Pine nodule extract and its component, SJ-2, have been reported to have an inhibitory effect on catecholamine secretion induced by acetylcholine (its physiological secretagogue) in cultured bovine adrenal medullary cells (J Pharmacol Sci., 2017).

　The present study aimed to determine the effect of pine nodule extract on the autonomic nervous activity induced by mental stress (Uchida-Kraepelin arithmetic test) in healthy young students. Autonomic nervous balance was measured by power spectral analysis of heart rate variability using a standard hexagonal radar chart. Four 15-min repetitions of an arithmetic task served as an acute mental stressor that caused an increase in sympathetic parameters in the placebo group, while no increases in sympathetic parameters were observed in the pine nodule extract group.

　The present study demonstrated that the assay system of the autonomic nervous balance detected increases in sympathetic nervous activity induced by acute arithmetic stress in the placebo group, but that those increases were cancelled in healthy young students who ingested pine nodule extract, suggesting that this pine nodule extract may an anti-stress effect.

Thyrotropin receptor antibody (TRAb) is a causative antibody of Graves' disease. Epstein-Barr virus (EBV) persists in human B cells, and occasionally reactivates. We have reported that both Graves' disease patients and healthy controls have EBV-infected B cells that have TRAb as their surface globulin (TRAb(+) EBV(+) cells). The peripheral blood mononuclear cells containing TRAb(+) EBV(+) cells produced TRAbs along with EBV reactivation. We proposed the EBV reactivation-induced Ig production as alternative system of Ig production.

The antibodies produced by EBV reactivation-induced system are IgM dominant, and skewed to be autoreactive. However, the class of thyroid stimulating TRAb is known to be IgG. We studied about the role of the IgM antibody.

We purified TRAb-IgM from culture medium of TRAb(+) EBV(+) cells. Then, we cultured porcine thyroid cells with the TRAb-IgM and complements, and then, measured cAMP and LDH levels to estimate thyroid stimulating effect and cell injury, respectively. We observed the increase of the levels of LDH, but could not detect cAMP.

We considered that TRAb-IgM did not have thyroid stimulating effect, but it could injure the thyroid cells and release thyroid antigens including TSH receptor antigen. The relevance of EBV reactivation to Graves' disease may have a possibility for the new therapy.

Maternal nutrition during pregnancy has been found to have a significant impact upon the health of offspring after maturation. However, strategies for modulation of metabolism without an adverse effect on the fetus have remained limited. It was recently shown that maternal high calorie diet induces obesity in later life of the offspring, and maternal oral administration of uncarboxylated osteocalcin (GluOC), which crosses the placenta, improves metabolic status in the offspring by unknown mechanism. 

We thus explored the molecular mechanisms for the effect of maternal high calorie diet and GluOC during gestation on metabolic properties of the offspring. From our results, a maternal high-fat, high-sucrose diet during pregnancy causes metabolic disorders in the liver of the offspring due to hypermethylation of the Pygl gene, encoding glycogen phosphorylase L, which mediates hepatic glycogenolysis. The lower expression of Pygl induced by the maternal diet causes the hepatic accumulation of glycogen and triglyceride in the offspring, which remains in adulthood. On the other hand, maternal GluOC upregulates Pygl expression in the offspring via both direct and indirect pathways to improve maternal diet-induced obesity and abnormal energy metabolism.

We propose that maternal high calorie diet is reflected in the hepatic glycogenolysis capacity of the offspring via epigenetic modification of Pygl and maternal oral administration of GluOC protects the offspring from metabolic disorders induced by maternal diet by regulation of glycogenolysis.

Rhamnan sulfate (RS) is a sulfate polysaccharide composed of L-rhamnose and sulfated L-rhamnose found in green algae such as Monostroma nitidum. It has been reported to have anticoagulant and antiviral effects in the last decade. In 2015, our group discovered the anti-obesity effect of RS using the diet-induced obesity model of zebrafish (Zang L, et al. J Funct Foods. 2015;17:364-370). In this study, we first administrated RS (0.25 mg/g food volume) orally to high-fat diet-treated mice for 4 weeks. RS increased fecal volume and calorie excretion with suppression of body weight increase, and decreased plasma lipids and fasting blood glucose levels, which was consistent with the results of our zebrafish study. Noting the increased excretion by RS, we administrated RS (100 mg/day) to subjects with low defaecation frequencies (3–5 times/week) for 2 weeks in a double-blind placebo-controlled manner. As a result, Firmicutes tended to decrease, while Bacteroides increased. It is known that the obese population has more Firmicutes and fewer Bacteroides depending on their body mass index. Thus, RS improved the intestinal microflora in the direction of anti-obesity. In addition, clostridia (Firmicutes), which produce medium-chain fatty acids that increase the absorption of water in the intestine (one of the causes of constipation), were reduced by the intake of RS. Furthermore, Metagenomics profiling using PICRUSt and KEGG pathway showed that RS intake activated the "cytochrome p450-mediated excretion of foreign substances" pathway, "biological defense against invasive bacteria," and the "biomolecular NAD synthesis" pathway, which has already been reported to have a therapeutic effect on constipation, suggesting a possible mechanism for the ameliorating effect of RS.

The combination of isobutylmethylxanthine, insulin and dexamethasone (MIX treatment) is widely used to differentiate 3T3-L1 fibroblasts to mature adipocytes. In the preliminary experiment performed to determine the role of glucocorticoid receptor, a single treatment with RU486, a glucocorticoid receptor antagonist, was counterintuitively found to be sufficient to induce adipogenesis with smaller lipid droplets than MIX treatment. Here we aimed to characterize the RU486-induced adipocytes by DNA microarray analysis and compare to those in the adipose tissues. Total RNAs isolated from the undifferentiated 3T3-L1 fibroblasts (control cells) and the adipocytes induced by 5-day treatment with 1 micromolar RU486 (RU cells) and those induced by MIX treatment (MIX cells) were subjected to analysis with an Affimetrix Clariom S Mouse DNA chip. The microarray data of the mouse epididymal and inguinal adipose tissues were obtained from GEO DataSet in NCBI, the National Institute of Health, USA. Transcriptome Analysis Console 4.0.2 (Thermo Fischer Scientific) was use to analyze the data. The primary component analysis revealed that the RU cells were closer to epididymal and inguinal adipose tissues in terms of primary component 2 and 3, respectively, than MIX cells or control cells. Forty four genes were found to be common between RU cells and epididymal adipose tissues. The findings indicate RU486 induces adipocytes closer to those of in vivo adipose tissues than MIX treatment. Single treatment with RU486 is suggested to be a novel method to induce normal adipogenesis.

Heparan sulfate (HS) is a highly sulfated glycosaminoglycan distributed on the cell surface. Our recent studies revealed that HS played an essential role in energy homeostasis through the regulation of insulin secretion from pancreatic β-cells and glucose sensitivity in adipose tissues. Although liver is one of the most important organs for energy homeostasis, the functions of HS in liver remain largely unknown. In the present study, we phenotyped hepatocyte-specific HS-deleted mice (cKO) to elucidate the roles of HS in hepatocytes.

Blood glucose testing showed that HS deletion in hepatocytes resulted in the lower glucose level of cKO after glucose challenge due to higher insulin sensitivity, indicating the augmented insulin signaling in cKO hepatocytes. Indeed, the phosphorylation level of Akt, which is one of the important molecules of insulin signaling, was robustly increased in cKO liver after insulin treatment. Biochemical assays indicated HS reduction led to the enhanced differentiation of hepatocytes due to attenuated TGFβ signaling which interrupts hepatocyte differentiation. These data suggests that HS in hepatocytes prevents the differentiation of hepatocytes and has a negative impact on insulin sensitivity.

【Subject】It is suggested that aging and excessive intake of fat may induce dysbiosis and intestinal inflammatory damage additively. Recent studies reported that molecular hydrogen has anti-inflammatory effect and antioxidant effect. Therefore, we analyzed the effects of molecular hydrogen on dysbiosis and intestinal injury induced by aging and excessive intake of fat.

