The Mechanism Of Action Of Neferine Down-regulating Androgen-mediated Epithelial-mesenchymal Transition Against Benign Prostatic Hyperplasia

Objective: Benign prostatic hyperplasia(BPH)is a common male urinary system disease.BPH leads to hyperplasia of prostatic stroma and glands,which in turn oppresses urethra to produce urinary retention and inflammation.In recent years,Chinese herbal medicine has been paid more and more attention to the treatment of prostate-related diseases.Neferine is a dibenzyl isoquinoline alkaloid extracted from natural lotus seed heart,which has many pharmacological actions,such as antioxidation,anti-inflammation,antidepressant,etc.,and has mild effects and little adverse reactions.However,the mechanism of action of neferine inhibiting prostatic hyperplasia has not been detail discovered.The purpose of this study is to examine whether neferine inhibits BPH and EMT(epithelial-mesenchymal transitions)process by regulating androgen and related signaling pathways.Methods: A mouse model of benign prostatic hyperplasia was established to observe the pharmacological effects of neferine on benign prostatic hyperplasia.Testosterone propionate induced benign prostatic hyperplasia mouse model.Different concentrations of neferine and finasteride were treated in BPH mice,and prostate tissues were collected after sacrificed in the 14 th or 28 th day.The changes of cell morphology were observed in different groups by H&E staining.Serum PSA(prostate spwecific antigen)content was detected by ELISA kit.In addition,normal human prostate stromal immortalized cell line WPMY-1 and human prostate cancer epithelial cell line LNCa P were used in this study.The anti-proliferation effect of neferine on androgen-induced stromal cells and cancer epithelial cells was detected by MTT assay.The effect of neferine on ROS was detected by flow cytometry.Western Blot was used to detect protein expression of neferine including 5α-reductase type Ⅱ,AR(androgen receptor),PSA,pro-caspase-3,cleaved caspase-3,uncleaved PARP,cleaved PARP,TGF-β,TGFBR2,p-Smad2/3 and Smad2/3,N-Cadherin,E-Cadherin and vimentin in mouse BPH model and in vitro BPH model(WPMY-1,LNCa P).In addition,Western Blot was used to detect TGF-β1-induced protein expression in cancer epithelial and stroma cells.Results: BPH model was induced by testosterone propionate(7.5mg/kg).Neferine(2,5mg/kg)and finasteride(10 mg/kg)decreased the prostate volume and prostate index.Serum PSA levels were decreased after neferine treatment.The results of H&E staining showed that the l thickness of epitheliawas reduced.Testosterone(1 mol/L)induced cell proliferation.Neferine(10 mol/L,100 mol/L)and finasteride(10 mol/L,100 mol/L)decreased cell viability in the prescence of testosterone.Compared with testosterone treatment group,the levels of ROS in stroma cells were decreased in neferine treatment group.In vivo,compared with the BPH group,the protein expressions of pro-caspase-3and uncleaved PARP were down-regulated,cleaved caspase-3 and cleaved PARP were up-regulated.In vitro and in vivo experiments,the results showed that the expression of AR pathway,TGF-β/Smads pathway and EMT were regulated,while the expression of5α-reductase type Ⅱ,AR and PSA decreased.The protein expression of Smad2/3 did not change significantly,but the expressions of TGF-β,TGFBR2 and p-Smad2/3 decreased.The protein expression of N-Cadherin and vimentin were inhibited,and the expression of E-Cadherin was induced.Neferine and finasteride regulated AR pathway,TGF-β/Smads pathway and EMT-related proteins in TGF-β1-treated WPMY-1 and LNCa P cells.Conclusion: This study reported the effects of neferine on BPH mice,epithelium and stroma cells.Neferine can reverse EMT process and prevent the progress of benign prostatic hyperplasia by blocking androgen /AR pathway and TGF-β/Smads pathway.Neferine is considered as a potential drug for the treatment of BPH.