山姜素对来曲唑诱导的多囊卵巢综合征模型大鼠的治疗作用及其机制

doi:10.13481/j.1671-587X.20220116

摘要/Abstract

摘要： 目的

探讨山姜素（ALP）对来曲唑诱导的多囊卵巢综合征（PCOS）模型大鼠的作用，并阐明其潜在机制。

方法

SD大鼠随机分为对照组、模型组、低剂量ALP组和高剂量ALP组。以1 mg·kg^－1·d^－1来曲唑连续口服21 d构建PCOS大鼠模型；低和高剂量ALP组大鼠在建模成功后，分别给予50和100 mg·kg^－1 ALP口服，每天1次，连续给药14 d。检测各组大鼠的体质量、卵巢质量/体质量比和动情周期，酶联免疫吸附测定（ELISA）法检测大鼠血清中睾酮和17β-雌二醇水平，HE染色观察各组大鼠卵巢组织病理形态表现，免疫组织化学染色观察各组大鼠卵巢组织中裂解的天冬氨酸特异性半胱氨酸蛋白酶3（Cleaved caspase-3）蛋白表达强度和分布情况，Western blotting法检测各组大鼠卵巢颗粒细胞中B细胞淋巴瘤2（Bcl-2）、Bcl-2相关X蛋白（Bax）、微管相关蛋白1轻链3-Ⅰ（LC3-Ⅰ）及微管相关蛋白轻链-Ⅱ（LC3-Ⅱ）蛋白表达水平并计算Bax/Bcl-2和LC3-Ⅱ/LC3-Ⅰ比值。

结果

与对照组比较，模型组大鼠体质量、卵巢质量/体质量比和发情间期时像百分率明显升高（P<0.01），血清中睾酮和17β-雌二醇水平明显升高（P<0.01）；与模型组比较，低和高剂量ALP组大鼠体质量、卵巢质量/体质量比、发情间期时像百分率和血清中睾酮水平明显降低（P<0.05或P<0.01），高剂量ALP组大鼠血清中17β-雌二醇水平明显降低（P<0.05）。对照组大鼠卵巢颗粒细胞层结构正常；模型组大鼠卵巢呈多囊性改变，出现充液性大囊性卵泡和闭锁性卵泡，颗粒细胞层排列紊乱且层数减少；低和高剂量 ALP 组大鼠卵巢中囊性卵泡和闭锁性卵泡数减少，颗粒细胞层结构趋向正常。对照组大鼠卵巢颗粒细胞层中Cleaved caspase-3蛋白呈阴性表达；模型组大鼠卵巢颗粒细胞层中Cleaved caspase-3蛋白呈阳性表达；与模型组比较，低和高剂量 ALP 组大鼠卵巢颗粒细胞层中Cleaved caspase-3表达强度降低。与对照组比较，模型组大鼠卵巢颗粒细胞中Bax/Bcl-2和LC3-Ⅱ/LC3-Ⅰ比值明显升高（P<0.01）；与模型组比较，低和高剂量ALP组大鼠卵巢颗粒细胞中Bax/Bcl-2和LC3-Ⅱ/LC3-Ⅰ比值明显降低（P<0.05或P<0.01）。

结论

ALP可能通过降低外周血中睾酮和17β-雌二醇水平和颗粒细胞中自噬性死亡相关蛋白水平来发挥对来曲唑诱导的PCOS模型大鼠的治疗作用。

关键词: 山姜素, 多囊卵巢综合征, 激素调节, 细胞自噬, 颗粒细胞

Abstract: Objective

To investigate the effect of alpinetin （ALP） in the letrozole-induced polycystic ovary syndrome （PCOS） model rats， and to elucidate its potential mechanism.