【Method】Senescence-accelerated mouse prone-8 (SAMP8) were fed control diet or high fat diet (HFD) for 14 weeks, and then the each group was fed placebo jelly or hydrogen-rich jelly for 4 weeks. After the treatment, small intestinal tissues were harvested for morphological examination as well as organoid analysis. Moreover, we analyzed alterations of microbiota composition in cecal feces by 16S rRNA gene analysis of microbiota profiling.

【Result & Conclusion】The treatment with hydrogen-rich jelly prevented intestinal morphological damage and suppressed the decreases of CDX2 and BrdU expression in HFD-loaded SAMP8 mice. The treatment also increased the number of cultured organoids derived from small intestine of HFD-loaded SAMP8 mice. The 16S rRNA gene sequencing analysis suggested that the treatment decreased the abundance of Proteobacteria phylum in HFD-loaded SAMP8 mice. These findings suggest that the treatment with molecular hydrogen may modify microbiota composition and suppress intestinal injury and regeneration dysfunction.

Donepezil is used for treatment of Alzheimer's disease, but it is associated with increased risk for gastrointestinal (GI) symptoms, such as anorexia, nausea, and vomiting. Their insufficient control affects the ability to continue the donepezil therapy. Rikkunshi-To (RKT), a traditional herbal Japanese medicine, has been prescribed for patients with various GI symptoms because it improves the GI function via the potentiation of ghrelin signaling pathway. In this study, we investigated the effects of RKT on the prevention of donepezil-induced anorexia, nausea, and vomiting in mice and the involvement of ghrelin in its therapeutic effect. We have reported that donepezil (5mg/kg, i.p.) induced anorexia and pica, kaolin ingestion behavior, could be used to evaluate nausea and vomiting in mice fed the normal diet. Mice fed the diet supplemented with RKT (1%) did not show donepezil-induced anorexia and pica, and this therapeutic effect was antagonized by pretreatment with the ghrelin receptor antagonist. Donepezil significantly suppressed the intestinal motility in mice fed the normal diet; however, RKT recovered the motility delay. Furthermore, the ghrelin receptor antagonist reduced the effect of RKT to improve the intestinal motility. These findings suggest that RKT is a candidate for the treatment of donepezil induced anorexia, nausea, and vomiting in human patients, and that the enhancement of ghrelin signaling is involved in its therapeutic effect.

Doxorubicin (Dox) is one of the practical anti-cancer agents against various tumor cell types. Unfortunately, the use of dox has been hampered due to the life-threatening cardiotoxic damage such as lethal cardiomyopathy it has caused. Mitochondria are double-membrane organelles that play a critical role in cellular homeostases, like ATP supply by oxidative phosphorylation. Many reports suggested that Dox impairs mitochondrial function that causes cardiomyopathy. However, the effect of Dox on oxidative phosphorylation has remained elusive. To confirm the effects of Dox on mitochondrial oxidative phosphorylation, we evaluated the effect of Dox on rat embryonic cardiomyoblast H9c2 cells by measuring oxygen consumption rate (OCR). Administration of 1 µM Dox caused significant decreases in the basal OCR. The addition of 1 µM Dox in the presence of oligomycin exhibited a significant increase of OCR compared to that without Dox, suggesting that Dox damaged regulation of the mitochondrial respiratory system. FCCP treatment with 1 µM Dox caused a significant reduction of the maximal respiratory. We also evaluated the extracellular acidification rate (ECAR). Administration of 1 µM Dox caused a significant increase or showed a tendency to increase in the basal ECAR and the presence of oligomycin, but not in the presence of FCCP. These results indicate that Dox impaired the mitochondrial respiration system and induced the metabolic shift of the cells in the presence of 1 µM of Dox occurred in the basal condition from oxidative phosphorylation to a glycolytic pathway to produce ATP.

Chemotherapy-induced peripheral neuropathy (CIPN) is one of the adverse events associated with the anticancer drugs, however, almost available analgesic drugs lack efficacy against CIPN. Previously our results using medical database, FAERS, suggested that HMG-CoA reductase inhibitors (statins) have the potential to ameliorate oxaliplatin-induced peripheral neuropathy (OIPN). In this study, we elucidated the effect and mechanism of statins to OIPN model mice and PC12 cell. Three statins (simvastatin, atorvastatin, and rosuvastatin) could not show the therapeutic and preventive effects against oxaliplatin-induced cold allodynia. On the other hand, repeated orally administration of each statins ameliorate development of oxaliplatin-induced mechanical allodynia and significantly suppressed already established allodynia induced by oxaliplatin. A gene-related database revealed that the expression of glutathione S-transferase (GST) family members is regulated by statins. Decreased survival rate of PC12 cells by treatment of oxaliplatin was canceled cotreatment of each statin for 24 hours. Furthermore, cell protective effect of statin was disappeared transfection of gstmu1 siRNA into PC12 cells. These our results suggest that statins might be one of the novel supportive care, which have neuroprotective effect to OIPN.

The ICH and the PMDA published their guideline S5(R3) for the evaluation of reproductive toxicity of medicinal products, and has started to develop zebrafish developmental toxicity testing in Japan. In this study, we attempted to establish a zebrafish quality control protocol, which is the most important accuracy basis for the zebrafish developmental toxicity test. We first analyzed 12,906 fertilized eggs and found that 297 (2.3%) eggs died by 3 hours after fertilization. And 2,104 (16.3%) had morphological abnormalities by 3 hours after fertilization. By 24 hours after fertilization, 4,646 eggs (36.0%) were found to be dead. However, the survival rates from 1 to 5 days after fertilization were stable compared to the rapid decline in survival rate at 24 hours after fertilization. On the other hand, the results of time-lapse imaging from 3 hours to 5 days after fertilization showed that abnormal egg imaging at 3 hours after fertilization could predict death or morphological abnormalities up to 5 dpf. As a result, we report that it was possible to remove these low-quality fertilized eggs before the start of compound exposure (6 hpf) for developmental toxicity testing, thus reducing the frequency of false positives and enhancing the accuracy of zebrafish developmental toxicity screening.

Due to the coronavirus pandemic situation, the pharmacological role-playing was conducted by online in 2020 and 2021. We reported consideration of pros and cons of the online pharmacology role-playing from results of a two-year student questionnaire survey. Two hundred twenty five 3rd-grade medical students joined the role-playing at Dokkyo Medical University. Twenty-four students were assigned as physicians or patients and played the informed consent in two cases; hypertension accompanying diabetes and dementia with polypharmacy. The remaining students were observers. All students answered the questionnaire with a score of one to five, regarding the usefulness of study on disease and pharmacotherapy, understanding the patients' feeling, improvement of motivation to become a doctor, and change of attitude to studying. The percentage of students who scored five or four was 63 to 83% for the players and 71 to 80% for the observers. The frequent answers regarding necessary points of the study were "quality of the study" and "communication ability" and "perspective from the patients" for the players, and "communication ability", "quality of the study" and "perspective from the patients" for the observers in order of frequency. Most students described positive impression in the free entry field that was considered as pons. A description of the difficulty of adjusting the online settings was considered as cons. Online pharmacological role-playing may be a useful approach for the medical students to learn the pharmacotherapy and doctor-patient relationship.

Glaucoma is the first cause of blindness in Japan, which is characterized by progressive degeneration of retinal ganglion cells (RGCs). Although an elevated intraocular pressure (IOP) is one of major risk factors, many Japanese glaucoma patients show normal level of IOP (i.e. normal tension glaucoma, NTG). We have recently discovered a novel NTG model (Astro-KO) mouse in which astrocytes lack the gene encoding ATP-binding cassette transporter A1 (ABCA1). The NTG mice at 3 months old (mo) showed no RGC damages but showed significant damages of RGCs and visual impairment at 12 mo. Although dysfunction of astrocytes was essential for NTG-like pathologies, molecular mechanisms remained unclear. To tackle this, we performed bulk and single-cell RNA-sequence of retina. We found that RGCs and retinal astrocytes up-regulate neuroinflammatory pathways including CXCR4 and CCR5 signaling. Immuonhistochemical analysis revealed that CXCL12 and CCL5, ligands for CXCR4 and CCR5, were up-regulated in retinal astrocytes of Astro-KO mice at 12 mo. CXCR4 and CCR5 in Astro-KO mice (12 mo) were expressed in the ganglion cell layer. Taken together, our data showed that lack of ABCA1 triggers neuroinflammation by astrocytes, which may cause RGC damages via CXCR4 and CCR5 activation.

