number of liver-infiltrated Treg was significantly increased in CD4CKO compared with CONT. Finally, naïve T cells isolated from CD4-CKO differentiate into Tregs more efficiently in vitro compared with CONT. These findings suggest that NRDC controls the pathogenesis of AIH via Treg.

While 5-Fluorouracil (5-FU) is the widely used chemotherapeutic agent, it often causes mucoenteritis with severe diarrhea, nausea, vomiting, and body weight loss. These conditions are related with impaired quality of life of patients with cancer. Glutamate (Glu) is a nonessential amino acid and the most important energy source in the small intestine. In this study, we aimed to evaluate the role of Glu on the 5-FU-induced mucoenteritis. Mucoenteritis was induced in male C57B/6N mice by repeated administration of 5-FU (50 mg/kg, i.p.). Glu was administered as a pretreatment for 7 days before the initial treatment of 5-FU. Disease severity was assessed by histological and physiological analysis. Moreover, cell proliferation, apoptosis, and intestinal permeability were assessed using immunohistochemistry. The effect of Glu on 5-FU-induced cell injury was also examined in IEC-6, rat intestinal epithelial cell line. The pretreatment with Glu significantly suppressed the histological changes, impairment of cell proliferation, and apoptosis by 5-FU. While the expression of excitatory amino acid transporters (EAAT) was decreased on the ileum tissue damaged by 5-FU, Glu treatment maintained a high expression level of EAAT. These results suggest that Glu prevents 5-FU-induced mucoenteritis via enhancement of Glu transporters. Thus, Glu administration may have protective effects on 5-FU-induced mucoenteritis.

Hepatic stellate cells (HSCs) are known to play a central role in liver fibrosis (LF), and considered to be a target for LF therapy. We have previously reported that caffeine (CAF) suppresses HSC activation via inhibition of adenosine receptors, while also observing that prostaglandin E₂ (PGE₂) facilitates the inhibitory effect of CAF on HSC activation. However, the mechanism remains to be elucidated. In the present study, we aimed to elucidate the molecular mechanism of the inhibitory effect of PGE₂ and CAF co-treatment. Despite sharing a common pathway for cAMP, intracellular cAMP levels showed no direct correlation with the inhibitory effect of PGE₂ and CAF co-treatment on HSC activation. RNA-seq analysis identified the transcription factor NRF2 as a candidate gene involved in the inhibitory effect of PGE₂ and CAF co-treatment on HSC activation. Reporter assays and immunostaining exhibited that the combination of PGE₂ and CAF resulted in a significant augmentation of NRF2 transcription activity and enhanced nuclear translocation of NRF2. These results suggest that PGE₂ facilitates the inhibitory effect of CAF on HSC activation by amplifying the transcriptional activity of NRF2.

Orphan G protein coupled receptor GPR35 is highly expressed in gastrointestinal tracts. We previously reported that GPR35 plays a protective role in the pathogenesis of colitis via promoting tissue repair and healing, but the detail mechanism is not fully understood. The present study investigated the protective role of GPR35 in the pathogenesis of dextran sulfate sodium (DSS)-induced colitis, especially in relation to epithelial barrier functions and inflammatory responses. Colitis was induced in male GPR35-deficient (GPR35KO) and wild-type (WT) mice by DSS treatment for 7 days. For intestinal epithelial barrier functions, mucus secretion and tight/adherence junction protein expression were examined. For inflammatory responses, cytokine expression was examined in bone marrow-derived macrophages (BMDM). DSS treatment produced severe colitis accompanied by body weight loss and diarrhea/bloody stool, but the severity was significantly aggravated in GPR35KO mice compared with WT mice. There is no difference in epithelial barrier functions between both mice. In contrast, LPS-induced upregulation of cytokine expression significantly enhanced in BMDM obtained from GPR35KO mice compared with WT mice. Further, lysophosphatidic acid, a GPR35 agonist, suppressed LPS-induced cytokine expression in BM from WT mice but not GPR35KO mice. These results suggest that GPR35 plays a protective role in the pathogenesis of DSS-induced colitis. This response may be mediated by attenuation of inflammatory responses but not regulating epithelial barrier functions.

