二甲双胍对多囊卵巢综合征大鼠胰岛素抵抗的影响机制研究

王鸣凯; 张新化; 李伟; 强萍

doi:10.7619/jcmp.20214796

二甲双胍对多囊卵巢综合征大鼠胰岛素抵抗的影响机制研究

王鸣凯¹,
张新化²,
李伟³,
强萍^4, ,

1.
南通大学附属医院分院药剂科, 江苏南通, 226001
2.
南通大学医学院人体解剖学系, 江苏南通, 226001
3.
江苏省南通市中医院肾内分泌科, 江苏南通, 226001
4.
苏州大学附属张家港医院妇产科, 江苏苏州, 215600

基金项目:

江苏省妇幼健康科研项目 F201843

详细信息

通讯作者:
强萍, E-mail: 30505543@qq.com

中图分类号: R711.7;R965
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出版历程
- 收稿日期: 2021-12-05
- 网络出版日期: 2022-05-06
- 发布日期: 2022-05-14

Study on action mechanism of metformin on insulin resistance in rats with polycystic ovary syndrome

1.
Department of Pharmacy, Division of Affiliated Hospital of Nantong University, Nantong, Jiangsu, 226001
2.
Department of Human Anatomy, Medical College of Nantong University, Nantong, Jiangsu, 226001
3.
Department of Renal Endocrinology, Nantong Hospital of Traditional Chinese Medicine of Jiangsu Province, Nantong, Jiangsu, 226001
4.
Department of Obstetrics and Gynecology, Zhangjiagang Hospital Affiliated to Suzhou University, Suzhou, Jiangsu, 215600

