In Japan, S-1 is used as adjuvant chemotherapy for pancreatic cancer. Neutropenia during S-1 chemotherapy is reported to be an independent predictor of prolonged survival in patients with advanced gastric cancer. However, this is unclear in pancreatic cancer. This study aimed to examine the effect of severe neutropenia caused by S-1 adjuvant chemotherapy on pancreatic cancer prognosis: overall survival (OS) and recurrence-free survival (RFS), and the potential effect of the duration from surgery to S-1 administration on OS and RFS. This single-center, retrospective, observational study included patients who newly received S-1 adjuvant chemotherapy after curative resection of pancreatic cancer at the Japanese Red Cross Kyoto Daini Hospital between January 1, 2016, and September 30, 2020. Of the 43 patients, 9 had grade 3 or higher neutropenia (G3 group) and had a significantly longer median OS than the other 34 (non-G3 group) did. The median RFS of the G3 group was longer than that of the non-G3 group. The median time from surgery to S-1 administration was significantly shorter in the G3 group than in the non-G3 group. Cox proportional hazards regression analysis revealed that duration from surgery to S-1 administration <51 d (hazard ratio: 0.375, 95% confidence interval: 0.154–0.914, p = 0.031) and occurrence of grade 3 neutropenia (hazard ratio: 0.198, 95% confidence interval: 0.046–0.860, p = 0.031) were significantly associated with prolonged OS. In conclusion, initiating S-1 adjuvant chemotherapy early after surgery and the occurrence of grade 3 neutropenia may improve pancreatic cancer prognosis.

Metallothionein (MT) is a small-molecule protein that functions in essential trace element homeostasis. Among MT isoforms, MT3 is involved in neuronal activity, and its expression is reported to be decreased in patients with neurodegenerative conditions such as Alzheimer’s disease; however, only a few effective drugs have been reported to induce MT3 expression. In this study, we evaluated existing drugs for the induction of MT3 expression in the neuronal cell line of ReNcell CX cells. Using recombinant proteins of MT isoforms with the 3× Flag tag, we performed Western blotting (WB) with the primary antibodies against MT3 or Flag tag, and this method of WB for MT3 was confirmed specifically to detect the MT3 protein. We treated ReNcell CX cells with several HIF-PH inhibitors and evaluated MT3 expression via real-time RT-PCR. We found that FG4592 significantly enhanced MT3 expression at both RNA and protein levels. FG4592 treatment increased the amount of hypoxia-inducible factor 1 alpha (HIF1α) binding to the MT3 promoter. These findings indicate that FG4592 induces MT3 expression via increased HIF1α. In conclusion, we found FG4592 to be an endogenous MT3 inducer in the cells of the nervous system in this study. The findings of this study are expected to lead to the development of new MT3-inducing drugs for neurodegenerative diseases based on FG4592.

Fluorescence imaging analysis was performed in cardiomyocytes from the sinus node, the orthotopic pacemaker, and the pulmonary vein, a potential ectopic pacemaker that may cause atrial fibrillation, focusing on the role of the Na⁺/Ca²⁺ exchanger (NCX). Isolated cardiomyocytes from the guinea pig pulmonary vein and sinus node showing automaticity were loaded with fluorescence probes for analysis. Inhibition of NCX by SEA0400 decreased the Ca²⁺ transient frequency in the pulmonary vein cardiomyocytes but not in the sinus node. The basal intracellular Ca²⁺ concentration, as well as the number of Ca²⁺ sparks in the subsarcolemmal region, was higher in the pulmonary vein cardiomyocytes than in the sinus node. By contrast, the intracellular Na⁺ concentration was not different between the pulmonary vein and sinus node cardiomyocytes. The equilibrium potential for NCX (E_NCX) was estimated to be less negative in the pulmonary vein cardiomyocytes than in the sinus node. In conclusion, the forward mode NCX is involved in spontaneous activity in the pulmonary vein cardiomyocytes but not in the sinus node; this is probably because the Ca²⁺ supply and the driving force for the forward mode NCX are both larger in the pulmonary vein cardiomyocytes than in the sinus node.

