Deletion of GPR81 activates CREB/Smad7 pathway and alleviates liver fibrosis in mice.

BACKGROUND: Enhanced glycolysis is a crucial metabolic event that drives the development of liver fibrosis, but the molecular mechanisms have not been fully understood. Lactate is the endproduct of glycolysis, which has recently been identified as a bioactive metabolite binding to G-protein-coupled receptor 81 (GPR81). We then questioned whether GPR81 is implicated in the development of liver fibrosis. METHODS: The level of GPR81 was determined in mice with carbon tetrachloride (CCl4)-induced liver fibrosis and in transforming growth factor beta 1 (TGF-ß1)-activated hepatic stellate cells (HSCs) LX-2. To investigate the significance of GPR81 in liver fibrosis, wild-type (WT) and GPR81 knockout (KO) mice were exposed to CCl4, and then the degree of liver fibrosis was determined. In addition, the GPR81 agonist 3,5-dihydroxybenzoic acid (DHBA) was supplemented in CCl4-challenged mice and TGF-ß1-activated LX-2 cells to further investigate the pathological roles of GPR81 on HSCs activation. RESULTS: CCl4 exposure or TGF-ß1 stimulation significantly upregulated the expression of GPR81, while deletion of GPR81 alleviated CCl4-induced elevation of aminotransferase, production of pro-inflammatory cytokines, and deposition of collagen. Consistently, the production of TGF-ß1, the expression of alpha-smooth muscle actin (α-SMA) and collagen I (COL1A1), as well as the elevation of hydroxyproline were suppressed in GPR81 deficient mice. Supplementation with DHBA enhanced CCl4-induced liver fibrogenesis in WT mice but not in GPR81 KO mice. DHBA also promoted TGF-ß1-induced LX-2 activation. Mechanistically, GPR81 suppressed cAMP/CREB and then inhibited the expression of Smad7, a negative regulator of Smad3, which resulted in increased phosphorylation of Smad3 and enhanced activation of HSCs. CONCLUSION: GPR81 might be a detrimental factor that promotes the development of liver fibrosis by regulating CREB/Smad7 pathway.

Immune response gene 1 deficiency impairs Nrf2 activation and aggravates liver fibrosis in mice.

A growing body of evidence suggests that metabolic events play essential roles in the development of liver fibrosis. Immune response gene 1 (IRG1) catalyzes the generation of itaconate, which function as a metabolic checkpoint under several pathological circumstances. In the present study, the hepatic level of IRG1 was determined in mice with carbon tetrachloride (CCl4)-induced liver fibrosis. And then the pathological significance of IRG1 and the pharmacological potential of 4-octyl itaconate (4-OI), a cell-permeable derivate of itaconate, in liver fibrosis were investigated in mice. The results indicated that the hepatic level of IRG1 was upregulated in mice with liver fibrosis. CCl4-induced formation of fibrotic septa and deposition of collagen was aggravated in IRG1 KO mice. IRG1 deletion also resulted in increased expression of transforming growth factor beta 1 (TGF-ß1), enhanced phosphorylation of Smad3, elevated level of alpha smooth muscle actin (α-SMA) and hydroxyproline, which were associated with compromised activation of nuclear erythroid 2-related factor 2 (Nrf2)-mediated antioxidant system and exacerbated oxidative stress. Interestingly, supplementation with 4-OI activated Nrf2 pathway, suppressed TGF-ß1 signaling and attenuated fibrogenesis. Our data indicated that upregulation of IRG1 might function as a protective response during the development of liver fibrosis, and 4-OI might have potential value for the pharmacological intervention of liver fibrosis.

Impacts of small-molecule STAT3 inhibitor SC-43 on toxicity, global proteomics and metabolomics of HepG2 cells.

