电针对睾酮低下老年大鼠睾丸间质细胞慢性炎性反应的影响
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摘要
目的:探讨电针对睾酮低下老年大鼠睾丸间质细胞(Leydig细胞)慢性炎性反应的影响及其抗雄性生殖衰老的可能机制。方法:SD老年大鼠随机分为电针组、药物组、老年对照组,每组8只,SD青年大鼠8只作为青年对照组。电针组取"肾俞""关元"穴予电针治疗,持续15min,每日1次,治疗5d休息2d,连续8周。药物组予腹部皮下注射丙酸睾酮注射液(7mg·kg~(-1)·3d~(-1)),连续8周。老年对照组和青年对照组给予腹部皮下注射0.9%氯化钠溶液,注射剂量及疗程同药物组。治疗前后观察大鼠力竭游泳时间,采用ELISA法检测大鼠血清睾酮(TT)和游离睾酮(FT)水平,免疫组化法检测Leydig细胞环氧化酶-2(COX-2)蛋白表达水平,Western blot法检测大鼠睾丸核因子-κB p 65(NF-κB p 65)、COX-2、白介素-1β(IL-1β)及肿瘤坏死因子-α(TNF-α)蛋白表达水平。结果:与青年对照组比较,老年对照组力竭游泳时间及血清TT、FT水平降低(P<0.01);治疗后,电针组与药物组力竭游泳时间及血清TT、FT水平高于老年对照组(P<0.01)。与青年对照组比较,老年对照组睾丸NF-κB p 65、COX-2及其下游炎性因子IL-1β、TNF-α表达明显上调(P<0.01);与老年对照组比较,电针组可显著下调睾丸NF-κB p 65、COX-2、IL-1β及TNF-α蛋白的表达水平(P<0.01)。结论:电针能改善睾酮低下老年大鼠血清TT及FT含量,并能降低老年大鼠睾丸组织的炎性反应,这可能是改善雄性生殖衰老的机制之一。
Objective To assess the effect of electroacupuncture(EA)on chronic inflammatory response of Leydig cells in aged rats with low testosterone,so as to investigate its underlying mechanism of anti-male reproductive aging.Methods Twenty-four 20 months old SD rats were randomly divided into EA,medication and aged control groups(n= 8 in each),and other 8 young SD rats(2 months of age)were used as the youth control group.EA(2 Hz/100 Hz,1 mA)was applied to"Guanyuan"(CV 4)and bilateral"Shenshu"(BL 23)for 15 min,once daily for 8 weeks except the weekends.The medication group received abdominal subcutaneous injection of testosterone propionate(mg·kg~(-1)·3d~(-1))for 8 weeks.The aged control group and the youth control group received subcutaneous injection of 0.9% normal saline,with the same dose and same treatment frequency as those of the medication group.The rats'physical power was assessed according to the exhausted swimming duration,and the levels of serum total testosterone(TT)and free testosterone(FT)were determined by ELISA.The pathological changes of the testis tissue were detected by using H.E.staining,and the immunoactivity of cyclooxygenase-2(COX-2)in Leydig cells was detected by immunohistochemistry.The expression levels of nuclear factor-κB p 65(NF-κB p 65),COX-2,interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)proteins in the testis tissues were determined by Western blot.Results Before and after treatment,the exhaustive swimming duration and the levels of serum TT and FT in the aged control group were significantly lower than those of the youth control group(P <0.01).After the treatment,the exhaustive swimming duration and serum TT and FT in the EA and medication groups were notably higher than those in the aged control group(P<0.01).HE staining showed that the incompleteness of basement membrane of spermatogenic tubules,reduction of spermatogenic cells and supporting cells and irregularity of Leydig cells in the testis tissue of the aged rats were relatively milder after EA intervention.Compared with the youth control group,the expression levels of NF-κB p 65 and COX-2,IL-1βand TNF-αin the testicular tissue were significantly higher in the aged control group(P<0.01),while in compared with the aged control group,the expression levels of NF-κB p 65,COX-2,IL-1βand TNF-αproteins were significantly down-regulated in the EA group(P<0.01).Conclusion EA intervention can improve the physical power of the aged rats with low testosterone,which may be related to its effects in up-regulating TT and FT levels,and in reducing chronic inflammatory response in the testis tissue.