Carboxylesterase (CES) plays an important role in the metabolism of ester-containing drugs such as prodrugs and is highly expressed in the human intestine and liver. The ideal prodrug is barely hydrolyzed by human intestinal CES (CES2A1) but is extensively converted to an active drug by human hepatic CES (CES1A). It is, therefore, important to evaluate CES2A1-mediated hydrolysis during intestinal absorption. Unfortunately, Caco-2 cells, the most common enterocyte model for drug permeability, are not suitable for permeability studies of prodrugs due to their high and extremely low expression of CES1A and CES2A1, respectively. Previously, we have prepared CES2/Caco-2^CES1KD cells with reduced human CES1A and highly expressed CES2A1. In the present study, the metabolic and transport properties of CES2/Caco-2^CES1KD cells were characterized. The expression of transporters and metabolizing enzymes other than CESs was similar in CES2/Caco-2^CES1KD and Caco-2 cells. However, the expression of CES2A1 in CES2/Caco-2^CES1KD was about 7–10 fold higher than that of CES1A in Caco-2 cells and comparable to levels found in the human intestine. Hydrolysis during transport across cell monolayers was analyzed using ethyl and butyl esters of p-aminobenzoic acid (PABA). Ethyl PABA, a better substrate for CES1A than CES2A1, was similarly hydrolyzed in Caco-2 and CES2/Caco-2^CES1KD cell monolayers due to the high expression of CES2A1 in CES2/Caco-2^CES1KD cells. Butyl PABA, a good substrate for CES2A1, was substantially hydrolyzed in CES2/Caco-2^CES1KD cell monolayers, in contrast to negligible hydrolysis in Caco-2 cell monolayers. N-Acetylation of PABA derived from PABA esters showed similar activity in Caco-2 and CES2/Caco-2^CES1KD cell monolayers.

The non-canonical phosphorylation of the receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) at Ser-897 plays crucial roles in tumor progression in a tyrosine kinase-independent manner. This phosphorylation is catalyzed by p90 ribosomal S6 kinase (RSK), a kinase downstream of extracellular signal-regulated kinase (ERK). We recently reported that stress-responsive kinase mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2), instead of ERK, regulates RSK under cellular stress conditions; however, the function of MK2 in ERK-activated cells is still unknown. We herein clarified that MK2 regulates the RSK-EphA2 axis in ERK-activated echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) lung cancer cells. In addition, an MK2 inhibitor blocked enhancements in cell motility induced by the constitutively activated RSK-EphA2 axis. The present results reveal the importance of MK2 in the ERK-activated non-canonical activation of EphA2.

Enhanced inflammatory and immune responses have been observed in patients with major depressive disorder, pointing to anti-inflammatory substances as potential seeds for developing novel antidepressants. Omega-3 polyunsaturated fatty acid metabolites, such as resolvin D and E series, maresins, and protectins (collectively known as specialized pro-resolving mediators) demonstrate anti-inflammatory effects. This study examined the antidepressant-like effects of maresin-1 (MaR1) on lipopolysaccharide (LPS)-induced depression-like behaviors in mice. Using the tail suspension test (TST) and the forced swim test (FST), we assessed depression-like behaviors 26 and 28 h after intraperitoneal injection of LPS (0.8 mg/kg), respectively. An open field test (OFT) was also conducted to evaluate locomotor activity 24 h after LPS injection. Intracerebroventricular (i.c.v.) injection of MaR1 (10 ng/mouse) immediately after the LPS challenge mitigated the increased immobility time in the TST and FST, without affecting locomotor activity in the OFT, indicating the preventive effects of MaR1 on LPS-induced depression-like behaviors. Furthermore, i.c.v. injection of MaR1 23 h after the LPS challenge reduced the immobility time in both tests, underscoring its therapeutic potential. These findings suggest that MaR1 could be a promising seed for developing novel antidepressants.

