Research On The Mechanisms And Relationship Between The Mechanism Of Reducing The Intestinal Flora And The Anti-Alcoholic Steatohepatitis Of Xiaozhitang

ObjectiveTo study the effect of Xiaozhitang on non-alcoholic steatohepatitis mouse model and its effect on intestinal flora,and in vitro experiments further explore the mechanism of Xiaozhitang on free fatty acids(FFAs)-induced nonalcoholic fat Hepatitis(NASH)cell model and its mechanism.MethodsThe mice in the experimental group(C57BL/6j mice)were randomly divided into normal group,positive group and model group.The normal and positive group were fed with normal diet.The model group was fed with D12482 high fat diet.The model group was randomly divided into model group,Xiaoqingtang high medium,low dose group and fenofibrate group after the model was success.The rats were sacrificed for 9 weeks.Total cholesterol(TC),triglyceride(TG)content in liver tissue and TC,TG,aspartase aminotransferase(AST),alanine aminotransferase(ALT),content in the serum was detected by automatic biochemical analyzer.The expression of tumor necrosis factor alpha(TNF-?)?interleukin 6(IL-6)andLpopolysaccharide(LPS)was detected by ELISA.Two liver tissues were collected for paraffin embedding,HE staining and frozen sections,and oil red 0 staining.The bacterial species of Prevotella,Bifidobacterium,Escherichia coli,Fusobacterium and Lactobacillus was examined by reverse transcription polymerase chain reaction(RT-PCR).Extraction of feces and colon contents of the total DNA,the use of high-throughput sequencing technology to detect NASH mice feces and intestinal mucosa.The expression of TLR4 and NF-?B p65 protein was detected by RT-PCR.The expression of TLR4 and NF-?B p65 protein was detected by Immunohistochemical staining.In vitro experiments were performed by 0.5?mol/L FFAs HepG2 cells were used to establish NASH cell model,and NASH cell model was treated with different concentrations of Xiaozhitang and fenofibrate.The intracellular lipid droplets were observed by oil red 0 staining.The contents of TNF-a and IL-6 in supernatant were detected by Elisa.The expression of NF-?B p65,IKK ?,Phospho-IKK ?/?(Ser176/180),Phospho-IRS-1(Ser307)and IRS-1 protein were detected by Western blotting.ResultsThe levels of serum TG,TC,ALT,AST,FBG increased in model group(P<0.05 or P<0.01)compared with the normal group,but there was no significant decrease in serum ALT and AST in the fenofibrate group.Compared with the model group,the FINS,HOMA-IR,LPS,TNF-a and IL-6 were significantly increased(P<0.01)in each experimental group,but there was no significant difference in the fenofibrate groups(P>0.05)-The relative expression of TLR4 and NF-?B mRNA in the liver tissue of the model group was significantly higher than that of the normal group(P<0.05,P<0.01).The expression of TLR4 and NF-?B mRNA in the liver tissue of Xiaozhitang high dose group was significantly lower than that in the control group(P<0.05,P<0.01),but the expression of NF-?B mRNA in the middle and low dose groups was not significantly different from that in the model group.Compared with the normal group,the values of TLR4 and NF-?B mean density were significantly higher in the model group(P<0.05,P<0.01),which indicated that the expression of TLR4 and NF-?B protein in the high dose group was lower than that in the low dose group.And fenofibrate group had no significant difference compared with the model group.Compared with normal and positive group,the number of Fusobacterium,Pseudomonas,Bifidobacterium and Lactobacillus in the model group was significantly decreased(P<0.01),while the number of Escherichia coli(P<0.01),while there was no significant difference between the normal group and the zhengxiao normal group.Compared with the model group,and the number of Escherichia coli was decreased(P<0.01),while the low dose of Xiaozhi Decoction group and fenofibrate group had no difference with the model group.The levels of TNF-? and IL-6 in the supernatant of the model group were significantly higher than those in the normal group(P<0.01).The expression of IKK ?,P-IKK ?/?(Ser176/180),P-IRS-1(Ser307),NF-?B p65 nucleoprotein increased significantly in model group compared with the normal group(P<0.01),while the expression of IKK,8,P-IKK ?/?(Ser176/180),P-IRS-1(Ser307)decreased significantly in each experimental group compared with the model group(P<0.01),especially in Xiaozhitang high dose group.The expression of P-IKK ?/?(Ser176/180)?P-IRS-1(Ser307)reduced significantly in Xiaozhitang high dose group compared with middle dose,low dose group and fenofibrate group(P<0.01)..The expression of IRS-1 protein in each administration group was significantly increased(P<0.05).There was no significant difference in NF-?B p65 total protein in all groups.ConclusionXiaozhitang can significantly improve the insulin resistance of NAFLD,reduce liver fat accumulation and inflammatory response,may be related to its adjustment of intestinal flora,promote Lactobacillus,Bifidobacterium and other advantages of the number of bacteria related to the growth.Xiaozhitang can improve the lipid deposition and inflammatory response in FFA-induced NASH cell model.The mechanism may be related to the inhibition of intracellular IKK ?/?(Ser176/180),IRS-1(Ser307)phosphorylation and IKK ? protein expression.