Astrocyte is the predominant type of glial cell that attracts attention as a potential target of drug discovery and therapeutics for neurodegenerative diseases. Astrocytic Ca²⁺ signals are enhanced by neuronal hyperactivities and/or bioactive substances released from injured brain cells. Ca²⁺ signals can trigger Ca²⁺-dependent processes including various gene expressions and secretion of neuro-protective/toxic molecules in astrocytes. Thus, analysis of astrocytic Ca²⁺ signals may provide clues for the regulation of neurodegenerative diseases. Because it remains elusive how the pathological conditions affect astrocytic Ca²⁺ activities, we are trying to establish a new method to analyze astrocytic Ca²⁺ activities in neurodegenerative diseases. We applied a drug-induced or genetical neurodegenerative disease model to a transgenic mouse line that expresses a genetically encoded Ca²⁺ sensor, YC-Nano50, in astrocytes. We found disorder-related changes in astrocytic Ca²⁺ activities from macroscopic Ca²⁺ imaging analysis in the cortical surface of these mice. This method may clarify astrocytic Ca²⁺ activities in brain pathology and contribute to the development of therapeutic strategies for neurodegenerative diseases.

Extracellular purines, including ATP and adenosine (ADO), are important neurotransmitter and neuromodulator in the central nervous system (CNS). Purine concentration is controlled by purine release and metabolisms. Astrocytes play important role in purine metabolisms in the CNS. In our previous study, we have shown that fibroblast growth factor 2 (FGF2) upregulates purine metabolic enzymes in rat spinal cord astrocytes. In this study, we investigated the effects of FGF2 on purine metabolisms in rat cortical astrocytes.

Cultured astrocytes from rat cerebral cortex were treated with FGF2. Enzymatic activity for purine metabolism was measured by incubation with extracellular solution containing ATP, AMP or ADO and measurement of those metabolites with HPLC. The expression levels of enzymes were measured by real-time PCR.

In cultured cortical astrocytes, FGF2 increased the mRNA and activity of ecto-5'-nucleotidase (CD73) and adenosine deaminase (ADA), and decreased those of ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2). An FGF receptor inhibitor, SU5402, inhibited the changes in the expression and activity of CD73, ADA and ENTPD2. U0126, a MEK inhibitor, and SP600125, a JNK inhibitor, inhibited the increase of CD73 and ADA, respectively. On the other hand, neither U0126 nor SP600125 inhibited the decrease of ENTPD2.

These results indicate that FGF2 modulates the expression and activity of CD73, ADA and ENTPD2 through FGF receptor. Furthermore, it is suggested that different intracellular signaling pathways are involved in modulation of each purine metabolic enzymes.

【Purpose】

Metastatic brain tumors emit various components, including cyclic 2'3'-GMP-AMP (cGAMP) to surrounding astrocytes, which may affect the tumor microenvironments. To identify the cGAMP targets, we analyzed transcriptome in cGAMP-incorporated astrocytes.

【Method】

cGAMP is encapsulated in lipid nanoparticles (ssPalm), then introduced into primary cultured astrocytes. After extracting the RNA, the fold change (FC) by cGAMP was investigated by microarray. Among the genes with high intrinsic expression levels, we searched for interacting factors with large FC in STRING's protein-protein interaction database. As an indicator of astrocyte responsiveness, changes in intracellular calcium concentration were observed by confocal time-lapse imaging.

【Result/Discussion】

As candidates of cGAMP targets, interferon-stimulated genes were determined, such as Viperin and Usp18. Cholecystokinin (CCK), which was reported to be involved in glutamate secretion, showed the largest FC(318-fold). Further, the intracellular calcium concentration increased when CCK was added to astrocytes. We have previously demonstrated that the introduction of cGAMP alters glutamine-glutamate metabolism in astrocytes. We continue to be interested in how these changes relate to each other and contribute to the malignant transformation of metastatic brain tumors.

Glial cells are vital for the modulation of synaptic connections and healthy brain development. They control the excitatory / inhibitory synaptic balance and assemble neural circuity by synaptic formation or elimination. We have recently revealed that astrocytes form excitatory synapses in the adult injured brain through mGluR5 signaling. However, astrocytic mGluR5 is, in health brain, expressed in the limited time-window of the postnatal developmental stage. Thus, we surveyed whether / how astrocytic mGluR5 destines the subsequent synaptic assembly using astrocyte-specific mGluR5 KO mice (cKO). Unexpectedly, the number of excitatory synapses did not alter much in cKO, instead, the number of inhibitory synapses decreased significantly in cKO throughout ages. Interestingly, microglia frequently engulfed inhibitory synaptic elements in cKO. Astrocytic mGluR5 expression was transient event in the critical period, however, behavioral dysfunction was observed even in adult cKO mice. Next, we surveyed the astrocytic molecule which regulates microglial engulfment. We focused on astrocytic IL7, which is decreased in cKO, as a candidate for such molecules. IL7 treatment decreased engulfment-related gene expression in microglia. Hence, we conclude that astrocytes organize inhibitory network in the critical period by modulating microglial phagocytic activity. It should be noted that although mGluR5 is only transiently expressed in astrocytes in the critical period, its function greatly affects the inhibitory neuronal networks throughout life.

We investigated the effects of esaxerenone, a novel, nonsteroidal, and selective mineralocorticoid receptor blocker, on cardiac function in Dahl salt-sensitive (DSS) rats. We provided 6-week-old DSS rats a high-salt diet (HSD, 8% NaCl). Following six weeks of HSD feeding (establishment of cardiac hypertrophy), we divided the animals into the following two groups: HSD or HSD + esaxerenone (0.001%, w/w). In survival study, all HSD-fed animals died by 24 weeks of age, whereas the esaxerenone-treated HSD-fed animals showed significantly improved survival. We used the same protocol with a separate set of animals to evaluate the cardiac function by echocardiography after four weeks of treatment. The results showed that HSD-fed animals developed cardiac dysfunction as evidenced by reduced stroke volume, ejection fraction, and cardiac output. Importantly, esaxerenone treatment decreased the worsening of cardiac dysfunction concomitant with a significantly reduced level of systolic blood pressure. In addition, treatment with esaxerenone in HSD-fed DSS rats caused a reduced level of cardiac remodeling as well as fibrosis. Furthermore, inflammation and oxidative stress were significantly reduced. These data indicate that esaxerenone has the potential to mitigate cardiac dysfunction in salt-induced myocardial injury in rats.

In the heart, the frequency of spontaneous electrical activity generated in the sinoatrial node (SAN) is determined by diastolic depolarization which is believed to be formed by ion channel-dependent membrane potential changes. It has also been proposed that intracellular Ca²⁺ may regulate spontaneous activity via electrogenic Na⁺/Ca²⁺ exchanger (NCX); Ca²⁺ clock theory. In this study, we investigated whether NCX contributes to spontaneous activity in guinea pig SAN.

Immunofluorescence staining showed the expression of NCX on the cell membrane of the SAN cells.

To evaluate the role of NCX in spontaneous activity and Ca²⁺ dynamics, high-speed Ca²⁺ imaging was performed on SAN cells and spontaneous Ca²⁺ transients were observed. The inhibition of NCX activity by perfusion with low Na⁺ solution resulted in an increase in the basal fluorescence intensity, but had little effect on the frequency of Ca²⁺ transients. Glass microelectrode recording of action potential revealed that low Na⁺ solution has no effect on the diastolic depolarization. Furthermore, SEA0400, an inhibitor of NCX, did not affect the frequency of spontaneous activity and the diastolic depolarization.

These results suggest that, although NCX is expressed in guinea pig SAN cells and is responsible for Ca²⁺ efflux, its contribution to spontaneous activity is very small.

