| ObjectiveTo study the effects of Xiaoyaosan on nonalcoholic steatohepatitis(NASH)rats with liver stagnation and spleen deficiency and macrophage inflammatory model via the Toll like receptor 4(TLR4)/TIR Domain containing adaptor protein inducing interferon-?(TRIF)pathway,to reveal the pathogenesis of NASH liver stagnation and spleen deficiency syndrome and the anti-inflammatory mechanism of Xiaoyaosan.MethodsPart one: The effects of Xiaoyaosan on rats of NASH with liver stagnation and spleen deficiency syndrome via TLR4/TRIF signaling pathwayForty male SD rats were randomly divided into blank control group(n=10),disease syndrome combined model group(n=30).The model of NASH liver stagnation and spleen deficiency was replicated by using high-fat and high-sugar diet and “chronic restraint + hunger disorder” method,and the modeling time was 14 w.The rats of disease syndrome combined model group are divided into model group(Model group,n=10),Xiaoyaosan treatment group(XYS group,n=10),and glycine treatment group(Glycine group,n=10),and the rats in the XYS and Glycine groups were treated with Xiaoyaosan and glycine for 4 weeks respectively.To obverve the weight and behavior changes of rats in each group.The content of triglycerides(TG),total cholesterol(TC)and alanine aminotransferase(ALT)in serum were detected.The endotoxin(ET)content of plasma was detected.The content of serotonin(5-HT)and norepinephrine(NE)in the hypothalamus of rats was detected.The D-xylose content was measured and the D-xylose excretion rate was calculated.Hematoxylin-eosin(HE)staining was used to observe the histopathological changes of the liver.Real-time quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA content of TLR4 and TRIF in liver tissue.Part two: The effect of Xiaoyaosan on macrophage inflammation model and its mechanism1.The optimal conditions for selecting the macrophage inflammation modelThe experiment was divided into groups which macrophages were stimulated with 5?g/mL lipopolysaccharide(LPS)for 0h,6h,12 h,24h.And tumor necrosis factor-?(TNF-?),interleukin-6(IL-6),interleukin-4(IL-4),interleukin-10(IL-10)were detected by ELISA.2.The effect of different concentrations of Xiaoyaosan on the proliferation of macrophage inflammation models detected by CCK-8The experiment was divided into 5% Xiaoyaosan-containing serum culture solution group(5%XYS group),10% Xiaoyaosan-containing serum culture solution group(10%XYS group),and 20% Xiaoyaosan-containing serum culture solution group(20%XYS group).The Xiaoyaosan-containing serum was prepared and the effects of different concentrations of Xiaoyaosan-containing serum culture solution for the cell proliferation of macrophage inflammation model after 0h,6h,12 h and 24 h were detected by CCK-8.3.The effect of Xiaoyaosan on macrophage inflammatory modelThe experiment was divided into normal group(Control group),model group(Model group),Xiaoyaosan group(XYS group).Xiaoyaosan and 5?g/mL LPS were combined on macrophages for 6h,and the contents of TNF-?,IL-6,IL-4 and IL-10 were detected by ELISA.The expression of TLR4 and TRIF proteins was detected by western blot and immunocytochemistry.ResultsPart one: The effects of Xiaoyaosan on rats of NASH with liver stagnation and spleen deficiency syndrome via TLR4/TRIF signaling pathway1.Compared with Control group,the urinary D-xylose excretion rate and the contents of 5-HT and NE in hippocampus from Model group were significantly decreased(p<0.01);the serum TG,TC and ALT levels were significantly increased(p<0.01);the plasma ET content was significantly increased(p<0.01);the results of HE staining showed that the hepatocytes were dominated by macrovesicular steatosis,with ballooning degeneration,destruction of hepatic lobule structure,scattered point-like necrosis and inflammatory cell infiltration,and the NAS score was significantly increased(p<0.01);real-time PCR results of liver tissue showed a significant increase in mRNA expression of TLR4 and TRIF(p<0.01).2.Compared with Model group,the urinary D-xylose excretion rate and 5-HT and NE contents in hippocampus of XYS group and Glycine group were significantly increased(p<0.01);the serum TG,TC and ALT levels were significantly decreased(p<0.01);the plasma ET content was significantly