A 3-dimensional (3D) cell culture is now being actively pursued to accomplish the in vivo-like cellular morphology and biological functions in cell culture. We recently obtained nano-fibrillated bacterial cellulose (NFBC). In this study, we developed a novel NFBC-based 3D cell-culture system, the OnGel method, and the Suspension method. HepG2 human liver cancer cells were cultured via these methods and formed spherical formulations in the optimized condition, 1.0% (w/v) of NFBC in the OnGel method, and 0.06–0.10% (w/v) of NFBC in the Suspension method. Non-cancerous cells such as human-induced pluripotent stem (iPS) cells and human mesenchymal stem cells (MSCs) also formed spherical formulations. It is noteworthy that both the size and cell viability of spheroids prepared via these methods were comparable to those cultured using commercially available 3D cell-culture systems. Both OnGel and Suspension methods are less complicated than the existing 3D cell-culture systems, which is an invaluable advantage for the preparation of cancer spheroids. The NFBC-based 3D cell-culture systems introduced here show great promise as a tool to prepare cultures for cell-derived spheroids for the progress of both in vitro and in vivo studies of the biological functioning of cells.

Pharmacological activation of G protein-coupled receptor 119 (GPR119) produces pleiotropic beneficial effects, including the promotion of insulin secretion from pancreatic β-cells, enhancement of glucagon-like peptide (GLP)-1 secretion from intestinal L cells, glucose-dependent insulin secretion, and food intake and body weight gain suppression. Thus, GPR119 has attracted attention as a promising new target for type 2 diabetes mellitus (T2DM) treatment. Here, we identified a new small GPR119 agonist, NCP-322. This compound showed potent enhancing effects on insulin and GLP-1 secretion, which played a role in pancreatic β-cells and intestinal L cells. In the oral glucose tolerance test, NCP-322 administration reduced glycemic excursions that were only exhibited during hyperglycemia. Furthermore, NCP-322 administration did not induce hypoglycemia, the main side effect of antidiabetic drugs. These results suggest the promising therapeutic potential of NCP-322 for T2DM treatment.

Oxidative stress and neuroinflammation accompanied by microglial activation are increased in Alzheimer’s disease (AD) and contribute to the pathogenesis of AD. Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) is a master transcription factor that acts as an endogenous defense mechanism against oxidative stress and inflammation and is a potential target for preventing AD. Psoraleae Semen (PS) reportedly has antioxidant and anti-inflammatory effects. This study aimed to examine the effects of PS extract (PSE) on Nrf2 activation and prevention of cognitive dysfunction in App^NL-P-F AD model mice. The effects of PSE on antioxidant response element (ARE) activity and cytoprotection in PC12 cells and on microglial activation in BV-2 cells were evaluated. PSE showed high ARE activity and prevented 6-hydroxydopamine-induced cytotoxicity in PC12 cells. Moreover, PSE suppressed lipopolysaccharide-induced nitric oxide production in BV-2 cells. Oral administration of PSE prevented cognitive dysfunction in App^NL-P-F mice without affecting motor function. Our results support that PSE can contribute to the development of new preventive and therapeutic agents for AD focusing on Nrf2 activation.

