通精灵对精索静脉曲张模型大鼠生精细胞凋亡及凋亡因子Cyt C、caspase-9的影响
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摘要
[目的]探讨通精灵对精索静脉曲张模型大鼠睾丸组织的保护作用及与凋亡因子细胞色素C(cytochrome C,Cyt C)、半胱氨酸蛋白酶-9(cysteine aspartic acid specific protease-9,caspase-9)表达的关系。[方法]72只SPF级Wistar大鼠,随机分成假手术组、模型组、通精灵低、中、高剂量组及左卡尼汀组。采用左肾静脉部分结扎法联合"夹尾激怒法"建立实验性精索静脉曲张合并中医肝气郁结证候复合大鼠模型。模型建立后,通精灵低、中、高剂量组分别给予浓度为1.6、3.2、6.4g·m L-1通精灵生药水煎液灌胃,假手术组和模型组予以0.9%氯化钠溶液灌胃,左卡尼汀组则予0.021g·mL-1左卡尼汀口服液灌胃,以上各组灌胃体积均为1mL/100g体质量,1次/d,持续8周。以HE染色观察睾丸组织结构;TUNEL染色和Annexin V-FITC法检测生精细胞凋亡情况;Western blot检测睾丸组织中Cyt C、caspase-9的表达。[结果]模型组中睾丸组织发生明显病理损伤,而通精灵各剂量组及左卡尼汀组中睾丸的病理损伤有不同程度改善。模型组中睾丸生精细胞凋亡率明显高于假手术组,差异有统计学意义(P<0.05),通精灵中、高剂量组及左卡尼汀剂量组中生精细胞凋亡率明显低于模型组,差异有统计学意义(P<0.05或P<0.01)。Western blot提示,模型组大鼠睾丸组织中Cyt C、caspase-9蛋白表达显著高于假手术组(P<0.01);通精灵中、高剂量组及左卡尼汀组中Cyt C、caspase-9表达低于模型组(P<0.05或P<0.01);高剂量组中Cyt C、caspase-9蛋白表达明显低于左卡尼汀组(P<0.05,P<0.01)。[结论]通精灵可以有效改善精索静脉曲张大鼠睾丸组织损伤,降低Cyt C和caspase-9的表达,从而调控生精细胞的凋亡。这可能是通精灵改善精索静脉曲张患者生育能力的机制之一。
[Objective] To explore the protective effect of Tongjingling(TJL) on testicular tissue in rats with varicocele and its relationship with the expression of apoptosis factors cytochrome C(Cyt C) and cysteine aspartic acid specific protease-9(caspase-9).[Methods] Seventy two male SPF Wistar rats were randomly divided into six groups of equal number: sham operation group, model group, low-dose TJL, middle-dose TJL and high-dose TJL group and L-carnitine group. The composite rat model of experimental varicocele(EVC) with TCM syndrome of stagnation of liver Qi was induced by partial ligation of the left renal vein of rats and the method of clamping-tail. After establishment of varicocele model of rats, low-dose TJL, middle-dose TJL and high-dose TJL groups were given decoction with different concentrations of crude drugs(1.6g·m L-1, 3.2g·m L-1, 6.4g·mL-1). Rats in sham operation group and model group were given 0.9% saline. Rats in L-carnitine group were given L-carnitine oral liquid(0.021g·m L-1). All the above groups were gavaged according to 1mL/100 g body weight, once a day for 8 weeks. The histological structure of the testis was observed by HE staining. TUNEL staining and Annexin V-FITC were used to detect the apoptosis of spermatogenic cells. The expression of Cyt C and caspase-9 in testicular tissue was detected by Western blot. [Results] In model group, the testicular tissue had obvious pathological damage, while the pathological damage of testicular tissue was improved in TJL groups and L-carnitine group. The apoptosis of testis spermatogonia in model group was significantly higher than that in sham operation group(P<0.05). The apoptosis of the spermatogenic cells in middle-dose, high-dose TJL groups and L-carnitine group was significantly lower than that in model group(P<0.05, P<0.01). Compared with sham operation group, the expression of Cyt C and caspase-9 in testicular tissue of model group increased significantly(P<0.01). Compared with model group, the expression of Cyt C and caspase-9 decreased with different degrees in middle-dose, high-dose TJL groups and L-carnitine group(P<0.05, P<0.01). Compared with L-carnitine group, the expression of Cyt C and caspase-9 protein in high-dose TJL group was significantly lower(P<0.05, P<0.01).[Conclusion] TJL can effectively improve testicular tissue damage in rats with varicocele, reduce the expression of Cyt C and caspase-9, and regulate the apoptosis of spermatogenic cells. Spermatogenic cells in rats with varicocele. This may be one of the molecular mechanism for improving the fertility of infertile patients with varicocele.