decreased(p<0.01);the results of HE staining showed that the degree of hepatocyte steatosis was significantly reduced,with only a few vesicular steatosis,inflammatory cell infiltration was reduced,and the NAS score was significantly reduced(p<0.01);real-time PCR results showed that the contents of TLR4 mRNA and TRIF mRNA in liver tissues were significantly reduced(p<0.01).3.Compared with Glycine group,the contents of TLR4 mRNA and TRIF mRNA in liver tissues of XYS group were significantly decreased(p<0.05,p<0.01);HE staining results showed that microvesicular steatosis was reduced;while there were no significant differences in urine D-xylose excretion rate,hippocampal 5-HT and NE content,plasma ET content and liver tissue NAS score(p>0.05).Part two: The effect of Xiaoyaosan on macrophage inflammation model and its mechanism1.The optimal conditions for selecting the macrophage inflammation modelThe extracellular TNF-? and IL-6 levels in the 6h group,12 h group and 24 h group were significantly higher than those in the group which macrophages were stimulated with 5?g/mL LPS for 0h(p<0.01).The content of TNF-? and IL-6 in 12 h group and 24 h group was significantly higher than that in 6h group(p<0.01).There was no significant difference in TNF-? and IL-6 content between 12 h group and 24 h group(p>0.05).Compared to the group with macrophages stimulated with 5?g/mL LPS for 0h,the extracellular IL-4 content in 6h group,12 h group and 24 h group was significantly increased(p<0.05,p<0.01,p<0.01);the extracellular IL-10 content in 6h group,12 h group and 24 h group was significantly increased(p<0.01,p<0.05,p<0.05);there was no significant difference between the other groups(p>0.05).2.The effect of different concentrations of Xiaoyaosan on the proliferation of macrophage inflammation models detected by CCK-8When treated with Xiaoyaosan-containing serum culture solution for 6h,the survival cells rates of 10% XYS group and 20% XYS group were significantly increased compared with 5% XYS group(p<0.01);while there was not significant between 10% XYS group and 20% XYS group(p>0.05).When treated with Xiaoyaosan-containing serum culture solution for 12 h,the proliferation of cells in each group was similar to that at 6h.There was no difference in cell proliferation between different concentration groups when the Xiaoyaosan-containing serum culture solution was used for 0h and 24 hours(p>0.05).The proliferation effect on macrophages cultured by drug-containing serum of the same concentration for different time: For 5% XYS group,there was no difference at each time point(p>0.05).For 10% XYS group,the cell viability of in 0h and 24 h was significantly lower than in 6h(p<0.05).For 20% XYS group,the cell viability in 0h and 24 h was significantly lower than in 6h(p<0.01);compared with the 12 h group,the cell proliferation rate was significantly lower in 24h(p<0.05).3.The effect of Xiaoyaosan on macrophage inflammatory modelCompared with the Control group,the extracellular content of TNF-? IL-6 in the Model group was significantly increased(p<0.01);IL-4 and IL-10 content was increased(p<0.05);the results of western blot showed that the proteins expressions of TLR4 and TRIF were significantly increased(p<0.01);immunocytochemical staining results showed that the expression of TLR4 and TRIF increased.Compared with the Model group,the extracellular contents of TNF-? and IL-6 in the XYS group were significantly decreased(p<0.01);the contents of IL-4 and IL-10 were increased(p<0.05,p<0.01),and the results of western blot showed that the proteins expressions of TLR4 and TRIF were significantly decreased(p<0.01);immunocytochemical staining revealed decreased expression of TLR4 and TRIF.Conclusion1.The pathogenesis of NASH liver stagnation and spleen deficiency may be related to TLR4/TRIF signaling pathway.2.The mechanism of Xiaoyaosan to improve endotoxemia and hepatic inflammatory reaction in rats with NASH liver depression and spleen deficiency syndrome may be related to the decrease of TLR4 mRNA and TRIF mRNA content.3.The anti-inflammatory effect of Xiaoyaosan on macrophage inflammatory model may be related to the decreased expression of TLR4 and TRIF proteins. |
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