Tandem mass tag-based proteomics analysis of type 2 diabetes mellitus with non-alcoholic fatty liver disease in mice treated with acupuncture

Objective: To explore the proteomics profiles of hepatocytes of mice treated with acupuncture for type 2 diabetes mellitus (T2DM) with non-alcoholic fatty liver disease (NAFLD). Methods: We used a Tandem mass tag (TMT)-based quantitative proteomics approach to identify proteins with potential molecular mechanisms associated with acupuncture interventions for T2DM with NAFLD. Results: Acupuncture effectively improved body weight, blood glucose, and insulin levels in T2DM with NAFLD mouse models and reversed steatosis within hepatocytes. Quantitative TMT- based proteomics analysis identified a total of 4710 quantifiable proteins and 1226 differentially expressed proteins (DEPs) in the model control group (MCG) compared to the normal control group (NCG). The Acupuncture Treatment Group (ATG) presented in 122 DEPs compared to the MCG group. We performed a bioinformatics analysis, which revealed that DEPs enriched in the KEGG pathway after acupuncture treatment were mainly involved in the PPAR signalling pathway, fatty acid biosynthesis, fatty acid metabolism, fatty acid elongation, fat digestion, and absorption. We used parallel reaction monitoring (PRM) technology to explore the association of aldehyde oxidase 1 (Aox1), acyl-coenzyme A thioesterase 2 (Acot2), perilipin-2 (Plin2), acetyl-CoA carboxylase 1 (Acc), NADP-dependent malic enzyme (Me1), fatty acid synthase (Fasn), ATP-citrate synthase (Acly), fatty acid binding protein (Fabp2) with lipid synthesis, fatty acid oxidation and hepatocyte steatosis. Conclusions: Our results show that acupuncture can regulate the protein expression of T2DM in the NAFLD mice model, and can effectively improve hepatocyte steatosis, and has potential benefits for the clinical treatment of this disease.

The Study of Yin-Chen-Hao-Tang Preventing and Treating Alcoholic Fatty Liver Disease through PPAR Signaling Pathway Based on Network Pharmacology and RNA-Seq Transcriptomics

Background. Alcoholic fatty liver disease (AFLD) is the first stage of the alcoholic liver disease course. Yin-Chen-Hao-Tang (YCHT) has a good clinical effect on the treatment of AFLD, but its molecular mechanism has not been elucidated. In this study, we tried to explore the molecular mechanism of YCHT in improving hepatocyte steatosis in AFLD mice through network pharmacology and RNA sequencing (RNA-Seq) transcriptomics. Methods. Network pharmacological methods were used to analyze the potential therapeutic signaling pathways and targets of YCHT on AFLD. Then, the AFLD mice model was induced and YCHT was administered concurrently. Liver injury was measured by serum alanine aminotransferase (ALT) activity and liver tissue H&E staining, and liver steatosis was determined by serum triglyceride (TG) level and liver tissue Oil Red staining. The molecular mechanism of YCHT on prevention and treatment of mice AFLD was investigated according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differential expression genes data obtained by liver tissue RNA-Seq. Finally, ethanol-induced AFLD AML12 hepatocyte model was established, YCHT with or without PPARα agonist pemafibrate or PPARγ inhibitor GW9662 was administered, Nile Red fluorescent staining was used to evaluate steatosis levels in AML12 hepatocytes, and qRT-PCR was used to detect PPARα and PPARγ gene expression. Results. The results of network pharmacology analysis showed that YCHT may exert its pharmacological effect on AFLD through 312 potential targets which are involved in many signaling pathways including the PPAR signaling pathway. AFLD mice experiments results showed that YCHT markedly decreased mice serum ALT activity and serum TG levels. YCHT also significantly improved alcohol-induced hepatic injury and steatosis in mice livers. Furthermore, KEGG pathway enrichment results of RNA-Seq showed that the PPAR signaling pathway should be the most relevant pathway of YCHT in the prevention and treatment of AFLD. AFLD hepatocyte model experiment results showed that YCHT could remarkably reduce hepatocyte steatosis through reducing PPARγ expression and increasing PPARα expression. Conclusions. Our study discovered that PPARγ and PPARα are the key targets and the PPAR signaling pathway is the main signaling pathway for YCHT to prevent and treat AFLD.