Objective To assess the effect of electroacupuncture(EA)on chronic inflammatory response of Leydig cells in aged rats with low testosterone,so as to investigate its underlying mechanism of anti-male reproductive aging.Methods Twenty-four 20 months old SD rats were randomly divided into EA,medication and aged control groups(n= 8 in each),and other 8 young SD rats(2 months of age)were used as the youth control group.EA(2 Hz/100 Hz,1 mA)was applied to"Guanyuan"(CV 4)and bilateral"Shenshu"(BL 23)for 15 min,once daily for 8 weeks except the weekends.The medication group received abdominal subcutaneous injection of testosterone propionate(mg·kg~(-1)·3d~(-1))for 8 weeks.The aged control group and the youth control group received subcutaneous injection of 0.9% normal saline,with the same dose and same treatment frequency as those of the medication group.The rats'physical power was assessed according to the exhausted swimming duration,and the levels of serum total testosterone(TT)and free testosterone(FT)were determined by ELISA.The pathological changes of the testis tissue were detected by using H.E.staining,and the immunoactivity of cyclooxygenase-2(COX-2)in Leydig cells was detected by immunohistochemistry.The expression levels of nuclear factor-κB p 65(NF-κB p 65),COX-2,interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)proteins in the testis tissues were determined by Western blot.Results Before and after treatment,the exhaustive swimming duration and the levels of serum TT and FT in the aged control group were significantly lower than those of the youth control group(P <0.01).After the treatment,the exhaustive swimming duration and serum TT and FT in the EA and medication groups were notably higher than those in the aged control group(P<0.01).HE staining showed that the incompleteness of basement membrane of spermatogenic tubules,reduction of spermatogenic cells and supporting cells and irregularity of Leydig cells in the testis tissue of the aged rats were relatively milder after EA intervention.Compared with the youth control group,the expression levels of NF-κB p 65 and COX-2,IL-1βand TNF-αin the testicular tissue were significantly higher in the aged control group(P<0.01),while in compared with the aged control group,the expression levels of NF-κB p 65,COX-2,IL-1βand TNF-αproteins were significantly down-regulated in the EA group(P<0.01).Conclusion EA intervention can improve the physical power of the aged rats with low testosterone,which may be related to its effects in up-regulating TT and FT levels,and in reducing chronic inflammatory response in the testis tissue.
引文
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[20]LI X,HALES K H,WATANABE G,et al.The effect of tumor necrosis factor-αand cAMP on induction of AP 1activity in MA 10tumor Leydig cells[J].Endocrine,1997,6(2):317-324.
[21]BASSIL N.Late-onset hypogonadism[J].Med Clin North Am,2011,95(3):507-523.
[22]刘建民,梁凤霞,李佳,等.电针强壮穴对衰老模型大鼠T细胞免疫调节功能的影响[J].针刺研究,2009,34(4):242-247.
[23]罗磊,许晓瑾.电针对力竭游泳大鼠肾脏线粒体自由基代谢和线粒体功能的影响[J].中国针灸,2001,21(6):366-368.
[24]杜艳军,孙国杰.艾灸对老年大鼠海马线粒体膜电位、神经元凋亡的影响[J].针刺研究,2005,30(4):212-214.
[25]关晨霞,高希言,梁杰.针灸对亚急性衰老小鼠脑组织中一氧化氮、丙二醛、超氧化物歧化酶的影响[J].针刺研究,2001,26(2):111-113.
[26]赵利华,文建军,杨柯,等.艾灸对D半乳糖致小鼠衰老模型抗衰老作用的研究[J].针刺研究,2008,33(4):255-257.
[2]FERNANDEZ-BALSELLS M M,MURAD M H,LANE M,et al.Clinical review 1:adverse effects of testosterone therapy in adult men:a systematic review and meta-analysis[J].J Clin Endocrinol Metab,2010,95(6):2560-2575.
[3]MORGENTALER A.Testosterone therapy in men with prostate cancer:scientific and ethical considerations[J].J Urol,2009,181(3):972-979.
[4]任毅,杨晓光,李学智,等.电针对中老年部分雄激素缺乏综合征雄性大鼠睾丸细胞色素P 450侧链裂解酶及17β-羟基类固醇脱氢酶3表达的影响[J].针刺研究,2015,40(1):13-19.
[5]REN Y,YANG X,ZHANG Y,et al.Effects and mechanisms of acupuncture and moxibustion on reproductive endocrine function in male rats with partial androgen deficiency[J].Acupunct Med,2016,34(2):136-143.