摘要

摘要:
目的基于白细胞介素-6(IL-6)/信号传导和转录激活因子3(STAT3)通路探讨二甲双胍改善多囊卵巢综合征(PCOS)大鼠胰岛素抵抗的作用机制。
方法取SD大鼠50只于颈背部皮下注射脱氢表雄酮建立PCOS模型，并随机分为模型组、二甲双胍组、STAT3抑制剂组、IL-6激活剂组、二甲双胍+IL-6激活剂组，每组10只；另取10只SD大鼠(于颈背部皮下注射油剂2 mL/kg)设为假手术组。检测各组大鼠血糖、雄激素睾酮、胰岛素水平，并计算胰岛素抵抗指数; 采用苏木精-伊红(HE)染色法观察大鼠卵巢形态; 采用免疫组化法检测IL-6阳性表达情况; 采用实时定量逆转录聚合酶链反应(qRT-PCR)法检测微小RNA-21(miR-21)水平; 采用蛋白质印迹法(Western blot)检测IL-6、STAT3、磷酸化STAT3(p-STAT3)及下游胰岛素受体底物-2(IRS-2)、信号转导途径抑制蛋白3(SOCS-3)、过氧化物酶体增殖物激活受体γ(PPARγ)蛋白表达量。
结果与假手术组相比，模型组大鼠胰岛素抵抗指数[(40.88±3.74)对比(15.23±1.00)]、睾酮分泌水平[(1.73±0.19) ng/mL对比(1.10±0.10) ng/mL]升高，卵巢组织中卵泡囊性改变、卵母细胞放射冠消失、黄体及颗粒层细胞减少等病理损伤严重, IL-6/STAT3通路处于活化状态，其介导的炎症、胰岛素抵抗、雄激素分泌等相关蛋白及基因表达升高，差异均有统计学意义(P<0.05)。与模型组相比，二甲双胍组、STAT3抑制剂组IL-6/STAT3通路活化均被抑制，大鼠胰岛素抵抗指数[(20.39±2.41)、(20.89±2.54)对比(40.88±3.74)]、睾酮分泌水平[(1.29±0.11)、(1.29±0.11) ng/mL对比(1.73±0.19) ng/mL] 降低，卵巢病理改变改善，差异有统计学意义(P<0.05)。与二甲双胍组相比，二甲双胍+IL-6激活剂组大鼠胰岛素抵抗指数[(39.87±3.88)对比(20.39±2.41)]、睾酮分泌水平[(1.70±0.18) ng/mL对比(1.29±0.11) ng/mL]升高，卵巢病理损伤恢复缓慢，差异均有统计学意义(P<0.05)。
结论二甲双胍可通过抑制IL-6/STAT3通路活化改善PCOS大鼠胰岛素抵抗、雄激素分泌及卵巢形态改变。
- 二甲双胍 /
- 多囊卵巢综合征 /
- 白细胞介素-6 /
- 信号传导和转录激活因子3 /
- 胰岛素抵抗
Abstract:
Objective To explore the action mechanism of metformin in improving insulin resistance of rats with polycystic ovary syndrome (PCOS) by interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) pathway.
Methods A total of 50 SD rats were injected subcutaneously with dehydroepiandrosterone into the back of the neck to establish a PCOS model, and were randomly divided into model group, metformin group, STAT3 inhibitor group, IL-6 activator group, and metformin+IL-6 activator group, with 10 rats per group. Another 10 SD rats (subcutaneously injected with oil 2 mL/kg on the back of the neck) were selected as sham operation group. The levels of blood glucose, testosterone (androgen), insulin level of rats in each group were measured, and the insulin resistance index was calculated; hematoxylin and eosin staining (HE) was performed to observe the ovarian morphology; immunohistochemical method was performed to measure the positive expression of IL-6; real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) method was performed to measure the level of microRNA-21 (miR-21); western blot was performed to measure the expressions of IL-6, STAT3, phosphorylated-STAT3 (p-STAT3) and downstream insulin receptor substrate-2 (IRS-2), signal transduction pathway inhibitor protein 3 (SOCS-3), peroxisome proliferator activation receptor γ (PPARγ) proteins.
Results Compared with the sham operation group, the insulin resistance index[(40.88±3.74) versus (15.23±1.00)] and androgen secretion level[(1.73±0.19) ng/mL versus(1.10±0.10) ng/mL] in the model group were increased, the follicles cystic changes, the disappearance of the oocyte radiation crown, the decrease of the corpus luteum and the granulosa cells and other pathological damages in the ovarian tissue were serious, the IL-6/STAT3 pathway was in an active state, leading to inflammation, insulin resistance, androgen secretion-related proteins and genes mediated increased (P<0.05). Compared with the model group, IL-6/STAT3 pathway activation in both metformin group and STAT3 inhibitor group were inhibited, and insulin resistance index[(20.39±2.41), (20.89±2.54) versus (40.88±3.74)] and androgen secretion level[(1.29±0.11) ng/mL, (1.29±0.11) ng/mL versus (1.73±0.19) ng/mL](P<0.05). Compared with the metformin group, insulin resistance index[(39.87±3.88) versus (20.39±2.41)], androgen secretion level[(1.70±0.18) versus (1.29±0.11)ng/mL] in the metformin+IL-6 activator group increased, and the recovery of ovarian pathological injury was slow, the differences were statistically significant (P<0.05).
Conclusion Metformin can inhibit IL-6/STAT3 activation to improve insulin resistance, androgen secretion and ovarian morphology changes of PCOS rats.
- metformin /
- polycystic ovary syndrome /
- interleukin-6 /
- signal transducer and activator of transcription 3 /
- insulin resistance

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图 1 各组大鼠卵巢组织HE染色图(放大倍数100倍)

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图 2 各组大鼠卵巢组织IL-6免疫组化染色图(放大倍数100倍)

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图 3 各组大鼠卵巢组织IL-6、p-STAT3/STAT3、SOCS-3、IRS-2和PPARγ蛋白Western blot结果

A: 假手术组; B: 模型组; C: 二甲双胍组; D: STAT3抑制剂组; E: IL-6激活剂组; F: 二甲双胍+IL-6激活剂组。

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表 1 各组大鼠空腹血糖、胰岛素、胰岛素抵抗指数、睾酮和IL-6水平比较(x±s)