OBJECTIVE: In this study, we aimed to investigate the cytotoxicity and potential mechanisms of SC-43 by analyzing the global proteomics and metabolomics of HepG2 cells exposed to SC-43. METHODS: The effect of SC-43 on cell viability was evaluated through CCK-8 assay. Proteomics and metabolomics studies were performed on HepG2 cells exposed to SC-43, and the functions of differentially expressed proteins and metabolites were categorized. Drug affinity responsive target stability (DARTS) was utilized to identify the potential binding proteins of SC-43 in HepG2 cells. Finally, based on the KEGG pathway database, the co-regulatory mechanism of SC-43 on HepG2 cells was elucidated by conducting a joint pathway analysis on the differentially expressed proteins and metabolites using the MetaboAnalyst 5.0 platform. RESULTS: Liver cell viability is significantly impaired by continuous exposure to high concentrations of SC-43. Forty-eight dysregulated proteins (27 upregulated, 21 downregulated) were identified by proteomics analysis, and 184 dysregulated metabolites (65 upregulated, 119 downregulated) were determined by metabolomics in HepG2 cells exposed to SC-43 exposure compared with the control. A joint pathway analysis of proteomics and metabolomics data using the MetaboAnalyst 5.0 platform supported the close correlation between SC-43 toxicity toward HepG2 and the disturbances in pyrimidine metabolism, ferroptosis, mismatch repair, and ABC transporters. Specifically, SC-43 significantly affected the expression of several proteins and metabolites correlated with the above-mentioned functional pathways, such as uridine 5'-monophosphate, uridine, 3'-CMP, glutathione, γ-Glutamylcysteine, TF, MSH2, RPA1, RFC3, TAP1, and glycerol. The differential proteins suggested by the joint analysis were further selected for ELISA validation. The data showed that the RPA1 and TAP1 protein levels significantly increased in HepG2 cells exposed to SC-43 compared to the control group. The results of ELISA and joint analysis were basically in agreement. Notably, DARTS and biochemical analysis indicated that SART3 might be a potential target for SC-43 toxicity in HepG2 cells. CONCLUSION: In summary, prolonged exposure of liver cells to high concentrations of SC-43 can result in significant damage. Based on a multi-omics analysis, we identified proteins and metabolites associated with SC-43-induced hepatocellular injury and clarified the underlying mechanism, providing new insights into the toxic mechanism of SC-43.

Small hepatitis B surface antigen interacts with and modulates enoyl-coenzyme A hydratase expression in hepatoma cells.

Enoyl-coenzyme A hydratase (ECHS1) is a key enzyme in the metabolism of fatty acids in mitochondria. We previously reported that hepatitis B surface antigen (HBsAg) interacted with ECHS1 in a yeast two-hybrid system. In the current study, we further examined their interaction by using GST pull-down and co-immunoprecipitation assays. The results confirmed that small hepatitis B surface antigen (SHBs) interacted with ECHS1. Furthermore, confocal imaging showed that SHBs and ECHS1 co-localized in HepG2 cells. To clarify the biological function of the interaction, human hepatoma cell lines that transiently and stably expressed SHBs were generated. The expression of SHBs led to a significant decrease in ECHS1 protein levels. ECHS1 protein levels were reduced to 48.44 ± 7.12 % in Huh7 cells transiently expressing SHBs, and to 54.97 ± 3.54 % in HepG2 cells stably expressing SHBs. In conclusion, our findings suggest that SHBs interacts with ECHS1 and regulates ECHS1 protein levels in hepatoma cells.

In Vitro Inhibition of Growth, Biofilm Formation, and Persisters of Staphylococcus aureus by Pinaverium Bromide.

Biofilm or persister cells formed by Staphylococcus aureus are closely related to pathogenicity. However, no antimicrobials exist to inhibit biofilm formation or persister cells induced by S. aureus in clinical practice. This study found that pinaverium bromide had antibacterial activity against S. aureus, with the MIC50/MIC90 at 12.5/25 µM, respectively. Pinaverium bromide (at 4 × MIC) showed a rapid bactericidal effect on S. aureus planktonic cells, and it was more effective (at least 1-log10 cfu/mL) than linezolid, vancomycin, and ampicillin at 4 h of the time-killing test. Pinaverium bromide (at 10 × MIC) significantly inhibited the formation of S. aureus persister cells (at least 3-log10 cfu/mL) than linezolid, vancomycin, and ampicillin at 24, 48, 72, 96, and 120 h of the time-killing test. Biofilm formation and adherent cells of S. aureus isolates were significantly inhibited by pinaverium bromide (at 1/2 or 1/4 × MICs). The fluorescence intensity of the membrane polarity of S. aureus increased with the treatment of pinaverium bromide (≥1 × MIC), and the MICs of pinaverium bromide increased by 4 times with the addition of cell membrane phospholipids, phosphatidyl glycerol and cardiolipin. The cell viabilities of human hepatocellular carcinoma cells HepG2 and Huh7, mouse monocyte-macrophage cells J774, and human hepatic stellate cells LX-2 were slightly inhibited by pinaverium bromide (<50 µM). There were 54 different abundance proteins detected in the pinaverium bromide-treated S. aureus isolate by proteomics analysis, of which 33 proteins increased, whereas 21 proteins decreased. The abundance of superoxide dismutase sodM and ica locus proteins icaA and icaB decreased. While the abundance of global transcriptional regulator spxA and Gamma-hemolysin component B increased. In conclusion, pinaverium bromide had an antibacterial effect on S. aureus and significantly inhibited the formation of biofilm and persister cells of S. aureus.

