title: Establishment of a novel model for studying the effects of extracts of Chinese herb medicine on human type II 5alpha-reductase in vitro.
abstract: Human steroid 5alpha-reductase type II (hSRD5A2) and dihydrotestosterone (DHT) play important roles in benign prostatic hyperplasia (BPH). The aim of our study was to establish a novel model to investigate the inhibitory effects of extracts and compounds of Chinese herb medicine on hSRD5A2. The gene, hSRD5A2, was artificially synthesized and cloned into pcDNA3.1(+) vector, which was transfected into CHO cells by liposome. Transfected cells were screened through G418 and MTX. The expressed protein of hSRD5A2 by cells was purified and detected by western blotting. A minimum reactive system comprising hSRD5A2 and testosterone (T) as substrate together with NADPH as hydrogen donor was established for screening inhibitors of hSRD5A2. The reaction system was optimized in the concentrations of T, NADPH, and hSRD5A2 and reaction temperature, time, and activity of hSRD5A2 were determined by the production of DHT. Furthermore, we screened some extracts and compounds of Chinese herb medicine using this model. The concentrations of T, NADPH, and hSRD5A2 were 0.02 microM, 0.8 mM, and 0.05 U/microl, respectively, in the model; maximum activity of hSRD5A2 was achieved at 37 degrees C and 60 min reaction, and mangiferin had significant inhibitory effect on the activity of hSRD5A2. The model in this study is convenient and reliable for screening and evaluation of inhibitors of hSRD5A2; mangiferin may be a potential medicine for the treatment of BPH.
authors.affiliation: Department of Pharmacy, Anhui University of Traditional Chinese Medicine, Hefei, PR China. xs_wang2003@163.com
authors.forename: Xingsheng
authors.initials: X
authors.lastname: Wang
authors.forename: Jianmin
authors.initials: J
authors.lastname: Liao
authors.forename: Dengke
authors.initials: D
authors.lastname: Yin
authors.forename: Fan
authors.initials: F
authors.lastname: Zhan
authors.forename: Sufei
authors.initials: S
authors.lastname: Dai
authors.forename: Guangyan
authors.initials: G
authors.lastname: Xie
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Sang
mesh_terms: D000818:Animals; D016466:CHO Cells; D042944:Cholestenone 5 alpha-Reductase / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004365:Drugs, Chinese Herbal / Q000494:pharmacology* / Q000627:therapeutic use; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008954:Models, Biological; D011470:Prostatic Hyperplasia / Q000188:drug therapy; D013696:Temperature; D013997:Time Factors; D044004:Xanthones / Q000494:pharmacology / Q000627:therapeutic use
chemical_list: D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D044004:Xanthones; C013592:mangiferin; D042944:Cholestenone 5 alpha-Reductase