Purpose. Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common urological disorder. Although ferroptosis is closely associated with inflammation, oxidative stress, and neuropathic pain, its role in CP/CPPS has not yet been elucidated. Therefore, we sought to explore the role and mechanism of ferroptosis in the prostatitis development. Methods. The experimental autoimmune prostatitis (EAP) was established through intradermal immunization of prostate extract. Iron chelator deferoxamine (DFO) and free radical scavenger edaravone (EDA) were applied to evaluate the effects of ferroptosis inhibition on oxidative stress, ferroptosis, inflammation, fibrosis, and mast cell activation in the context of CP/CPPS. Results. Increased generation of lipid peroxidation products (ROS and MDA) and decreased activities of antioxidant enzymes (SOD and CAT) suggested an aberrant oxidative stress status in EAP model. Elevated iron concentration was observed in the EAP model. Meanwhile, we discovered significant biological performances associated with ferroptosis in CP/CPPS, including the downregulation of the system Xc^-/GPX4 axis and the upregulation of the ACSL4/LPCAT3 axis. EAP rats performed serious leukocyte infiltration, advanced inflammatory grade, and abnormal expression of inflammatory mediators. Abundant collagen deposition, enhanced RhoA, ROCK1, and α-SMA protein levels indicated that EAP rats were prone to suffer from stromal fibrosis compared with control group. An elevated number of degranulated mast cells and corresponding marker TPSB2 represented that mast cell-sensitized pain was amplified in the EAP model. Furthermore, reduction of NRF2/HO-1 indicated a vulnerability of EAP towards ferroptosis response. However, application of DFO and EDA had partially reversed the adverse influences mentioned above. Conclusion. We first demonstrated that ferroptosis might be a crucial factor of chronic prostatitis progression. Inhibition of ferroptosis using DFO and EDA represented a promising approach for treating prostatitis by ameliorating inflammation, fibrosis, and mast cell activation.

Objective. The aim of this study was to investigate whether tissue kallikrein (KLK1) can protect the prostate from inflammatory damage and the mechanism involved in it. Methods. A total of 50 male Wistar rats were used in this study. Initially, 20 rats were sacrificed to obtain the prostate antigen to induce experimental autoimmune prostatitis (EAP), and the remaining 30 rats were randomly divided into 5 experimental groups (normal control group (NC group), NC+KLK1 group (NCK group), EAP group, EAP+KLK1 group (EAPK group), and EAP+KLK1+HOE140 group (EAPKH group); n = 6). It should be explained that KLK1 mainly exerts its biological effects through bradykinin, and HOE140 is a potent and selective bradykinin receptor B2 (BDKRB2) antagonist. EAP was induced by intradermal injection of 15 mg/ml prostate antigen and complete Freund’s adjuvant on days 0, 14, and 28. KLK1 was injected via tail vein at a dose of 1.5 × 10⁻³ PAN U/kg once a day, and HOE140 was administered by intraperitoneal injection at 20 μg/kg once every two days. Rats were sacrificed on day 42. The RNA and protein of the rat prostate were extracted to analyze the expression differences of KLK1, as well as the inflammation‐, fibrosis‐, and oxidative stress‐related genes. The inflammatory cell infiltration and microvessel density of the prostate were also analyzed by pathological examination. In addition, pathological analysis was performed on prostate samples from patients undergoing benign prostate hyperplasia (BPH) surgery. Results. The expression of KLK1 in the prostate decreased in the EAP group as well as BPH patients with obvious inflammation. KLK1 administration significantly inhibited inflammatory cell infiltration and reduced the production of inflammatory cytokines in the EAPK group. Prostate samples from the EAP group showed increased infiltration of T cells and macrophages, as well as gland atrophy, hypoxia, fibrosis, and angiogenesis. KLK1 administration upregulated endothelial nitric oxide synthase (eNOS) expression and suppressed oxidative stress, as well as transforming growth factor β1 (TGF‐β) signaling pathways and the proangiogenic vascular endothelial growth factor (VEGF) in the EAPK group. However, in the EAPKH group in which HOE140 blocked BDKRB2, the beneficial effects of KLK1 were all cancelled. In addition, KLK1 intervention in normal rats had no obvious side effects. Conclusion. The KLK1 expression is inhibited in the inflamed prostates of humans and rats. Exogenous KLK1 restored endothelial function via a BDKRB2‐dependent way and then played a role in improving microcirculation and exerted anti‐inflammatory, antifibrotic, and antioxidative stress effects in the rat chronic‐inflamed prostate.