Aim To investigate the influence of dosing time and the role of glucagon-like peptide-1 receptor (GLP-1r) in the central nervous system (CNS) on the hepatic clock-modulating effect of exendin-4 (Exe-4). Methods Male B6 mice and CNS-specific GLP-1r knockout mice were maintained under a 12 h/12 h light/dark cycle, with 4-h time restricted feeding (TRF) or daytime-feeding (DF). Exe-4 or vehicle was administered repeatedly at the beginning of the active phase (ZT12) or rest phase (ZT0) for 4 or 5 days. After the last dosing, liver samples were collected every 6 h for measuring mRNA expression levels of the clock genes. Results The DF induced a substantial phase shift of the hepatic clock, and Exe-4 administered at ZT12 almost completely inhibited this effect of DF. On the other hand, Exe-4 administered at ZT0 had a minimal effect on the DF-induced phase shift, but enhanced the amplitude of hepatic clock. Under the TRF condition, Exe-4 did not affect the phase of the hepatic circadian clocks regardless of the dosing time. In the CNS-specific GLP-1r knockout mice, the counteracting effect of Exe-4 on DF-induced phase shift disappeared. Conclusion These results indicate that the effect of Exe-4 on the hepatic clock is dosing time-dependent and mediated by GLP-1r in CNS.

“Disuse syndrome” is the declining state of physical and mental functions due to physical inactivity, and it became more familiar probrem for the elderly in fraility as well as for young people living in “hikikomori". Our laboratory was the first to discover that skeletal muscle atrophy induced memory deficit in mice and its responsible myokine, hemopexin (Nagase T, Tohda C. 2021). We also identified other myokine which transferred to the central nervous system to promote axon elongation (Kodani A, et al. 2019). These studies suggest a relationship between muscle atrophy and brain function and the involvement of myokines in the phenomenone. The aim of this study was to explore effective drugs for the muscle atrophy-induced memory deficit.

We focused on Schisandra chinensis (SC), because many reports indicated that SC extract and its constituents increased skeletal muscle mass and muscle strength in mice and also humans by oral treatment. However, no studies investigated effects of SC on myokine-mediated regulation for cognitive function.

This study examined the effect of intramuscular injection of SC extract on memory deficit in a mouse model of disuse syndrome. To identify SC extract-induced myokines, atrophied skeletal muscle was isolated from mice and treated by SCextract ex vivo. Several candidates were obtained on silver stained PAGE. Identification and functional contribution of those proteins to memory are under investigation. We clarify the potential of SC extract and its molecular mechanism for disuse-elicited cognitive deficit.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by deposition of Aβ. Our therapeutic strategy for AD is to regenerate neural circuits in the brain to recover memory function. We previously found diosgenin as a candidate drug for regenerating axons in the brain and improving memory impairment in AD model (5XFAD) mouse. In addition, diosgenin-driven axonal regeneration is mediated by changes in expression levels of several proteins (e.g. SPARC, Galectin-1) in neurons. In this study, we aimed to explore key candidate molecules that regulate the expression of these axonal regeneration-related proteins and clarify their contributions to axonal regeneration and memory recovery.

RNA-seq analysis revealed that 7-day treatment of diosgenin (1 μM) to hippocampal neurons (ddY mice, P7) drastically upregulated the expression of Rn7sk, one of the well-known transcription factors. Overexpression of Rn7sk significantly promoted axonal growth and upregulated the expression of SPARC in primary cultured hippocampal neurons, and also recovered memory deficits in 5XFAD mice. This study suggests that Rn7sk may be a responsible molecule for diosgenin-induced axonal regeneration, which proposes a novel therapeutic target for AD treatment.

Hepatic stellate cells (HSCs) are activated in response to liver injury and secrete huge amounts of collagen, the primary cause of liver fibrosis (LF). Thus, the regulation of trans-differentiation of HSCs, both activation of quiescent HSCs and reversion of activated HSCs, is crucial for therapeutic strategy for LF. However, few compounds have been reported to have such effects and no definitive therapy is available. Here, we elucidate the effect of DIF-1, a compound inhibiting HSC activation we previously reported, on activated HSC and LF mouse model. DIF-1 reduced the expression of type I collagen α 1 (Col1a1) and α-smooth muscle actin, markers of activated HSCs, even when treated after HSC activation. We further performed in silico analysis utilizing the relation between structural transition and HSC reversion effect of several DIF-1 analogs to identify molecular target of DIF-1. DIF-1 reduced the expression of activated HSC marker genes (Acta2, Col1a1, Pdgfrb), while it increased that of a quiescent HSC marker gene (Lrat) in thioacetamide-induced LF mouse model. Moreover, DIF-1 reduced the amount of collagen fiber in liver tissue. Taken together with our previous report, we propose that DIF-1 is a useful compound for LF treatment, acting on both quiescent and activated HSCs.

【Background and Purpose】

We are focusing on the veterinary clinical application of Ligilactobacillus animalis isolated from healthy canine intestinal microbiota. Our previous findings have indicated that oral administration of L. animalis significantly inhibited the development of atopic dermatitis in a mouse model. The objective of this presentation is to clarify the relevance of IL-10 production from dendritic cells in the suppression of skin allergies by L. animalis.