[Objective] To explore the protective effect of Tongjingling(TJL) on testicular tissue in rats with varicocele and its relationship with the expression of apoptosis factors cytochrome C(Cyt C) and cysteine aspartic acid specific protease-9(caspase-9).[Methods] Seventy two male SPF Wistar rats were randomly divided into six groups of equal number: sham operation group, model group, low-dose TJL, middle-dose TJL and high-dose TJL group and L-carnitine group. The composite rat model of experimental varicocele(EVC) with TCM syndrome of stagnation of liver Qi was induced by partial ligation of the left renal vein of rats and the method of clamping-tail. After establishment of varicocele model of rats, low-dose TJL, middle-dose TJL and high-dose TJL groups were given decoction with different concentrations of crude drugs(1.6g·m L-1, 3.2g·m L-1, 6.4g·mL-1). Rats in sham operation group and model group were given 0.9% saline. Rats in L-carnitine group were given L-carnitine oral liquid(0.021g·m L-1). All the above groups were gavaged according to 1mL/100 g body weight, once a day for 8 weeks. The histological structure of the testis was observed by HE staining. TUNEL staining and Annexin V-FITC were used to detect the apoptosis of spermatogenic cells. The expression of Cyt C and caspase-9 in testicular tissue was detected by Western blot. [Results] In model group, the testicular tissue had obvious pathological damage, while the pathological damage of testicular tissue was improved in TJL groups and L-carnitine group. The apoptosis of testis spermatogonia in model group was significantly higher than that in sham operation group(P<0.05). The apoptosis of the spermatogenic cells in middle-dose, high-dose TJL groups and L-carnitine group was significantly lower than that in model group(P<0.05, P<0.01). Compared with sham operation group, the expression of Cyt C and caspase-9 in testicular tissue of model group increased significantly(P<0.01). Compared with model group, the expression of Cyt C and caspase-9 decreased with different degrees in middle-dose, high-dose TJL groups and L-carnitine group(P<0.05, P<0.01). Compared with L-carnitine group, the expression of Cyt C and caspase-9 protein in high-dose TJL group was significantly lower(P<0.05, P<0.01).[Conclusion] TJL can effectively improve testicular tissue damage in rats with varicocele, reduce the expression of Cyt C and caspase-9, and regulate the apoptosis of spermatogenic cells. Spermatogenic cells in rats with varicocele. This may be one of the molecular mechanism for improving the fertility of infertile patients with varicocele.
引文
[1]Jensen C F S,stergren P,Dupree J M,et al.Varicocele and male infertility[J].Nat Rev Urol,2017,14(9):523-533.
[2]Lundy S D,Sabanegh E S Jr.Varicocele management for infertility and pain:a systematic review[J].Arab J Urol,2018,16(1):157-170.
[3]Sedaghatpour D,Berookhim B M.The role of varicocele in male factor subfertility[J].Curr Urol Rep,2017,18(9):73.
[4]徐锋,陈赟,陈海,等.大鼠精索静脉曲张模型及其高位结扎术后睾丸生精细胞凋亡及其IL-1和NO含量的变化[J].中华男科学杂志,2016,22(3):200-204.XU Feng,CHEN Yun,CHEN Hai,et al.Impact of varicocele and varicocelectomy on the apoptosis of spermatogenic cells and the levels of nitrogen monoxidum and interleukin-1 in the rat testis[J].National Journal of Andrology,2016,22(3):200-204.
