温阳生精汤含药血清对肾阳虚大鼠睾丸间质细胞中雌激素分泌的影响

作者	单位
刘镘利	第四军医大学西京医院中医科
王宗仁	第四军医大学西京医院中医科
南亚昀	第四军医大学西京医院中医科
马亮	第四军医大学西京医院中医科
马静	第四军医大学西京医院中医科

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中文摘要:

目的观察温阳生精汤含药血清对腺嘌呤致雄性肾阳虚不育大鼠睾丸间质细胞中雌二醇(es-tradiol,E2)合成以及影响其合成的芳香化酶(cytochrome P450aromatase,P450arom)活性、其编码基因CYP19表达的影响,从蛋白和mRNA水平探讨温阳生精汤含药血清治疗肾阳虚不育的机制。方法将实验大鼠随机分为4组,每组5只,即正常组及温阳生精汤高、中、低浓度组。各组大鼠分别以生理盐水和高、中、低浓度中药连续灌胃10天。末次灌胃给药2h后,心脏采血,离心后分离血清。从肾阳虚大鼠体内分别分离并纯化睾丸间质细胞,经鉴定后原代培养,随机分为5组,即空白对照组,模型组,中药高、中、低浓度(1.2、1.0、0.8g/mL)组,用放射免疫法测定细胞培养液中E2含量,氚水释放实验检测P450arom在细胞中的活性,同时用Western blot法和荧光定量PCR法分别测定CYP19蛋白和mRNA表达。结果与空白对照组比较,模型组中间质细胞上清中E2的含量明显减少,同时伴随P450arom活性抑制,以及CYP19蛋白和mRNA表达的显著减少(P<0.01,P<0.05)。与模型组比较,经温阳生精汤含药血清干预后可显著增加间质细胞中E2合成量,上调P450arom活性,同时在蛋白和mRNA水平上调细胞中CYP19表达,并具有部分剂量依赖性(P<0.01,P<0.05)。结论温阳生精汤含药血清可以有效提高肾阳虚大鼠睾丸间质细胞中E2的合成,并可能部分通过上调P450arom活性,提高CYP19基因表达实现,这可能是其治疗男性肾阳虚不育的机制之一。

英文摘要:

Objective To study the effects of Wenyang Shengjing Decoction(WSD) containing serum on the estradiol(E2) secretion,the synthesized cytochrome P450 aromatase(P450arom) activities,as well as the expression of its encode gene CYP19 in Leydig cells of male sterile rats of adenine induced Shen-yang deficiency(SYD).Methods Experimental rats were randomly divided into 4 groups,i.e.,the normal control group,the high,middle,and low dose WSD groups,5 in each group.The normal saline,low,middle,and high dose WSD were respectively given to rats of all groups for 10 successive days.Blood was drawn from rats′ heart 2 h after the last gastrogavage.The serum was separated after centrifuge.Leydig cells isolated and purified from SYD rats were primary cultured in vitro and divided into 5 groups in random,i.e.,the blank control group,the model group,the high,middle,and low dose WSD groups(1.2,1.0,and 0.8 g/mL,respectively).The content of E2 released in the culture supernate was determined by radioimmunoassay.The P450arom activity was detected by tritium release assay.Meanwhile,the mRNA and protein expressions of CYP19 were analyzed using fluorescent quantitative PCR and Western blot respectively.Results Compared with the blank control group,the E2 secretion of the supernate of Leydig cells obviously decreased in the model groups,accompanied with the inhibition of P450arom activities,significant decreased protein and mRNA expressions of CYP19(P<0.01,P<0.05).Compared with the model group,after intervened by WSD containing serum,the E2 secretion in the Leydig cells could be significantly increased,the P450arom activities up-regulated,the CYP19 expressions up-regulated at the protein and mRNA levels partially in a dose-dependent manner(P<0.01,P<0.05).Conclusions WSD containing serum could effectively elevate the E2 secretion in Leydig cells,which might be partially achieved through up-regulating P450arom activities and enhancing the gene expression of CyP19.This might be one of its mechanisms of action for treating male infertility of SYD.

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