[Background] Autophagy is a self-degradation system of intracellular organelles and found in failing heart. We investigated whether angiotensin II (ANG II) enhanced myocyte autophagy and the role of autophagy in ANG II-induced injury. [Methods and Results] Neonatal rat cardiac myocytes were treated with ANG II (1–100 nmol/L). ANG II dose dependently increased autophagy, assessed by microtubule-associated protein 1 light chain (LC) 3-II expression. It also enhanced the intracellular reactive oxygen species (ROS), detected by H2DCFDA staining. NADPH oxidase- and mitochondria-derived ROS production was increased by ANG II, while ANG II-induced autophagy was suppressed by inhibitors of these sources of ROS. ROS-producing mitochondria colocalized with lysosomes after ANG II stimulation. Myocyte apoptosis was observed using nuclear staining with DAPI. A 6-hour stimulation with ANG II did not affect myocyte apoptosis, while a co-treatment with 3-methyl-adenine (3MA), an autophagy inhibitor, increased apoptosis. A longer ANG II stimulation for 24 hours induced apoptosis, while the co-treatment with 3MA did not lead to further increase. [Conclusion] ANG II enhanced intracellular ROS production, leading to autophagy in myocytes. Autophagy was beneficial because it removed damaged mitochondria, which suppressed myocyte apoptosis in the early stages of the ANG II stimulation, while the longer ANG II stimulation itself induced apoptosis that was not effectively suppressed by autophagy.

We previously reported the 2,5-dimethylcelecoxib (DM-C) attenuated cardiac remodeling in different types of cardiac hypertrophy model. However, it remains unclear whether DM-celecoxib attenuates fibroblast-to-myofibroblast transformation (FMT) which plays the key role in cardiac fibrosis after myocardial infarction (MI). Therefore, we investigated the effect of DM-C on FMT using cryoinjury induced myocardial infarction (CMI) mouse model and TGF-β1-stimulated cardiac fibroblasts. We found that DM-celecoxib attenuated deterioration of left ventricular ejection fraction after CMI by decreasing cardiac fibrosis. Analysis of the expression level of α-smooth muscle actin, a marker for myofibroblast, indicated that DM-celecoxib decreased FMT in cardiac injured site. In the cardiac fibroblasts, DM-celecoxib suppressed expression of α-SMA and phosphorylation levels of Smad 2/3 and GSK-3, indicating that DM-celecoxib suppressed α-SMA expression by inhibiting TGF-β signaling pathway via activation of GSK-3. These results suggested that DM-celecoxib attenuated cardiac fibrosis via suppressing fibroblast-to-myofibroblast transformation in injured site after CMI by suppressing TGF-β signaling pathway via activation of GSK-3. Thus, DM-celecoxib has a potential as a novel anti-fibrotic agent after MI in clinical setting.

After myocardial infarction (MI), various kinds of cytokines are produced from pro- and/or anti- inflammatory cells that infiltrated into myocardium and contribute to scar formation and/or tissue repair. Among these cytokines, it is widely accepted that TGFβ family performs multiple functions, such as cell proliferation and fibrosis; however, it remains to be fully clarified whether there are functional differences among TGFβ1, 2, and 3. The aim of this study is to elucidate the pathophysiological significance of TGFβ3 after MI. MI was generated by coronary ligation. We measured the expression of TGFβ3 over time after MI by quantitative RT-PCR, and found that the expression of TGFβ3 mRNA peaked 7 days after MI (17.8±12.9 fold v.s non-MI). Quantitative RT-PCR and immunohistological staining showed that TGFβ3 was mainly expressed at the border region of infarction. To examine the effects of TGFβ3 on post-infarct remodeling, we　administered TGFβ3 neutralizing antibody (TGFβ3 nAb) intravenously after MI. Echocardiographic analysis revealed that TGFβ3 nAb reduced cardiac dysfunction (fractional shortening: control IgG;29.8±7.3%, TGFβ3 nAb; 36.9±5.8%). In addition, Masson trichrome staining showed that neutralizing antibodies inhibited cardiac fibrosis after MI. TGFβ3 could promote adverse cardiac remodeling after MI as a novel therapeutic target.

Inflammatory bowel disease (IBD) is a multifactorial chronic inflammatory disorder of intestinal tract with intermittent episodes of remission and relapse. Although sustained clinical remission has recently been recognized as an important goal of IBD therapy, there are not many treatment options to maintain long-term remission. We have proposed that the enhancement of interleukin (IL)-10 production in the intestinal macrophages has the potential to be a novel therapeutic mechanism for maintaining the remission of IBD and found berberine as a candidate compound for IL-10 enhancer. In the present study, we developed a useful experimental animal model to evaluate the relapse phase of IBD and investigated the effect of berberine on this model. Berberine significantly attenuated colitis symptoms during the relapse phase of IBD relapse model. To further investigate how berberine enhances IL-10 production in intestinal macrophages, we explored target protein for berberine that controls this response. We detected fatty acid synthase (FAS) as a candidate target protein. FAS inhibitor suppressed the IL-10 production of macrophages enhanced by berberine in a concentration-dependent manner. This study suggests that FAS activators such as berberine may be useful in developing a novel therapeutic strategy aimed at preventing relapse in IBD.

Macrophage polarization is critical for liver tissue repair following acute liver injury. However, the underlying mechanisms of macrophage phenotype switching are not well defined. Invariant natural killer T (iNKT) cells orchestrate tissue inflammation and tissue repair by regulating cytokine production. Herein, we examined whether iNKT cells played an important role in liver repair after hepatic ischemia-reperfusion (I/R) injury by affecting macrophage polarization. To this end, we subjected male C57BL/6 mice to hepatic I/R injury, and mice received an intraperitoneal (ip) injection of α-galactosylceramide (α-GalCer) or vehicle. Compared with that of the vehicle, α-GalCer administration resulted in the promotion of liver repair accompanied by acceleration of macrophage polarization and by increases in the numbers of Ly6C^high pro-inflammatory macrophages and Ly6C^low reparative macrophages. Treatment with anti-IL-4 antibodies delayed liver repair, which was associated with an increased number of Ly6C^high macrophages and a decreased number of Ly6C^low macrophages. Treatment with anti-IFN-γ antibodies promoted liver repair, associated with reduced the number of Ly6C^high macrophages, but did not change the number of Ly6C^low macrophages. Bone marrow-derived macrophages up-regulated the expression of genes related to both a pro-inflammatory and a reparative phenotype when co-cultured with activated iNKT cells. Anti-IL-4 antibodies increased the levels of pro-inflammatory macrophage-related genes and decreased those of reparative macrophage-related genes in cultured macrophages, while anti-IFN-γ antibodies reversed the polarization of macrophages. These results suggest that the activation of iNKT cells by α-GalCer facilitated liver repair after hepatic I/R injury by both IL-4-and IFN-γ-mediated acceleration of macrophage polarization.

Liver fibrosis is a common pathway leading to cirrhosis and liver cancer in chronic liver disease, and has a great influence on the prognosis of the disease. Activated stellate cells, which are induced by various stimuli, overproduce extracellular matrix such as collagen, and play a central role in the development of liver fibrosis.

Prostanoids, consisting of prostaglandins (PGs) and thromboxane, exert a variety of actions in the body by binding to their specific receptors. Previously, we have reported that prostaglandin I₂ suppresses the development of diet-induced nonalcoholic steatohepatitis in mice (FASEB J, 2018.). However, role of prostanoids in the function of hepatic stellate cells remain unclear. Therefore, we examined the role of prostanoids using primary cultured hepatic stellate cells.

RT-qPCR analysis of prostanoid receptor mRNA expression in hepatic stellate cells prepared from wild-type mice revealed high expression of PGE₂ receptor EP₂. We next stimulated hepatic stellate cells with TGF-beta and IL-1beta to activate them. Activated hepatic stellate cells showed increased expression of fibrosis-related factors mRNA such as type 1 collagen and CTGF, however, pretreatment with an EP₂ agonist significantly suppressed the increased expression of these mRNAs, and this inhibitory effect of EP₂ agonist disappeared in EP₂^–/– hepatic stellate cells.

