The Impact And Mechanism Of Peptidoglycan On Non-alcoholic Fatty Liver Disease

Background and Objective Non-alcoholic fatty liver disease(NAFLD)is not a common benign lesion but a early symptom of many liver diseases which could result in fibrosis,recurrence of liver transplantation even hepatocarcinoma.Intestinal flora disturbance is one of the most important factors contributing to NAFLD.The propotion of intestinal firmicutes steeply increases in patients with NAFLD.PGN(peptidoglycan),the major component of plasma membrane of most firmicutes,induces the secretion of various inflammatory cytokines through the activation of NF ? B(nuclear factor kappa-light-chain-enhancer of activated B cells)through TLR2(toll like receptor 2)and NLRs(nucleotide-binding oligomerization domain-like receptors).The excessive activation of NF ? B induces energy metabolic disturbance.The lipid metabolism of liver mainly includes lipogenesis,lipolysis and fatty acids transportation.PPAR ?(peroxisome proliferator-activated receptor)is a significant transcription factor of lipid metabolism.Under inflammatory condition,the expression of SREBP1c(sterol regulatory element binding protein 1c)required for lipid production and CD36(C luster of D ifferentiation 36)required for fatty acids transportation are significantly upregulated.To date,little is known about the effect and mechinism of PGN on NAFLD,thus the aim of the present study is to identify the role of PGN in NAFLD and investigate PGN for the mode of action in lipogenesis and lipid transportation as well as mitochondrial function with both cell and animal models,which contributes to providing creative theoretical basis for prevention of NAFLD occurrence and therapy.Methods1.Establish C57BL/6 mice and cell models of NAFLD with PGN.2.Immunohistochemistry was applied to observe morphological changes of animal and cell models.3.WB(Western blotting)and QPCR(Real-time Quantitative PCR Detecting System)were applied to identify the expression of metabolic and immunological related genes of cell4.and animal models.5.Next generation DNA sequencing technology was applied to analyze the composition of gut microbiota from animal model.6.The MSD SCALE DISCOVERY platform was applied to quantify the amount of inflammatory factors in liver and serum of animal models.7.Use the Seahorse XF24 analyzer to examine mitochondrial function.8.Knockdown of TLR2 and overexpression of NOD2 in LO2 cells were applied to identify the effect of both receptors in lipid metabolism.9.8.The Cross-binding experiment between NF?B and PPAR? was applied to identify the effect of NF?B on the activity of PPAR?Results1.Hypertriglyceridemia and hypercholesterolemia were observed in mice injected with PGN and both glucose tolerance and insulin tolerance were deteriorated in the manner independent of food intake in mice fed with either standard chow or high-fat diet.PGN significantly increased lipid droplets measured by oil red O staining,as well as basal and oleic acid-stimulated levels of triglyceride content in PHCs and LO2 cells.2.Significant differences in liver and adipose tissue between treatment group and control group were observed by HE staining(hematoxylin-eosin staining),oil-red O staining and immunohistochemistry.3.The expression of lipogenesis and fatty acids transportation related genes were upregulated after PGN treatment in both cell and animal models.4.Intraperitoneal infusion of PGN significantly altered gut mirobiota constitution in mice.The propotion of bacteroidaceae decreased while the proportion of firmicutes significantly rose that is consistent with the clinical subjects.5.The increasing amount of inflammatory factors including IL-6,MCP-1 and TNF-alpha was observed in both liver and serum.6.PGN significantly inhibited mitochondrial function in LO2 cells.7.PGN-induced triglyceride accumulation in LO2 cells of knockdown TLR2 was not improved.The expression of NOD2/NF ? B/PPAR ? /CD36 signaling pathway was upregulated by overexpression of NOD2 in LO2 cells.Conclusions1.PGN contributes to insulin resistance,fatty liver and liver damage.2.PGN plays an important role in inflammatory liver and intestinal flora disturbance.3.The machanism of PGN-induced triglyceride accumulation in hepatocytes is involved in lipogenesis,FFAs uptake and mitochondrial function.