【Methods】

L. animalis was cultured to 1×10⁹ CFU/mL according to the established method (Cultured in MRS medium for 24 hours). A mouse-derived dendritic cell line (DC2.4 cells) was co-cultured with L. animalis viable bacteria (1×10⁸ CFU/mL) for 24 hours, and the levels of IL-10 and TNF-α in culture supernatant was measured by ELISA. Phosphorylation of p65, a key transcription factor in NF-κB signaling, in dendritic cells 24 hours after co-culture with L. animalis was also detected by Western blotting.

【Results and Discussion】

Co-culture with L. animalis significantly increased the production of IL-10 and TNF-α compared to the untreated group. p65-phosphorylation was also significantly enhanced by treatment of L. animalis compared to the untreated group. Previous studies demonstrated that there is a direct or indirect relationship between stimulation of NF-κB and IL-10 production. Our findings imply that pre-treatment of L. animalis activate the NF-κB signaling and promote the IL-10 production from dendritic cells, which resulted in the inhibition of allergic diseases.

Neovascular age-related macular degeneration (nAMD) is an ocular disease characterized by choroidal neovascularization (CNV). For nAMD treatment, the intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs is commonly used. However, it can be highly invasive and burdensome for nAMD patients. Arctigenin is a component of the herbal medicine burdock root and known to exhibit anti-tumor and vascular normalizing effects. Here, we investigated the effect of oral administration of arctigenin on CNV formation. The murine CNV model was created by laser photocoagulation and arctigenin at 100 mg/kg was orally administrated once a day. CNV area, vascular leakage and the endothelial cell proliferation were evaluated. Oral administration of arctigenin suppressed CNV formation and vascular leakage. In the CNV lesion, the number of proliferating endothelial cells was reduced in the arctigenin-treated group. Furthermore, the effects of arctigenin were confirmed using in vitro experimental systems using human retinal microvascular endothelial cells (HRMECs). Arctigenin at 30 µM attenuated VEGF-induced HRMECs proliferation and migration. These findings suggest that oral administration of arctigenin has beneficial effects on choroidal neovascularization of nAMD.

Pulmonary arterial hypertension (PAH) causes chronical increase in pulmonary artery pressure rises due to pulmonary vasoconstriction and vascular remodeling. Although several PAH drugs have been recently developed, no curative treatment has yet been achieved. Sustained elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) is closely associated to PAH pathogenesis such as enhanced contraction and excessive proliferation of pulmonary artery smooth muscle cells (PASMCs). [Ca²⁺]_cyt is regulated by membrane potentials which are partly regulated by voltage-dependent Cl^- channels. In the present study, the functional expression of voltage-dependent Cl^- channels (ClC family) was examined using PASMCs from normal subjects and idiopathic PAH (IPAH) patients. Expression analysis showed that ClC3 channel expression was increased in IPAH-PASMCs compared to normal-PASMCs. Swelling-activated Cl^- channel currents were larger in IPAH-PASMCs than in normal-PASMCs, which were attenuated by ClC3 siRNA. The growth of IPAH-PASMCs was inhibited by Cl^- channel blockers, niflumic acid and DIDS. ClC3 siRNA also decreased the proliferation of IPAH-PASMCs. In conclusion, the expression of ClC3 channels was upregulated in IPAH-PASMCs, resulting in excessive cell proliferation, which contributes to the pathogenesis of PAH.

The majority of peripheral serotonin (5-HT) is produced in the digestive tract and plays a crucial role in controlling intestinal peristalsis and energy metabolism. The serotonin transporter (SERT), expressed in intestinal epithelial cells, regulates the available amount of 5-HT. Recently, a functional CpG site (CpG3) was identified in the SERT gene promoter region, and its expression may be subject to epigenetic regulation. Coffee is the most widely consumed beverage worldwide and exhibits a U- or J-shaped relationship with the risk of a variety of diseases. While caffeine has traditionally been recognized as a key component of coffee, the physiological activity of dietary polyphenols, which can alter DNA methylation patterns, has recently gained attention.

In this study, the impact of habitual excessive coffee consumption on intestinal SERT expression was investigated using the Caco-2 cell line, an intestinal epithelial cell model. The uptake of 5-HT by SERT and its mRNA levels significantly and dose-dependently decreased following exposure to regular coffee and decaffeinated coffee for 24 to 48 hours. On the other hand, exposure to caffeine did not affect SERT mRNA levels. Additionally, well-known compounds of coffee such as chlorogenic acid did not exhibit any effects. Finally, the influence of coffee on the CpG3 methylation state was evaluated using pyrosequencing. Coffee exposure induced hypermethylation at CpG3.

This study reports the potential of excessive coffee consumption to decrease intestinal SERT expression and disrupt peripheral 5-HT regulation.