[5]Belardin L B,Del Giudice P T,Camargo M,et al.Alterations in the proliferative/apoptotic equilibrium in semen of adolescents with varicocele[J].J Assist Reprod Genet,2016,33(12):1657-1664.
[6]Shiraishi K,Matsuyama H,Takihara H.Pathophysiology of varicocele in male infertility in the era of assisted reproductive technology[J].Int J Urol,2012,19(6):538-550.
[7]吴骏,崔云,陈建伟,等.“通精灵”方对精索静脉曲张致男性不育患者精子DNA碎片率的影响[J].浙江中医药大学学报,2016,40(5):356-360.WU Jun,CUI Yun,CHEN Jianwei,et al.The Prescription of"Tongjingling"on the DFI caused by varicocele in male infertility[J].Journal of Zhejiang Chinese Medical University,2016,40(5):356-360.
[8]崔云,冯奕,郑武,等.通精灵对实验性精索静脉曲张大鼠生精细胞凋亡及Caspase-3 Hsp60表达的影响[J].中华中医药学刊,2008,26(12):2593-2597.CUI Yun,FENG Yi,ZHENG Wu,et al.Effect of Tongjing ling Decoction on spermatogenic cell apoptosis and the expression of caspase-3 and Hsp60 in the rat with expermiental varicocele[J].Chinese Archives of Traditional Chinese Medicine,2008,26(12):2593-2597.
[9]Tian R H,Ma M,Zhu Y,et al.Effects of aescin on testicular repairment in rats with experimentally induced varicocele[J].Andrologia,2014,46(5):504-512.
[10]杜宝昕,郑军状,刘冰,等.通精灵对实验性精索静脉曲张大鼠精子DNA完整性与睾丸组织氧化应激的影响[J].中华男科学杂志,2017,23(11):1025-1031.DU Baoxin,ZHENG Junzhuang,LIU Bing,et al.Tongjingling improves sperm DNA integrity and reduces oxidative stress in the testis of experimental varicocele rats[J].National Journal of Andrology,2017,23(11):1025-1031.
[11]秦川.实验动物学[M].北京:中国协和医科大学出版社,2016:167.QIN Chuan.Laboratory Animal Science[M].Beijing:Peking Union Medical College Press,2016:167.
[12]中国左卡尼汀临床应用专家共识编写组.左卡尼汀在男性不育中临床应用专家共识[J].中华男科学杂志,2015,21(1):82-85.China's expert consensus group on clinical application of L-carnitine.Expert consensus on clinical application of L-carnitine in male infertility[J].National Journal of Andrology,2015,21(1):82-85.
[13]陈实功.外科正宗[M].沈阳:辽宁科学技术出版社,1997:54.CHEN Shigong.Waike Zhengzong[M].Shenyang:Liaoning Science and Technoloy Press,1997:54.
[14]陈士铎.洞天奥旨[M].北京:中医古籍出版社,1992:138.CHEN Shiduo.Dongtian Aozhi[M].Beijing:TCM Ancient Books Press,1992:138.
[15]陈士铎.石室秘录[M].北京:人民军医出版社,2009:265.CHEN Shiduo.Shishi Milu[M].Beijing:People's Military Medical Press,2009:265.
[16]张锡纯.医学衷中参西录[M].石家庄:河北科学技术出版社,2002:358.ZHANG Xichun.Intergrating Chinese and Western Medicine[M].Shijiazhuang:Hebei Science and Technology Press,2002:358.
[17]崔云,郑武,万舟,等.通精灵结合精索内静脉高位断流术治疗不育症37例临床观察[J].浙江中医学院学报,1998,22(6):30-31.CUI Yun,ZHENG Wu,WAN Zhou,et al.The clinical observation of Tongjingling combined with the spermatic cord vein high block treated the infertility 37 cases[J].Journal of Zhejiang College of TCM,1998,22(6):30-31.
