不同针灸疗法对老年雄性大鼠下丘脑-睾丸中生殖激素调控系统相关基因表达的影响
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摘要
目的:探讨不同针灸疗法对老年大鼠血清性激素水平的影响及延缓衰老作用的基因调控途径。方法:SD老年大鼠随机分为老年组、温针灸组、温和灸组、电针组、药物组,每组8只,另取SD青年大鼠8只作为青年组。温针灸组、温和灸组及电针组取"肾俞""关元"穴治疗,每日1次,每次15min;药物组给予腹部皮下注射丙酸睾酮注射液(7mg·kg~(-1)·3d~(-1))。各组均治疗8周。ELISA法检测大鼠血清睾酮(T)、促性腺激素释放激素(GnRH)含量,实时荧光定量PCR法检测大鼠下丘脑中KISS-1、G蛋白耦联受体(GPR)54、RF酰胺相关肽-3(RFRP-3)、GPR147和睾丸中GnRH-R、GPR54、GPR147mRNA的表达。结果:老年组大鼠血清T、GnRH含量,下丘脑中KISS-1、GPR54、RFRP-3、GPR147mRNA表达及睾丸组织中GnRH-R、GPR54mRNA表达低于青年组(P<0.05),睾丸组织GPR147mRNA表达量高于青年组(P<0.05)。与老年组比较,温针灸组、温和灸组、电针组、药物组血清T含量升高(P<0.05),温针灸组、温和灸组、电针组血清GnRH含量,下丘脑KISS-1、GPR54、RFRP-3、GPR147 mRNA表达升高(P<0.05),睾丸组织GnRH-R、GPR54mRNA表达量升高,GPR147mRNA表达量降低(P<0.05)。温针灸组、温和灸组、电针组各指标改善均优于药物组(除血清T含量外,P<0.05)。结论:温针灸、温和灸和电针均能显著提高老年大鼠血清T水平,温针灸、温和灸、电针与丙酸睾酮补充疗法对于血清T水平的提高程度接近,但药物并不能明显激活GnRH合成分泌通路,故温针灸、温和灸和电针更适合在临床中作为长期治疗的方法。
Objective To observe the effect of warm needle moxibustion(WNM),mild moxibustion and electroacupuncture(EA)of"Shenshu"(BL23)and"Guanyuan"(CV4)on serum gonadal hormone levels and related gene expression in the hypothalamus and testis in aged rats,so as to explore their underlying mechanisms in anti-aging.Methods Forty male SD male rats were randomly divided into aged control,WNM,mild moxibustion,EA and medication groups(n = 8 in each group),and other 8 male young adult SD rats were used as the youth control group.WNM(acupuncture plus moxibustion),mild moxibustion or EA(2 Hz/100 Hz,1 mA)was separately applied to CV4 and bilateral BL23 for 15 min,once daily,5 days a week for 8 weeks.Rats of the medication group received subcutaneous injection of testosterone propionate injection(7 mg/kg),once every 3 days,and those of the aged and youth groups received the same dose of 0.9% NaCl solution for 8 weeks.The levels of serum testosterone(T)and gonadotropin-releasing hormone(GnRH)were measured by ELISA.The expression of Kisspeptin(KISS-1),G protein-coupled receptors 54(GPR54),RFamide-related peptide-3(RFRP-3)and GPR147 mRNAs in the hypothalamus and GnRH-R,GPR54 and GPR147 mRNAs in the testis tissues were detected by quantitative real-time PCR.Results The levels of serum T and GnRH,and the expressions of KISS-1,GPR54,RFRP-3 and GPR147 mRNAs in the hypothalamus and GnRH-R and GPR54 mRNAs in the testis in the aged rats were significantly lower than those in the youth rats(P<0.05),while the expression level of GPR147 mRNA of the testis tissue was obviously higher in the aged group than that in the youth group(P<0.05).Following the treatment,the lowered serum T and GnRH contents,the down-regulated expression levels of hypothalamic KISS-1,GPR54,RFRP-3 and GPR147 mRNAs and testicle GnRH-R and GPR54 mRNAs,as well as the up-regulated testicle GPR147 mRNA expression in the aged rats were reversed in the WNM,mild moxibustion and EA groups(not the medication group except serum T)in comparison with the aged control group(P<0.05).The therapeutic effects of WNM,mild moxibustion and EA were significantly superior to those of medication in up-regulating serum GnRH content and expression levels of KISS-1,GPR54,RFRP-3 and GPR147 mRNAs in the hypothalamus and GnRH-R,GPR54 and GPR147 mRNAs in the testis(P<0.05).No significant differences were found among the WNM,mild moxibustion and EA groups in serum T and GnRH contents,and the expression levels of KISS-1,GPR54,RFRP-3 and GPR147 mRNAs in the hypothalamus and GnRH-R and GPR54 and GPR147 mRNAs in the testis(P>0.05).Conclusion The WNM,mild moxibustion and EA therapies have an anti-aging effect by increasing serum T and GnRH levels in aged rats,which is possibly related to their effects in up-regulating the expression of hypothalamic KISS-1,GPR54,RFRP-3 and GPR147 mRNAs and testicle GnRH-R and GPR54 mRNAs,as well as down-regulating testicle GPR147 mRNA expression.The therapeutic effect of WNM,mild moxibustion and EA is evidently superior to that of medication.
