ABSTRACT
Objective::To observe the effect of Shenling Baizhusan(SBS)on the mammalian target of rapamycin complex 1 (mTORC1)/signal transducers and activators of transcription 3 (STAT3) pathway in liver hepatocyte of nonalcoholic fatty liver disease(NAFLD)rats induced by high fat diet, in order to reveal the mechanism of SBS against rat NAFLD from the perspective of inflammation. Method::Totally 80 SD rats were randomly divided into 4 groups, normal control group, model group, high-dose SBP group(30 g·kg-1), and low-dose SBS group(10 g·kg-1), with 20 rats in each group. The rats of NAFLD model were established by being fed with high-fat diets for 8 weeks, and the treatment groups were fed with high or low dose of SBS respectively. After treatment for 8 weeks, blood and liver samples of rats were collected. Alanine aminotransferase (ALT), aspartate aminotransferase(AST), total cholesterol (TC), triglyceride(TG), high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C)levels in blood serum were detected with automatic biochemical analyzer. The liver tissues were observed by oil red O and hematoxylin-eosin (HE) staining. Hepatocytes were isolated by type Ⅳ collagenase perfusion in vitro. Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-5 and IL-6 in hepatocytes were detected by enzyme-linked immunosorbent assay (ELISA), and the relevant gene and proteins expressions of mTORC1 and STAT3 in hepatocytes were detected by Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) and Western blot detection respectively. Result::Compared with the normal control group, the serum levels of TG, TC, AST, ALT and LDL-C were increased significantly, the levels of TNF-α, IL-1β, IL-5 and IL-6 in hepatocytes were increased significantly, and the expression levels of mTORC1, STAT3 mRNA and proteins in hepatocytes were increased significantly(P<0.01). Compared with the model group, the hepatic lipid accumulation of the medicine intervention group was relieved significantly, the serum levels of AST, ALT, TG and LDL-C were decreased significantly, the expression levels of TNF-α, IL-1β, IL-5 and IL-6 of hepatocytes were decreased significantly, and the expressions of mTORC1, STAT3 mRNA and proteins in hepatocytes were decreased significantly(P<0.05, P<0.01). In the high-dose SBS group, the effects in improving the lipid accumulation and inhibiting the inflammatory reaction were better than those of the low-dose SBS group, and the expressions of mTORC1 and STAT3 genes and proteins in hepatocytes were significantly decreased (P<0.05, P<0.01). Conclusion::SBS can improve the fat metabolism disorder and reduce liver lipid accumulation and inflammatory reaction in NAFLD rats induced by high-fat diet. The mechanism may be correlated with the inhibition of mTORC1/STAT3 pathway relating to genes and protein expression in hepatocytes.
ABSTRACT
Objective To identify the molecular mechanism of phosphatidylinositol-3-kinase (PI3K) in mediating necroptosis induced by tumor necrosis factor alpha (TNFα).Methods RNA interference mediated by lentivirus short hairpin RNA(shRNA) was used to downregulate p110α in L929 cells and Western blotting was used to determine the knockdown efficiency.In addition,Western blotting was used to detect the phosphorylation level of RIP1,RIP3 and MLKL in L929 cells treated with or without TNFα plus Z-VAD.Duolink assay kit was used to detect the interactions between different proteins or the same proteins.Results p110α knockdown was efficiently mediated by the lentivirus shRNA,which significantly suppressed the phosphorylation of RIP1,RIP3 and MLKL in the absence or presence of TNFα plus Z-VAD stimulation.Moreover,the interactions between p110α and RIP3,but not RIP1,increased in a time-dependent manner during the process of necroptosis,and p110α knockdown significantly inhibited the formation of necrosome and RIP1 homodimer or RIP3 homodimer.Conclusion PI3K mediates the TNFα-induced necroptosis by promoting the activation of RIP1/RIP3/MLKL signaling pathway.Given the increasing direct interactions between p110α and RIP3 during the process of necrosome formation,PI3K may regulate RIP3 kinase activity via direct phosphorylation of RIP3.