杜仲补天素胶囊改善环磷酰胺诱导的小鼠生精障碍研究
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摘要
目的采用环磷酰胺(cyclophosphamide,CP)诱导的小鼠生精障碍模型,探究杜仲补天素胶囊(Duzhong Butiansu Capsule,DBC)的生殖保护作用及其机制。方法采用昆明种小鼠ipCP(60mg/kg)制备生精障碍模型,连续5d,第8天起ig给予DBC高、低剂量(1.388、0.694 g/kg)干预4周,给药结束后检测各组小鼠体质量、脏器指数变化,HE染色检测睾丸病理结构变化,ELISA法测定血清睾酮(T)、促卵泡激素(FSH)、促黄体生成素(LH)、丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)水平;Western blotting及免疫组化法分析睾丸组织中转录因子NF-E2相关因子2(Nrf2)、血红素氧合酶-1(HO-1)、醌氧化还原酶1(NQO1)、组蛋白脱乙酰基酶2(HDAC2)与磷酸化蛋白激酶C(p-PKC)表达。结果与模型组比较,DBC对小鼠体质量下降有极显著干预作用,能够增加睾丸、附睾及肾脏指数,改善睾丸病理形态损伤,增加精子数、提高精子活力、降低精子畸形率,提高T水平,降低LH、FSH水平,降低MDA含量,提高SOD、GSH-Px活性,提高Nrf2、HO-1、NQO1、HDAC2与p-PKC蛋白的表达水平(P<0.05、0.01)。结论 DBC能够改善CP诱导的小鼠生精障碍,其机制可能与调节氧化应激相关的Nrf2/抗氧化作用元件(ARE)信号通路有关。
Objective To investigate the reproductive protective effect of Duzhong Butiansu Capsule(DBC) by using cyclophosphamide induced spermatogenic disorder model, and explore its mechanism. Methods The model of spermatogenic disorder was established by intraperitoneal injection of cyclophosphamide(60 mg/kg) for 5 d. Drug intervention at high and low doses(1.388 g/kg and 0.694 g/kg) was given for 4 weeks from the 8 th day. The body weight and organ index of each group were measured. The pathological structure of testis was detected by HE staining. ELISA method was used to detect the levels of T, FSH,LH, MDA, SOD, GSH-Px. Western blotting and immunohistochemistry were used to analyze the expression of Nrf2/ARE signaling pathway related factors Nrf2, HO-1, NQO1, HDAC2, and p-PKC in testicular tissue. Results Compared with the model group,DBC significantly reduced the weight of mice, increased the index of testis, epididymis and kidney, improved the pathological morphology of testis, increased the number of spermatozoa, increased the motility of sperm, decreased the rate of abnormal sperm,increased the level of T and decreasd the level of LH and FSH, increased the content of MDA, decreased the content of SOD and GSH-Px, increased the expression of Nrf2, HO-1, NQO1, HDAC2, and p-PKC protein, and increased the area of positive expression of Nrf2, HO-1 protein(P < 0.05 or P < 0.01). Conclusion DBC can obviously improve the spermatogenic disorder induced by cyclophosphamide, and the mechanism may be related to the regulation of Nrf2/ARE signal pathway associated with oxidative stress.
Objective To investigate the reproductive protective effect of Duzhong Butiansu Capsule(DBC) by using cyclophosphamide induced spermatogenic disorder model, and explore its mechanism. Methods The model of spermatogenic disorder was established by intraperitoneal injection of cyclophosphamide(60 mg/kg) for 5 d. Drug intervention at high and low doses(1.388 g/kg and 0.694 g/kg) was given for 4 weeks from the 8 th day. The body weight and organ index of each group were measured. The pathological structure of testis was detected by HE staining. ELISA method was used to detect the levels of T, FSH,LH, MDA, SOD, GSH-Px. Western blotting and immunohistochemistry were used to analyze the expression of Nrf2/ARE signaling pathway related factors Nrf2, HO-1, NQO1, HDAC2, and p-PKC in testicular tissue. Results Compared with the model group,DBC significantly reduced the weight of mice, increased the index of testis, epididymis and kidney, improved the pathological morphology of testis, increased the number of spermatozoa, increased the motility of sperm, decreased the rate of abnormal sperm,increased the level of T and decreasd the level of LH and FSH, increased the content of MDA, decreased the content of SOD and GSH-Px, increased the expression of Nrf2, HO-1, NQO1, HDAC2, and p-PKC protein, and increased the area of positive expression of Nrf2, HO-1 protein(P < 0.05 or P < 0.01). Conclusion DBC can obviously improve the spermatogenic disorder induced by cyclophosphamide, and the mechanism may be related to the regulation of Nrf2/ARE signal pathway associated with oxidative stress.