Objective To observe the effect of warm needle moxibustion(WNM),mild moxibustion and electroacupuncture(EA)of"Shenshu"(BL23)and"Guanyuan"(CV4)on serum gonadal hormone levels and related gene expression in the hypothalamus and testis in aged rats,so as to explore their underlying mechanisms in anti-aging.Methods Forty male SD male rats were randomly divided into aged control,WNM,mild moxibustion,EA and medication groups(n = 8 in each group),and other 8 male young adult SD rats were used as the youth control group.WNM(acupuncture plus moxibustion),mild moxibustion or EA(2 Hz/100 Hz,1 mA)was separately applied to CV4 and bilateral BL23 for 15 min,once daily,5 days a week for 8 weeks.Rats of the medication group received subcutaneous injection of testosterone propionate injection(7 mg/kg),once every 3 days,and those of the aged and youth groups received the same dose of 0.9% NaCl solution for 8 weeks.The levels of serum testosterone(T)and gonadotropin-releasing hormone(GnRH)were measured by ELISA.The expression of Kisspeptin(KISS-1),G protein-coupled receptors 54(GPR54),RFamide-related peptide-3(RFRP-3)and GPR147 mRNAs in the hypothalamus and GnRH-R,GPR54 and GPR147 mRNAs in the testis tissues were detected by quantitative real-time PCR.Results The levels of serum T and GnRH,and the expressions of KISS-1,GPR54,RFRP-3 and GPR147 mRNAs in the hypothalamus and GnRH-R and GPR54 mRNAs in the testis in the aged rats were significantly lower than those in the youth rats(P<0.05),while the expression level of GPR147 mRNA of the testis tissue was obviously higher in the aged group than that in the youth group(P<0.05).Following the treatment,the lowered serum T and GnRH contents,the down-regulated expression levels of hypothalamic KISS-1,GPR54,RFRP-3 and GPR147 mRNAs and testicle GnRH-R and GPR54 mRNAs,as well as the up-regulated testicle GPR147 mRNA expression in the aged rats were reversed in the WNM,mild moxibustion and EA groups(not the medication group except serum T)in comparison with the aged control group(P<0.05).The therapeutic effects of WNM,mild moxibustion and EA were significantly superior to those of medication in up-regulating serum GnRH content and expression levels of KISS-1,GPR54,RFRP-3 and GPR147 mRNAs in the hypothalamus and GnRH-R,GPR54 and GPR147 mRNAs in the testis(P<0.05).No significant differences were found among the WNM,mild moxibustion and EA groups in serum T and GnRH contents,and the expression levels of KISS-1,GPR54,RFRP-3 and GPR147 mRNAs in the hypothalamus and GnRH-R and GPR54 and GPR147 mRNAs in the testis(P>0.05).Conclusion The WNM,mild moxibustion and EA therapies have an anti-aging effect by increasing serum T and GnRH levels in aged rats,which is possibly related to their effects in up-regulating the expression of hypothalamic KISS-1,GPR54,RFRP-3 and GPR147 mRNAs and testicle GnRH-R and GPR54 mRNAs,as well as down-regulating testicle GPR147 mRNA expression.The therapeutic effect of WNM,mild moxibustion and EA is evidently superior to that of medication.
