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Exploration of Mechanism of Xiaonang Tiaojing Decoction in Treating PCOS-IR Model Rats Based on AMH/AMHR ⅡSignaling Pathway
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 29, Issue 9, Pages: 137-145(2023)
- 作者机构：
1.河南省中医院/河南中医药大学 第二附属医院，郑州 450002
2.河南中医药大学，郑州 450046
3.黄河科技学院，郑州 450000
- 作者简介：
- 基金信息：
DOI：10.13422/j.cnki.syfjx.20230568
CLC： R22;R28;R96;R588Published：05 May 2023，
Published Online：11 January 2023，
Received：03 November 2022，
- 稿件说明：
扫 描 看 全 文
周艳艳,袁俊俊,黄旭博等.基于AMH/AMHRⅡ信号通路探讨消囊调经汤治疗PCOS-IR模型大鼠的作用机制[J].中国实验方剂学杂志,2023,29(09):137-145.
ZHOU Yanyan,YUAN Junjun,HUANG Xubo,et al.Exploration of Mechanism of Xiaonang Tiaojing Decoction in Treating PCOS-IR Model Rats Based on AMH/AMHRⅡ Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(09):137-145.
周艳艳,袁俊俊,黄旭博等.基于AMH/AMHRⅡ信号通路探讨消囊调经汤治疗PCOS-IR模型大鼠的作用机制[J].中国实验方剂学杂志,2023,29(09):137-145. DOI： 10.13422/j.cnki.syfjx.20230568.
ZHOU Yanyan,YUAN Junjun,HUANG Xubo,et al.Exploration of Mechanism of Xiaonang Tiaojing Decoction in Treating PCOS-IR Model Rats Based on AMH/AMHRⅡ Signaling Pathway[J].Chinese Journal of Experimental Traditional Medical Formulae,2023,29(09):137-145. DOI： 10.13422/j.cnki.syfjx.20230568.
摘要
目的
2
基于抗苗勒氏管激素（AMH）/抗苗勒氏管激素Ⅱ型受体（AMHRⅡ）信号通路，探讨消囊调经汤改善多囊卵巢综合征伴胰岛素抵抗（PCOS-IR）模型大鼠的作用机制。
方法
2
48只成年雌性SD大鼠随机分为空白组、模型组、消囊调经汤组（11.4 g·kg
-1
·d
-1
）、达英-35组（0.21 g·kg
-1
·d
-1
），除空白组外各组大鼠通过高脂高糖饮食联合来曲唑构建PCOS-IR模型，给药组灌胃给予相应剂量药物15 d，每给药4 d间隔1 d，空白组及模型组灌胃给予等体积生理盐水。治疗期间每天记录大鼠动情周期，治疗结束后记录体质量、Lee's指数，采用酶联免疫吸附测定法（ELISA）测定AMH、促黄体生成素（LH）、LH/卵泡刺激素（FSH）、睾酮（T）、雌二醇（E
2
）水平，血糖仪检测空腹血糖（FPG），放射免疫分析法（RIA）检测空腹胰岛素（FINS）水平，计算胰岛素抵抗指数（HOMA-IR）、胰岛素敏感性指数（QUICKI），全自动生化检测仪测定甘油三酯（TG）、总胆固醇（TC）水平，苏木素-伊红（HE）染色观察卵巢组织形态学变化，免疫组织化学法（IHC）检测卵巢组织内AMHRⅡ、骨形态发生蛋白-15（BMP-15）、Smad5蛋白水平，蛋白免疫印迹法（Western blot）分析AMHRⅡ、BMP-15、Smad5蛋白表达水平。
结果
2
与空白组大鼠比较，模型组大鼠阴道脱落细胞可见大量白细胞，以动情间期为主；体质量、Lee's指数、糖脂代谢指标（FPG、FINS、HOMA-IR、TG、TC）、AMH及性激素（LH、LH/FSH、T）水平显著上升（
P
<
0.01），QUICKI及E
2
水平显著下降（
P
<
0.01）；卵巢表面囊性突起较多、湿重较大，卵巢组织有较多闭锁状、囊性扩张性卵泡，颗粒细胞层厚度减少，不见卵母细胞；卵巢组织中AMHRⅡ蛋白表达水平显著上升（
P
<
0.01），BMP-15、Smad5蛋白表达水平显著下降（
P
<
0.01）。与模型组大鼠比较，消囊调经汤组大鼠阴道脱落细胞于治疗第5~6天起见角化细胞，逐渐出现稳定的动情周期；体质量、Lee's指数、糖脂代谢指标（FPG、FINS、HOMA-IR、TG、TC）、AMH及性激素（LH、LH/FSH、T）水平明显下降（
P
<
0.05，
P
<
0.01），QUICKI及E
2
水平显著升高（
P
<
0.01）；卵巢表面较光滑、湿重较轻，卵巢组织可见卵母细胞及成熟卵泡，颗粒细胞层厚度增加，形态完整；卵巢组织中BMP-15、Smad5蛋白表达水平显著上升（
P
<
0.01），AMHRⅡ蛋白表达水平显著下降（
P
<
0.01）。
结论
2
消囊调经汤可能通过下调AMH/AMHRⅡ的同时介导PCOS-IR大鼠卵巢组织中BMP-15、Smad5上调，从而改善大鼠卵巢功能、性激素及糖脂代谢水平。
Abstract
Objective
2
To investigate the mechanism of Xiaonang Tiaojing decoction（XNTJD）in improving polycystic ovary syndrome with insulin resistance（PCOS-IR）model rats based on anti-Müllerian hormone（AMH）/AMH type Ⅱ receptor（AMHRⅡ）signaling pathway.
