Study On The Molecular Mechanism Of Warming And Tonifying Spleen And Kidney In The Treatment Of Ulcerative Colitis Model Of Spleen And Kidney Yang Deficiency Based On The Next-generation Sequencing

ObjectiveScreening of differentially expressed genes in colon of rats with spleen-kidney yangdeficiency ulcerative colitis based on next-generation sequencing.To observe the changes of expression of CXCL1,CXCL2,CXCL6,CCL7 and CCL12 in the chemokine signaling pathway and MMP3 MMP8 MMP13 in colon tissues,and the effect of the prescription of tonifying spleen and kidney on the expression of these factors,screening spleen-kidney yang deficiency UC Specific targets,and used as diagnostic markers,objective indexes of therapeutic effect and prognosis judgement.MethodsA total of 96 SPF Wistar rats were randomly divided into blank group,model groups,Chinese medicine high-dose group,middle-dose group,low-dose group,and SASP group.Except the blank group,the other groups were treated with drugs after successful modeling.(The model group was not treated).The colonic tissue morphology was observed with naked eye.The colon tissue of blank group and model group were selected for next-generation sequencing,differential gene expression analysis,differentially expressed genes GO enrichment analysis and Pathway enrichment analysis.Real-time PCR was used to detect the expression of CXCL1,CXCL2,CXCR2,CXCL6,CCL7,CCL12,MMP3,MMP8 and MMP13.Western blot was used to detect the expression of MMP3,MMP8 and MMP13 proteins in rat colon tissue.Results1.Analysis of differentially expressed genes in colonic tissues between blank group and model group: compared with model group,216 genes were screened according to q-value 0.05 Fold-change ≥ 1.5.There were 166 down adjustments and 84 up adjustments.Compared with the model group,the down-regulated genes were significantly more than the up-regulated genes,and the down-regulated genes were 1.96(166/84)times higher than the up-regulated genes.2.GO and Pathyway analysis of differentially expressed genes: The differentially expressed genes were selected for statistical analysis of biological processes(BP),cellular components(CC),and molecular functions(MF).BP were 3158,CC were 1863,and MF were 695.At the same time,related signal pathways were found.3.The m RNA expression of CXCL1,CXCL2,CXCR2,CXCL6,CCL7,and CCL12 in colon tissue of rats was detected by RT-qPCR: Compared with the colon tissue of the blank group,the expression levels of CXCL1,CXCL2,CXCR2,CXCL6,CCL7,and CCL12 in the model group were significantly increased.The difference was statistically significant(P<0.01).Compared with the colon tissue of the model group,the expression levels of CXCL1,CXCL2,CXCR2,CXCL6,CCL7,and CCL12 were significantly decreased in the treatment groups,and the difference was statistically significant(P< 0.01);consistent with the sequencing results.4.The mRNA expression of MMP3,MMP8 and MMP13 in colon tissue of rats was detected by RT-qPCR: Compared with the blank group,the expression of MMP3,MMP8,and MMP13 in the colon tissue of the model group was up-regulated,showing significant differences.The difference was statistically significant.Significance(P<0.01);compared with the model group,the expression of MMP3,MMP8,and MMP13 in the colon tissues of each treatment group was down-regulated,with significant differences(P<0.01),which was consistent with the sequencing results.5.The expression of MMP3,MMP8 and MMP13 protein in rat colon tissue was detected by Western blotting.Western blot showed that the expression of MMP3,MMP8 and MMP13 in the model group increased compared with the blank group,with significant differences.The difference was statistically significant(P<0.01).Compared with the model group,the relative expression levels of MMP3,MMP8,and MMP13 protein in each treatment group were downregulated,with significant differences(P<0.01).Consistent with the sequencing results.Conclusion1.The combined model of spleen and kidney yang deficiency UC rats is feasible,reasonable and scientific.NGS is one of the important methods in the objective Research of traditional Chinese Medicine2.The expression of CXCL1,CXCL2,CXCR2,CXCL6,CCL7,and CCL12 in the chemokine signaling pathway of the spleen-kidney-yang-deficiency ulcerative colitis was significantly up-regulated.The Granules of Lizhong Soup and Sishen Pills can effectively down-regulate the expression of these factors,slow down the inflammatory reaction and promote the repair of injured colonic mucosa.3.The method of warming spleen and kidney can significantly down-regulate the overexpression of MMP3 / MMP8 / MMP13 gene and protein in colonic mucosa of UC rats with deficiency of spleen and kidney yang,can be used as a specific marker for the diagnosis of UC and an evaluation index for the therapeutic effect of UC.