Mechanism Study Of PIEZO1 Regulation On Collagen Lysine Hydroxylation In Urethral Fibrosis

BackgroundUrethral stricture is a common condition in urology,and the main treatment method is surgery.However,surgical treatment is characterized by its high difficulty,high recurrence rate,and multiple complications.Therefore,finding new treatment methods is an urgent issue for urologists.The formation of urethral scar is the initial step in the development of urethral stricture,and the deposition of extracellular matrix proteins,mainly type I collagen(COL-I),is the main cause of its progression.Therefore,exploring the mechanism of abnormal deposition of collagen is of great significance for the prevention and treatment of urethral stricture.Post-translational hydroxylation modification of COL-I,especially lysine hydroxylation modification,plays an important role in maintaining the stability of collagen structure and promoting collagen deposition.This study aims to explore the role of lysine hydroxylation of collagen in urethral fibrosis and to reveal its mechanism of promoting fibrosis by increasing the deposition of COL-I,activating the piezoelectric mechanosensitive ion channel component 1(PIEZO1)through increased extracellular matrix stiffness,and subsequently increasing the expression of lysine hydroxylase(procollagen-lysine,2-oxoglutarate 5-dioxygenases,PLODs)through the calcium-cam-dependent protein kinase II(CAMKII)/hypoxia-inducible factor 1-alpha(HIF-1α)signaling pathway,leading to a vicious cycle in the fibrosis process and promoting the progressive development of fibrosis-re-fibrosis.This study will provide a new perspective for the research field of urethral fibrosis and lay a solid foundation for the prevention and treatment of urethral stricture.Method1.Using atomic force microscopy(AFM),the stiffness of narrowed urethral tissue and normal urethral tissue matrix was quantified.Liquid chromatography tandem mass spectrometry/mass spectrometry(LCMS/MS)was used to detect and analyze the level of collagen hydroxylation modification in 3 cases of narrowed urethral tissue and 3 cases of normal urethral tissue.2.The regulatory mechanism of post-translational modification of collagen in urethral fibrosis was investigated.Transcriptional sequencing was performed on 3 cases of narrowed urethral tissue and 3 cases of normal urethral tissue to identify differential genes between the two tissues.Bioinformatics methods were used to predict the regulatory mechanism of collagen lysine hydroxylation modification in urethral fibrosis.3.Tissue immunofluorescence and western blotting were used to detect the expression levels of PIEZO1,PLODs,p CAMKII,HIF-1α,and fibrosis markers COL-I and α-smooth muscle actin(α-SMA)in narrowed and normal urethral tissue.Western blotting,cell immunofluorescence,bladder urethral imaging,and other methods were used to validate whether PIEZO1 increased collagen lysine hydroxylation levels by up-regulating the CAMKII/HIF-1α signaling pathway and participated in urethral fibrosis.4.Literature was searched for lysine hydroxylation inhibitors.CCK8 experiments were used to screen for the optimal effective concentration and time of action of the lysine hydroxylation inhibitor 2,2’-dipyridine.Western blotting,hematoxylin-eosin staining,Masson staining,and bladder urethral imaging were used at the cellular and animal levels to validate the effectiveness of 2,2’-dipyridine in delaying urethral fibrosis progression.Results1.AFM measurements showed that the stiffness of normal human urethral tissue matrix was 4.2±1.92 k Pa,while the stiffness of narrowed urethral tissue matrix was 40.0±2.32 k Pa.LC-MS/MS detection of collagen extracted from narrowed and normal urethral tissue revealed a total of 1082 hydroxylation modification sites,among which 976 sites were hydroxylated on lysine residues and 106 sites were hydroxylated on proline residues.Lysine hydroxylation accounted for 90.2% of all modifications.Among the 49 lysine hydroxylation sites with statistically significant differences,42 sites showed increased expression while 7 sites showed decreased expression.Furthermore,the deposition of COL-I in the narrowed urethral tissue was significantly increased compared to normal urethral tissue.2.Transcriptomic analysis of human narrowed and normal urethral tissue identified 1983 differentially expressed m RNAs,with 546 m RNAs upregulated and 1437 m RNAs downregulated.The expression of lysyl hydroxylases PLOD1,PLOD2,and PLOD3 was upregulated,along with PIEZO1,HIF-1α,and fibrosis markers COL-I and α-SMA.Bioinformatic prediction suggested that PIEZO1 may regulate the expression of PLODs by modulating CAMKII/HIF-1α,affecting the stability of collagen and playing a role in the progression of urethral fibrosis.3.Immunofluorescence and Western blot analysis revealed higher expression levels of PLODs,PIEZO1,p CAMKII,HIF-1α,as well as fibrotic markers COL-Ⅰ and α-SMA in narrowed urethral tissue from both humans and rats compared to normal urethral tissue.Using a human urethral fibroblast model,it was found that increased matrix stiffness could activate PIEZO1,and the activated PIEZO1 could phosphorylate CAMKII to regulate HIF-1α expression,leading to increased expression of PLODs and enhanced stability of collagen.4.Using a human urethral fibroblast model,it was discovered that 2,2’-dipyridine could inhibit the expression of PLODs and fibrotic markers COL-Ⅰ and α-SMA.In a rat model of urethral stenosis,local injection of2,2’-dipyridine could delay the occurrence of urethral narrowing.ConcussionPIEZO1 promotes the progression of urethral fibrosis by upregulating the CAMKII/HIF-1α signaling pathway to increase collagen lysine hydroxylation levels.The progression of fibrosis promotes an increase in matrix stiffness,which positively regulates the expression and activation of PIEZO1.