Effect Of TLR4 Gene Methylation On The Pathogenesis Of Alcoholic Steatohepatitis With Phlegm And Blood Stasis Syndrome

Objective:Toll-like receptor 4（TLR4）gene promoter methylation level and TLR4/interferon TIR domain adaptor protein（TRIF）signaling pathway in the liver tissue of rats with alcoholic steatohepatitis（ASH）syndrome of phlegm and blood stasis The study of the expression level,the cell model of steatohepatitis and the study of the methylation level of the TLR4promoter after the intervention of hawthorn leaf total flavonoids（HLF）,to explore the phlegm and blood stasis syndrome and the partial pathogenesis of steatohepatitis,which is the clinical of ASH The treatment provides experimental basis.Method:1.To observe the change of TLR4 gene promoter methylation level in liver tissue of ASH model rats with phlegm and blood stasis syndromeThe 48 male SD rats were divided into the following four groups according to the random number table method,namely:normal group（control group,n=12）,alcoholic steatohepatitis phlegm and blood stasis model 2 weeks group（2w group,n=12）,alcoholic steatohepatitis phlegm and blood stasis syndrome model 3 weeks group（3w group,n=12）,alcoholic steatohepatitis phlegm and blood stasis syndrome model 4 weeks group（4w group,n=12）.The rats in the normal group were fed ordinary feed and drank tap water without other intervention stimulation.After one week of adaptive feeding,the rats in the other model groups were modeled with multiple factors,fed with a feed composed of 80%cornmeal,19.5%lard,0.5%cholesterol,and 30%alcohol as a beverage.On the first day of the experiment,rats in each model group were injected subcutaneously with 0.25m L/100g carbon tetrachloride（CCl₄）,and then 0.25m L/100g CCl₄（40%vegetable oil solution）was injected every 3 days.At the weekend,at the end of the 3rd,and at the 4th weekend,the blood and liver tissues of the rats in each group were collected to observe the formation of the ASH phlegm and blood stasis syndrome.During the experiment,the body weight,hair,tongue,mobility,and tail of rats in each group were observed,and frozen sections of rat liver tissues were prepared.The morphological changes,steatosis and inflammatory cell infiltration of rat liver cells were observed by pathological staining.;The enzyme-linked immunosorbent assay（ELISA）method was used to detect alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,triglycerides（TG）and total cholesterol in the serum of each group of rats.（TC）content and interleukin-10（IL-10）,transforming growth factor-β（TGF-β）,tumor necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）expression;Western blot（WB）was used to detect the protein content of TLR4,TRIF,and CD68 in the liver tissues of rats in each group;real-time fluorescent quantitative polymerase chain reaction（q RT-PCR）technology was used to detect the content of TLR4m RNA,TRIFm RNA,and CD68m RNA;Bisulfite sequencing PCR（BSP）was used to detect the methylation level of TLR4 gene in liver tissue.2.To observe the changes of TLR4 gene methylation level in the fatty hepatitis cell model and the total flavonoids of hawthorn leaves after interventionThe rat BLR-3A cell line was selected,and palmitic acid（PA）with different concentrations of 200μM,300μM,and 400μM was selected for modeling.The intervention time was 24 hours.After modeling,the supernatant was collected for use;cells were collected for use.Oil red O staining determines the optimal concentration for establishing steatohepatitis.CCK-8 was used to detect the effects of different HLF concentrations of 0,10,30,60,90,120,150μg/m L on cell survival,and to determine the optimal therapeutic dose of HLF for the treatment of cellular steatohepatitis cell models.The BRL-3A cell lines were divided into normal group（control group）,model group（PA group）,treatment group（HLF group）,PA group and HLF group were intervened with the optimal concentration of PA for 24 hours,and the HLF group was treated with the optimal amount of treatment Intervene for 24 hours,observe the results of cell oil red O staining in each group,and determine the levels of ALT and AST;use ELISA to detect the expression of TNF-α,IL-1β,IL-10,and TGF-βin the cell supernatant of each group;Bisulfite sequencing PCR method（BSP）was used to determine the expression of TLR4 promoter methylation in each group.Result:1.Observe the change of TLR4 gene promoter methylation level in liver tissue of ASH model rats with phlegm and blood stasis syndrome（1）Pathological observation results:Observe the HE stained and Oil Red O stained sections of the liver tissues of each group of rats.Compared with the control group,rats in each ASH group showed increased hepatocyte volume,and there may be a large amount of liver cells in the ASH group.Lipid droplet vacuoles.Compared with the 2w group and the3w group,the 4w group was more obvious,and there was also inflammatory cell infiltration accompanied by a small amount of liver fibrosis.