Objective. The aim of the present study was to investigate the protective effects and mechanisms of KLK1 on aging‐related prostate alterations and search clues about the application of KLK1 to the treatment of human BPH. Methods. Thirty‐six rats including 26 male wild‐type SD rats and 10 transgenic rats were fed to 3‐ or 18‐month‐old and divided into three groups: young WTR (yWTR) as the control (n = 16), aged WTR (aWTR) (n = 10), and aged TGR (aTGR) (n = 10). The prostates of the three groups of rats (10 rats per group) were harvested to evaluate the levels of KLK1 expression, oxidative stress, fibrosis, and involved signaling pathways, such as NO/cGMP, COX‐2/PTGIS/cAMP, and TGF‐β1/RhoA/ROCK1, via quantitative PCR, Western blot, histological examinations, and ELISA. Moreover, the remaining 6 yWTRs were sacrificed to obtain primary prostate fibroblast and aortic endothelial cells, and a coculture system was built with the cells for the verification of above signaling pathways in vitro. And the direct effects of bradykinin on prostate cells were detected by MTT experiment. Prostate specimens of 47 patients (age from 48 to 92 years) undergoing BPH surgery were collected after approval. Histological examinations and KLK1 IHC were preformed to analyze the relationship between KLK1 expression and age and prostate fibrosis. Results. The human KLK1 gene only existed and was expressed in aTGR. The prostate of young rats expressed more KLK1 than the aged and the expression of KLK1 in prostate decreased with age in humans (r = −0.347, P = 0.018). Compared to the aWTR group, the yWTR and aTGR groups showed milder fibrosis, less oxidative stress, upregulated NO/cGMP, and COX‐2/PTGIS/cAMP signaling pathways and inhibited TGF‐β1/RhoA/ROCK1 signaling pathway. In the coculture system, KLK1 suppressed TGF‐β1‐mediated fibroblast‐to‐myofibroblast transdifferentiation via cleaving LMWK to produce the BK which upregulate eNOS expression and NO production in endothelial cells. BK not only slightly stimulated the proliferation ability of prostatic stromal cells but also upregulated iNOS and inhibited TGF‐β1 expression in them. Conclusion. KLK1 protects prostate from oxidative stress and fibrosis via amplified NO/cGMP signal in aged rats. The decrease of KLK1 expression with aging is laying the groundwork for the application of KLK1 to the treatment of human BPH. The current experimental data showed that the side effects of KLK1 on the prostate cell were not obvious.

Lin D, et al. World J Mens Health. 2026 Feb;44:e15. https://doi.org/10.5534/wjmh.250222

Background: RNA activation, as a method of regulating gene expression at the transcriptional level, is far less widely used than RNA interference because of the insufficient understanding of the mechanism and the unstable success rate. It is necessary to analyze the failure cases of RNA activation to promote the application of RNA activation. When we validated the saRNAs designed to induce KLK1 expression, we found that saKLK1-374 can up-regulate KLK1 expression in prostate tumor cell lines, but failed in normal prostate cell lines. In addition, we also found that saKLK1-374 inhibited the growth of prostate cancer cells, which seems to be the opposite of the function of KLK1. This article is about experimental research and analysis of these two issues.Methods: To determine whether the phenomenon that the RNA activation of normal cells is difficult to succeed is only valid when the target gene is KLK1, we used p21^WAF1/CIP1 as the target gene to perform RNA activation experiments in normal prostate cells and prostate cancer cells. Next, to determine whether the above phenomenon exists in other tissues, we also performed RNA activation experiments with KLK1 and p21^WAF1/CIP1 as target genes in normal cell lines and tumor cell lines derived from the bladder. We have also extended the time from transfection to the detection of target gene expression to evaluate whether a longer saRNA action time can change the phenomenon that saRNA fails to up-regulate target gene expression in normal cells. In terms of mechanism research, we used fluorescently labeled dsRNA to evaluate the transfection efficiency, and also detected the expression of Ago2 and IPO8 proteins. In another issue of saKLK1-374 inhibiting prostate cancer cells, we tested the ROS content and apoptosis levels of prostate cancer cells after saKLK1-374 transfection. We used recombinant KLK1 protein to directly interfere with prostate cancer cells as a positive control for KLK1 function research. In turn, we also used siRNA to inhibit the expression of KLK1 in prostate cancer cells to compare the growth of prostate cancer cells when KLK1 mRNA was up-regulated and reduced.Results: The p21^WAF1/CIP1 gene could be significantly upregulated by saRNA in prostate cancer cell lines, but not in normal prostate cell lines. The expression of KLK1 in bladder-derived cell lines was extremely low and could not be induced by saRNA. The p21^WAF1/CIP1 gene could be up-regulated by saRNA to a higher extent in bladder cancer cell lines, while it was up-regulated by saRNA in normal urothelial cell line to a lower extent. Prolonging the action time of saRNA could not change that saRNA failed to induce the expression of target genes in normal cell lines. Compared with tumor cell lines, normal cell lines had lower transfection efficiency or lower expression of Ago2 and IPO8. After being transfected with saKLK1-374, prostate cancer cells had increased ROS and increased levels of apoptosis. The recombinant KLK1 protein did not increase ROS in prostate cancer cells, nor did it inhibit their growth. Even though saKLK1-374 up-regulated the expression of KLK1 in prostate cancer cells, siRNA still suppressed the expression of KLK1 below the baseline level, and in this case, the growth of prostate cancer cells was still at a suppressed level.Conclusion: Normal cell lines may be more difficult to be successfully induced target gene expression than tumor cells due to low transfection efficiency or low Ago2 and IPO8 expression. In addition, although saKLK1-374 is designed to up-regulate the expression of KLK1, the reason that it inhibits the proliferation of prostate cancer cells is irrelevant to the up-regulated expression of KLK1.