Vitamin D plays a crucial role in immune system function. Several studies have indicated that genetic variations in the vitamin D receptor (VDR) and vitamin D binding protein (VDBP, encoded by GC gene) increase the risk of developing asthma. However, the effect of these variations on the prognosis and clinical outcomes of asthma remains unclear. This study, involving 152 adult patients with asthma, aimed to assess the influence of VDR and GC polymorphisms on asthma severity and its exacerbation. Gene polymorphisms previously associated with asthma risk were analyzed, and VDR mRNA expression levels were evaluated in peripheral blood mononuclear cells. The AA genotype of the VDR rs2228570 polymorphism was associated with an elevated risk of severe asthma compared to the AG/GG genotype (odds ratio, 3.20; 95% confidence interval [CI], 1.24–8.28). Furthermore, patients with the rs2228570 AA genotype showed an elevated risk of exacerbation during the 1-year follow-up period (hazard ratio, 4.01; 95% CI, 1.75–9.15). VDR mRNA expression was significantly reduced in patients with the AA genotype. Furthermore, the mRNA expression levels of GLCCI1, HDAC2, NR3C1, and NFE2L2, which are associated with steroid response, were reduced in patients with the AA genotype. Our findings indicate that patients with the AA genotype of VDR rs2228570 are more likely to experience severe asthma and exacerbations. This polymorphism has the potential to reduce vitamin D efficacy by altering VDR function and expression, potentially resulting in increased inflammation and reduced steroid responsiveness in patients with asthma.

Statins are cholesterol-lowering drugs often used for the treatment of dyslipidemia. Statins also exert anti-cancer effects by inhibiting hydroxymethylglutaryl-CoA reductase (HMGCR), a rate-limiting enzyme in cholesterol synthesis. We previously reported that the susceptibility to statin treatment differs among cancer cells and that functional E-cadherin expression on the plasma membrane could be a biomarker of statin sensitivity in cancer cells. However, the detailed qualitative and molecular differences between statin-sensitive and statin-resistant cancer cells remain unclear. Here, we explored novel parameters related to statin sensitivity by comparing gene expression profiles and metabolite contents between statin-sensitive and statin-resistant lung cancer cell lines. We found that the expression of most cholesterol synthesis genes was lower in the statin-sensitive cancer cell line, HOP-92, than in the statin-resistant cancer cell line, NCI-H322M. Moreover, HOP-92 cells originally exhibited lower levels of CoA and HMG-CoA. Additionally, atorvastatin decreased the mRNA expression of PANK2, a rate-limiting enzyme in CoA synthesis. Atorvastatin also reduced the mRNA levels of the cholesterol esterification enzyme SOAT1, which was consistent with a decrease in the ratio of cholesterol ester to total cholesterol in HOP-92 cells. Our data suggest that the cholesterol synthetic flow and CoA content may be limited in statin-sensitive cancer cells. We also suggest that CoA synthesis and cholesterol storage may fluctuate with atorvastatin treatment in statin-sensitive cancer cells.

Sudachitin, a polymethoxyflavone found in sudachi peel, has been reported to improve hyperlipidemia in humans, and is thus attracting research attention. However, its effect on cardiac function remains unclear. We investigated the mechanisms underlying the chronotropic and inotropic effects of sudachitin on rat atria. Sudachitin (0.3–30 µM) produced concentration-dependent positive chronotropic and inotropic effects. Other polymethoxyflavones, including demethoxysudachitin (0.3–30 µM) and nobiletin (0.3–30 µM), also produced positive chronotropic and inotropic effects; however, the maximum efficacy of all polymethoxyflavones, including sudachitin, was lower than that of isoproterenol. Propranolol (0.1 µM) did not affect the positive chronotropic and inotropic effects of sudachitin. The concentration–response curves for the chronotropic and inotropic effects of dibutyryl-cAMP (1–100 µM) were shifted to the left upon pretreatment with sudachitin (3, 10 µM). Phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine 1 µM or milrinone 10 µM) alone, sudachitin alone (10, 30 µM), and a combination of phosphodiesterase inhibitors and sudachitin exhibited positive chronotropic and inotropic effects, whereas the lack of any interaction between each phosphodiesterase inhibitor and sudachitin indicated an additive effect of the two substances. These results suggest that sudachitin-induced positive chronotropic and inotropic effects similar to those of other polymethoxyflavones, but its maximum efficacy was lower than that of isoproterenol. Both demethoxysudachitin and nobiletin exhibited similar positive chronotropic and inotropic effects, indicating that these effects are not specific to sudachitin, but are common to polymethoxyflavones. The mechanism of action of sudachitin was associated with the enhancement of cAMP-dependent pathways, without the involvement of β-adrenoceptors.

