Activated Autophagy Restored The Impaired Frequency And Function Of Regulatory T Cells In Chronic Prostatitis

Background Chronic prostatitis or chronic pelvic pain syndrome is the most common type of prostatitis which accounted for 90%of all prostatitis diagnoses.Chornic prostatitis presented with complex clinical symptoms such as the pain above the suprapubic area and blow the waist,including the bladder area,frequent urination and sexual dysfunction such as erectile dysfunction.At present,the pathogenesis of chronic prostatitis is not clear and effective therapeutic drugs in clinical practice are limited.The patients always suffer many times of disease recurrence after the treatment.Previous studies have suggested that the etiology of chronic prostatitis is related to various factors such as neuromuscular effects,microbial factors,endocrine disorders.In recent years,more and more studies have found that immune disorders are closely related to the occurrence of chronic prostatitis,the local pain symptoms,mental symptoms and hyperalgesia.Studies also confirmed that patients with chronic prostatitis have imbalances in immune cells and impaired inflammatory cytokines.For example,studies have shown that inhibition of Treg cell could increase the susceptibility of prostatitis in mice.It had also been put that inhibiting the function of Treg cells and increasing the number of Th1 and Th17 cells could aggravate the local pain symptoms in mice with prostatitis,indicating that the activation of Th1 and Th17 cells and the inhibition of Treg cells jointly promote the occurrence of pelvic pain.Meanwhile,small molecules such as miRNA are also reported to be associated with the incidence of erectile dysfunction in chronic prostatitis.Therefore,immune factors play important regulatory roles in the pathogenesis of chronic prostatitis.Objective To further understand the immune pathogenesis of chronic prostatitis and to clarify the regulatory role of immune cells or immune-related signaling pathways in the disease,we conducted this research,the first part is designed to detect differentially expressed miRNA in chronic nonbacterial prostatitis,and enrich several signaling pathways related to disease.In the second part,the autoimmune prostatitis rodent model was conducted to further discussed the regulating mechanisms of previous enriched pathways in prostatitis.Methods In the first research,CNP(Chronic nonbacterial prostatitis,CNP)rat models were established through the intraprostatic injection of carrageenan into the prostate.Then,next-generation sequencing was performed to explore the miRNA expression profile in CNP.GO(Gene Ontology,GO)and KEGG(Kyoto Encyclopedia of Genes and Genomes,KEGG)bioinformatical analyses were conducted to reveal the enriched biological processes,molecular functions,and cellular components and signaling pathways.For the second part,in order to further clarify the role of immune factors in chronic prostatitis,EAP models was constructed through subcutaneous injection of prostate antigen extraction and verified with HE straining.And the numbers and suppressive functions of Treg cells in EAP group and Control group were tested.Then,CD4~+CD25~-cells from the two groups were separated with flow sorting technology and cultured to observe whether the cells could differentiate into CD4~+CD25~+Foxp3~+Treg cells under the condition of autophagy activation.Meanwhile,the GATA-3 expression in the cells was detected.Furthermore,CD4~+CD25~-cells and CD4~+CD25~+cells from the two groups were also magnetically sorted,and co-cultured to observe the changes in cell supressive function under the condition of autophagy activating.Meanwhile the porportation of GATA-3~+FOXP3~+and CTLA-4~+FOXP3~+in CFSE~-cells were detected at the same time.Finally,in vivo experiment,rapamycin was intraperitoneal injected into EAP mice for 4 weeks to observe whether the activation of autophagy could alleviate local pain symptoms and inflammation levels,and restore the number and function of Treg cells in EAP mice.Results As a result,84 miRNAs with significantly different expression levels were identified compared to the control group.Compared with previously reported miRNAs with altered expression in various inflammatory diseases,the majority of deregulated miRNAs in CNP,such as miR-146b-5p,miR-155-5p,miR-150-5p,and miR-139-5p,showed similar expression patterns.Moreover,bioinformatics analyses have enriched“mitogen-activated protein kinase(MAPK),cyclic adenosine monophosphate(cAMP),endocytosis,mammalian target of rapamycin(mTOR),autophagy and forkhead box O(FoxO)”signaling pathways.These pathways were all involved in immune responses,which indicates the critical regulatory role of the immune system in CNP initiation and progression.As for Treg cells,we found that the numbers and functions of Treg cells in the EAP group was diminished along with decreased expressions of GATA-3 and CTLA-4 in the CD4~+CD25~+Foxp3~+Treg cells.Meanwhile,the tolerance degree of pain in EAP mice was also decreased compared to the normal controls.Moreover,autophagy activator rapamycin could promote the transformation of CD4~+CD25~-cells to CD4~+CD25~+Foxp3~+Treg cells.Similarly,it could also restore the suppressive functions of Treg cells in vivo together with the up-regulated proportion of GATA-3~+Foxp3~+cells and CTLA-4~+Foxp3~+cells in CFSE~-cells.Furthermore,the vitro administration of autophagy activator rapamycin had the similar effects on recovering the frequency and functions of Treg cells as well as the expressions of GATA-3 and CTLA-4.Besides,the IL-1?expression within the prostate and the pain symptoms were also alleviated following the use of rapamycin.Conclusion We concluded that miRNAs could have possible regulatory effect in the development of CNP.Meanwhile,several immune-related signal pathways such as autophagy and FoxO signaling pathways were enriched with KEGG analysis,indicating the possible immunological regulatory functions in CNP progression.Meanwhile,the impaired frequency and functions of Treg cells may contribute to the progression of CPPS,and restoring the numbers and functions of Treg through activating autophagy could alleviate the inflammation of the prostate and pain symptoms.Our study provides a new way to further explore the immune pathogenesis of chronic prostatitis and to improve the therapeutic effects of chronic prostatitis.