The Effects Of LPS On The Progression Of Hepatic Fibrosis And The Signal Pathway Of TLR-4/NF-? Bin NAFLD Rats

Objective:To study the effects of lipopolysaccharide(LPS)on the progression of liver fibrosis and the signal pathway of TLR-4/NF-K B in rats with non-alcoholic fatty liver disease(NAFLD)induced by choline-deficient L-amino acid-defined(CDAA)diet.Method:Thirty-six male Sprague-Dawley rats,6 weeks of age and weighing 140-150g were obtained and fed a week to adapt to the environment.They were randomly divided into two groups:18 rats were fed with choline-supplemented L-amino acid-defined(CSAA)diet,18 rats were fed with choline-deficient L-amino acid-defined(CDAA)diet as model group.At the end of the 4th week,6 rats were randomly killed in the two groups,and the right lobe of liver was retained for pathological examination to prove the model of rats with non-alcoholic fatty liver disease was success.On the basis of the original diet,the remaining 24 rats were daily supplementary given intraperitoneal injection of normal saline(NS)or LPS(0.25 mg/kg).That is,6 rats in the CSAA group intraperitoneal injection of NS 0.25mg/kg,the residual 6 rats in the CSAA fed group intraperitoneal injection of LPS 0.25 mg/kg respectively.6 rats in the CDAA fed group intraperitoneal injection of NS 0.25 mg/kg,the residual 6 rats in the CDAA fed group intraperitoneal injection of LPS 0.25 mg/kg respectively.At the end of the 10th week,all the subjects were weighed under ether anesthesia.Blood was collected from abdominal aorta and serum was isolated.Aspartate aminotransferase(AST),alanine aminotransferase(ALT),total cholesterol(TC)and triglyceride(TG)were determined by biochemical method.ELISA double antibody sandwich assay was used to determine the levels of lipopoly saccharide binding protein(LBP),tumor necrosis factor-a(TNF-?)and interleukin-6(IL-6).A portion of the right lobe of the liver was taken and the histopathological changes of the liver were observed by HE stain.Picrosirius red staining and immunohis to chemical staining were perphomed to detect alpha smooth muscle actin(?-SMA).The expression of genes related to LPS-TLR signaling pathway was detected by PCR microarray(PCR-Array).The effect of LPS on the process of liver fibrosis and the TLR-4/NF-?B signaling pathway in NAFLD rats was further investigated.Results:1.At the end of 4th week,HE staining showed that there were obvious fatty changes in liver tissue of rats in CDAA group.At the end of 10th week,in CSAA+LPS group,the liver structure was normal,and a small number of inflammatory cells could be found in the liver.In CDAA+LPS group and CDAA NS group,steatosis,hepatic lobular structure disordered and inflammatory cell infiltration were found in liver tissue,but CDAA+LPS group was more obviously.2.Compared with the other groups,the CDAA+LPS group rats body weight lost singnificantly(P<0.05);liver weight increased(P<0.01)and liver index increased(P<0.01).3.The contents of aspartate aminotransferase(AST),alanine aminotransferase(ALT),total cholesterol(TC)and triglyceride(TG)in serum of CDAA+LPS group were higher than those of other groups(P<0.01).4.Compared with the other groups,the content of IL-6,TNF-? and LBP increased significantly(P<0.01),in the group of CDAA+LPS.5.Sirius red staining and immunohistochemical a-SMA analysis showed that the percentage of perivascular fibrosis area in CDAA+LPS group increased significantly compared with the other groups(P<0.01).6.The PCR Array result suggests that compared with the CDAA+NS group,the expression of regulated to up or down more than 2 times the gene in CSAA+LPS group,and CDAA +LPS group.Namely after intraperitoneal injection of LPS rat liver tissue high expression of inflammatory cytokines:IL-6?TNF a?IIb;Inflammatory cell chemokines:Ccl2?Cxcl10;Apoptosis related factors:Fadd?Rela?Ripk2;Genes associated with MAPK signaling pathway:Map3k1?Map4k1;Initiation and Regulation of Gene expression of NF-?B Pathway:Nfkbl?Nfkb2;Tumor and cancer-promoting genes Ptgs2;Interferon factor Irf1.Low expression of anti-inflammatory factor:lfnb1?IL2;Granulocyte colony stimulating factor:Csf3.Conclusions:1.LPS promoted hepatic fibrosis induced by CDAA feed in NAFLD rats.2 LPS acts on TLR-4 and activates downstream NF-?B and MAPK signal transduction pathways,regulates the transcription of inflammatory factors,expresses and releases various inflammatory factors,accelerates and expands inflammatory reaction,activated Kupffer cells and hepatic stellate cells promotes liver fibrosis.