五子衍宗丸干预线粒体通透性转换孔抑制精子凋亡的机制  

Effect of Wuzi Yanzong Wan on Sperm Apoptosis by Intervening with Mitochondrial Permeability Transition Pore

作　　者：刘红娟 吴德玲[1] 童小慧 李莉[1] 王桐生[1] LIU Hong-juan;WU De-ling;TONG Xiao-hui;LI Li;WANG Tong-sheng(Anhui Province Key Laboratory of Chinese Medicinal Formula,Anhui University of Chinese Medicine,Hefei 230012,China)

机构地区：[1]安徽中医药大学,中药复方安徽省重点实验室,合肥230012

出　　处：《中国实验方剂学杂志》2020年第21期34-39,共6页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81473674)。

摘　　要：目的:研究五子衍宗丸对电压依赖性阴离子通道1(VDAC1),腺嘌呤核苷酸转位酶(ANT),环孢菌素A结合蛋白D(CypD)等蛋白表达影响,分析其干预精子线粒体通透性转换孔(mPTP)开放的机制。方法:将40只大鼠随机分为正常组,模型组,阳性组(生精胶囊,1.6 g·kg-1·d-1),五子衍宗丸组(4.0 g·kg-1·d-1)。除正常组外其余各组灌服雷公藤多苷(30 mg·kg-1),连续8周,建立少弱精子症模型,从造模的第5周开始,各组按剂量灌胃给药,连续给药4周,末次给药后禁食12 h,3%水合氯醛麻醉动物,摘取睾丸和附睾组织。蛋白免疫印迹法(Western blot)检测大鼠睾丸组织VDAC1,ANT,CypD,B淋巴细胞瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax),半胱氨酸蛋白酶-3(Caspase-3),半胱氨酸蛋白酶-9(Caspase-9)蛋白表达;透射电镜观察精子线粒体的超微结构;原位末端标记(TUNEL)法检测大鼠睾丸生殖细胞凋亡情况。结果:与正常组比较,模型组大鼠VDAC1,CypD,Caspase-3,Caspase-9蛋白表达显著升高,Bax/Bcl-2显著升高(P<0.01);与模型组比较,生精胶囊组、五子衍宗丸组的VDAC1,CypD,Caspase-3,Caspase-9蛋白表达显著下降,Bax/Bcl-2显著降低(P<0.01);各组间ANT,Bcl-2蛋白表达无明显变化。电镜检查显示,模型组大鼠精子线粒体大小不一,排列紊乱,出现大量肿胀和空泡,生精胶囊组和五子衍宗丸组精子线粒体结构较完整,排列较整齐,肿胀和空泡现象明显减少;TUNEL检测结果显示,与正常组比较,模型组大鼠生精细胞凋亡率显著增加(P<0.01);与模型组比较,生精胶囊组、五子衍宗丸组细胞凋亡率显著降低(P<0.01)。结论:五子衍宗丸可能通过抑制VDAC1,CypD,Bax蛋白表达,降低mPTP的通透性,阻止Caspase凋亡蛋白家族的级联激活反应,发挥抗生殖细胞凋亡作用。Objective: To study the effect of Wuzi Yanzong Wan on the expressions of voltagedependent anion channel 1(VDAC1),adenine nucleotide transposase(ANT),cyclophilin D(CypD)and other proteins, and analyze its mechanism in intervening with sperm mitochondrial permeability transition pore(mPTP)opening. Method: Forty rats were randomly divided into 4 groups,namely normal group,model group,positive group(Shengjing capsule,1.6 g·kg-1·d-1),and Wuzi Yanzong Wan group(4.0 g·kg-1·d-1),with10 rats in each group. Except for the normal group,tripterygium wilfordii glycosides(GTW,30 mg·kg-1)was intragastrically administered for 8 weeks to establish the oligozoospermia model. After the 4 thweek,each group was given drugs through intragastric administration for 4 weeks,and fasted for 12 h after the last administration.These rats were anesthetized with 3% chloral hydrate,and their testis and epididymis tissues were collected.Western blot was used to determine the protein expressions of VDAC1,ANT,CypD,B-cell lymphoma/leukemia2(Bcl-2),Bcl-2 associated X protein(Bax),Caspase-3,Caspase-9 in rat testis,Testicular tissue and its ultrastructure were observed under electron microscopy. The apoptosis in spermatogenic cells was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL). Result: Western blot results showed that compared with the normal group,the expressions of VDAC1,CypD,Caspase-3,Caspase-9 and Bax/Bcl-2 in the model group were increased(P<0.01). The expressions of VDAC1,CypD,Caspase-3,Caspase-9 and Bax/Bcl-2 were significantly decreased in the positive group and the Wuzi Yanzong Wan group compared with the model group(P<0.01). There was no significant change in the expressions of ANT and Bcl-2 protein between the groups. Testicular ultrastructural evaluation showed different sizes and disordered arrangement of sperm mitochondria and a large number of swelling and vacuoles in the model group,while complete structure and neat arrangement of sperm mitochondria and much less swelling and vacuole in p

关 键 词：五子衍宗丸 少弱精子症 生殖细胞凋亡 线粒体通透性转换孔 

分 类 号：R2-0[医药卫生—中医学] R22