Although medication oversupply results in waste of medications, triggers of medication oversupply remain unclear. This nationwide retrospective cohort study aimed to identify associated factors and causes of chronic disease medication oversupply in Japan by quantitative and qualitative analyses. Data of financial year 2019 from a large insurance claims database were used. Excess days, which represent medication oversupply in days, were calculated for each of the major five classes of chronic disease medications. For each class, the two cohorts were formed, consisting of individuals aged ≥55 years with either excessive oversupply or normal supply according to excess days. Oversupply-associated factors were subjected to quantitative analyses using logistic regression models, which included excessive oversupply vs. normal supply as an outcome variable. A qualitative analysis to identify causes of oversupply was performed by reviewing the medication history of 50 individuals randomly selected from each excessive oversupply cohort, and causes were classified into seven categories. Oversupply-associated factors in all classes were greater frequency of early supply (≥6 vs. <3 times/10 supplies, odds ratio (OR) 5–9 for all classes), inpatient prescription (included vs. not included, OR 3–5), and higher number of concomitant ingredients (≥16 vs. 1–5 ingredients, OR 3–5). The most common category of causes for oversupply in all classes was the “early supply of medications prescribed by a single facility.” The factors and causes of oversupply might reflect the unique features, rules, and customs of Japan’s healthcare system. This finding might help in developing measures for reducing medication oversupply.

Excessive inflammatory responses to viral infections, known as cytokine storms, are caused by overactivation of endolysosomal Toll-like receptors (TLRs) (TLR3, TLR7, TLR8, and TLR9) and can be lethal, but no specific treatment is available. Some quinoline derivatives with antiviral activity were tried during the recent coronavirus disease 2019 (COVID-19) pandemic, but showed serious toxicity, and their efficacy for treating viral cytokine storms was not established. Here, in order to discover a low-toxicity quinoline derivative as a candidate for controlling virally induced inflammation, we synthesized a series of derivatives of amodiaquine (ADQ), a quinoline approved as an antimalarial, and tested their effects on TLRs-mediated production of inflammatory cytokines and cell viability in vitro. In J774.1 murine macrophages, ADQ inhibited interleukin-6 (IL-6) production induced by TLR3 agonist poly(I:C), TLR7 agonist imiquimod, and TLR9 agonist cytosine-phosphate-guanosine oligodeoxynucleotide (CpG ODN) with IC₅₀ values of 2.43, 3.48, and 0.0359 µM, respectively, indicating that ADQ has a high inhibitory selectivity for TLR9 signaling. A structure–activity relationship study revealed that an appropriately substituted amino group on the phenol moiety and a halogen substituent on quinoline are important for potent anti-inflammatory activity and low cytotoxicity. ADQ analogs bearing N-butylethyl, N-3-fluoropiperidinyl, and N-4-fluoropiperidinyl groups in place of the N-diethyl group exhibited more potent activity and lower cytotoxicity than ADQ. ADQ and its analogs appear to inhibit the activity of TLRs recognizing pathogen nucleic acids via alkalinization of endolysosomes. Our results suggest that ADQ analogs are promising candidates as therapeutic agents for cytokine storms mediated by TLRs recognizing pathogen nucleic acid with reduced side effects.