Immune response gene 1 deficiency aggravates high fat diet-induced nonalcoholic fatty liver disease via promotion of redox-sensitive AKT suppression.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder worldwide. Immune response gene 1 (IRG1) catalyzes the production of bio-active itaconate, which is actively involved in the regulation of signal transduction. A recent study has found that the expression of IRG1 was significantly down-regulated in obesity-associated fatty liver, but the potential roles of IRG1 in the development NAFLD remain unclear. The present study found that genetic deletion of IRG1 aggravated high fat diet (HFD)-induced metabolic disturbance, including obesity, dyslipidemia and insulin resistance. In addition, HFD induced more severe liver steatosis and higher serum ALT and AST level in IRG1 KO mice, which were accompanied with altered expression of genes involved in lipid uptake, synthesis and catabolism. RNA-seq and immunoblot analysis indicated that deficiency of IRG1 is associated with suppressed activation of AKT, a master metabolic regulator. Mechanistically, IRG1/itaconate enhanced the antioxidative NRF2 pathway and prevented redox-sensitive suppression of AKT. Interestingly, supplementation with 4-octyl itaconate (4-OI), a cell-permeable derivate of itaconate, alleviated HFD-induced oxidative stress, AKT suppression and liver steatosis. Therefore, IRG1 probably functions as a protective regulator in the development of NAFLD and the cell-permeable 4-OI might have potential value for the pharmacological intervention of NAFLD.

Negative regulation of pro-apoptotic AMPK/JNK pathway by itaconate in mice with fulminant liver injury.

Accumulating evidence indicates that metabolic responses are deeply integrated into signal transduction, which provides novel opportunities for the metabolic control of various disorders. Recent studies suggest that itaconate, a highly concerned bioactive metabolite catalyzed by immune responsive gene 1 (IRG1), is profoundly involved in the regulation of apoptosis, but the underlying mechanisms have not been fully understood. In the present study, the molecular mechanisms responsible for the apoptosis-modulatory activities of IRG1/itaconate have been investigated in mice with lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced apoptotic liver injury. The results indicated that LPS/D-Gal exposure upregulated the level of IRG1 and itaconate. Deletion of IRG1 resulted in exacerbated hepatocytes apoptosis and liver injury. The phospho-antibody microarray analysis and immunoblot analysis indicated that IRG1 deletion enhanced the activation of AMP-activated protein kinase (AMPK)/c-jun-N-terminal kinase (JNK) pathway in LPS/D-Gal exposed mice. Mechanistically, IRG1 deficiency impaired the anti-oxidative nuclear factor erythroid-2 related factor 2 (Nrf2) signaling and then enhanced the activation of the redox-sensitive AMPK/JNK pathway that promotes hepatocytes apoptosis. Importantly, post-insult supplementation with 4-octyl itaconate (4-OI), a cell-permeable derivate of itaconate, resulted in beneficial outcomes in fulminant liver injury. Therefore, IRG1/itaconate might function as a negative regulator that controls AMPK-induced hepatocyte apoptosis in LPS/D-Gal-induced fulminant liver injury.

[Sustained efficacy of alpha-interferon therapy combined with Yixuesheng Capsule in treatment of chronic hepatitis B].

