“劳倦过度，房室不节”致肾阳虚及补肾治疗对小鼠HPA轴及睾丸端粒酶活性影响的研究
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摘要
目的:探讨“劳倦过度、房室不节”所致肾阳虚小鼠下丘脑-垂体-肾上腺轴功能变化,研究肾阳虚证及补肾治疗对睾丸端粒酶活性和端粒酶催化亚基(Tert)表达的影响,为肾本质研究及“肾主生殖”理论提供现代科学依据。
方法:复制“劳倦过度、房室不节”肾阳虚小鼠模型。采用电镜、光镜、放射免疫分析、酶联免疫分析、免疫组织化学分析、原位末端标记(TUNEL)、反转录PCR (RT-PCR)、活性光密度酶联免疫测定(TRAP-ELISA)等方法,对正常、肾阳虚、补肾阳药物三组小鼠进行肾上腺皮质的组织形态学及超微结构观察、肾上腺轴激素含量测定、肾上腺组织凋亡基因表达及凋亡率测定、生殖能力检测、睾丸组织端粒酶活性及端粒酶催化亚基检测。
结果:“劳倦过度、房室不节”肾阳虚小鼠肾上腺脏器系数下降,肾上腺组织形态及超微结构改变,下丘脑-垂体-肾上腺轴激素水平下降,肾上腺组织Caspase3、Fas基因表达异常及凋亡率下降;生殖力减弱,睾丸端粒酶Tert表达下降,端粒酶活性降低。金匱肾气丸对这种异常有一定反向调节作用。
结论:“劳倦过度、房室不节”肾阳虚小鼠下丘脑-垂体-肾上腺轴功能紊乱,肾上腺组织形态及超微结构被破坏;睾丸内精原细胞及精母细胞端粒酶催化亚基的表达和端粒酶活性下降,生殖力降低。金匱肾气丸可以提高下丘脑-垂体-肾上腺轴激素水平、改善肾上腺形态结构、增强端粒酶活性表达,改善下丘脑-垂体-肾上腺轴、性腺轴功能紊乱,提高小鼠生殖力。
Objective : This experimental study focused on functional variation of hypothalamic-pituitary- adrenal axis in Kidney-Yang deficiency syndrome mice model, and the effects of kidney-Yang deficiency syndrome and kidney invigorating on telomerase activation and Tert expression in testis, so as to provide scientific basis for research on kidney essence and the theory of kidney controlling reproduction.
Methods : The mice model of kidney-Yang deficiency syndrome caused by overfatigue and sexual overstrain was established.The mice were divided into three groups, normal control, kidney-Yang deficiency syndrome group, and JINGUISHENQI bolus group, to detect morphological changes,ultrastructural variation of adrenal cortex, the content of plasma adrenal axis hormone, apoptosis gene expression and apoptosis rate of adrenal tissue, reproductive activity,and telomerase and catalytic subbase telomerase in testis by means of light microscopy,electron microscopy,radioimmunoassay, enzymoimmunoassay, immunohistochenistry,TUNEL, RT-PCR and active optical density enzymoimmunoassay(TRAP-ELISA).
Results : The degression of mice adrenal organ factor, morphological and ultrastructural changes of adrenal tissue,decrease of plasma adrenal axis hormone level,abnormal expression of Caspase3, Fas genes in adrenal tissue reduction of apoptosis rate;descline of reproductive activity,descending expression of telomerase Tert of testis and decline of telomerase activity were observed in mice with Kidney Yang deficiency syndrome caused by overfatigue and sexual overstrain. Jinguishenqi bolus had certain modulation of these abnomalities.
Conclusion : Overfatigue and sexual overstrain caused the tectological and ultractructural changes of mice adrenal tissue , reduced apoptosis, and led to dysfunction of hypothalamic-pituitary- adrenal axis, which may be related to decrease of adrenal cells, weakening proliferation, reduction of newborn cells, and functional compensation of extant adrenal cells. The reduced reproduction was connected with the reduced expression and activity of telomerase of spermatogonia and spermatoctes,thus it could reduce spermatogenesis. Jinkuishenqi bolus could modify the morphology and constructure of adrenal tissue improve the hormone level of hypothalamic-pituitary-adrenal axis, and regulate the expression of apoptosis gene and the activity of telomerase, through which it would ameliorate dysfunction of hypothalamic-pituitary- adrenal axis and improve reproduction in mice.