[18]吕逸清,陈斌.精索静脉曲张致不育机制研究进展[J].中华男科学杂志,2008,14(5):454-458.LYU Yiqing,CHEN Bin.Progress in researches on the mechanism of varicocele-induced male infertility[J].National Journal of Andrology,2008,14(5):454-458.
[19]朱少明,程帆.精索静脉曲张致不育机制的研究进展[J].安徽医药,2017,21(11):1945-1948.ZHU Shaoming,CHENG Fan.A review:the mechanism of male sterility caused by varicocele[J].Anhui Med Pharm J,2017,21(11):1945-1948.
[20]Sheehan M M,Ramasamy R,Lamb D J.Molecular mechanisms involved in varicocele-associated infertility[J].J Assist Reprod Genet,2014,31(5):521-526.
[21]武政华,柯鑫文,冯少勇,等.茶多酚对精索静脉曲张大鼠睾丸生精细胞凋亡的影响[J].中华男科学杂志,2015,21(8):702-707.WU Zhenghua,KE Xinwen,FENG Shaoyong,et al.Tea polyphenols reduces the apoptosis of spermatogenic cells in rats with experimental varicocele[J].National Journal of Andrology,2015,21(8):702-707.
[22]张长城,周安方,张茂林,等.实验性精索静脉曲张对大鼠睾丸生精细胞凋亡的影响[J].中华男科学杂志,2003,9(7):507-509.ZHANG Changcheng,ZHOU Anfang,ZHANG Maolin,et al.Effects of experimental varicocele on the apoptosis of spermatogenic cells in rats[J].National Journal of Andrology,2003,9(7):507-509.
[23]崔云,冯奕,郑武,等.通精灵对实验性精索静脉曲张大鼠生精细胞凋亡及Bcl-2表达的影响[J].中国中西医结合外科杂志,2009,15(2):173-177.CUI Yun,FENG Yi,ZHENG Wu,et al.Effect of Tongjingling Decoction on spermatogenic cell apoptosis and expression of Bcl-2 in rats with experimental varicocele[J].Chinese Journal of Surgery of Integrated Traditional and Western Medicine,2009,15(2):173-177.
[24]XU Ming,ZHANG Hong,YIN Xiuting,et al.The effect of electroacupuncture on the protein expression levels of Cyt-C,Caspase-3 and Caspase-9 in the bladder tissue of rats with neurogenic bladder after suprasacral spinal cord injury[J].World Journal of Acupuncture-Moxibustion,2017,27(3):49-56.
[25]王力,陈东,闵泽,等.益精方对不育症大鼠睾丸生精细胞凋亡及Bcl-2、Bax蛋白表达的影响[J].中国中西医结合杂志,2014,34(5):602-605.WANG Li,CHEN Dong,MIN Ze,et al.Effect of Yijing Recipe on apoptosis of testis spermatogenic cells and the expression of Bcl-2 and Bax protein in infertility rats[J].Chinese Journal of Integrated Traditional and Western Medicine,2014,34(5):602-605.
[26]Kulikov A V,Shilov E S,Mufazalov I A,et al.Cytochrome C:the Achilles’heel in apoptosis[J].Cell Mol Life Sci,2012,69(11):1787-1797.
[27]Monian P,Jiang X.Clearing the final hurdles to mitochondrial apoptosis:regulation post cytochrome C release[J].Exp Oncol,2012,34(3):185-191.
[28]朱愿,马勇,陈立军.Caspase与细胞凋亡[J].武警医学院学报,2004,13(4):346-348.ZHU Yuan,MA Yong,CHEN Lijun.Caspase and apoptosis[J].Acta Academiae Medicinae CPAPF,2004,13(4):346-348.
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[2]Lundy S D,Sabanegh E S Jr.Varicocele management for infertility and pain:a systematic review[J].Arab J Urol,2018,16(1):157-170.
[3]Sedaghatpour D,Berookhim B M.The role of varicocele in male factor subfertility[J].Curr Urol Rep,2017,18(9):73.
