Screening for differentially expressed genes in mice with autoimmune hepatitis and effect of saikosaponin-d on the expression of several differentially expressed genes

• Main Author: CHEN Hao
• Format: Article
• Language: zho
• Published: Editorial Department of Journal of Clinical Hepatology 2020-04-01
• Series: Linchuang Gandanbing Zazhi
• Online Access: http://www.lcgdbzz.org/qk_content.asp?id=10685
• ISSN: 1001-5256; 1001-5256

ObjectiveTo investigate the differentially expressed genes in autoimmune hepatitis (AIH) mice by microarray screening, the effect of saikosaponin-d (SS-d) on several differentially expressed genes, the pathogenesis of AIH, and the therapeutic mechanism of SS-d. MethodsA total of 40 healthy female specific pathogen-free C57BL/6 mice (with a body weight of 20±2 g) were divided into gene chip group with 8 mice and SS-d treatment group with 32 mice. The mice in the gene chip group were further divided into normal control group and model group, with 4 mice in each group; the mice in the normal control group were given normal feeding, and those in the model group were given injection of concanavalin A (Con A) via the caudal vein at a dose of 15 mg/kg; the mice were sacrificed 12 hours later to collect liver tissue samples, the differentially expressed genes were screened out according to the instructions of Agilent chip, and some differentially expressed genes were verified by quantitative real-time PCR. The mice in the SS-d treatment group were randomly divided into normal control group (routine feeding), model group (injection of Con A via the caudal vein at a dose of 15 mg/kg), and low- and high-dose SS-d groups (pretreated with intraperitoneal injection of SS-d at doses of 2.5 mg/kg and 5.0 mg/kg, respectively, followed by the injection of Con A via the caudal vein at a dose of 15 mg/kg 8 hours later), with 8 mice in each group. Venous blood samples were collected 12 hours later, and ELISA was used to measure serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Liver tissue samples were collected under sterile conditions; some samples were fixed by paraformaldehyde to prepare sections, and HE staining was performed; some liver tissue samples were used to extract RNA, and quantitative real-time PCR was used to measure the changes in the mRNA expression of several differentially expressed genes (interferon γ ［IFN γ］, interleukin-4 ［IL-4］, interleukin-5 ［IL-5］, interleukin-13 ［IL-13］, and interleukin-17 ［IL-17］). A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. The t-test was used between two groups. ResultsA total of 11 512 differentially expressed genes were screened out for the gene chip group, with 5189 upregulated genes and 6323 downregulated genes, which were significantly enriched in 138 signaling pathways. The results of quantitative real-time PCR showed that increases in the mRNA expression of IFN γ and IL-17 and reductions in the mRNA expression of IL-4, IL-5, and IL-13(all P＜0.01), which was consistent with the results of chip screening. Compared with the normal control group, the SS-d treatment group had significant increases in the serum levels of ALT and AST (all P＜0.01) and the mRNA expression of IFN γ and IL-17 (all P＜0.05), significant reductions in the mRNA expression of IL-4, IL-5, and IL-13 (all P＜0.05), and infiltration of a large number of lymphocytes in liver tissue. Compared with the model group, the low- and high-dose SS-d groups had significant reductions in the serum levels of ALT and AST (all P＜0.05) and the degree of lymphocyte infiltration in liver tissue, as well as significant reductions in the mRNA expression of IFN-γ and IL-17 (all P＜0.05) and significant increases in the mRNA expression of IL-4, IL-5, and IL-13 (all P＜0.05), and there were significant differences in the changes in these genes between the high- and low-dose SS-d groups (P＜0.05). ConclusionThe development and progression of AIH is the result of abnormal expression of a large number of genes, among which IFN γ, IL-4, IL-5, IL-13, and IL-17 are closely associated with the pathogenesis of AIH and are the main biological targets for SS-d to exert an immunoregulatory effect and a protective effect against liver injury.