| Objective:Establishing a mouse model of autoimmune hepatitis(AIH)induced by Concanavalin A(Con A),using high-throughput gene chips to screen differentially expressed genes in AIH mice,for the occurrence of AIH The mechanism research provides research targets;observe the effect of saikosaponin D(SS-D)on the expression of some differentially expressed genes,and provide a laboratory basis for the clinical treatment of SS-D for AIH.Methods:1.Thirty-six C57BL/6 female mice were randomly divided into 6 groups(n=6):normal control group,AIH 6h group,AIH 12h group,AIH 24h group,AIH 48h group,AIH168h group.The mice in the normal control group were given a saline injection through the tail vein;the remaining groups of mice were injected with Con A solution through the tail vein at a dose of 15 mg·kg~-11 body weight to establish the AIH mouse model,and at the corresponding time after injection kill the mice.The liver and serum of mice in each experimental group were collected,and HE staining and ELISA were used to observe the degree of liver cell necrosis and inflammatory cell infiltration in the liver,and the changes of serum ALT and AST content.2.Eight C57BL/6 female mice were randomly divided into normal control group(n=4)and model group(n=4).The normal control group is reared routinely,and the model group mice are established with the above-mentioned method to establish the AIH model.The mice in the model group were sacrificed 12 hours after modeling.The liver tissue was extracted under sterile and low temperature conditions.The total RNA of the liver tissue was extracted,and the differentially expressed genes were screened according to the Agilent-085631 chip manual.Set analysis.At the same time,qRT-PCR technology was used to verify some of the differentially expressed genes.3.Twenty-four C57BL/6 female mice were randomly divided into normal control group,model group and treatment group(n=8).The normal control mice were given tail saline injection of normal saline,and the model group and the treatment group established AIH mouse models as described above.Among them,the mice in the model group were sacrificed after 12 hours of modeling;the mice in the treatment group were given SS-D 5.0mg·kg-1 pretreatment 12 hours before modeling,and were sacrificed 12 hours after modeling.The liver and peripheral blood of mice in each experimental group were collected under sterile and low temperature conditions.Some livers were subjected to conventional HE staining and some were used to extract total RNA,and qRT-PCR was used to observe the differentially expressed genes(including IFN-?mRNA,IL-4 mRNA,IL-5 mRNA,IL-13 mRNA and IL-17 mRNA)content changes;observe the changes in serum ALT and AST content of mice in each experimental group.Results:1.In the AIH mouse model induced by Con A,the serum ALT and AST contents showed a time-dependent increase,and reached a certain time point(ALT:6h,AST:24h)and began to decline;hepatic cell degeneration and necrosis(typically Necrosis of the plate),inflammatory cell infiltration is obvious(mainly lymphocytes).The above lesions are time-related,that is,at 6 hours after modeling,lymphocyte activation occurs around the central vein of liver lobule in mice,and acute hepatocyte injury begins to appear;at 12hours,the lymphocyte infiltration around the central vein of liver lobule and within the hepatic sinusoids is significant Increased;abnormal activation of lymphocytes at 24h,the degree of liver tissue infiltration reached a peak,accompanied by significant aggravation of liver cell damage;typical interface necrosis at 48h,accompanied by lymphocyte infiltration;and at 168h,the mouse liver lymph The cell infiltration disappeared,but the left liver parenchymal cells were extensively degenerated and necrotic.It is suggested that the model is suitable for studying the mechanism of early immune disorder of AIH at 12h.2.Compared with normal control mice,the gene chip group screened a total of11,512 differentially expressed genes,of which 5 189 were up-regulated and 6 323 were down-regulated.Among them,781 expression up-regulated genes can be annotated to 292signaling pathways,and the top three signaling pathways with the most significant differences are Cytokine-cytokine receptor interaction signaling pathway,TNF signaling pathway,NOD-like receptor signaling pathway;789 expression down-regulation The genes can be annotated to 306 signaling pathways,and the top three signaling pathways with the most significant differences are Metabolic signaling pathway,Retinol metabolism signaling pathway and Peroxisome signaling pathway.The qRT-PCR detection results of IFN-?mRNA,IL-4 mRNA,IL-5 mRNA,IL-13 mRNA and IL-17 mRNA content are consistent with the results of the chip screening,which not only indicates the credibility of the chip detection results,but also shows the expression changes of the above genes are closely related to the pathogenesis of AIH mice.3.Compared with the model group,the content of alt,AST in serum and the infiltration degree of lymphocytes in liver in the SS-D treatment group were significantly reduced;meanwhile,the changes of IFN-?mRNA,IL-4 mRNA,IL-5 mRNA,IL-13mRNA and IL-17 mRNA among the experimental groups were statistically significant.It is suggested that SS-D can reduce the immune damage of the liver of AIH mice,and its mechanism is related to the expression of IFN-?,IL-4,IL-5,IL-13 and IL-17.Conclusion:Con A induced time-dependent immune injury in the liver of AIH mice.In the 12h group,hepatitis cell infiltration was significant and liver cell damage was slight,which can be used as an ideal model for studying the mechanism of early immune disorder in AIH.It is complicated and is the result of the common action of a large number of abnormal expressions of genes,and is related to the abnormal activity of multiple signal transduction pathways;SS-D can reduce the immune inflammatory response in the liver of AIH mice.IL-4,IL-5,IL-13,IL-17 and other differentially expressed genes are related. |
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