These results indicate a possibility that a selective EP₂ agonist become a therapeutic drug that suppress the development of liver fibrosis mediated by activated hepatic stellate cells.

Asthma is a disease characterized by symptoms, such as chronic airway inflammation, airway hyperresponsiveness (AHR) and mucus hypersecretion. Although steroids are the most effective anti-inflammatory therapy for asthma, they don't fully inhibit AHR and mucus production in a group of patients. IL-17A and IL-17F have been suggested to correlate with the severity of asthma, and we have previously shown the increased expression of IL-17A and MUC5AC, which did not respond to steroids, in house dust mite extract (HDM)-sensitized mice. In this study, we investigated the roles of these cytokines in both asthma pathogenesis and steroid sensitivity, using IL17A and IL17F-deficient mice (IL17A, IL-17F KO mice). Sensitization of HDM in IL17A, IL-17F KO mice markedly increased airway hypersensitivity and MUC5AC production compared to wild-type (WT) mice, whereas there was no change in inflammatory markers. In addition, dexamethasone attenuated inflammation, AHR and MUC5AC expression in IL17A, IL-17F KO mice, whereas it was not effective to MUC5AC expression in WT mice. These results suggest that IL-17A and IL-17F are involved in steroid-resistant mucus production. Though the mechanisms for this IL-17-induced steroid-resistance is being investigated, these data may provide new strategy to treat mucin in asthmatic patients.

Angiotensin-converting enzyme 2 (ACE2) is a receptor for cell entry of SARS-CoV-2, and recombinant soluble ACE2 protein inhibits SARS-CoV-2 infection as a decoy. ACE2 is a carboxypeptidase that degrades angiotensin II (Ang II) and thereby improves the pathologies of acute lung injury. To address whether the carboxypeptidase activity of ACE2 is protective in COVID-19, we investigated the effects of B38-CAP, an ACE2-like enzyme, on SARS-CoV-2-induced lung injury. Expression of endogenous ACE2 protein was significantly downregulated in the lungs of SARS-CoV-2-infected hamsters or human ACE2 transgenic mice, leading to elevation of Ang II levels. In vivo administration of recombinant SARS-CoV-2 Spike trimer also downregulated ACE2 expression, elevated Ang II levels and considerably worsened the symptoms of acute lung injury in hamsters exposed to acid aspiration. Despite its ACE2-like catalytic core, B38-CAP neither bound to Spike nor neutralized cell entry of SARS-CoV-2. However, treatment with B38-CAP improved the pathologies of Spike-augmented acid-induced lung injury. In SARS-CoV-2-infected hamsters, B38-CAP significantly improved lung edema and pathologies of lung injury without affecting viral RNA loads. Moreover, in human ACE2 transgenic mice, B38-CAP also attenuated SARS-CoV-2-induced lung edema and pathologies and improved lung functions. These results provide the first experimental in vivo evidence that increasing ACE2-like enzymatic activity is a potential therapeutic strategy to alleviate lung pathologies in COVID-19.

Glioblastoma (GBM), the most malignant form of primary brain tumor, has a very dismal prognosis. Glioma stem cells (GSCs) contribute to the initiation and progression of GBM. Extracellular signal-regulated kinase 5 (ERK5) has been shown to regulate tumorigenicity, metastasis and drug resistance of cancer stem cells (CSCs). However, the functional role and underlying mechanisms of ERK5 expressed in GSCs on gliomagenesis are still poorly understood in vitro and in vivo. Silencing ERK5 by shRNA significantly attenuated the self-renewal potential and tumorigenicity of patient-derived GSCs, which can be rescued by the overexpression of STAT3. In addition, the pharmacological inhibition of ERK5 significantly suppressed the self-renewal potential and tumorigenicity of GSCs. Bioinformatics analyses demonstrated that ERK5 expression was significantly higher in human GBM tissues, and ERK5 pathway was positively regulated in GSCs. Moreover, ERK5 expression was associated with poor survival in GBM patients. These results highlight the importance of the ERK5-STAT3 axis in maintaining stemness and tumorigenicity in GSCs, thereby demonstrating a potential target for GSC-directed therapy.

Glioblastoma multiforme (GBM), a lethal brain tumor, contains a small population of GBM stem cells (GSCs), which potentially cause chemotherapeutic resistance and tumor recurrence. Recent studies have reported that incorporation of ribosomes and ribosomal proteins into somatic cells promote lineage trans-differentiation toward multipotency. In this study, we sought to investigate the mechanism underlying stemness acquisition in GBM cells by focusing on 40S ribosomal protein S6 (RPS6). RPS6 was significantly up-regulated in high-grade glioma and localized at perivascular, perinecrotic, and border niches in GBM tissues. siRNA-mediated RPS6 knock-down significantly suppressed the characteristics of GSCs, including their tumor-sphere potential and GSC marker expression. In contrast, RPS6 overexpression enhanced the tumor-sphere potential of GSCs and these effects were attenuated by STAT3 inhibitor (AG490). Moreover, RPS6 expression was significantly correlated with SOX2 expression in different glioma grades. Immunohistochemistry data herein indicated that RPS6 was predominant in GSC niches, concurrent with the data from IVY GAP databases. Furthermore, RPS6 and other ribosomal proteins were up-regulated in GSC-predominant areas in this database. The present results indicate that, in GSC niches, ribosomal proteins play crucial roles in the development and maintenance of GSCs and are clinically associated with chemo-radio-resistance and GBM recurrence.

Angiotensin II (Ang II) reportedly facilitates distal hematogenous metastasis in various murine intravenous metastasis models. However, it is unclear whether Ang II accelerates the initial processes of metastasis that begins in primary tumors. We examined the effects of Ang II on primary tumors and lung metastasis lesions using a murine spontaneous metastasis model, in which triple negative breast cancer 4T1 cells were injected into the mammary fat pad of BALB/c mice. Ang II administration significantly accelerated primary tumor growth and lung metastasis formation, and Ang II type 1 receptor blocker valsartan attenuated them. Ang II increased the expression of proteins related to cell proliferation and epithelial-to-mesenchymal transition (EMT) in primary tumors, and valsartan attenuated them. However, Ang II did not change the proliferation, migration, invasion, or the protein expressions in 4T1 cells in vitro. In contrast, when 4T1 cells were co-cultured with fibroblasts, Ang II significantly accelerated cell migration and increased the expression levels of EMT-related proteins in 4T1 cells. And moreover, Ang II significantly increased the mRNA expression level of IL-6 in fibroblasts co-cultured with 4T1 cells. These results suggested that Ang II accelerates interaction between cancer cells and surrounding fibroblasts by soluble factors such as IL-6 to promote EMT and cell migration, which result in the initiation of cancer metastasis.

Metastasis predicts poor prognosis in patients with esophageal squamous cell carcinoma, whereas the regulatory mechanism of metastasis remains largely unknown. In this study, by using murine tumor metastasis model and spatial transcriptome analysis, we aimed to decipher the molecular basis of metastasis. Implantation of NRS1M squamous cell carcinoma cells into C3H/HeN mice gave rise to the growth of primary tumor, and elicited lymph node and lung metastasis. We isolated enhanced metastatic cells (em-NRS1M cells) by in vivo passage. RNA-Seq analysis revealed that cytokines network was perturbed in em-NRS1M cells compared with parental cells, implicating intrinsic immunomodulation in em-NRS1M cells. Immunohistochemical staining demonstrated that in vivo NRS1M tumors exhibited focal immunosuppressive areas featured by decreased CD8-positive T cells and CD11c-positive dendritic cells. We performed spatial transcriptome analysis (Visium) with tissue sections of em-NRS1M tumors and identified the tumor areas with gene signatures of immunosuppression, in which antigen presentation and interferon response were downregulated. Importantly, metastasis-related genes were significantly upregulated in the areas. Furthermore, we identified Galectin-X as a novel metastasis-driving factor. Our data suggest that immunosuppression-associated Galectin-X plays a crucial role in tumor metastasis and may be a potential target of cancer immunotherapy.