引文
[1]陈坚,潘钦石,王瑜敏,等.中老年男性体检人群生殖激素水平的动态变化及相关因素分析[J].中华男科学杂志,2017,23(2):131-136.
[2]蓝碧云,李芳萍,郑晓彬,等.中老年男性血清睾酮降低与腹型肥胖的相关性研究[J].中华男科学杂志,2017,23(8):763-765.
[3]肖海英,卢艳慧,龚燕平,等.老年男性血清性激素水平与代谢综合征的相关性[J].中华医学杂志,2016,96(9):702-706.
[4]帕力达·阿不力孜,艾克拜·艾合麦提,周晓辉,等.新疆老年男性骨转化生化标志物及性激素与骨质疏松症的相关性研究[J].中国骨质疏松杂志,2016,22(10):1241-1245.
[5]曹于,张丽,赵丹阳,等.董氏针灸改善多囊卵巢综合征患者卵巢功能:随机对照预试验[J].中国针灸,2017,37(7):710-714.
[6]任丽娜,郭丽红,马文珠,等.针刺治疗多囊卵巢综合征:随机对照试验的系统综述和Meta分析[J].针刺研究,2014,39(3):238-246.
[7]金春兰,魏立新,赵吉平,等.电针与达因-35治疗多囊卵巢综合征的疗效对比[J].中国针灸,2014,34(12):1174-1178.
[8]周建勇,张小月,于美玲,等.经皮穴位电刺激对多囊卵巢综合征大鼠激素及卵巢甾体激素代谢酶表达的影响[J].针刺研究,2016,41(1):11-17.
[9]蒋希荣,任路,李春日.电针对围绝经期抑郁症大鼠下丘脑-垂体-卵巢轴的影响[J].针刺研究,2017,42(1):45-49.
[10]杨宝芝,崔薇,李静.电针对多囊卵巢综合征患者卵子质量及妊娠结局的影响[J].针刺研究,2015,40(2):151-156.
[11]郑艳华,丁涛,叶丹凤,等.低频电针对多囊卵巢综合征大鼠糖代谢及氧化应激的影响[J].针刺研究,2015,40(2):125-130.
[12]柳向荣,臧志伟,李晓玲,等.电针子宫、天枢治疗围绝经期综合征疗效观察[J].针刺研究,2016,41(3):247-250.
[13]丁海珍,于宝生.下丘脑Kisspeptin表达与青春期发育的关系[J].国际儿科学杂志,2010,37(5):531-533.
[14]THOMPSON E L,PATTERSON M,MURPHY K G,et al.Central and peripheral administration of kisspeptin-10stimulates the hypothalamic-pituitary-gonadal axis[J].J Neuroendocrinol,2004,16(10):850-858.
[15]IWASA T,MATSUZAKI T,MURAKAMI M,et al.Developmental changes in the mammalian gonadotropin-inhibitory hormone(GnIH)ortholog RFamide-related peptide(RFRP)and its cognate receptor GPR147in the rat hypotha lamus[J].Int J Dev Neurosci,2012,30(1):31-37.
[16]HENDRIKSE M.Interactions of GPR54and GPR147receptors with RF-amide ligands[J].Emerg Med,2014,15(1):49-53.
[17]QI Y,OLDFIELD B J,CLARKE I J.Projections of RFamide-related peptide-3neurones in the ovine hypothalamus,with special reference to regions regulating energy balance and reproduction[J].J Neuroendocrinol,2009,21(8):690-697.
[18]RICCIUTI A,TRAVISON T G,DI DALMAZI G,et al.Asubset of men with age-related decline in testosterone have gonadotroph autoantibodies[J].J Clin Endocrinol Metab,2016,101(4):1535-1541.
[19]NEAVES W B,JOHNSON L,PORTER J C,et al.Leydig cell numbers,daily sperm production,and serum gonadotropin levels in aging men[J].J Clin Endocrinol Metab,1984,59(4):756-763.
[20]王军,孙蕾,张涛,等.Kisspeptin在初情期前母羊下丘脑的分布定位[J].中国农学通报,2012,28(20):102-105.