[6]FRANCESCHI C,CAMPISI J.Chronic inflammation(inflammaging)and its potential contribution to age-associated diseases[J].J Gerontol,2014,69(Suppl 1):S 4-S 9.
[7]BACHER S,SCHMITZ M L.The NF-kappa B pathway as a potential target for autoimmune disease therapy[J].Curr Pharmaceut Des,2004,10(23):2827-2837.
[8]CHUNG H Y,SUNG B,JUNG K J,et al.The molecular inflammatory process in aging[J].Antioxid Red Signal,2006,8(3/4):572-581.
[9]CHUNG H Y,LEE E K,CHOI Y J,et al.Molecular inflammation as an underlying mechanism of the aging process and age-related diseases[J].J Dent Res,2011,90(7):830-840.
[10]KUMAR A,TAKADA Y,BORIEK A M,et al.Nuclear factor-kappaB:its role in health and disease[J].J Molecul Med,2004,82(7):434-448.
[11]DONATO A,BLACK A K,GANO L,et al.Aging is associated with greater nuclear NF-kappa B,reduced I kappa B alpha,and increased expression of proinflammatory cytokines in vascular endothelial cells of healthy humans[J].Aging Cell,2008,7(6):805-812.
[12]ZHAO X,BIAN Y,SUN Y,et al.Effects of moderate exercise over different phases on age-related physiological dysfunction in testes of SAMP 8mice[J].Exp Gerontol,2013,48(9):869-880.
[13]SYNTIN P,CHEN H,ZIRKIN B R,et al.Gene expression in Brown Norway rat Leydig cells:effects of age and of age-related germ cell loss[J].Endocrinology,2001,142(12):5277-5285.
[14]WANG X,SHEN C L,DYSON M T,et al.Cyclooxygenase-2regulation of the age-related decline in testosterone biosynthesis[J].Endocrinology,2005,146(10):4202-4208.
[15]FRUNGIERI M B,GONZALEZ-CALVAR S I,PARBORELL F,et al.Cyclooxygenase-2and prostaglandin F 2alpha in Syrian hamster Leydig cells:inhibitory role on luteinizing hormone/human chorionic gonadotropin-stimulated testosterone production[J].Endocrinology,2006,147(9):4476-4485.
[16]BEATTIE M C,ADEKOLA L,PAPADOPOULOS V,et al.Leydig cell aging and hypogonadism[J].Expl Gerontol,2015,68:87-91.
[17]WANG D L,NAGPAL M L,CALKINS J H,et al.Interleukin-1beta induces interleukin-1alpha messenger ribonucleic acid expression in primary cultures of Leydig cells[J].Endocrinology,1991,129(6):2862-2866.
[18]HALES D B.Interleukin-1inhibits Leydig cell steroidogenesis primarily by decreasing 17 alpha-hydroxylase/C 17-20lyase cytochrome P 450expression[J].Endocrinology,1992,131(5):2165-2172.
[19]LI X,YOUNGBLOOD G L,PAYNE A H,et al.Tumor necrosis factor-alpha inhibition of 17alpha-hydroxylase/C 17-20lyase gene(Cyp 17)expression[J].Endocrinology,1995,136(8):3519-3526.
[20]LI X,HALES K H,WATANABE G,et al.The effect of tumor necrosis factor-αand cAMP on induction of AP 1activity in MA 10tumor Leydig cells[J].Endocrine,1997,6(2):317-324.
[21]BASSIL N.Late-onset hypogonadism[J].Med Clin North Am,2011,95(3):507-523.
[22]刘建民,梁凤霞,李佳,等.电针强壮穴对衰老模型大鼠T细胞免疫调节功能的影响[J].针刺研究,2009,34(4):242-247.
[23]罗磊,许晓瑾.电针对力竭游泳大鼠肾脏线粒体自由基代谢和线粒体功能的影响[J].中国针灸,2001,21(6):366-368.
[24]杜艳军,孙国杰.艾灸对老年大鼠海马线粒体膜电位、神经元凋亡的影响[J].针刺研究,2005,30(4):212-214.
[25]关晨霞,高希言,梁杰.针灸对亚急性衰老小鼠脑组织中一氧化氮、丙二醛、超氧化物歧化酶的影响[J].针刺研究,2001,26(2):111-113.
[26]赵利华,文建军,杨柯,等.艾灸对D半乳糖致小鼠衰老模型抗衰老作用的研究[J].针刺研究,2008,33(4):255-257.