Hepatic stellate cells (HSCs) are largely involved in hepatic fibrosis associated with liver diseases such as non-alcoholic steatohepatitis. An increase in cytosolic [Ca²⁺] in HSCs facilitates hepatic fibrosis, however, the regulatory mechanism is unclear. T-type voltage-dependent Ca²⁺ channels (T-VDCCs) contribute to neuronal transmission and cardiac rhythm in excitable cells. Recently, the pathophysiological roles of T-VDCCs in non-excitable cells such as cancer cells and immune cells have been focused. Under whole-cell patch-clamp configurations, transient inward currents were observed in mouse activated HSCs, but not in quiescent HSCs. Quantitative real-time PCR analysis revealed that the mRNA expression of T-VDCCs was significantly higher in activated HSCs than in quiescent HSCs. The viability of activated HSCs was significantly reduced by the treatment with T-VDCC inhibitors. These results suggest that the upregulation of T-VDCC expression in activated HSCs contributes to the regulation of Ca²⁺ signaling and cell proliferation. This study may contribute to the comprehensive understanding of HSC functions in hepatic fibrosis.

Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺-permeable, non-selective cation channel that is activated by oxidative stress such as reactive oxygen species (ROS) and pyridine nucleotides such as adenosine diphosphate (ADP) ribose. TRPM2 channels are widely expressed in leukocytes, and TRPM2-mediated ROS-sensitive Ca²⁺ signaling play a crucial role in a number of cellular processes and functions. In this study, transcript of TRPM2 was cloned from canine peripheral blood mononuclear cells (PBMCs) and some of functional properties of canine TRPM2 were analyzed in vitro. In canine TRPM2 sequence, the basic structure of TRP channel and the binding sites for ADP ribose in MHR1/2 and NUDT9-H domains were conserved. We analyzed the reactivity of heterologously expressed canine TRPM2 to hydrogen peroxide (H₂O₂) in Ca²⁺-imaging experiments. Canine TRPM2 was activated by H₂O₂ in a concentration-dependent manner. This reaction was inhibited by perfusion of TRPM2 inhibitor 2-APB or Ca²⁺-free extracellular solution. Next, canine PBMCs were incubated with H₂O₂ and were subjected to RT-qPCR analysis. The expression of an anti-inflammatory cytokine IL-10 was not enhanced, but the expression of inflammatory cytokines IL-1β, TNFα, and IL-8 (CXCL8) were tended to be enhanced after incubation with H₂O₂. Our results suggest that TRPM2 plays an important role in activation of canine leukocytes, and that TRPM2 is a possible target for regulation of leukocytes functions.

Opioid receptors (ORs) belong to a member of GPCRs and are receptive to compounds that exert analgesic effects, such as morphine. Many opioid ligands contain a morphinan structure, and their pharmacological activity (efficacy) varies, including agonists and antagonists. It is important in drug discovery to elucidate the binding mechanism of ligands to ORs and the relationship between their efficacy. Here, we applied vibrational spectroscopy-based GPCR-ligand interaction studies to κ-opioid receptor (KOR) to elucidate differences in the binding mechanism of various ligands to the receptor, which share a common morphinan structure but exhibit different efficacy. The agonist binding spectra showed the spectral down-shift in amide-I band reflecting to weakening the hydrogen bond between C=O and N-H pairs of peptide backbone, whereas the antagonist bound spectra showed the opposite change, which indicates the different conformational changes that occur between an agonist and antagonist binding to KOR. Furthermore, differences in the vibrational bands derived from functional group of amino acids were also observed for different ligand efficacy. The mechanism by which differences in pharmacological efficacy arise from a common morphinan structure will be discussed, based on protein backbone and functional group of amino acid changes.

Cystic fibrosis (CF) is an autosomal recessive disease in which mutations in the CFTR gene cause various symptoms through its channel malfunction. Among over 1900 gene mutations, the most common mutation is ΔF508 which causes a trafficking defect of CFTR to plasma membrane (classified as ‘class II’ ). CF is quite rare in Japanese and listed in ‘Intractable Diseases’ by MHLW, Japan. Q98R mutation is the third most frequent disease associated mutation found in Japanese CF patients following H1085R and L441P. The Q98R mutation is in the class II same as H1085R, L441P and ΔF508 mutations.

Some pharmaceutical companies have been developing several expression correctors, e.g., lumacaftor, galicaftor, tezacaftor and elexacaftor, and channel function potentiators, e.g., ivacaftor, for ΔF508 mutation.

In this study we characterized the effects of the Q98R mutation on CFTR processing and investigated the effects of the Caucasian CF drugs on the expressions and functions of Q98R-CFTR.

From our in vitro data, it is suggested that Orkambi, a combination of lumacaftor and ivacaftor, is a potential candidate in the pharmaceutical therapy for Japanese CF patients with the Q98R CFTR mutation.