Icariin improves busulfan-and cyclophosphamide-induced reproductive function damage in male mice

WU Yao; SHI Shan-shan; LIU Hai-yan; HUANG Hao; SHI Xing-hua; HOU Jing

doi:10.13263/j.cnki.nja.2025.01.004

Abstract：Objective:To comprehensively evaluate the effect of icariin in alleviating reproductive function damage(RFD)in male mice via in vitro and in vivo experiments.Methods:We isolated Leydig cells from 60 KM male mice in vitro,and examined the toxic effect of icariin on the Leydig cells using Cell Counting Kit-8(CCK-8).We equally randomized the mice into six groups:nor-mal control,RFD model control(made by intraperitoneal injection of busulfan at 10 mg/kg combined with cyclophosphamide(CP)at 120 mg/kg),positive control,and low-,medium-and high-dose icariin.After modeling,we treated the mice in the positive control group with Wuziyanzong Pills and those in the low-,medium-and high-dose icariin groups by intragastrical administration of icariin at 20,40 and 80 mg/kg-1,respectively,for 30 successive days.Then we obtained the weight and visceral coefficients of the reproductive organs,calculated the sperm count,observed the pathological changes in the testis tissue by HE staining,measured the serum testos-terone(T)level by ELISA,determined the indexes of testicular oxidative stress and nitric oxide(NO)signaling pathway by colorimet-ric assay,and detected the expression levels of the pro-apoptotic genes Fas and Bax by qRT-PCR.Results:CCK-8 assay confirmed that icariin had no toxic effect on the isolated Leydig cells of the mice,and could effectively reduce busulfan-and CP-induced cytotoxic-ity and promote the secretion of serum T.Icariin at 80 mg/kg significantly increased the visceral coefficient of the testis and promoted spermatogenesis(P＜0.05),but had little effect on the visceral coefficient of the epididymis in the RFD model mice.Testicular histo-morphometric observation revealed significantly improved testis structure,intact boundary membrane of seminiferous tubules and in-creased numbers of various types of spermatogenic cells of the model mice after treated with icariin.Compared with the mice in the model control group,those treated with high-dose icariin showed a significantly reduced content of malondialdehyde(MDA)(by 35.3％,P＜0.01),elevated total antioxidant capacity(TAOC)and superoxide dismutase(T-SOD)activity(P＜0.05),and de-creased NO content and nitric oxide synthase(NOS)activity in the testis tissue(P＜0.01).In addition,icariin exhibited an evident inhibitory effect on the expressions of the pro-apoptotic genes Bax and Fas.Conclusion:Icariin can ameliorate oxidative stress-in-duced damage to the testicular function and protect spermatogenesis of male mice by elevating TAOC,decreasing NOS activity,inhibi-ting the NO level in the testis,and suppressing busulfan-and CP-induced apoptosis of testicular cells.