The Inhibition Of Arsenic-induced Malignant Transformation In Bladder Epithelial Cells By Matrine Via NOX 2

Objective:Arsenic is a metalloid element widely existing in nature,World Health Organization reported that about 140 million people in 70 countries are at risk of arsenic exposure in drinking water,which has created an important public health issue around the world.NOX2 is the most characterized and widely distributed of NADPH Oxidases.NOX2 can limit inflammation and injury by modulating key signaling pathways that affect neutrophil accumulation and clearance.ROS generated by NOX2 can also promote tumorigenesis and development through an immunological pathway.NOX2 may be a key target for the treatment of tumors.For a long time,natural products have been considered the key potential drugs to treat a series of fatal diseases,including cancer.A variety of natural products isolated from Chinese herbs are also widely used in cancer treatment for their excellent antiproliferation,pro-apoptosis,anti-metastasis,autophagy,and immune regulation activities.Matrine(C₁₅H₂₄N₂O)is one of the key tetracyclo-quinolizindine alkaloids isolated from the roots of Sophora flavescens（Kushen）.It has been reported to possess anti-inflammatory,antifibrotic,antitumor and analgesic pharmacological effects.However,it is unclear whether matrine can inhibit arsenic-induced bladder epithelial cell proliferation,migration and angiogenesis through NOX2.Therefore,our study suggests that the action of matrine on SV-HUC-1 cells via the NOX2 pathway can provide a potential therapeutic strategy for arsenite-induced bladder cancer.Method:1.To ensure continuous exposure to Na As O₂,SV-HUC-1 cells were cultured in a medium containing 0 or 0.5μM Na As O₂ for about 40 weeks（T40W）and were added fresh medium every day.To evaluate the effect of matrine,SV-HUC-1 cells were treated with matrine（2mg/m L,diluted by F-12K medium）for 24 h.The cell viability was ascertained by the CKC-8 assay.BD flow cytometer was employed to analyze the cell cycle.Analysis of wound healing determined the migration of SV-HUC-1 cells.The level of ROS was determined by ROS fluorescent.Real time PCR was employed to ascertain the m RNA expression levels of Cyclin D1,PCNA,E-cadherin,Vimentin,VEGF,NLRP3,IL-1β,Caspase 1,IL-18,NOX2.Western blot was used to detect the protein expression levels of CCND,PCNA,E-cadherin,Vimentin,VEGF,NLRP3,IL-1β,Caspase 1,IL-18,NOX2.2.Thirty Wistar rats were randomly divided into five groups:control group,low arsenite-treated group（10 mg/L Na As O₂）,high arsenite-treated group（50 mg/L Na As O₂）with three different groups.Control group rats were treated with distilled water for twelve weeks.Low arsenite-treated rats were supplied with distilled water containing 10 mg/L Na As O₂for twelve weeks.High arsenite-treated rats were provided with distilled water containing50 mg/L Na As O₂ for twelve weeks,and two of three groups individually irrigate 20 mg/Kg apocynin（APO）and 20 mg/Kg matrine（MAT）once per day in the ninth week for three weeks.The pathological changes in rat bladder epithelial cells was detected by HE staining assay.Immunohistochemical assay was using to detect the rat bladder epithelial cells expression level in E-cadherin,Vimentin,NLRP3,IL-1βand Cyclin D1.Using immunofluorescence assay to discovered the expression level of VEGF and NOX2 in rat bladder epithelial cells.Results:1.Long-term arsenite exposure can promote proliferation,migration,and angiogenesis in SV-HUC-1 Cells.2.Chronic arsenite-treated activates the expressions of NLRP3 inflammasome in SV-HUC-1 cells.3.Chronic arsenite-treated activates the expressions of ROS and NOX2 in SV-HUC-1 cells.4.Matrine suppressed the expressions of NLRP3 inflammasome via inhibiting NOX2 in vivo.5.Matrine decreased the proliferation and cell viability in bladder epithelial cells.6.Matrine regulated E-cadherin and Vimentin and diminished the ability of migration in bladder epithelial cells.7.Matrine declined the angiogenesis in bladder epithelial cells.Conclusions:1.Chronic arsenic exposure induced bladder epithelial cell alterations with tumor cell characteristics and increased expression of NLRP3 inflammation-related factors,ROS and NOX2.2.Matrine inhibits malignant transformation of bladder epithelial cells induced by chronic arsenic exposure via the NOX2 pathway.