[4]徐锋,陈赟,陈海,等.大鼠精索静脉曲张模型及其高位结扎术后睾丸生精细胞凋亡及其IL-1和NO含量的变化[J].中华男科学杂志,2016,22(3):200-204.XU Feng,CHEN Yun,CHEN Hai,et al.Impact of varicocele and varicocelectomy on the apoptosis of spermatogenic cells and the levels of nitrogen monoxidum and interleukin-1 in the rat testis[J].National Journal of Andrology,2016,22(3):200-204.
[5]Belardin L B,Del Giudice P T,Camargo M,et al.Alterations in the proliferative/apoptotic equilibrium in semen of adolescents with varicocele[J].J Assist Reprod Genet,2016,33(12):1657-1664.
[6]Shiraishi K,Matsuyama H,Takihara H.Pathophysiology of varicocele in male infertility in the era of assisted reproductive technology[J].Int J Urol,2012,19(6):538-550.
[7]吴骏,崔云,陈建伟,等.“通精灵”方对精索静脉曲张致男性不育患者精子DNA碎片率的影响[J].浙江中医药大学学报,2016,40(5):356-360.WU Jun,CUI Yun,CHEN Jianwei,et al.The Prescription of"Tongjingling"on the DFI caused by varicocele in male infertility[J].Journal of Zhejiang Chinese Medical University,2016,40(5):356-360.
[8]崔云,冯奕,郑武,等.通精灵对实验性精索静脉曲张大鼠生精细胞凋亡及Caspase-3 Hsp60表达的影响[J].中华中医药学刊,2008,26(12):2593-2597.CUI Yun,FENG Yi,ZHENG Wu,et al.Effect of Tongjing ling Decoction on spermatogenic cell apoptosis and the expression of caspase-3 and Hsp60 in the rat with expermiental varicocele[J].Chinese Archives of Traditional Chinese Medicine,2008,26(12):2593-2597.
[9]Tian R H,Ma M,Zhu Y,et al.Effects of aescin on testicular repairment in rats with experimentally induced varicocele[J].Andrologia,2014,46(5):504-512.
[10]杜宝昕,郑军状,刘冰,等.通精灵对实验性精索静脉曲张大鼠精子DNA完整性与睾丸组织氧化应激的影响[J].中华男科学杂志,2017,23(11):1025-1031.DU Baoxin,ZHENG Junzhuang,LIU Bing,et al.Tongjingling improves sperm DNA integrity and reduces oxidative stress in the testis of experimental varicocele rats[J].National Journal of Andrology,2017,23(11):1025-1031.
[11]秦川.实验动物学[M].北京:中国协和医科大学出版社,2016:167.QIN Chuan.Laboratory Animal Science[M].Beijing:Peking Union Medical College Press,2016:167.
[12]中国左卡尼汀临床应用专家共识编写组.左卡尼汀在男性不育中临床应用专家共识[J].中华男科学杂志,2015,21(1):82-85.China's expert consensus group on clinical application of L-carnitine.Expert consensus on clinical application of L-carnitine in male infertility[J].National Journal of Andrology,2015,21(1):82-85.
[13]陈实功.外科正宗[M].沈阳:辽宁科学技术出版社,1997:54.CHEN Shigong.Waike Zhengzong[M].Shenyang:Liaoning Science and Technoloy Press,1997:54.
[14]陈士铎.洞天奥旨[M].北京:中医古籍出版社,1992:138.CHEN Shiduo.Dongtian Aozhi[M].Beijing:TCM Ancient Books Press,1992:138.
[15]陈士铎.石室秘录[M].北京:人民军医出版社,2009:265.CHEN Shiduo.Shishi Milu[M].Beijing:People's Military Medical Press,2009:265.
[16]张锡纯.医学衷中参西录[M].石家庄:河北科学技术出版社,2002:358.ZHANG Xichun.Intergrating Chinese and Western Medicine[M].Shijiazhuang:Hebei Science and Technology Press,2002:358.