（2）Test results of liver index and blood lipids of rats in each group:Compared with the control group,the body weight of each model group was significantly reduced（P<0.01）,and the liver index in the 2w and 3w groups was significantly increased（P<0.05）,4w The group is especially obvious（P<0.01）.Compared with the control group,the AST and ALT of the 2w group were slightly higher,but there was no statistical significance;the3w group AST expression was statistically significant（P<0.01）,while the ALT expression was not significant;the 4w group AST（P<0.01）and ALT（P<0.05）expressions were all increased,both of which were statistically significant.Compared with the control group,the expression of TC and TG in each group of the model increased,and the increase in the4w group was significant（P<0.01）.（3）Test results of cytoinflammatory factors in the serum of rats in each group:Compared with the control group,the expression of TNF-α,IL-10,and TGF-βin the 2w group did not change significantly,and there was no statistical significance（P>0.05）,The expression of IL-1βincreased,which was statistically significant（P<0.05）.IL-1βand TNF-αwere significantly increased in the 3w and 4w groups,while IL-10 and TGF-βwere significantly decreased,both of which were statistically significant.Significance（P<0.01）.（4）The detection results of the target protein content in the liver tissue of rats in each group:Compared with the normal group,the protein content of TLR4,TRIF and CD68 in the liver tissue of the ASH group increased（P<0.01）.（5）Detection results of the relative content of target gene m RNA in the liver tissue of each group:Compared with the control group,the relative content of TLR4,TRIF,and CD68 m RNA in the liver tissue of each group of ASH rats increased significantly（P<0.01）.（6）Detection of TLR4 gene promoter methylation levels in rat liver tissues of each group:Compared with the control group,With the prolongation of modeling weeks,the methylation level of TLR4 gene in the liver tissue of rats in each model group decreased significantly（P<0.01）.2.To observe the changes of TLR4 gene methylation level in the fatty hepatitis cell model and the total flavonoids of hawthorn leaves after intervention.（1）The optimal concentration of PA to stimulate BRL-3A cells to establish a steatohepatitis model is 400μM.（2）The optimal therapeutic dose of HLF for the treatment of steatohepatitis cell model is 30μg/m L.（3）Oil red O staining results:Compared with the control group,the lipid droplets in the cytoplasm of the PA group were significantly increased;compared with the PA group,the lipid droplets in the cytoplasm of the HLF group were significantly reduced.（4）The detection results of ALT and AST in the cells of each group:Compared with the control group,the expression levels of ALT and AST in the cell supernatant of the PA group were significantly increased（P<0.05）;compared with the PA group,the HLF group had two The level of those who had declined（P<0.05）.（5）Detection results of inflammatory factors in the cell supernatant of each group:Compared with the control group,IL-1βand TNF-αin the PA group were significantly increased（P<0.01）,and the contents of IL-10 and TGF-βwere significantly decreased（P<0.01）;Compared with the PA group,the expression levels of IL-1βand TNF-αin the HLF group decreased（P<0.05）,while the expression levels of IL-10 and TGF-βincreased significantly（P<0.05）.（6）Detection of TLR4 gene methylation level in cells:Compared with the control group,the TLR4 gene methylation level of the PA group was significantly reduced（P<0.05）;compared with the PA group,the HLF group was significantly increased（P<0.05）.Conclusion:1.The model of ASH phlegm and blood stasis mutual syndrome can be established by using compound factors to model for 4 weeks.2.Part of the pathogenesis of ASH rat model with phlegm and blood stasis syndrome may be related to down-regulating the level of TLR4 methylation and affecting the expression of TLR4/TRIF pathway proteins and genes.3.Part of the mechanism of HLF in the treatment of cellular steatohepatitis may be related to the up-regulation of the level of TLR4 methylation,thereby improving the degree of steatosis and inflammation in the cell.