IL17 Pathway In Chronic Nonbacterial Prostatitis Based On RNA-seq Technology

Objective:The study aim to establish an effective animal model of chronic non-bacterial prostatitis in SD rats by intra-prostatic injection of Xiaozhiling solution.Biological characteristics were studied by measuring the hematological parameters of rats.RNA-seq technology was used to screen differentially expressed genes,and bioinformatics methods were used to analyze the enrichment of differentially expressed genes.Then,we validated and analyzed the genes involved in the signal pathway and preliminarily explored the possible mechanism of the development of chronic nonbacterial prostatitis.Methods:1.Thirty healthy male Sprague-Dawley rats weighing 200-250g were randomly divided into 3 groups:normal group,sham group and model group.2.Establishment of animal models:An effective animal model of chronic nonbacterial prostatitis was established by injecting Xiaozhiling in prostate.3.The pathological changes of prostate tissue to evaluate the success of the model were observed.The serum levels of Ca²⁺,Mg²⁺,Zn²⁺,IgG,CRP,and testosterone were measured,and the changes in biological characteristics were observed.4.RNA-seq technology was used to sequence rat prostate RNA and screen out differentially expressed mRNA.5.Application of DIVAD and KEGG to analysis KEGG Pathway of differentially expressed genes and using GO functional annotation analysis to identify biological pathways involved in differentially expressed genes.6.Western blot,RT-PCR were used to verify the expression levels of differentially expressed genes in the IL17 signaling pathway and to compare the expression of differential factors in the two groups.Results:1.In the animal model of chronic non-bacterial prostatitis in SD rats,the glandular cavities in the prostate tissue of rats were significantly damaged,the interstitial fibroblasts proliferated,lymphocytes and mononuclear cells infiltrated,showing significant chronic inflammation.2.Compared with the normal group,there was no significant difference in the expression of each indicator in the sham operation group（P>0.05）.The white blood cells,serum Mg²⁺,and testosterone of the rats in the modeling group were elevated,but the difference was not statistically significant（P>0.05）.Compared with the normal group and the sham group,the serum Zn²⁺level in the model group decreased,but the difference was not statistically significant（P>0.05）.The serum levels of Ca²⁺,serum IgG,and serum CRP in the model group were significantly higher than those in the normal group and the sham group（P<0.05）.3.The 5425differential genes were screened by RNA-seq sequencing.Using the log2（Fold Change）absolute value greater than 1 and P<0.05 as criteria,129 genes with significantly different expressions were screened out,among which 103 were up-regulated and 26 genes down-regulated.4.The differentially expressed genes were subjected to GO enrichment analysis.The markedly enriched GO functional categories were:positive regulation of biological processes,regulation of stimulatory responses,regulation of cellular constituent organization,progression of immune system,organization of cell components,defense response,response to external stimuli,inflammation,wound healing,immune response,etc.Significantly enriched pathways are metabolic pathways,PI3K-Akt signaling pathways,cancer pathways,IL-17 signaling pathways,tight junctions,Jak-STAT signaling pathways,protein processing in the endoplasmic reticulum,proteoglycans in cancer,TNF signaling pathways Leukocytes transendothelial migration,phagosomes,cytokine-cytokine receptor interactions,regulation of actin cytoskeleton,AMPK signaling pathway,chemokine signaling pathway,Ras signaling pathway,complement and coagulation cascades.Conclusions:1.Xiaozhiling injection into the rat prostate can be used to establish a chronic non-bacterial prostatitis rat model.2.Ca²⁺,IgG,CRP have obvious changes in the course of chronic nonbacterial prostatitis.3.There are significant differences in the expression of nine genes such as CXCL1,CXCL3,CCL2,CCL7,IL6,COX2,LCN2,MMP3,and USP25 in IL17 signaling pathway in the development of chronic nonbacterial prostatitis,indicating that these factors are involved in the disease.Which lay the foundation for the further study of the pathogenesis of chronic nonbacterial prostatitis.