引文
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[2]Camargo M,Intasqui P,Lima C B,et al.MALDI-TOFFingerprinting of seminal plasma lipids in the study of human male infertility[J].Lipids,2014,49(9):943-956.
[3]Rana K P,Thaper D L,Prabha V J.Is there a role for Serratia marcens in male infertility:An experimental study[J].Microb Pathog,2017,105(4):13-18.
[4]韦凤华.环磷酰胺临床应用研究进展[J].中国药事,2013,27(3):324-326.
[5]董良.环磷酰胺在男性不育动物模型中的应用[A]//中国中西医结合学会男科专业委员会.第十二次全国中西医结合男科学术大会暨全国中西医结合男科诊疗技术研修班暨2017上海市中西医结合学会上海市中医药学会泌尿男科专业委员会学术年会讲义论文资料汇编[C].北京:中国中西医结合学会,2017.
[6]张曦文,李乐乐,江素鑫,等.环磷酰胺生殖毒性的研究进展[J].中国研究型医院,2018,5(1):27-32.
[7]杨群芳.环磷酰胺对雄性生殖系统毒性损伤的研究进展[J].儿科药学杂志,2013,19(3):62-65.
[8]赵罗娜,刘明,张永萍,等.杜仲补天素片对雄性动物促生育作用的研究[J].中草药,2017,48(16):3419-3424.
[9]陈指龙,张晓春,薛立群,等.环磷酰胺致小鼠生精障碍作用研究[J].动物医学进展,2017,38(5):34-38.
[10]罗少波,胡海翔,贾金铭.腹腔注射环磷酰胺对小鼠精子凋亡的影响[J].空军总医院学报,2010,26(1):32-34.
[11]陈振文,谷龙杰.精液分析标准化和精液质量评估--WHO《人类精液检查与处理实验室手册》(第5版)出版[J].中国计划生育学杂志,2012,20(1):58-62.
[12]李文洁.影响免疫组化染色的因素及相关注意事项[J].世界最新医学信息文摘,2018,18(93):125.
[13]刘秋梅.白消安与环磷酰胺在家兔体内药动学及相互作用研究[D].太原:山西医科大学,2018.
[14]乐小炎.乌贼墨多糖干预环磷酰胺介导睾丸氧化应激损伤的Nrf2/ARE调控机制研究[D].湛江:广东海洋大学,2014.
[15]侯晓鸿,郝卫东.下丘脑-垂体-性腺轴调节睾酮合成的研究进展[J].癌变·畸变·突变,2018,30(6):483-485.
[16]Jin Y,Miao W,Lin X,et al.Acute exposure to3-methylcholanthrene induces hepatic oxidative stress via activation of the Nrf2/ARE signaling pathway in mice[J].Envir Toxicol,2014,29(12):1399-1408.
[17]李延森,李照见,贾潇潇,等.环境高温诱导巴马香猪睾丸细胞凋亡并激活Nrf2/HO-1抗氧化信号通路[J].畜牧兽医学报,2018,49(6):1154-1162.
[18]路阳.Nrf2蛋白的O-GlcNAcylation修饰在抗氧化应激中的作用和机制研究[D].长春:东北师范大学,2017.
[19]Mercado N,Thimmulappa R,Thomas C M R,et al.Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress[J].Biochem Biophys Res Commun,2011,406(2):292-298.
[20]Murphy K,Llewellyn K,Wakser S,et al.Mini-GAGR,an intranasally applied polysaccharide,activates the neuronal Nrf2-mediated antioxidant defense system[J].Biol Chem,2018,293(47):18242-18269.
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