Despite decreasing number of gastric cancer patients, prognoses of advanced gastric cancer are still poor. Some Rho GEF expressions are supposed to be higher in invasive cancer. Based on this hypothesis, we narrow down 72 Rho-GEF genes to 9 candidate genes by in silico method. Then we focused on FARP1 since it has never been reported to have clinical significance in cancers. From an immunohistochemical study of 91 cases of invasive gastric cancer, the overall survival of the patients with higher FARP1 expression cancer was significantly shorter than that in the patients with lower FARP1 cancer (P = 0.025). FARP1 expression is related to lymphatic metastasis (N) (P = 0.012), lymphatic invasion (P = 0.025) and recurrence rate (P = 0.002). In vitro study FARP1 high expression promoted gastric cancer cell motility, invasion with filopodium formation, and increase of GTP/CDC42, but did not increase proliferation. Integrinβ5 coprecipitated with FARP1 using anti-flag antibody. In the presence of 0.3 nM SB273005 Integrinβ5 inhibitor, filopodia formation and GTP/CDC42 activation in FARP1 expressing cells decreased. From these results, the Integrinβ5-FARP1-CDC42 cascade is related to the prognoses of advanced gastric cancer patients through the increase of cell motility and a new target of invasive cancer therapy.

Kamebakaurin (KA), isolated from Rabdosia excisa, has been reported to inhibit the productions of pro-inflammatory mediators. Mast cells are known to have a central role in allergic and inflammatory diseases. In this study, we investigated the effect of KA on the release of inflammatory mediators using mouse bone marrow-derived mast cells (BMMCs). The BMMCs were incubated without or with KA. The cells were stained with trypan blue to evaluate cell viability. The IgE-sensitized cells were activated by IgE cross-linking using dinitrophenyl-human serum albumin following incubation without or with KA. After antigen challenge, degranulation was evaluated by β-hexosaminidase assay, histamine was quantified by HPLC, and leukotriene B₄ (LTB₄) was assayed by ELISA. The phosphorylation of Syk and Gab2, known as the signal transduction molecules downstream of FcεRI, were evaluated by Western blot analysis. The 30 μM or less concentrations KA treated did not affect cell viability. We next investigated the effect of 30 μM or less concentrations of KA in the release of chemical mediators. The 10 or 30 µM KA significantly reduced degranulation and LTB₄ production. In addition, the exposure of these KA concentrations also tended to decrease histamine release. Further, KA decreased the phosphorylation of Syk and Gab2. These findings indicate KA blocks mast cells activation without affecting the cell viability through the inhibition of Syk and/or Gab2 phosphorylation, and KA may have the potential to become a prototype for anti-allergic and anti-inflammatory drug.

It was reported that ILC2 were involved in induction of steroid-resistant allergic airway inflammation, one of endotypes of intractable asthma. We have reported that asthmatic responses including airway remodeling in 5 and 500 µg ovalbumin-induced models of BALB/c mice were sensitive and resistant, respectively, to dexamethasone, and that the steroid resistance was cancelled by anti-IL-5 mAb treatment. We also suggested that the major cellular source of IL-5 was ILC2 in the lung. In this study, it was analyzed whether function and molecular expression in ILC2 isolated from the steroid-resistant model were altered in comparison with those of the steroid-sensitive model. 1) In vitro IL-5-producing activity of ILC2 isolated from the lung of the steroid-resistant model in response to TSLP/IL-33 was considerably higher than those of the steroid-sensitive model. 2) FACS analyses revealed that expression of TSLP receptors (TSLPR) was up-regulated on the cell surface of ILC2 of the steroid-resistant model. 3) RNA-seq analyses revealed significant up-regulation of STAT5A mRNA expression in ILC2 of the steroid-resistant model. In conclusion, pro-inflammatory function of ILC2 was up-regulated in the intractable asthma. The up-regulation could be due to increases in TSLPR and STAT5A expression.

Ozone has a strong oxidation effect and has been used for disinfection and sterilization. In addition, ozone generators have increasingly been installed at some hospitals, where they have frequently been used for infection control against COVID-19.

Therefore, this study aimed to evaluate the development of acute lung injury (ALI) and allergic asthma symptoms in untreated mice with their development in each mouse model with and without exposure to ozone. To confirm whether low concentrations of ozone influence the clinical condition of the respiratory system, we first measured oxygen saturation as measured by pulse oximetry (SpO2). Ozone exposure significantly reduced the amount of SpO2. Inflammatory cytokines such as MCP-1 and RANTES in the ALI were increased in low ozone exposure compared with normal mice. IgE levels in the allergic asthma mouse model were also increased in low ozone exposure.

Our results indicate that ozone at low concentrations, such as the currently acceptable concentration (0.1 ppm), has adverse effects on ALI and asthma model mice. These findings suggest that controlling chemicals at low concentrations such as ozone may improve the treatment of respiratory diseases such as ALI and asthma.

Food allergy is a phenomenon in which adverse immune reactions are caused after intake of foods. Oral tolerance is the state of unresponsiveness of the immune system to foods administered via an oral route. The mechanism for the failure of acquiring oral tolerance in food allergy patients remains unclear. Recently, the hypothesis, that allergic sensitization results from cutaneous exposure and that tolerance occurs as results of oral exposure to food, has been proposed. In this study, we investigated the balance of the relationship between oral tolerance and sensitization via skin to use murine food allergy models. We have already reported the food allergy model using ovalbumin (OVA) in which BALB/c mice were sensitized by intraperitoneal injection of OVA (IP-model). Oral tolerance was induced by oral administration of OVA before the sensitization. The induced oral tolerance in IP-model prevented increasing OVA-specific IgE and inhibited decreasing body temperature and diarrhea as symptoms of food allergy. In another food allergy model, the mice were conducted epicutaneous sensitization for OVA(EC-model). Oral tolerance in EC-model was broken and OVA-specific IgE increased slightly. These results might indicate that percutaneous load of allergen could break oral tolerance for food.

Natural killer group 2 member D (NKG2D) expressed in natural killer (NK) cells and T cells is a receptor activated by NKG2D ligands such as MICA and MICB. In patients with gastric cancer, expression of NKG2D in tumor-infiltrating immune cells correlates with good prognosis. Because these tumor-infiltrating cells were mainly CD8⁺ T cells, we hypothesized that NKG2D⁺ T cells had proficient anti-tumor response. In the study, we investigated whether MICA or MICB activated T cells via NKG2D in vitro by using coculture system. Panc1, a human pancreatic cancer cell line, expressed MICA and MICB among eight NKG2D ligands. We sorted cells with low or high NKG2D expression from activated T cells and cocultured each of them with Panc1 cells that had been treated with siRNA of MICA and/or MICB.  After 3 days of coculture, we isolated T cells from coculture and measured interferon gamma (INFg) mRNA as an activation marker. INFg expression of NKG2D^low T cells were augmented by downregulation of MICB, suggesting that MICB was a negative regulator.  In contrast, NKG2D^high T cells remained expressing INFg regardless of downregulation of MICA and/or MICB. These results suggest that NKG2D^high T cells were an elite population that could be activated independently of NKG2D ligands expression in cancer cells.

Immune checkpoint inhibitors (ICIs) are potentially able to be strong strategy to treat the Head and Neck Cancer, however they lack enough their efficacy, so the search for factors that can improve their efficacy is needed. The gut microbiota and its metabolites have been found to play an important role via modification tumor microenvironment with the treatment of ICIs. It was recently reported that inosine, one of the gut bacterial metabolites, modulates cancer immunity, however the detail of the mechanism is still unclear. In the present study, we attempt to clarify the direct effects of inosine to cancer cells and immune cells in vitro. In the equilibrium phase of cancer immunoediting, it is important to make the immune side dominant in order to control cancer, thus inosine was added directly to co-cultures of T cells and cancer cells reproducing the equilibrium phase between cancer and immunity. These cells are harvested and divided into cancer cell and immune cell subsets followed by immunological analysis. We also investigated the in vivo efficacy of inosine utilizing BALB/c mice with CT26 murine colon cancer model. As a result, it was found that there is an optimal concentration of inosine that inhibits cancer cell growth in co-cultures of T cells and cancer cells. The anti-tumor effect differs depending on the concentration and route of administration in a tumor-bearing mouse model. In the future, we will find the conditions for inhibiting tumor growth and activating T cells, which will lead to the development of tumor immunotherapy against Head and Neck Cancer using inosine.