方法:复制“劳倦过度、房室不节”肾阳虚小鼠模型。采用电镜、光镜、放射免疫分析、酶联免疫分析、免疫组织化学分析、原位末端标记(TUNEL)、反转录PCR (RT-PCR)、活性光密度酶联免疫测定(TRAP-ELISA)等方法,对正常、肾阳虚、补肾阳药物三组小鼠进行肾上腺皮质的组织形态学及超微结构观察、肾上腺轴激素含量测定、肾上腺组织凋亡基因表达及凋亡率测定、生殖能力检测、睾丸组织端粒酶活性及端粒酶催化亚基检测。
结果:“劳倦过度、房室不节”肾阳虚小鼠肾上腺脏器系数下降,肾上腺组织形态及超微结构改变,下丘脑-垂体-肾上腺轴激素水平下降,肾上腺组织Caspase3、Fas基因表达异常及凋亡率下降;生殖力减弱,睾丸端粒酶Tert表达下降,端粒酶活性降低。金匱肾气丸对这种异常有一定反向调节作用。
结论:“劳倦过度、房室不节”肾阳虚小鼠下丘脑-垂体-肾上腺轴功能紊乱,肾上腺组织形态及超微结构被破坏;睾丸内精原细胞及精母细胞端粒酶催化亚基的表达和端粒酶活性下降,生殖力降低。金匱肾气丸可以提高下丘脑-垂体-肾上腺轴激素水平、改善肾上腺形态结构、增强端粒酶活性表达,改善下丘脑-垂体-肾上腺轴、性腺轴功能紊乱,提高小鼠生殖力。
Objective : This experimental study focused on functional variation of hypothalamic-pituitary- adrenal axis in Kidney-Yang deficiency syndrome mice model, and the effects of kidney-Yang deficiency syndrome and kidney invigorating on telomerase activation and Tert expression in testis, so as to provide scientific basis for research on kidney essence and the theory of kidney controlling reproduction.
Methods : The mice model of kidney-Yang deficiency syndrome caused by overfatigue and sexual overstrain was established.The mice were divided into three groups, normal control, kidney-Yang deficiency syndrome group, and JINGUISHENQI bolus group, to detect morphological changes,ultrastructural variation of adrenal cortex, the content of plasma adrenal axis hormone, apoptosis gene expression and apoptosis rate of adrenal tissue, reproductive activity,and telomerase and catalytic subbase telomerase in testis by means of light microscopy,electron microscopy,radioimmunoassay, enzymoimmunoassay, immunohistochenistry,TUNEL, RT-PCR and active optical density enzymoimmunoassay(TRAP-ELISA).
Results : The degression of mice adrenal organ factor, morphological and ultrastructural changes of adrenal tissue,decrease of plasma adrenal axis hormone level,abnormal expression of Caspase3, Fas genes in adrenal tissue reduction of apoptosis rate;descline of reproductive activity,descending expression of telomerase Tert of testis and decline of telomerase activity were observed in mice with Kidney Yang deficiency syndrome caused by overfatigue and sexual overstrain. Jinguishenqi bolus had certain modulation of these abnomalities.
Conclusion : Overfatigue and sexual overstrain caused the tectological and ultractructural changes of mice adrenal tissue , reduced apoptosis, and led to dysfunction of hypothalamic-pituitary- adrenal axis, which may be related to decrease of adrenal cells, weakening proliferation, reduction of newborn cells, and functional compensation of extant adrenal cells. The reduced reproduction was connected with the reduced expression and activity of telomerase of spermatogonia and spermatoctes,thus it could reduce spermatogenesis. Jinkuishenqi bolus could modify the morphology and constructure of adrenal tissue improve the hormone level of hypothalamic-pituitary-adrenal axis, and regulate the expression of apoptosis gene and the activity of telomerase, through which it would ameliorate dysfunction of hypothalamic-pituitary- adrenal axis and improve reproduction in mice.
引文
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