抗苗勒氏管激素在免疫性卵巢早衰中的发病意义及左归丸的作用机制

Effect of Anti-mullerian Hormone in Immune Premature Ovarian Failure and Mechanism of Zuogui Wan

目的:研究抗苗勒氏管激素(AMH)在免疫性卵巢早衰(POF)中的发病意义及左归丸对POF小鼠AMH及其信号转导通路[AMH/活化素蛋白激酶2(ALK2)/Smad1]的影响,探讨左归丸的作用机制。方法:8周龄Balb/c雌性小鼠随机分为正常组、模型组、戊酸雌二醇组、左归丸组。以小鼠透明带3多肽建立POF动物模型,予左归丸及戊酸雌二醇进行治疗。电化学发光法检测小鼠血清AMH,卵泡刺激素(FSH)的水平,光学显微镜检测小鼠生长卵泡比率,免疫组化法及蛋白质免疫印迹(Western blot)法检测小鼠卵巢AMH及其信号转导蛋白ALK2,Smad1蛋白的表达。结果:与正常组比较,模型组小鼠血清AMH水平明显降低,FSH水平明显升高(P<0.05);与模型组比较,左归丸组明显升高小鼠血清AMH水平,明显降低FSH水平(P<0.05)。与正常组比较,模型组小鼠卵巢生长卵泡比率明显减少(P<0.01),与模型组比较,左归丸组生长卵泡比率明显增高(P<0.05)。AMH,ALK2,Smad1蛋白均在卵泡颗粒细胞胞浆表达。与正常组比较,模型组小鼠卵巢AMH,ALK2,Smad1蛋白表达明显下降(P<0.05);与模型组比较,左归丸组小鼠卵巢AMH蛋白表达明显升高(P<0.05),免疫组化检测显示ALK2,Smad1表达均有上升,但无统计学差异,Western blot检测显示ALK2,Smad1表达也均明显升高(P<0.05)。结论:POF的发生与AMH分泌不足、卵巢AMH蛋白表达低下有关,其通过影响AMH/ALK2/Smad1信号转导,影响始基卵泡的募集,从而使卵泡消耗过快,卵巢功能过早衰竭,卵巢处于衰老状态。左归丸可促进POF小鼠AMH分泌,上调卵巢AMH蛋白的表达,并通过影响AMH/ALK2/Smad1信号转导,抑制始基卵泡的过快募集和消耗,改善卵巢功能。

Objective： To study the role of anti-mullerian hormone（ AMH） in immune premature ovarian failure（ POF）,investigate the effects of Zuogui Wan（ ZGW）,which is the kidney tonifying recipe,on AMH and its signal transduction pathway AMH / ALK2 / Smad1 in POF mice,and explore the mechanism of ZGW on treating immune POF. Method： The 8 weeks old Balb / c female mice were randomly divided into normal group,model group,Estradiol valerate（ trade name： progynova） group and ZGW group. POF animal models were established by zona pellucida 3 polypeptides and then were treated with ZGW or positive control medicine of progynova. The levels of AMH and follicule-stimulating hormone（ FSH） in serum of mice were detected byelectrochemical luminescence method. The follicle growth rate of the mice was detected by optical microscope. The expression levels of AMH,ALK2 and Smad1 in mouse ovary were detected by immunohistochemistry and Western blot assay. Result： As compared with the normal group,the AMH level was decreased and FSH level was increased in serum of model group mice（ P 0. 05）. As compared with the model group,the AMH level was increased and FSH level was decreased in serum of ZGW group（ P 0. 05）. As compared with the normal group,follicle growth rate was significantly decreased in model group mice（ P 0. 01）. As compared with the model group,follicle growth rate was increased in ZGW group（ P 0. 05）. As compared with the normal group,the protein expression levels of AMH,ALK2 and Smad1 were decreased in mice ovarian of model group（ P 0. 05）.As compared with the model group,the protein expression levels of AMH were increased in mice ovarian of ZGW group（ P 0. 05）; the expression levels of ALK2 and Smad1 were increased in the immunohistochemistry method,but the difference was not significant; the expression levels of both ALK2 and Smad1 were significantly increased in Western blot assay（ P 0. 05）. Conclusion： The occurrence of POF was related to the insufficient secretion of AMH and poor expression of ovarian AMH protein. It affected the primordial follicle recruitment by affecting AMH /ALK2 / Smad1 signal transduction,leading to fast follicle consumption,premature ovarian failure,and ovarian aging. ZGW can promote the secretion of AMH in POF mice,up-regulate AMH protein expression in ovary,inhibit the rapid collection and consumption of primordial follicles and improve ovarian function by affecting AMH / ALK2 /Smad1 signal transduction.