| Objective:Changes in toll-like receptor 4(TLR4)gene promoter methylation level,expression level of TLR4/ interferon TIR domain binding protein(TRIF)/p65 signaling pathway and intervention of Xiaoyao San in liver tissues of rats with liver depression and spleen deficiency syndrome model of non-alcoholic steatohepatitis(NASH)were studied,to explore the partial pathogenesis of NASH liver depression and spleen deficiency syndrome and the therapeutic mechanism of Xiaoyao San,to provide theoretical and experimental basis for NASH clinical research,and to reveal part of the biological connotation of “liver master drainage”.Methods:1.To investigate the changes of TLR4 gene promoter methylation level in liver tissue of rats with NASH liver depression and spleen deficiency syndromeSixty-five male SD rats were randomly divided into normal group(Normal group,n=13),non-alcoholic steatohepatitis model group(NASH group,n=13),low-dose treatment group(XYS-Lgroup,n=13),medium-dose treatment group(XYS-M group,n=13)and high-dose treatment group(XYS-H group,n=13).Rats in the normal group were fed with normal feed and drank tap water without other intervention stimulation.Rats in the NASH group and the XYS-L,XYS-M and XYS-H groups were fed high-fat diet,drank30% fructose solution,and underwent chronic stress restraint stimulation and hunger disorder stimulation daily.The model was validated after 24 weeks.Rats in each group were fed for 4 weeks according to the previous method,and rats in the normal group and NASH group were given normal saline(NS)2ml/(kg·d)by gavage,and rats in XYS-L,XYS-M and XYS-H groups were given 6.48g/(kg·d),3.24g/(kg·d),and 1.62g/(kg·d)by gavage of Xiaoyao San.At the end of 28 weeks,blood,urine,hippocampal and liver tissues of rats in each group were collected,and the weight of liver tissues was measured for the statistics of liver index.During the experiment,the body weight,hair,tongue and mobility of each group of rats were observed and recorded,which was used for the statistics of the syndrome score of liver depression and spleen deficiency.Frozen sections of rat liver were prepared,and morphological changes,steatosis and infiltration degree of inflammatory cells were observed by pathological staining.Serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride(TG)and total cholesterol(TC),urinary D-xylose excretion rate and noradrenaline(NE)and 5-HT contents were detected by enzyme-linked immunosorbent assay(ELISA).TLR4 methylation levels in liver tissues were detected by bisulfite sequencing PCR(BSP).2.To investigate the expression changes of TLR4/TRIF/p65 signaling pathway in liver tissues in the model of NASH liver depression and spleen deficiency syndromeAfter collecting the liver tissues of each group at the end of 28 weeks,the expressions of IL-10?TGF-??TNF-??IL-1? and nitric oxide(NO)in the serum of each group were detected by ELISA.The protein contents of TLR4,TRIF and p65 in rat liver were detected by western blot(WB).Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)was used to detect the mRNA contents of TLR4,TRIF and p65.The correlation between TLR4 mRNA relative expression and methylation level in rat liver was analyzed.Results:1.To investigate the changes of TLR4 gene promoter methylation level in liver tissue of rats with NASH liver depression and spleen deficiency syndrome(1)Pathological sections: The results of HE staining and oil red O staining in the liver tissues of each group showed that,compared with the normal group,the volume of liver cells in the NASH group increased,a large number of fatty vacuoles appeared in the liver tissues,and inflammatory cell infiltration was obvious,accompanied by obvious liver fibrosis.Compared with the NASH group,the volumes of liver cells in the XYS-L,XYS-M and XYS-H groups all decreased,and the levels of fatty vacuoles,inflammatory cell infiltration and liver fibrosis in the liver tissues all decreased,among which,the XYS-M group showed the most significant reduction.(2)Liver index and serum indexes: Compared with the normal group,the liver index of the NASH group increased(P<0.01),and the contents of AST(P<0.05),ALT,TG and TC in the serum increased significantly(P<0.01).Compared with NASH group,liver index,XYS-L and XYS-M group decreased significantly(P<0.05),XYS-L group of ALT in serum(P<0.05),TG(P<0.01)and TC(P<0.01)content were decreased,the AST XYS-M group(P<0.01),the ALT(P<0.01),TG(P<0.05)and TC(P<0.01)were decreased,the content of TG XYS-H group(P<0.01)the content of all down.