Role Of PEBP4 In Chronical Non-bacterial Prostatitis And Related Mechanism

ObjectivePhosphatidylethanolamine-binding protein 4（PEBP4）is a member of the PEBP family and is involved in many biological processes including neural development,myocyte differentiation,spermatogenesis,cell membrane synthesis and apoptosis.However,whether PEBP4 affects inflammation has not been reported at both inside and outside the country.Method1.Verify the expression of PEBP4 in CNPThe expression level of PEBP4 protein in prostate tissues of patients with BPH and patients with BPH combined with CNP was detected by immunohistochemistry,and the relationship between the expression level of PEBP4 protein and clinicopathological characteristics of patients with prostatitis was analyzed combined with clinical data of patients.2.Construction and validation of PEBP4^-/-mouse stable genetics（1）In order to further observe the relationship between PEBP4 gene and CNP,we planned to construct PEBP4^-/-transgenic mice.C57BL/6 is a commonly used model of CNP.Based on the reliability and operability of experiments,we chose C57BL/6N as the animal strain source for constructing transgenic mice to explore the relationship between PEBP4 and CNP.Construction of PEBP4 knockout mice was entrusted to Guangzhou Cyagen Biosciences Inc.According to the design,using the Cre-LOXP system,coding exon knockout,the company cultivated to obtain F0 heterozygous,we further cultured to obtain F1 heterozygous and homozygous.（2）PEBP4^-/-transgenic mice were successfully constructed and validated by genotyping PCR and Western Blot.（3）To observe the relationship between PEBP4 and serum androgen and litter size per fetus.（1）The pathological changes of prostate and seminal vesicle in mice were observed by naked eye.（2）H&E staining was used to observe the inflammatory cell infiltration in prostate,seminal vesicle and testis of mice.3.Explore the effect of PEBP4 on inflammatory cell infiltration of CNP（1）The pathological changes of prostate and seminal vesicle in mice were observed by naked eye.（2）H&E staining was used to observe the inflammatory cell infiltration in prostate,seminal vesicle and testis of mice.4.To explore the molecular mechanism of CNP induced by PEBP4（1）Using CRISPR-Cas9 technology,PEBP4 was knocked out from human prostate RWPE-1 cells,stable cell culture was screened,and total protein was extracted.Western blot assay was used to detect the gene knockdown efficiency of PEBP4.（2）RWPE-1 and PEBP4KO human prostate cells were stimulated by TNF-α,a stimulant of NF-κB pathway.The key signaling molecules p65 and IκB of NF-κB pathway and their phosphorylation molecules p-p65 and p-IκB were detected by Western blot.The ubiquitination level of the key signal molecule TRAF6 was detected.Result1.Immunohistochemical experiments showed that PEBP4 was highly expressed in prostate tissues of BPH patients,while it was low expressed in prostate tissues of BPH patients with CNP.Patients with BPH with high PEBP4 expression had a higher incidence of CNP than those with low PEBP4 expression during 2-year follow-up.In addition,patients with BPH combined with CNP had a higher recurrence rate of CNP after discharge in patients with high PEBP4 expression than those with low PEBP4expression.2.Genotyping PCR was performed to amplify the target genes,followed by agarose gel electrophoresis,and the bands were obtained after exposure.As shown in the figure,the PCR product size of homozygous mice was 252 bp,and that of wild-type mice was about 429 bp.If it is heterozygous,there are 2 bands,i.e.252bp and 429bp double bands.To further prove the function of this gene after knockout,we selected mouse testicular tissue for Western-blot verification.It was found that PEBP4 was almost not expressed in the testes of PEBP4^-/-mice,while PEBP4 was normally expressed in the testes of PEBP4^+/+mice.PCR and Western-blot results showed that PEBP4gene knockout mice were successfully constructed.In addition,macroscopic observation showed that the seminal vesicle tissues of PEBP4^-/-mice were dark in color,hyperemic and edema.3.A large number of inflammatory cells were observed in the prostate,seminal vesicle and testis of PEBP4^-/-mice by H&E staining.4.After the PEBP4 gene was knocked down in RWPE-1 cells by crisp-Cas9 technique,the protein level of PEBP4 decreased in Western blot assay,indicating that the RWPE-1 cells with PEBP4 knockdown were successfully constructed.5.Western Blot analysis showed that after PEBP4 knockdown,NF-κB protein level of RWPE-1 and ubiquitination level of TRAF6 increased.The phosphorylation of p65and IκB increased after stimulation with different concentrations of TNF-αConclusion1.PEBP4 is underexpressed in patients with CNP,and the low expression level is associated with higher incidence of CNP and higher recurrence rate in patients with CNP.2.Knockdown of PEBP4 can promote inflammatory cell infiltration in prostate, seminal vesicle and testicular tissues of mice,and may play a role in the recurrence and development of CNP.3.PEBP4 can promote the infiltration of CNP inflammatory cells by activating NF-κB signaling pathway.PEBP4 may be a potential biomarker of CNP and a potential target for prevention,diagnosis and treatment of CNP.