Astragaloside Attenuates Atherosclerosis Coupled Inflammation Through miR-101/Mitogen-Activated Protein Kinase Phosphatase-1 (MKP-1)/p38 Signaling in Mice

This study aimed at elucidating the effect of astragaloside on atherosclerosis coupled inflammation and potential mechanism in mice. C57BL/6J mice were maintained in high-fat diet (HFD) for 12 weeks to induce atherosclerosis, with or without treatment with astragaloside (50 mg/kg). In turn, serum biochemical parameters in mice were also evaluated. Multiple tissue stain assay, including HE, were employed to assess the pathological alterations in arteries, and blood inflammation mediators were examined using ELISA. Expressions of microRNA101 (miR-101), p-p38 and mitogen-activated protein kinase phosphatase-1 (MKP-1) in the arteries were evaluated by qPCR and Western blot. Finally, AML-193 cells were transfected by miR-101 mimics and inhibitors. Expression of miR-101, MKP-1 and downstream inflammation cytokines were then analyzed. High-fat diet (HFD) mice with astragaloside treatment exhibited reduced atherosclerotic plaques size evaluated by oil red o, improved hepatocyte steatosis, and increased collagen fibers in atherosclerotic plaques for more stable plaque. Further, astragaloside treatment suppressed miR-101 transcription and enhanced MKP-1 expression, thus restraining the secretion of inflammation factors in vitro. Moreover, the inhibited impact of astragaloside in inflammatory factors production was ineffective in the presence of miR-101 mimics in AML-193 cells stimulated by LPS. Astragaloside exerted an anti-inflammatory role through miR-101/MKP-1/p38 signaling, for reducing atherosclerotic plaques and alleviate inflammation damage in mice and AML-193 cell.

PATHOMORPHOGENESIS OF LIVER STEATOSIS IN PATIENTS WITH OPIOID DEPENDENCE

Background. One of the significant factors in the progression of fibrotic changes in the liver is hepatocyte steatosis, that persists in drug addicted patients even after the elimination of the hepatitis C virus and cessation of drug use. Analysis of the pathomorphogenesis of hepatic steatosis in opioid dependence (OZ) will make it possible to assess the factors that affect ultrastructural changes in hepatocytes and the processes of lipid granule (LH) degradation. Objective. Assessment of ultrastructural changes in LH in the liver tissue of patients with OZ. Material and methods. Histological preparations of liver tissue from 20 patients with OZ aged 21 to 40 years (18 men and 2 women) with different duration of OZ and opioid tolerance. Results. There was established the following dependence of ultrastructural changes in the liver in patients with different duration of OZ and opioid tolerance. The most pronounced changes were noted in the group of patients with prolonged (more than 6 years) opioid intoxication and high tolerance to opioids in the liver tissue, in whom, along with severe steatosis, there were more significant violations of the mechanisms of LH degradation, destruction of cristae in mitochondria, a decrease in the number of lipophagosomes and PH with signs of superficial degradation than in the group of patients with OZ duration up to 6 years, as well as with moderate and high opioid tolerance. Conclusions. Ultrastructural changes in hepatocytes in the form of progression of steatosis in the centrilobular and periportal zones, a decrease in the activity of LH degradation, gross morphological changes in mitochondria, a decrease in the activity of surface LH degradation depend on the activity and duration of opioid dependence and are more pronounced with long-term (more than 6 years) highly progressive opioid dependence.

Novel duck reovirus exhibits pathogenicity to specific pathogen-free chickens by the subcutaneous route

To study the pathogenicity of new duck reovirus (NDRV) to chickens, eighty 3-day-old SPF chickens were equally divided into two groups. The experimental group was inoculated with a NDRV challenge strain of 100 μL (10–5.00 ELD50/0.1 mL) by the subcutaneous (s.c.) route, and the control group was inoculated with 100 μL of sterile phosphate-buffered saline (PBS) by the same route. In the experimental group, chickens exhibited introflexion of claws, performing of splits, stunting syndrome, weight loss and death. Gross lesions such as enlargement and yellowish-white focal necroses were observed in the liver and spleen. Microscopic changes were typical including varying degrees of hepatocyte steatosis and necrosis, splenic lymphocyte necrosis, interstitial pneumonia. Viral loads were detected in lung, liver, heart, spleen, duodenum, burse and kidney. The liver and spleen viral loads remained a much higher level and maintained for a longer time, suggesting that these tissues might be the target organs. In summary, NDRV can cause systemic infections and death in chickens, which indicated that chickens may be infected by NDRV in poultry production.