(3)Syndrome score of liver depression and spleen deficiency syndrome and spleendeficiency:Compared with the normal group,the syndrome score of liver depression and spleen deficiency syndrome in NASH group was significantly increased(P<0.01).Compared with NASH group,the syndrome scores of XYS-L,XYS-M and XYS-H groups all decreased(P<0.01).(4)Urine D-xylose excretion rate and detection of 5-HT and NE contents in hippocampal tissue in rats: Compared with normal group,urine D-xylose excretion rate and 5-HT and NE contents in hippocampal tissue in NASH group decreased(P<0.01).Compared with NASH group,the urine D-xylitose excretion rate(P<0.01),5-HT(P<0.01)and NE(P<0.05)in XYS-L group increased,and the urine D-xylitose excretion rate and 5-HT and NE content in hippocampus in XYS-M and XYS-H groups increased significantly(P<0.01).(5)Detection of the methylation level of TLR4 gene in liver tissues: Compared with the normal group,the methylation level of TLR4 gene in liver tissues of NASH group was decreased(P<0.01).Compared with NASH group,the methylation level of TLR4 gene in XYS-L(P<0.05)and XYS-M(P<0.01)groups increased.2.To investigate the expression changes of TLR4/TRIF/p65 signaling pathway in liver tissues in the model of NASH liver depression and spleen deficiency syndrome(1)Results of serum cytokines in rats: Compared with the normal group,the serum contents of IL-10 and TGF-? in the NASH group decreased significantly(P<0.01),and the contents of TNF-?,IL-1? and NO in the NASH group increased significantly(P<0.01).Compared with NASH group,XYS-L group of IL-10 in serum(P<0.01)expression increases obviously,IL-1?,and NO expression is significantly decreased(P<0.01),XYS-M group serum IL-10(P<0.01),the TGF-?(P<0.05)higher expression,TNF-?,and IL-1??NO content decreased significantly(P<0.01),XYS-H group IL-10 in serum(P<0.01)expression increases obviously,IL-1? and NO content decreased significantly(P<0.01).(2)Detection results of TLR4,TRIF and p65 protein in rat liver: Compared with the normal group,the protein content of TLR4,TRIF,and p65 in liver tissues of the NASH group increased(P<0.01);Compared with NASH group,the protein contents of TLR4(P<0.01),TRIF(P<0.01)and p65(P<0.05)in XYS-L liver tissues decreased.The protein contents of TLR4,TRIF and p65 in liver tissues of XYS-M and XYS-H groups weresignificantly reduced(P<0.01).(3)The relative mRNA levels of TLR4,TRIF and p65 in rat liver were detected:Compared with the normal group,the relative levels of TLR4,TRIF,and p65 mRNA in liver tissue of rats in the NASH group increased significantly(P<0.01);Compared with the NASH group,in the XYS-L liver tissue Relative mRNA levels of TLR4(P<0.01),TRIF(P<0.01),and p65(P<0.05)decreased,mRNA levels of TLR4,TRIF,and p65 in liver tissues of the XYS-M and XYS-H groups were significantly reduced(P<0.01).(4)Correlation analysis of TLR4 mRNA relative content and methylation level in liver tissues: The methylation level of TLR4 and its relative mRNA expression were negatively correlated(P<0.01).Conclusion:1.The pathogenesis of NASH rat model of liver depression and spleen deficiency may be related to the down-regulation of methylation level of TLR4 promoter in liver tissue.2.The pathogenesis of NASH rat model of liver depression and spleen deficiency is related to the increase of TLR4 / TRIF / p65 signaling pathway expression.3.The Xiaoyao San middle-dose treatment group can better treat NASH rats with liver depression and spleen deficiency syndrome.4.Xiaoyao San improves the degree of fatty tissue degeneration,inflammation and liver in liver tissues by up-regulating the methylation level of TLR4 gene and down-regulating the expression of TLR4 /TRIF /p65 signaling pathway in a rat model of non-alcoholic steatohepatic liver stagnation and spleen deficiency syndrome.Symptoms of depression and spleen deficiency may be a new mechanism for Xiaoyao San to treat non-alcoholic steatohepatitis with liver depression and spleen deficiency. |
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