益肾生精方治疗少弱精子症的药效评价及作用机制

Efficacy and Mechanism of Yishen Shengjing Prescription in Treatment of Oligoasthenospermia

目的:采用环磷酰胺致大鼠少弱精子症模型,研究益肾生精方对少弱精子症的治疗作用及作用机制。方法:随机选取8只SD雄性大鼠为正常组,其余大鼠连续5 d腹腔注射环磷酰胺(35 mg·kg^(-1)·d^(-1))制备大鼠少弱精子症模型,而后随机分为模型组、益肾生精方低、中、高剂量组(2.91、5.83、11.66 g·kg^(-1)),五子衍宗丸组(1.03 g·kg^(-1)),左卡尼汀组(0.17 g·kg^(-1)),每组8只。药物干预28 d后,记录大鼠体质量、睾丸质量和指数;采用计算机辅助精子分析(CASA)系统检测附睾中精子质量;苏木素-伊红(HE)染色观察睾丸组织形态;比色法检测睾丸组织中丙二醛(MDA)、超氧化物歧化酶(SOD)水平;酶联免疫吸附测定法(ELISA)检测血清中卵泡刺激素(FSH)、黄体生成素(LH)、睾酮(T)的水平;原位末端标志法(TUNEL)检测睾丸组织中细胞的凋亡情况;蛋白免疫印迹法(Western blot)检测睾丸组织中B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、活化的胱天蛋白酶-3(cleaved Caspase-3)蛋白表达水平。结果:与正常组比较,模型组大鼠体质量、睾丸质量和指数、精子浓度和活力显著降低(P<0.01),睾丸病理评分显著升高(P<0.01);与模型组比较,益肾生精方中、高剂量组、五子衍宗丸组和左卡尼汀组可提高体质量、睾丸质量和指数,并提高精子浓度和活力;降低睾丸病理评分。氧化应激水平检测结果显示,与正常组比较,模型组大鼠睾丸组织中SOD水平显著降低(P<0.01),MDA水平显著升高(P<0.01);与模型组比较,益肾生精方高剂量组、五子衍宗丸组和左卡尼汀组可以明显提高SOD活性,降低MDA水平(P<0.05,P<0.01)。血清激素水平检测结果显示,与正常组比较,模型组T水平显著降低(P<0.01),FSH和LH水平显著升高(P<0.01);与模型组比较,益肾生精方组、五子衍宗丸组和左卡尼汀组可以显著提高T水平(P<0.01),降低LH水平(P<0.05,P<0.01)。TUNEL结果显示,与正常组比较,模型组生精细胞凋亡率显著增加(P<0.01);与模型组比较,益肾生精方中剂量组、五子衍宗丸组和左卡尼汀组可显著降低凋亡率(P<0.01)。Western blot结果显示,与正常组比较,模型组Bcl-2蛋白水平明显降低(P<0.05)、Bax和cleaved Caspase-3蛋白水平明显升高(P<0.05,P<0.01);益肾生精方高剂量组、五子衍宗丸组和左卡尼汀组可以明显升高Bcl-2蛋白水平,降低cleaved Caspase-3蛋白水平(P<0.05,P<0.01)。结论:益肾生精方可改善少弱精子症模型大鼠的精子质量,其机制可能与抗氧化、调节性激素水平以及抑制生精细胞凋亡等多个途径有关。

Objective:We aimed to investigate the efficacy and mechanism of Yishen Shengjing Prescription(YSP)in the treatment of oligoasthenospermia in rats.Method:The oligoasthenospermia rat model was established by injection with cyclophosphamide(35 mg·kg^(-1)·d^(-1))for 5 consecutive days.Rats were randomly assigned into control group(without treating with cyclophosphamide),model group,low-(YSP-L),medium-(YSP-M),and high-(YSP-H)dose(2.91,5.83,and 11.66 g·kg^(-1),respectively)groups,Wuzi Yanzongwan(WYW,1.03 g·kg^(-1))group,and L-carnitine(0.17 g·kg^(-1))group,with 8 rats in each group.After28 days of drug intervention,the body weight,testicular weight,and testicular index of rats were recorded.The sperm quality in epididymis was detected by computer-assisted sperm analysis(CASA)system.Hematoxylineosin(HE)staining was employed for observation of testicular tissue morphology.The levels of malondialdehyde(MDA)and superoxide dismutase(SOD)in testicular tissue were detected by colorimetry.The levels of follicle-stimulating hormone(FSH),luteinizing hormone(LH),and testosterone(T)in serum were determined by enzyme-linked immunosorbent assay(ELISA).TdT-mediated dUTP nick-end labeling(TUNEL)was employed to detect the apoptosis of testicular cells.The protein levels of B cell lymphoma-2(Bcl-2),Bax,and cleaved Caspase-3 in testicular tissue were detected by Western blot.Result:Compared with the control group,the model group showed decreased body weight,testicular weight and index,sperm concentration and motility(P<0.01)and increased testicular pathological score(P<0.01).Compared with the model group,the YSP-M,YSP-H,WYW,and L-carnitine groups showed increased body weight,testicular weight,testicular index,sperm concentration and motility and decreased testicular pathological score.After modeling,the SOD level decreased(P<0.01)while the MDA content increased(P<0.01)in the testicular tissue.YSP-H,WYW,and L-carnitine reversed the SOD and MDA level changes caused by modeling.Compared with the control group,the model group exhibited declined T level(P<0.01)and increased FSH and LH levels(P<0.01).Compared with the model group,YSP,WYW,and L-carnitine increased the T level(P<0.01)and decreased the LH level(P<0.05,P<0.01).The apoptosis rate of spermatogenic cells in the model group was higher than that in the control group(P<0.01),whereas YSP-M,WYW,and L-carnitine reversed such changes(P<0.01).The model group rats showed decreased expression of Bcl-2(P<0.05)and increased expression of Bax and cleaved Caspase-3(P<0.05,P<0.01).Compared the model group,YSP-M,YSP-H,WYW,and L-carnitine up-regulated the Bcl-2 expression and down-regulated the cleaved Caspase-3 expression(P<0.05,P<0.01).Conclusion:YSP improved the sperm quality of oligoasthenospermia model rats by regulating the antioxidant system and sex hormone levels and inhibiting the apoptosis of spermatogenic cells.