Method
2
Forty-eight adult female SD rats were randomly divided into the blank group， model group， XNTJD group（11.4 g·kg
-1
·d
-1
） and Diane-35 group（0.21 g·kg
-1
·d
-1
）， PCOS-IR model was established by high-fat and high-sugar diet combined with letrozole in rats of all groups except the blank group， rats in the administration groups were given the corresponding dose of drugs by gavage for 15 days with an interval of 1 d every 4 d， normal saline of the same volume was given to the blank group and the model group. Estrous cycle was recorded daily during treatment. At the end of treatment， body weight and Lee's index were recorded， AMH， luteinizing hormone（LH）， LH/follicle stimulating hormone（FSH）， testosterone（T）and estradiol（E
2
）levels were measured by enzyme-linked immunosorbent assay（ELISA）， fasting plasma glucose（FPG）was measured by glucometer， fasting insulin（FINS） level was measured by radioimmunoassay（RIA）， and the insulin resistance index（HOMA-IR） and insulin sensitivity index（QUICKI）were calculated， triglyceride（TG）and total cholesterol（TC）levels were measured by automatic biochemical analyzer， hematoxylin-eosin（HE）staining was used to observe the morphological changes of the ovary， the levels of AMHRⅡ， bone morphogenetic protein-15（BMP-15）and Smad5 in ovarian tissues were detected by immunohistochemistry（IHC），Western blot was used to analyze the protein expression levels of AMHRⅡ， BMP-15 and Smad5.
Result
2
Compared with the blank group， a large number of leukocytes were observed in the vaginal exfoliated cells of rats in the model group， mainly in diestrus， the levels of body weight， Lee's index， glucose-lipid metabolism indexes（FPG， FINS， HOMA-IR， TG， TC）， AMH and sex hormones（LH， LH/FSH， T）were significantly increased（
P
<
0.01）， and QUICKI and E
2
levels were significantly decreased（
P
<
0.01）， there were more cystic bulges on the ovarian surface， more wet weight， more atretic and cystic dilated follicles in the ovarian tissues， and the thickness of granulosa cell layer was reduced without oocytes， the expression level of AMHRⅡ protein in ovarian tissues was significantly increased（
P
<
0.01）， and the expression levels of BMP-15 and Smad5 proteins were significantly decreased（
P
<
0.01）. Compared with the model group， the exfoliated cells in the vagina of rats treated with XNTJD group showed keratinocytes from the 5
th
to 6
th
day of treatment， and a stable estrous cycle gradually appeared， body weight， Lee's index， glucose-lipid metabolism indexes（FPG， FINS， HOMA-IR， TG， TC）， AMH and sex hormones（LH， LH/FSH， T）levels were significantly decreased（
P
<
0.05，
P
<
0.01）， QUICKI and E
2
levels were significantly increased（
P
<
0.01）， ovarian surface was smoother and lighter in wet weight， oocytes and mature follicles were observed in ovarian tissues， the thickness of granulosa cell layer increased and the morphology was intact， the expression levels of BMP-15 and Smad5 proteins were significantly increased（
P
<
0.01）and the expression level of AMHRⅡ protein was significantly decreased（
P
<
0.01）in ovarian tissues.
Conclusion
2
XNTJD may mediate the up-regulation of BMP-15 and Smad5 in ovarian tissues of PCOS-IR rats by down-regulating AMH/AMHRⅡ， thereby improving ovarian function， sex hormones and glucose-lipid metabolism levels in PCOS-IR rats.
关键词
多囊卵巢综合征（PCOS）胰岛素抵抗（IR）消囊调经汤抗苗勒氏管激素（AMH）/抗苗勒氏管激素Ⅱ型受体（AMHRⅡ）信号通路糖脂代谢骨形态发生蛋白-15（BMP-15）Smad5
Keywords
polycystic ovary syndrome（PCOS）insulin resistance（IR）Xiaonang Tiaojing decoctionanti-Müllerian hormone（AMH）/AMH type Ⅱ receptor（AMHRⅡ）signaling pathwayglucose-lipid metabolismbone morphogenetic protein-15（BMP-15）Smad5
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