Mucociliary clearance (MCC) is a host defense mechanism of the respiratory system. Beating cilia plays a crucial role in the MCC process and ciliary beat frequency (CBF) is activated by several factors including elevations of the intracellular cAMP concentration ([cAMP]_i), intracellular Ca²⁺ concentration ([Ca²⁺]_i), and intracellular pH (pH_i). In this study, we investigated whether an artichoke-extracted component cynaropicrin could be a beneficial compound for improving MCC. We found that cynaropicrin increased [cAMP]_i using A549 cells bearing Pink Flamindo. Then, we also confirmed that cynaropicrin elevates CBF using airway epithelial ciliated cells (AECCs). We next investigated the effects of cynaropicrin on the alternation of [Ca²⁺]_i, and pH_i. Cynaropicrin increased [Ca²⁺]_i, but not pH_i. Further experiments also found that cynaropicrin increased [cAMP]_i primarily by raising [Ca²⁺]_i. To elucidate the mechanisms of cynaropicrin to increase [Ca²⁺]_i, we investigated the alternation of the effects of cynaropicrin on [Ca²⁺]_i using several compounds. BTP-2 and ruthenium red (RuR) inhibited cynaropicrin-induced [Ca²⁺]_i increase and RuR reduced also [cAMP]_i. These results suggest that cynaropicrion increased [Ca²⁺]_i by augmenting the Ca²⁺ influx and that the increase of [cAMP]_i by cynaropicrin was induced by [Ca²⁺]_i elevation. Interestingly, cynaropicrin decreased the Ca²⁺ concentration in the endoplasmic reticulum following inhibition of sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA). SERCA activator CDN1163 abolished this effect. Furthermore, RuR and Ca²⁺-free conditions suppressed the increase of CBF. In conclusion, cynaropicrin inhibits SERCA, induces store-operated calcium entry, and thereby increases CBF.

Depressed patients may exhibit glucocorticoid hypersecretion, suggesting that elevated levels of glucocorticoids may play an important role in the pathophysiology of depression. Some postmortem brain studies have shown decreased pH and increased lactate levels in psychiatric patients, implying involvement of these factors in the pathogenesis. To investigate the effects of glucocorticoids on brain pH and lactate levels, and their roles in depressive symptoms, brain pH and lactate were examined in mice treated with corticosterone (CORT), the major bioactive glucocorticoid in rodents. A single administration of CORT decreased hippocampal pH after 24 h. Three weeks of CORT treatment decreased pH in the prefrontal cortex (PFC), striatum, and hippocampus (HC), whereas intake of pH 9.0 drinking water increased pH in these brain regions. pH and lactate levels were correlated in the PFC and HC of mice treated with CORT for 3 weeks. The suppression of body weight gain and decrease in adrenal weight observed after 3 weeks of CORT treatment were not alleviated by pH 9.0 water. However, an increase in immobility time in the forced swim test and a decrease in neurogenesis in the hippocampus were alleviated. The decrease in brain pH and increase in immobility time in the forced swim test and a decrease in neurogenesis in the hippocampus induced by CORT treatment were abolished by co-treatment with the glucocorticoid receptor (GR) antagonist mifepristone. These findings indicate that decreased brain pH via GRs may be related to glucocorticoid-induced depression-like behavior and decreased hippocampal neurogenesis.

Therapeutic drug monitoring (TDM) is a routine clinical practice used to individualize drug dosing to maintain drug efficacy and minimize the consequences of overexposure. TDM is applied to many drug classes, including immunosuppressants, antineoplastic agents, and antibiotics. Considerable effort has been made to establish routine TDM practices for each drug. However, because TDM has been developed within the context of specific drugs, there is insufficient understanding of historical trends within the field of TDM research as a whole. In this study, we employed text-mining approaches to explore trends in the TDM research field. We first performed a PubMed search to determine which drugs and drug classes have been extensively studied in the context of TDM. This investigation revealed that the most commonly studied drugs are tacrolimus, followed by cyclosporine and vancomycin. With regard to drug classes, most studies focused on immunosuppressants, antibiotics, and antineoplastic agents. We also subjected PubMed records of TDM-related studies to a series of text-mining pipelines. Our analyses revealed how TDM research has evolved over the years, thereby serving as a cornerstone for forecasting future research trends.