[17]崔云,郑武,万舟,等.通精灵结合精索内静脉高位断流术治疗不育症37例临床观察[J].浙江中医学院学报,1998,22(6):30-31.CUI Yun,ZHENG Wu,WAN Zhou,et al.The clinical observation of Tongjingling combined with the spermatic cord vein high block treated the infertility 37 cases[J].Journal of Zhejiang College of TCM,1998,22(6):30-31.
[18]吕逸清,陈斌.精索静脉曲张致不育机制研究进展[J].中华男科学杂志,2008,14(5):454-458.LYU Yiqing,CHEN Bin.Progress in researches on the mechanism of varicocele-induced male infertility[J].National Journal of Andrology,2008,14(5):454-458.
[19]朱少明,程帆.精索静脉曲张致不育机制的研究进展[J].安徽医药,2017,21(11):1945-1948.ZHU Shaoming,CHENG Fan.A review:the mechanism of male sterility caused by varicocele[J].Anhui Med Pharm J,2017,21(11):1945-1948.
[20]Sheehan M M,Ramasamy R,Lamb D J.Molecular mechanisms involved in varicocele-associated infertility[J].J Assist Reprod Genet,2014,31(5):521-526.
[21]武政华,柯鑫文,冯少勇,等.茶多酚对精索静脉曲张大鼠睾丸生精细胞凋亡的影响[J].中华男科学杂志,2015,21(8):702-707.WU Zhenghua,KE Xinwen,FENG Shaoyong,et al.Tea polyphenols reduces the apoptosis of spermatogenic cells in rats with experimental varicocele[J].National Journal of Andrology,2015,21(8):702-707.
[22]张长城,周安方,张茂林,等.实验性精索静脉曲张对大鼠睾丸生精细胞凋亡的影响[J].中华男科学杂志,2003,9(7):507-509.ZHANG Changcheng,ZHOU Anfang,ZHANG Maolin,et al.Effects of experimental varicocele on the apoptosis of spermatogenic cells in rats[J].National Journal of Andrology,2003,9(7):507-509.
[23]崔云,冯奕,郑武,等.通精灵对实验性精索静脉曲张大鼠生精细胞凋亡及Bcl-2表达的影响[J].中国中西医结合外科杂志,2009,15(2):173-177.CUI Yun,FENG Yi,ZHENG Wu,et al.Effect of Tongjingling Decoction on spermatogenic cell apoptosis and expression of Bcl-2 in rats with experimental varicocele[J].Chinese Journal of Surgery of Integrated Traditional and Western Medicine,2009,15(2):173-177.
[24]XU Ming,ZHANG Hong,YIN Xiuting,et al.The effect of electroacupuncture on the protein expression levels of Cyt-C,Caspase-3 and Caspase-9 in the bladder tissue of rats with neurogenic bladder after suprasacral spinal cord injury[J].World Journal of Acupuncture-Moxibustion,2017,27(3):49-56.
[25]王力,陈东,闵泽,等.益精方对不育症大鼠睾丸生精细胞凋亡及Bcl-2、Bax蛋白表达的影响[J].中国中西医结合杂志,2014,34(5):602-605.WANG Li,CHEN Dong,MIN Ze,et al.Effect of Yijing Recipe on apoptosis of testis spermatogenic cells and the expression of Bcl-2 and Bax protein in infertility rats[J].Chinese Journal of Integrated Traditional and Western Medicine,2014,34(5):602-605.
[26]Kulikov A V,Shilov E S,Mufazalov I A,et al.Cytochrome C:the Achilles’heel in apoptosis[J].Cell Mol Life Sci,2012,69(11):1787-1797.
[27]Monian P,Jiang X.Clearing the final hurdles to mitochondrial apoptosis:regulation post cytochrome C release[J].Exp Oncol,2012,34(3):185-191.
[28]朱愿,马勇,陈立军.Caspase与细胞凋亡[J].武警医学院学报,2004,13(4):346-348.ZHU Yuan,MA Yong,CHEN Lijun.Caspase and apoptosis[J].Acta Academiae Medicinae CPAPF,2004,13(4):346-348.
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