[21]IRWIG M S,FRALEY G S,SMITH J T,et al.Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1mRNA in the male rat[J].Neuroendocrinology,2004,80(4):264-272.
[22]王雪松,张才田,居晓斌,等.大鼠下丘脑KiSS-1与GnRHmRNA的表达[J].中华男科学杂志,2007,13(4):315-319.
[23]HINUMA S,SHINTANI Y,FUKUSUMI S,et al.New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals[J].Nat Cell Biol,2000,2(10):703-708.
[24]KRIEGSFELD L J,MEI D F,BENTLEY G E,et al.Identification and characterization of a gonadotropin-inhibitory system in the brains of mammals[J].Proc Natl Acad Sci USA,2006,103(7):2410-2415.
[25]RIZWAN M Z,POLING M C,CORR M,et al.RFamide-related peptide-3receptor gene expression in GnRH and kisspeptin neurons and GnRH-dependent mechanism of action[J].Endocrinology,2012,153(8):3770-3779.
[26]JOHNSON M A,TSUTSUI K,FRALEY G S.Rat RFamide-related peptide-3stimulates GH secretion,inhibits LH secretion,and has variable effects on sex behavior in the adult male rat[J].Horm Behav,2007,51(1):171-180.
[27]POLING M C,QUENNELL J H,ANDERSON G M,et al.Kisspeptin neurones do not directly signal to RFRP-3neurones but RFRP-3may directly modulate a subset of hypothalamic kisspeptin cells in mice[J].J Neuroendocrinol,2013,25(10):876-886.
[28]DUCRET E,ANDERSON G M,HERBISON A E.RFamide-related peptide-3,a mammalian gonadotropin-inhibitory hormone ortholog,regulates gonadotropin-releasing hormone neuron firing in the mouse[J].Endocrinology,2009,150(6):2799-2804.
[29]陶晓倩,柳海燕,时姗姗,等.大鼠膜联蛋白5的重组表达及其对人精子体外运动功能的影响[J].中华男科学杂志,2010,16(5):400-404.
[30]陶晓倩,王大勇,柳海燕,等.膜联蛋白5对大鼠睾丸间质细胞睾酮分泌及3β-羟基类固醇脱氢酶表达的影响[J].医学研究生学报,2010,23(4):342-346.
[2]蓝碧云,李芳萍,郑晓彬,等.中老年男性血清睾酮降低与腹型肥胖的相关性研究[J].中华男科学杂志,2017,23(8):763-765.
[3]肖海英,卢艳慧,龚燕平,等.老年男性血清性激素水平与代谢综合征的相关性[J].中华医学杂志,2016,96(9):702-706.
[4]帕力达·阿不力孜,艾克拜·艾合麦提,周晓辉,等.新疆老年男性骨转化生化标志物及性激素与骨质疏松症的相关性研究[J].中国骨质疏松杂志,2016,22(10):1241-1245.
[5]曹于,张丽,赵丹阳,等.董氏针灸改善多囊卵巢综合征患者卵巢功能:随机对照预试验[J].中国针灸,2017,37(7):710-714.
[6]任丽娜,郭丽红,马文珠,等.针刺治疗多囊卵巢综合征:随机对照试验的系统综述和Meta分析[J].针刺研究,2014,39(3):238-246.
[7]金春兰,魏立新,赵吉平,等.电针与达因-35治疗多囊卵巢综合征的疗效对比[J].中国针灸,2014,34(12):1174-1178.
[8]周建勇,张小月,于美玲,等.经皮穴位电刺激对多囊卵巢综合征大鼠激素及卵巢甾体激素代谢酶表达的影响[J].针刺研究,2016,41(1):11-17.
[9]蒋希荣,任路,李春日.电针对围绝经期抑郁症大鼠下丘脑-垂体-卵巢轴的影响[J].针刺研究,2017,42(1):45-49.
[10]杨宝芝,崔薇,李静.电针对多囊卵巢综合征患者卵子质量及妊娠结局的影响[J].针刺研究,2015,40(2):151-156.
[11]郑艳华,丁涛,叶丹凤,等.低频电针对多囊卵巢综合征大鼠糖代谢及氧化应激的影响[J].针刺研究,2015,40(2):125-130.