Recently, immune checkpoint inhibitors (ICIs) have emerged as a new option for the treatment of head and neck cancer. However, improving their efficacy remains a challenge, and approaches to the tumor microenvironment are important. While intestinal bacteria and their metabolites, short-chain fatty acids (SCFAs), have attracted much attention, the effects of SCFAs on cancer and immune cells are still unclear. In this study, we decided to focus on SCFA-A, one of the SCFAs that have the strongest effect on immune activity in preliminary experiments, to clarify its effects on cancer and immune cells. By co-culturing cancer cells and T cells at different cell concentrations, we reproduced the three sequential phases of cancer immunoediting: cancer and T cells in equilibrium, cancer-dominant, and T cell-dominant. SCFA-A was directly added to the co-cultures of cancer cells and T cells. In addition, SCFA-A was orally administered to a tumor-bearing mouse model to clarify the mechanism of the anti-tumor effect of SCFA-A in the tumor microenvironment. SCFA-A inhibited tumor growth in the co-culture of cancer cells and T cells, and oral administration of SCFA-A to a tumor-bearing mouse model increased anti-PD-1 activity. This study is expected to improve the efficacy of the treatment of ICIs and may lead to drug discovery for cancer immunotherapy and the discovery of new biomarkers.

Tumor blood vessel structure is different from normal blood vessel features in terms of short lumen diameter, serpentine course and poor tight junction formation. These phenomena lead to form specific tissue environment, so called tumor microenvironments (TME). The TME polarizes tumor-infiltrating macrophages towards tumor supportive phenotype. We have previously reported that prolyl-hydroxylase inhibitor (PHDi) improved blood flow, hypoxia and TME in tumor tissues. The PHDi action mechanism is PHD inhibition, resulting in increasing hypoxia-inducing factors (HIFs) expression level. But the detail of its mechanism is unclear. In this study, we investigated the effect of HIFs in tumor infiltrating macrophage on tumor growth of PHD inhibitors on tumor progression.

In this study, we have used Lewis lung carcinoma (LLC) syngeneic tumor mouse models and used macrophage-specific HIF-1 and HIF-2 knockout mice. tumor was measured and calculated the tumor volume. Tumor tissues were collected at day16 and analyzed tumor vessels and immune cells by immunofluorescence staining.

In macrophage-specific HIF-2 knockout mice, tumor growth was inhibited by PHDi administration, but in macrophage-specific HIF-1 knockout mice were not inhibited it. In addition, macrophage-specific HIF-2 knockout mice were observed blood vessel normalization after PHDi administration, whereas macrophage-specific HIF-1 knockout mice were not observed it.

These results suggest that the inhibition of tumor growth by PHDi is associate with the HIF-1 in tumor infiltrating macrophages.

Trophoblast cell surface antigen 2 (TROP2) has become one of the effective therapeutic targets by antibody-drug conjugates (ADCs) such as sacituzumab govitecan which US FDA approved in 2020. TROP2 is reported to be overexpressed, and involved in tumor cell proliferation, invasion, metastasis, and poor prognosis in several types of solid tumor. Several clinical trials of anti-TROP2 ADCs are ongoing worldwide in patients with breast and lung cancer. Here, using a Cell-Based Immunization and Screening (CBIS) method, we developed a novel anti-TROP2 monoclonal antibody (clone TrMab-6; mouse IgG_2b, kappa). TrMab-6 was found to be applicable for many experiments, including flow cytometry, Western blotting, and immunohistochemistry. Furthermore, we investigated the potential of TrMab-6 for in vitro antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) activities, and in vivo antitumor activities. TrMab-6 strongly induced anti-tumor effects against breast cancer cell lines, including MCF7, MDA-MB-231, and MDA-MB-468 in vitro. In vivo experiments revealed that TrMab-6 significantly reduced tumor growth on breast cancer xenograft models. These results indicated that TrMab-6 could be a promising treatment option for TROP2-expressing breast cancers.

Background: Human epidermal growth factor receptor 3 (HER3) has been found to be overexpressed in many cancers such as lung, breast, and colon cancers. High expression of HER3 is thought to be a negative prognostic factor in several solid tumors including colorectal cancer. Moreover, HER3 is associated with drug resistance to EGFR- and HER2-targeted therapy in some cancers. Therefore, HER3 is thought to be an important therapeutic target.

Purpose: We aimed to develop a novel anti-HER3 monoclonal antibody (mAb), and investigated its antitumor activities against colorectal cancer.

Methods: We employed the Cell-Based Immunization and Screening (CBIS) method using HER3-overexpressed CHO-K1 cells for producing anti-HER3 mAbs. Anti-HER3 mAbs were screened using flow cytometry (FCM). Then, we examined the antibody‑dependent cellular cytotoxicity (ADCC) and the complement‑dependent cytotoxicity (CDC) activities of anti-HER3 mAbs for Caco‑2 (a human colorectal adenocarcinoma cell line). Moreover, a mouse xenograft model of Caco-2 was used for examining the antitumor activity.

Results: We developed an anti-HER3 mAbs, H₃Mab-17 (IgG_2a, kappa) using CBIS method. H₃Mab-17 reacted with HER3-expressing cells in FCM. In vitro analysis demonstrated that H₃Mab-17 showed the ADCC and CDC activities against Caco-2. H₃Mab-17 significantly reduced tumor development in Caco-2 xenograft compared with control mouse IgG.

Conclusion: We have successfully established a novel anti-HER3 mAb (H₃Mab-17), which could be a useful antibody-based therapy for patients with HER3-expressing colorectal cancers.

Background: Tranditonal Japanese Kampo medicine have been used for various diseases, however, there is few reports regarding Kampo medicine to exert renal protective action. In the present study, we investigated renal protective effect of Kampo medicine by using the model with cisplatin-induced nephrotoxicity (CIN).

Methods: We used and examined four Kampo formulas (A-D), which are considered to have an efficacy of anti-inflammation. We evaluated the effect of them via in culture cells and a mouse model with CIN.

Results: First, we checked whether those Kampo formulas exerted a preventive action of cisplatin-induced cell injury by using renal proximal tubular cells, and only C inhibited cell death by cisplatin. Mice administered cisplatin developed kidney injury with renal dysfunction, augmented oxidative stress, increased apoptosis, and elevated inflammatory cytokines; however, most of these symptoms were suppressed by treatment with C. Additionally, C did not interfere with its anti-tumor effect against tumor cells in in vitro experiments.

Conclusion: These findings suggest that Kampo formula C has a renal protective action.

Theanine is an amino acid found especially in green tea leaves. Theanine acts on the nervous system to relieve stress and assist in the maintenance of natural sleep. Theanine is also reported to have mild anti-cancer effects. However, the molecular mechanisms underlying these potential effects of theanine remain unknown. We evaluated the effect of theanine on cell growth of several neuronal cell lines (NSC-34, Neuro2A, SH-SY5Y), glial cell lines (C8-D1A, U-251MG) and vascular endothelial cell lines (bEnd.3, and HUVEC). Among them, we found that the growth of proliferative NSC-34 mouse motor neuron–like hybrid cells was the most sensitive to be inhibited by theanine and that this inhibition was highly correlated with the expressions of two solute carriers suspected of being theanine transporters: solute carrier family 38 member 1 (Slc38a1, a glutamine transporter) and solute carrier family 7 member 5 (Slc7a5; a glutamine/leucine exchanger). Theanine-mediated inhibition of cell growth in NSC-34 cells was completely mitigated by co-exposure with leucine, suggesting that theanine inhibited cell growth by disrupting Slc7a5-mediated glutamine/leucine exchange.

The increase of obesity and diabetic patients comes to be a problem, and it is said that excessive sugar intake is one of the reasons. In this study, we focused on glucose and sucrose, and examined changes in body weight, feeding behavior, and glucose metabolism of mice during long-term intake of beverages containing these sweeteners.