The blood–brain barrier (BBB) is a dynamic interface controlling the compound translocation between the blood and the brain, thereby maintaining neural homeostasis. There is cumulative evidence that BBB impairment during diabetes mellitus (DM) takes part in the progression of cognitive dementia. As tight junction proteins and ATP-binding cassette (ABC) transporters regulate substance exchange between the circulating blood and brain, the expression and function of these molecules under DM should be fully clarified. To understand the alteration of ABC transporter function in the BBB under DM, in vitro multicellular rat BBB spheroids consisting of conditionally immortalized rat brain capillary endothelial cells, astrocytes, and pericytes were newly developed. Immunostaining and permeability analysis of paracellular transport markers suggested the construction of tight junctions on the surface of the BBB spheroids. Transport analyses using fluorescence substrates of P-glycoprotein (P-gp), the breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 4 (MRP4) indicate the functional expression of these transporters in the spheroids. After treatment with advanced glycation end-products (AGEs), involved in various signals during DM, the mRNA expression of tight junction molecules and ABC transporters in the BBB spheroids was upregulated. Furthermore, the functional changes in P-gp and BCRP in the BBB spheroids exposed to AGEs were canceled by the inhibitors of the receptor for AGEs (RAGE). These results suggest that AGE-RAGE interaction upregulates P-gp and BCRP function in the BBB.

Nurr1 (NR4A2) is a member of nuclear receptor superfamily that regulates gene transcription in midbrain dopaminergic neurons and also inhibits nuclear factor-κB-mediated inflammatory responses in brain microglial cells. To date, various compounds have been reported to stimulate transcriptional activity of Nurr1 on neuronal genes, but their anti-inflammatory actions are poorly characterized. The present study examined the effects of three kinds of Nurr1 ligands, amodiaquine, 1,1-bis(3′-indolyl)-1-(p-chlorophenyl)-methane (C-DIM12) and 5-chloronaphthalen-1-amine (5-CNA), on inflammatory responses of microglial BV-2 cells. Lipopolysaccharide (LPS)-induced upregulation of interleukin-1β mRNA and tumor necrosis factor α mRNA was inhibited by all three compounds, whereas upregulation of interleukin-6 mRNA and inducible nitric oxide synthase (iNOS) mRNA was significantly inhibited only by 5-CNA. On the other hand, LPS-induced nuclear translocation of p65 subunit of nuclear factor-κB was prevented only by amodiaquine. C-DIM12 increased nuclear localization of Nurr1 and transiently upregulated Nurr1 protein expression, whereas amodiaquine and 5-CNA had no effect on these parameters. Notably, inhibitory effect of 5-CNA on iNOS mRNA upregulation was reversed by co-application of amodiaquine. Conversely, inhibitory effect of amodiaquine on p65 nuclear translocation was cancelled by 5-CNA. These results reveal distinct characteristics of anti-inflammatory actions of Nurr1 ligands.

Cellular aging causes declining cell functionality, gradually disrupting cellular homeostasis. Mitochondria are crucial in numerous metabolic processes, including the electron transport chain and fatty acid β-oxidation. Mitochondrial dysfunction is closely linked to aging-related liver dysfunction because it impairs fatty acid metabolism, potentially leading to nonalcoholic fatty liver disease. We demonstrated that neferine-induced autophagy suppressed the aging phenotype in proliferative and replicative aging-induced cells and aging liver tissue by reactivating mitochondrial function. Pharmacological analyses revealed that neferine-induced autophagy via the death-associated protein kinase 1 (DAPK1) and c-Jun N-terminal kinase (JNK) signaling pathways despite the lack of AMP activated protein kinase (AMPK) signaling activation. Furthermore, neferine stimulated ATP production and β-oxidation activity in aging cells. Our in vivo experiments demonstrated that oral administration of neferine rejuvenated aging liver tissue, suppressed fatty acid accumulation in the liver, and reduced senescence-associated β-galactosidase activity. Thus, neferine rejuvenated aging cells and liver tissue by inducing autophagy to reactivate mitochondrial function.