[12]柳向荣,臧志伟,李晓玲,等.电针子宫、天枢治疗围绝经期综合征疗效观察[J].针刺研究,2016,41(3):247-250.
[13]丁海珍,于宝生.下丘脑Kisspeptin表达与青春期发育的关系[J].国际儿科学杂志,2010,37(5):531-533.
[14]THOMPSON E L,PATTERSON M,MURPHY K G,et al.Central and peripheral administration of kisspeptin-10stimulates the hypothalamic-pituitary-gonadal axis[J].J Neuroendocrinol,2004,16(10):850-858.
[15]IWASA T,MATSUZAKI T,MURAKAMI M,et al.Developmental changes in the mammalian gonadotropin-inhibitory hormone(GnIH)ortholog RFamide-related peptide(RFRP)and its cognate receptor GPR147in the rat hypotha lamus[J].Int J Dev Neurosci,2012,30(1):31-37.
[16]HENDRIKSE M.Interactions of GPR54and GPR147receptors with RF-amide ligands[J].Emerg Med,2014,15(1):49-53.
[17]QI Y,OLDFIELD B J,CLARKE I J.Projections of RFamide-related peptide-3neurones in the ovine hypothalamus,with special reference to regions regulating energy balance and reproduction[J].J Neuroendocrinol,2009,21(8):690-697.
[18]RICCIUTI A,TRAVISON T G,DI DALMAZI G,et al.Asubset of men with age-related decline in testosterone have gonadotroph autoantibodies[J].J Clin Endocrinol Metab,2016,101(4):1535-1541.
[19]NEAVES W B,JOHNSON L,PORTER J C,et al.Leydig cell numbers,daily sperm production,and serum gonadotropin levels in aging men[J].J Clin Endocrinol Metab,1984,59(4):756-763.
[20]王军,孙蕾,张涛,等.Kisspeptin在初情期前母羊下丘脑的分布定位[J].中国农学通报,2012,28(20):102-105.
[21]IRWIG M S,FRALEY G S,SMITH J T,et al.Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1mRNA in the male rat[J].Neuroendocrinology,2004,80(4):264-272.
[22]王雪松,张才田,居晓斌,等.大鼠下丘脑KiSS-1与GnRHmRNA的表达[J].中华男科学杂志,2007,13(4):315-319.
[23]HINUMA S,SHINTANI Y,FUKUSUMI S,et al.New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals[J].Nat Cell Biol,2000,2(10):703-708.
[24]KRIEGSFELD L J,MEI D F,BENTLEY G E,et al.Identification and characterization of a gonadotropin-inhibitory system in the brains of mammals[J].Proc Natl Acad Sci USA,2006,103(7):2410-2415.
[25]RIZWAN M Z,POLING M C,CORR M,et al.RFamide-related peptide-3receptor gene expression in GnRH and kisspeptin neurons and GnRH-dependent mechanism of action[J].Endocrinology,2012,153(8):3770-3779.
[26]JOHNSON M A,TSUTSUI K,FRALEY G S.Rat RFamide-related peptide-3stimulates GH secretion,inhibits LH secretion,and has variable effects on sex behavior in the adult male rat[J].Horm Behav,2007,51(1):171-180.
[27]POLING M C,QUENNELL J H,ANDERSON G M,et al.Kisspeptin neurones do not directly signal to RFRP-3neurones but RFRP-3may directly modulate a subset of hypothalamic kisspeptin cells in mice[J].J Neuroendocrinol,2013,25(10):876-886.
[28]DUCRET E,ANDERSON G M,HERBISON A E.RFamide-related peptide-3,a mammalian gonadotropin-inhibitory hormone ortholog,regulates gonadotropin-releasing hormone neuron firing in the mouse[J].Endocrinology,2009,150(6):2799-2804.
[29]陶晓倩,柳海燕,时姗姗,等.大鼠膜联蛋白5的重组表达及其对人精子体外运动功能的影响[J].中华男科学杂志,2010,16(5):400-404.
[30]陶晓倩,王大勇,柳海燕,等.膜联蛋白5对大鼠睾丸间质细胞睾酮分泌及3β-羟基类固醇脱氢酶表达的影响[J].医学研究生学报,2010,23(4):342-346.