Mice were divided into three groups: water-group, glucose beverage-group, and sucrose beverage-group. Beverages and foods were made accessible at any time for two months. We measured body weight, amounts of 24-hour beverages intake and food intake once a week. After the experiment, oral glucose tolerance test (OGTT) and oral sucrose tolerance test (OSTT) were conducted to compare blood glucose levels and insulin secretion levels.

As a result, although the total calorie intake was the same among the three groups, the body weight of the sucrose beverage-group was significantly higher than that of the water-group and the glucose beverage-group. From the OGTT results, insulin was hardly elevated even though the blood glucose level of each sweet beverage-group rose sharply. From the OSTT results, the blood glucose level of the sucrose beverage-group was significantly higher than that of the glucose beverage-group, but no significant difference was observed in the insulin secretion amount.

Therefore, it was suggested that long-term ingestion of sweet beverages causes a decrease in insulin secretory capacity, and in sucrose, absorption of disaccharides and decomposition into monosaccharides are activated, which may cause weight gain.

Background: The goal of the role-play for pharmacological education (RPPE) is to acquire fundamental competences for drug therapy. These competences are based on presentation and communication skills as well as understanding of pharmacology and pathophysiology. The conventional face-to-face RPPE exhibited much higher learning effects in players than audiences, although audiences also established efficient learning through observation and discussion. However, the learning outcomes of the online RPPE (oRPPE) has not been determined. In this study, we evaluated oRPPE learning effects.

Methods: Participants: 4^th grade medical student at Tohoku Medical and Pharmaceutical University who took oRPPE in 2020 and 2021. Measurements: The questionnaire was used to evaluate learning effects. Four following items were asked on a five-point scale after the course completion. 1) Knowledge. Was oRPPE effective in understanding drug therapy? 2) Patients. Was oRPPE effective in understanding the patient's feelings? 3) Motivation. Has your motivation as a medical student improved through oRPPE? 4) Behavioral changes. Will oRPPE bring you a change in learning attitude? Analysis: Each item was compared between players and audiences. Differences were considered significant at P < 0.01.

Results: Total participants were 194 students (Players: 110, Audiences: 84) who completed the course. We collected questionnaire from players (n=106) and audiences (n=72). Knowledge, Motivation and Behavioral changes were significantly higher in players [Mean score (95% CI). Players vs. Audiences. Knowledge 4.509 (4.317-4.702) vs. 4.139 (3.948-4.329) (p<0.001); Motivation 4.434 (4.244-4.624) vs. 3.972 (3.746-4.199) (p<0.001); Behavioral changes 4.283 (4.107-4.459) vs. 4.042 (3.847-4.237) (p=0.0098)]. On the other hand, Patients was not statistically significant between players and audiences [4.377 (4.178-4.577) vs. 4.208 (4.007-4.409) (p=0.0216)].

Conclusions: Players showed much higher learning effects in Knowledge, Motivation and Behavioral changes. Learning as an audience, however, also established a better understanding of Patient's feeling.

Endoplasmic reticulum (ER) stress is caused by the accumulation of misfolded/unfolded proteins in the ER lumen, which leads to the activation of unfolded protein response (UPR), a set of signal transduction pathways. UPR coordinates protein homeostasis through the regulation of protein quality, synthesis, and degradation. Accumulating evidence suggests that the UPR plays an important role in neuropathological conditions such as neurodegenerative diseases and brain insults. However, the cell types and timing of UPR induction in pathogenesis are not fully understood. In this study, we examined the cellular and temporal patterns of the UPR after brain injury using the stab injury model in ER stress-activated indicator (ERAI) mice, which can monitor the UPR by detecting the activity of an ER stress transducer with green fluorescence. The fluorescent signals increased over time in the ipsilateral cortex from 6 hours to 7 days after brain injury in ERAI mice. The reporter signals were observed in injured neurons in the early stages after brain injury. However, the major cells positive for the fluorescent signals were non-neuronal cells such as vascular cells and astrocytes throughout the period analyzed after brain injury. These results suggest that UPR may play important roles not only in neurons but also in the non-neuronal cells in the course of neurological diseases including brain injury.

Polygalae Radix (PR) is traditionally used for dementia. Extracts of PR reportedly improved memory in elderly human and dementia mouse models. We previously found that the water extract of PR induced axonal growth in amyloid β-treated cultured neurons, and increased M2 microglia that showed anti-inflammatory and neuroprotective effects. These effects might contribute to memory recovery in the mouse models. In this study, we aimed to identify active constituents in the PR extract and analyze mechanisms of the constituents.

Thirty minutes after oral administration of the PR extract to normal mice, sibiricose A5 (SA5) was detected in the brains by LC-MS analysis. SA5 at 10 μM significantly induced axonal growth after amyloid β induced axonal degeneration in cultured cortical neurons. In cultured cortical microglia, amyloid β increased inflammatory M1 microglia. After that, SA5 at 1 nM tended to increase M2 predominance but not significant. Continuous intracerebroventricular injection of SA5 at 10 μM significantly improved memory impairment in 5XFAD Alzheimer's disease model mice, but SA5 at 1 nM did not. SA5 injection at 10 μM significantly decreased degenerated axon in the perirhinal cortex of 5XFAD mice. DARTS analysis showed a candidate of direct binding protein with SA5.

Above results indicate that SA5 affects neurons, not microglia, in the brain, induces axonal growth, and then recovers memory in 5XFAD mice. Now, we are investigating whether the protein identified by DARTS analysis is involved in mechanisms of SA5.

Neuroinflammation is a key pathological component of neurodegenerative disease and is characterized by microglial activation and the secretion of proinflammatory mediators. We previously reported that a surge in prostaglandin D₂ (PGD₂) production and PGD₂-induced microglial activation could provoke neuroinflammation. We also reported that a lipid sensor GPR120, which is expressed in intestine, could be activated by polyunsaturated fatty acids, thereby mediating secretion of glucagon-like peptide-1 (GLP-1). To reveal the relationship between PGD₂-microglia-provoked neuroinflammation and intestinal PUFA/GPR120 signaling, we investigated neuroinflammation and neuronal function in GPR120 knockout (KO) mice.

In the current study, we discovered notable neuroinflammation (increased PGD₂ production and microglial activation) and neurodegeneration (declines in neurogenesis, hippocampal volume, and cognitive function) in GPR120 KO mice. We also found that inhibition of PGD₂ production and potentiation of GLP-1 bioactivity reduced PGD₂-microglia-provoked neuroinflammation and further neurodegeneration in GPR120 KO mice.

These observations raise the possibility that intestinal GLP-1 secretion, stimulated by intestinal GPR120, may remotely contributed to suppress PGD₂-microglia-provoked neuroinflammation in the hippocampus.

Chronic widespread pain is a serious problem in our life; fibromyalgia is a well-known disease that causes chronic widespread pain, which might be ascribed to central sensitization. Repeated injection of reserpine, which depletes monoamines in the nervous system, has been used as an animal model of fibromyalgia. Repeated subcutaneous injection of reserpine induces behaviors associated with pain. Microglia in the spinal cord is one of the conceivable factors for the mechanism of central sensitization. However, it is still unclear whether and how microglia in the specific region of the brain affect pain perception.

Using CX3CR1-CreERT2 mice, we generated microglia-selective knockout mice of IRF8 or MafB, both of which have been previously confirmed to exhibit reduced symptoms of nerve injury-induced neuropathic pain. We found that these mice showed significant suppression of reserpine-induced mechanical hypersensitivity. Since no apparent morphological activation of microglia was seen in the spinal cord and the prefrontal cortex at 5 days after reserpine injection, we performed single-cell RNA sequencing with 10X Genomics Chromium platform on CD11b- and P2Y12-positive cells in the prefrontal and anterior cingulate cortex. Through a non-biased graph-based clustering analysis, we detected populational change among detected clusters. These results support the application of a concept of microglial regulation of reserpine-induced mechanical pain and also suggest functional alteration of prefrontal microglia in the symptom.