Methods

The SD rats were randomly divided into control group， model group， low dose of ALP group and high dose of ALP group.The PCOS rat models were constructed by oral administration of 1 mg·kg^－1·d^－1 letrozole for 21 d， and the rats in low and high doses of ALP groups were orally administrated with 50 and 100 mg·kg^－1 ALP once a day for 14 consecutive days after modeling. The body weights， ovary weight/body weight ratios and oestrous cycle of the rats in various groups were detected. The levels of testosterone and 17β-estradiol in serum of the rats were detected by ELISA. The pathomorphology of ovary tissue of the rats in various groups was observed by HE staining. The expression intensities and distribution of Cleaved cysteine-containing aspartate-specific protease-3 （Cleaved caspase-3） protein in ovary tissue of the rats in various groups were observed by immunohistochemical staining.The expression levels of B-cell lymphoma-2（Bcl-2）， Bcl-2 associated X protein （Bax）， and microtubule associated protein 1 light chain 3 （LC3）-Ⅱ（LC3-Ⅱ）and LC3-Ⅰ proteins in granulosa cells of the rats in various groups were detected by Western blotting method.The Bax/Bcl-2 and LC3-Ⅱ/LC3-Ⅰ ratios were calculated.

Results

Compared with control group， the body weight， the ovary weight/body weight ratio， and the percentage of diestrous phase of the rats in model group were increased（P<0.01），and the levels of testosterone and 17β-estradiol in serum were increased （P<0.01）； compared with model group， the body weights， the ovary weight/body weight ratios， the percentage of diestrous phase and the serum testosterone level in low and high doses of ALP groups were significantly decreased（P<0.05 or P<0.01）， and the serum 17β-estradiol levels in high dose of ALP group were significantly decreased（P<0.05）. The structure of granulosa cell layer in ovary of the rats in control group was normal； the ovary of the rats in model group showed polycystic changes， the fluid-filled large cystic follicles and atretic follicles were found， and the granulosa cell layer was disordered and the number of layers was reduced； the number of cystic follicles and atretic follicles in ovary of the rats in low and high doses of ALP groups was decreased， the structure of granulosa cell layer tended to be normal.The expression of Cleaved caspase-3 in the granulosa cell layer of the rats in control group；the expression of Cleaved caspase-3 in granulose cell layer of the rats in model group was posititive，and the expression intensities of Cleaved caspase-3 in granulosa cell layer in low and high doses of ALP groups were decreased compared with model group.Compared with control group， the ratios of Bax/Bcl-2 and LC3-Ⅱ/LC3-Ⅰ in granulosa cells of the rats in model group were significantly increased （P<0.01）； compared with model group， the ratios of Bax/Bcl-2 and LC3-Ⅱ/LC3-Ⅰ in granulosa cells of the rats in low and high doses of ALP groups were significantly decreased （P<0.05 or P<0.01）.

Conclusion

ALP exerts the therapeutical effects in the letrozole-induced PCOS model rats which might be due to its ability to decrease the levels of testosterone and 17β-oestradiol in the peripheral blood and the levels of autophagic death-related proteins in granulosa cells.

Key words: Alpinetin, Polycystic ovary syndrome, Hormone regulation, Autophagy, Granulosa cells

中图分类号:

R285.5

孙艳敏,胡俊攀,王冰玉,傅金英. 山姜素对来曲唑诱导的多囊卵巢综合征模型大鼠的治疗作用及其机制[J]. 吉林大学学报(医学版), 2022, 48(1): 129-135.

Yanmin SUN,Junpan HU,Bingyu WANG,Jinying FU. Therapeutic effect of alpinetin in letrozole-induced polycystic ovary syndrome model rats and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2022, 48(1): 129-135.

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参考文献 21

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Group	Body weight(m/g)	Ovary weight/body weight ratio (η/%)	Percentage of diestrous phase (η/%)
Control	195.35±23.42	0.21±0.03	59.36±5.77
Model	228.29±27.49^*	0.35±0.08^*	85.21±11.48^*
ALP
Low dose	211.52±8.91^△	0.29±0.03^△	75.16±14.19^△
High dose	202.16±15.21^△△	0.26±0.06^△△	70.53±6.76^△△

Group	Testosterone	17β-estradiol
Control	0.52±0.14	27.36±4.02
Model	1.54±0.17^*	63.49±11.00^*
ALP
Low dose	1.28±0.20^△	57.32±11.65
High dose	0.89±0.23^△△	40.76±14.90^△

Group	Bax/Bcl-2	LC3-Ⅱ/LC3-Ⅰ
Control	96.73±29.58	102.75±16.35
Model	312.69±24.29^*	464.16±50.42^*
ALP
Low dose	244.32±13.74^△	354.75±66.20^△
High dose	197.54±17.65^△△	206.83±43.16^△△

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