OBJECTIVE: To observe the difference between the combination therapy of alpha-interferon (IFN-alpha) therapy Yixuesheng Capsule and the monotherapy of IFN-alpha in treatment of chronic hepatitis B. METHOD: A total of 288 patients with HBeAg-positive chronic hepatitis B proven by liver biopsy were included in this study. During the individualized therapy, they received hypodermic injection of IFN-alpha 1b, with 5 MU x time(-1) and three times x w(-1). Of them, 125 patients received combination therapy with Yixuesheng Capsule for three months, with 1.0 g/time and three times/d; and 163 patients received only IFN-alpha 1b (the IFN-alpha monotherapy group). After the course of therapy, all patients were followed up for at least 24 months. The intention-to-treat analysis was adopted for statistic analysis. RESULT: The two groups showed no statistical significance by gender, age, liver necroinflammation grading, liver fibrosis staging, serum ALT levels, serum HBV DNA levels and IFN-alpha therapy course. The whole course and the 24-month follow-up visit cover all of 112 patients in the combination treatment group and 141 cases in the IFN-alpha monotherapy group. The response rates of the combination treatment group and the IFN-alpha monotherapy group were 48.0% (60/125) and 35.0% (57/163) (x = 4.980, P = 0.026) at the end of treatment, respectively, 45.6% (57/125) and 33.1% (54/163) (x2 = 4.645, P =0.031) at the end of 12-month-follow-up period, respectively, and 38.4% (48/125) and 32.5% (53/163) (x2 = 1.076, P = 0.300) at the end of 24-month follow-up period, respectively. CONCLUSION: The combination treatment with IFN-alpha and Yixuesheng Capsule shows a slightly better sustained efficacy on HBeAg-positive chronic hepatitis B patients compared with IFN-alpha monotherapy.

Therapeutic benefits of apocynin in mice with lipopolysaccharide/D-galactosamine-induced acute liver injury via suppression of the late stage pro-apoptotic AMPK/JNK pathway.

The excessive generation of reactive oxygen species (ROS) plays crucial roles in the development of acute liver injury. Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is responsible for the robust production of ROS under inflammatory circumstance, but the pathological roles of NOX and the pharmacological significance of NOX inhibitor in acute liver injury remains unclear. In the present study, the potential roles of NOX in acute liver injury were investigated in a mouse model with lipopolysaccharide (LPS)/D-galactosamine (D-Gal)-induced acute liver injury. The results indicated that LPS/D-Gal exposure time-dependently increased the level of ROS in liver tissue. Pretreatment with the NOX inhibitor apocynin suppressed LPS/D-Gal induced upregulation of ROS, 8-hydroxy-2-deoxyguanosine (8-OH-dG), protein carbonyl content and thiobarbituric acid reactive substances (TBARS). Pretreatment with apocynin also suppressed LPS/D-Gal-induced elevation of aminotransferase, alleviated histological abnormalities, inhibited the production of pro-inflammatory cytokine tumor necrosis factor α (TNF-α), blocked the activation of caspase cascade, reduced the count of TUNEL-positive cells and prevented LPS/D-Gal-induced mortality. Interestingly, post insult treatment with apocynin also suppressed LPS/D-Gal-induced oxidative stress, hepatocyte apoptosis, liver damage but improved the survival rate. Mechanistically, posttreatment with apocynin prohibited LPS/D-Gal-induced activation of the late stage pro-apoptotic AMP-activated protein kinase (AMPK)/c-Jun N-terminal kinase (JNK) pathway. Post-insult treatment with the antioxidant N-acetylcysteine also resulted in suppressed activation of AMPK/JNK, mitigated apoptosis and alleviated liver injury. These data suggest that NOX-derived ROS might be a crucial late stage detrimental factor in LPS/D-Gal-induced acute liver injury via promoting the activation of the pro-apoptotic AMPK/JNK pathway, and the NOX inhibitor might have important value in the pharmacological intervention of inflammation-base liver damage.

Comprehensive analysis of transcriptomics and metabolomics to understand triptolide-induced liver injury in mice.

Triptolide, a major active component of Triptergium wilfordii Hook. f, is used in the treatment of autoimmune disease. However, triptolide is associated with severe adverse reactions, especially hepatotoxicity, which limits its clinical application. To examine the underlying mechanism of triptolide-induced liver injury, a combination of dose- and time-dependent toxic effects, RNA-seq and metabolomics were employed. Triptolide-induced toxicity occurred in a dose- and time-dependent manners and was characterized by apoptosis and not necroptosis. Transcriptomics profiles of the dose-dependent response to triptolide suggested that PI3K/AKT, MAPK, TNFα and p53 signaling pathways were the vital steps in triptolide-induced hepatocyte apoptosis. Metabolomics further revealed that glycerophospholipid, fatty acid, leukotriene, purine and pyrimidine metabolism were the major metabolic alterations after triptolide exposure. Finally, acylcarnitines were identified as potential biomarkers for the early detection of triptolide-induced liver injury.