组别	n	空腹血糖/(mmol/L)	胰岛素/(mIU/L)	胰岛素抵抗指数	睾酮/(ng/mL)	IL-6/(ng/L)
假手术组	10	5.35±0.30	64.06±6.04	15.23±1.00	1.10±0.10	7.54±0.90
模型组	10	7.84±0.45^*	117.33±8.16^*	40.88±3.74^*	1.73±0.19^*	46.38±4.09^*
二甲双胍组	10	5.96±0.36^#	76.81±7.06^#	20.39±2.41^#	1.29±0.11^#	11.77±1.24^#
STAT3抑制剂组	10	6.06±0.40^#	77.52±7.11^#	20.89±2.54^#	1.29±0.11^#	12.96±1.33^#
IL-6激活剂组	10	8.96±0.81^#△	207.54±9.04^#△	82.68±8.11^#△	2.18±0.19^#△	96.58±9.35^#△
二甲双胍+IL-6激活剂组	10	7.80±0.50^△	115.01±7.19^△	39.87±3.88^△	1.70±0.18^△	44.97±4.37^△
IL-6: 白细胞介素-6; STAT3: 信号传导和转录激活因子3。与假手术组比较, *P<0.05; 与模型组比较, #P<0.05; 与二甲双胍组比较, △P<0.05。

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表 2 各组大鼠卵巢组织IL-6阳性表达比较(x±s)

组别	n	IL-6/(平均光密度/mm²)
假手术组	10	0.16±0.01
模型组	10	1.17±0.10^*
二甲双胍组	10	0.46±0.04^#
STAT3抑制剂组	10	0.49±0.04^#
IL-6激活剂组	10	1.99±0.19^#△
二甲双胍+IL-6激活剂组	10	1.19±0.11^△
IL-6: 白细胞介素-6; STAT3: 信号传导和转录激活因子3。与假手术组比较, * P<0.05; 与模型组比较, #P<0.05; 与二甲双胍组比较, △P<0.05。

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表 3 各组大鼠组织miR-21表达比较(x±s)

组别	n	miR-21/U6
假手术组	10	1.02±0.09
模型组	10	2.06±0.20^*
二甲双胍组	10	1.29±0.12^#
STAT3抑制剂组	10	1.33±0.11^#
IL-6激活剂组	10	2.77±0.21^#△
二甲双胍+IL-6激活剂组	10	2.00±0.19^△
IL-6: 白细胞介素-6; STAT3: 信号传导和转录激活因子3; miR-21: 微小RNA-21。与假手术组比较, * P<0.05; 与模型组比较, #P<0.05; 与二甲双胍组比较, △P<0.05。

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表 4 各组大鼠卵巢组织IL-6、p-STAT3/STAT3、SOCS-3、IRS-2、PPARγ蛋白表达比较(x±s)

组别	n	IL-6/β-actin	p-STAT3/STAT3	SOCS-3/β-actin	IRS-2/β-actin	PPARγ/β-actin
假手术组	10	1.00±0.08	1.11±0.11	1.08±0.09	1.16±0.09	1.17±0.09
模型组	10	1.99±0.19^*	2.09±0.20^*	2.18±0.21^*	0.44±0.04^*	0.38±0.03^*
二甲双胍组	10	1.37±0.14^#	1.40±0.14^#	1.50±0.15^#	0.85±0.08^#	0.79±0.07^#
STAT3抑制剂组	10	1.40±0.13^#	1.45±0.13^#	1.53±0.14^#	0.81±0.08^#	0.74±0.07^#
IL-6激活剂组	10	2.69±0.21^#△	2.75±0.24^#△	2.99±0.23^#△	0.17±0.01^#△	0.10±0.01^#△
二甲双胍+IL-6激活剂组	10	1.90±0.18^△	2.00±0.20^△	2.09±0.20^△	0.46±0.04^△	0.40±0.04^△
IL-6: 白细胞介素-6; STAT3: 信号传导和转录激活因子3; p-STAT3: 磷酸化STAT3; SOCS-3: 信号转导途径抑制蛋白3; IRS-2: 胰岛素受体底物-2; PPARγ: 过氧化物酶体增殖物激活受体γ。与假手术组比较, * P<0.05; 与模型组比较, #P<0.05; 与二甲双胍组比较, △P<0.05。

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