Ferulic Acid Ameliorates Atherosclerotic Injury by Modulating Gut Microbiota and Lipid Metabolism

Atherosclerosis is a leading cause of death worldwide. Recent studies have emphasized the significance of gut microbiota and lipid metabolism in the development of atherosclerosis. Herein, the effects and molecular mechanisms involving ferulic acid (FA) was examined in atherosclerosis using the ApoE-knockout (ApoE-∕-, c57BL/6 background) mouse model. Eighteen male ApoE−/− mice were fed a high-fat diet (HFD) for 12 weeks and then randomly divided into three groups: the model group, the FA (40 mg/kg/day) group and simvastatin (5 mg/kg/day) group. As results, FA could significantly alleviate atherosclerosis and regulate lipid levels in mice. Liver injury and hepatocyte steatosis induced by HFD were also mitigated by FA. FA improved lipid metabolism involving up-regulation of AMPKα phosphorylation and down-regulation of SREBP1 and ACC1 expression. Furthermore, FA induced marked structural changes in the gut microbiota and fecal metabolites and specifically reduced the relative abundance of Fimicutes, Erysipelotrichaceae and Ileibacterium, which were positively correlated with serum lipid levels in atherosclerosis mice. In conclusion, we demonstrate that FA could significantly ameliorate atherosclerotic injury, which may be partly by modulating gut microbiota and lipid metabolism via the AMPKα/SREBP1/ACC1 pathway.

Olanzapine leads to nonalcoholic fatty liver disease through the apolipoprotein A5 pathway

ABSTRACTThe antipsychotic drug olanzapine was reported to induce nonalcoholic fatty liver disease (NAFLD), whereas the underlying mechanism remains incompletely understood. This study investigated whether apolipoprotein A5 (apoA5) and sortilin, two interactive factors involved in NAFLD pathogenesis, are implicated in olanzapine-induced NAFLD. In the present study, at week 8, olanzapine treatment successfully induced hepatic steatosis in male C57 BL/6J mice, even when fed a normal chow diet, which was independent of animal body weight gain. Likewise, olanzapine effectively mediated hepatocyte steatosis in human HepG2 cells in vitro, characterized by substantially elevated intracellular lipid droplets. Increased plasma triglyceride concentration and decreased plasma apoA5 levels were observed in mice treated for 8 weeks with olanzapine. Surprisingly, olanzapine markedly enhanced hepatic apoA5 protein levels in mice, without a significant effect on rodent hepatic ApoA5 mRNA expression. Our in vitro study showed that olanzapine reduced apoA5 protein levels in the medium and enhanced apoA5 protein expression in hepatocytes, whereas this drug exerted no effect on hepatocyte APOA5 mRNA expression. By transfecting APOA5 siRNA into HepG2 cells, it was demonstrated that APOA5 knockdown effectively reversed olanzapine-induced hepatocyte steatosis. In addition, olanzapine drastically increased sortilin mRNA and protein levels in vivo and in vitro. Interestingly, SORT1 knockdown reduced intracellular apoA5 protein expression and increased medium apoA5 protein levels in vitro, without affecting intracellular APOA5 mRNA expression. Furthermore, SORT1 knockdown greatly ameliorated hepatocyte steatosis in vitro. This study provides the first evidence that sortilin inhibits the hepatic apoA5 secretion that is attributable to olanzapine-induced NAFLD, which provides new insight into effective strategies against NAFLD for patients with schizophrenia administered olanzapine.

Empagliflozin Alleviates Hepatic Steatosis by Activating the AMPK-TET2-Autophagy Pathway in vivo and in vitro

Background: Metabolic associated fatty liver disease (MAFLD), characterized by hepatic lipid accumulation and fatty degeneration, is intertwined with obesity and type 2 diabetes mellitus (T2DM). Empagliflozin is a sodium-glucose cotransporter-2 inhibitor that effectively lowers blood glucose, but its effect on MAFLD and associated mechanisms are not fully understood.Methods: Eight-week-old db/db mice, an in vivo model, were administered empagliflozin or saline intragastrically. A hepatocyte steatosis model was established by inducing HL7702 cells with high glucose and palmitic acid and then treated with or without empagliflozin. The autophagy inhibitor (3-methyladenine, 3-MA) and AMP-activated protein kinase (AMPK) activator (AICAR)/inhibitor (Compound C) were used to determine the involvement of AMPK and autophagy in the regulation of lipid accumulation by empagliflozin. Ten-eleven translocation 2 (TET2) knockdown was achieved by siRNA transfection. Hepatic steatosis was evaluated by Oil Red O staining and triglyceride quantification. Immunohistochemistry, immunofluorescence, and western blot were performed to assess protein levels.Results: Empagliflozin alleviated liver steatosis in db/db mice and reduced triglyceride content and lipid accumulation in the hepatocyte steatosis model. Empagliflozin elevated autophagy, accompanied by an increase in p-AMPK and TET2. Both 3-MA and Compound C abolished the ability of empagliflozin to induce autophagy and reduce hepatic steatosis, while these effects could be recapitulated by AICAR treatment. TET2 knockdown resulted in autophagy inhibition and lipid accumulation despite empagliflozin treatment.Conclusion: Empagliflozin improves hepatic steatosis through the AMPK-TET2-autophagy pathway. The use of empagliflozin as a treatment for preventing and treating MAFLD in patients with T2DM warrants further study.