| Objective:To establish a mouse model of autoimmune hepatitis（AIH）induced by Concanavalin A（Con A）,and verify the stability of the model.At the same time,microarray chips are used to screen the differentially expressed micro RNAs（mi-RNAs,mi RNAs）of liver injury models,and the target genes of mi RNAs are predicted and functionally analyzed through bioinformatics methods,in order to explore the potential role of mi RNAs in the pathogenesis of AIH,to provide a new target for the integrated Chinese and Western medicine treatment of clinical AIH.Methods:1.Sixteen male SPF-grade C57BL/6 mice（6 weeks old）with a body weight of（20±2）g were randomly divided into model group and control group,with 8 mice in each group.The mice in the normal control group were injected with normal saline through the tail vein;the mice in the model group were injected with Con A solution through the tail vein at a dose of 15 mg·kg-1body weight to establish an AIH mouse model.All mice were sacrificed 8 hours after modeling,and venous blood,liver total protein and total RNA were collected under hypothermic aseptic conditions.ELISA method was used to detect serum glutalanine aminotransferase（ALT）,aspartate aminotransferase（AST）,interleukin（IL）-10and IL-17 levels,and Western blotting to detect cytotoxic T lymphocyte related antigens（CTLA）-4 protein expression level,q RT-PCR technology to detect IL-10,IL-17 and CTLA-4 m RNA expression level to verify the stability of the model.2.Eight male SPF-grade C57BL/6 mice were randomly divided into model group and control group,with 4 mice in each group.The mice in the model group were injected with Con A by tail vein at a dose of 15 mg/kg,while the mice in the control group were given the same dose of normal saline.After 8 hours of modeling,all mice were killed under anesthesia,and the liver tissues was taken out under low temperature and aseptic conditions.The total RNA of mouse liver tissues were extracted.According to the standard process of microarray,the quantitative,integrity detection and labeling of total RNA,hybridization and elution of microarray,scanning,collection and sorting of data were carried out.Feature extraction software is used to process the original image and extract the original data.Genespring GX software is used to standardize the original data.The standardized data were filtered,and at least one group of 75%samples labeled as"detected"were retained for subsequent analysis.The differentially expressed mi RNAs were screened and verified by QRT PCR;the target genes of differentially expressed mi RNAs were predicted by mirwalk and mirdb database,and then the biological functions of the target genes were explored by go enrichment,KEGG enrichment pathway analysis and mi RNA m RNA interaction network construction.Results:1.The liver cells of the mice in the normal group were normal,the structure of liver lobules was complete and clear,and the liver cords were arranged in an orderly order;in the model group,swelling,degeneration,and necrosis of liver cells were seen,with typical interface necrosis,accompanied by a large number of inflammatory cell infiltration.Compared with the normal control group,the serum ALT and AST of the model group were significantly increased（P<0.05）;compared with the normal group:IL-17 m RNA expression increased,IL-10 m RNA and CTLA-4 m RNA expression decreased;serum The level of IL-17 increased,the level of serum IL-10 decreased,and the expression of CTLA-4 protein in liver tissue decreased,suggesting the successful establishment of the AIH model.The above results are all statistically significant（P<0.05）.2.In the model group,31 up-regulated and 18 down-regulated mi RNAs were detected,which have regulatory relationships with 959（601 up-regulated,358 down-regulated）target genes.The results of q RT-PCR verification showed that compared with the control group,mmu-mi R-21a-3p（20.69 times）,mmu-mi R-1934-3p（16.40 times）and mmu-mi R-188-5p（19.54 times）m RNA expression increased（all P values<0.01）,while mmu-mi R-22-5p（3.49 times）,mmu-mi R-7055-3p（2.61 times）and mmu-mi R-126a-5p The expression of m RNA（4.34 times）was down-regulated（both P values<0.01）,which was statistically significant and consistent with the chip screening results.GO analysis showed that compared with the control group,the target genes expressing up-regulated mi RNAs in the model group mainly possess molecular functions such as"DNA binding",participate in biological processes such as"transcription,DNA-templated",and are mainly enriched in"neuronal cell body""And other cellular components.The target genes of down-regulated mi RNAs mainly have molecular functions such as"RNA polymerase II proximal promoter sequence-specific DNA binding",participate in biological processes such as"regulation of transcription,DNA-templated",and are mainly enriched in cellular components such as"nucleoplasm".The results of KEGG pathway analysis showed that the up-regulated targets mainly involved"Endocytosis",and the down-regulated targets mainly involved the"Hippo signaling pathway".The above functional analysis results were statistically significant（P<0.05）.Conclusion:1.Mouse tail vein injection of Con A to prepare mouse liver injury model,which can be used as an ideal model for studying the mechanism of AIH immune disorder.2.The balance of Th17 and Treg may be related to the pathogenesis of AIH,and changes in the expression of CTLA-4,IL-10 and IL-17 play a key role in the occurrence and development of AIH.3.There are differential expressions of mi RNAs in the pathogenesis of AIH,and as a special biomarker they play an important role in the pathogenesis of AIH.These differentially expressed mi RNAs may provide new targets for the prevention and treatment of AIH. |
