淫羊藿苷调节SDF-1/CXCR4信号通路对多囊卵巢综合征大鼠卵巢颗粒细胞凋亡的影响

doi:10.11958/20231504

摘要/Abstract

摘要：

目的探讨淫羊藿苷调节基质细胞衍生因子-1（SDF-1）/CXC型趋化因子受体4（CXCR4）信号轴对多囊卵巢综合征（PCOS）大鼠卵巢颗粒细胞凋亡的影响。方法将从PCOS模型组大鼠卵巢组织中成功分离的颗粒细胞分为PCOS组、淫羊藿苷低剂量组、淫羊藿苷中剂量组、淫羊藿苷高剂量组、重组大鼠SDF-1蛋白（Rr-SDF-1）组、淫羊藿苷高剂量+Rr-SDF-1组，另取从正常对照组大鼠卵巢组织中成功分离的颗粒细胞作为对照组。酶联免疫吸附试验（ELISA）检测卵巢颗粒细胞上清液中卵泡刺激素（FSH）、睾酮（T）、黄体生成素（LH）水平；CCK-8法、EdU染色检测颗粒细胞增殖；流式细胞术检测颗粒细胞凋亡；Western blot检测颗粒细胞中Bcl-2相关X蛋白（Bax）、裂解的胱天蛋白酶-3（Cleaved Caspase-3）、SDF-1、CXCR4蛋白表达。结果与对照组比较，PCOS组FSH水平、光密度（OD）₄₅₀值、EdU阳性细胞率降低，T、LH水平、细胞凋亡率、Bax、Cleaved Caspase-3、SDF-1、CXCR4蛋白表达水平升高（P<0.05）。与PCOS组比较，淫羊藿苷低、中、高剂量组FSH水平、OD₄₅₀值、EdU阳性细胞率均依次升高，T、LH水平、细胞凋亡率、Bax、Cleaved Caspase-3、SDF-1、CXCR4蛋白表达水平均依次降低；Rr-SDF-1组对应指标变化趋势与上述相反（P<0.05）。与淫羊藿苷高剂量组比较，淫羊藿苷高剂量+Rr-SDF-1组FSH水平、OD₄₅₀值、EdU阳性细胞率降低，T、LH水平、细胞凋亡率、Bax、Cleaved Caspase-3、SDF-1、CXCR4蛋白表达水平升高（P<0.05）。结论淫羊藿苷可能通过抑制SDF-1/CXCR4信号通路抑制PCOS大鼠卵巢颗粒细胞凋亡。

关键词: 淫羊藿苷, 基质细胞衍生因子-1/CXC型趋化因子受体4信号通路, 多囊卵巢综合征, 颗粒细胞, 增殖, 凋亡

Abstract:

Objective To investigate the effect of icariin on apoptosis of ovarian granulosa cells in rats with polycystic ovarian syndrome (PCOS) by regulating stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor-4 (CXCR4) signal axis. Methods Granular cells successfully isolated from ovarian tissue of rats in the PCOS model group were grouped into the PCOS group, the low-dose icariin group, the medium-dose icariin group, the high-dose icariin group, the Rr-SDF-1 group and the high-dose icariin+Rr-SDF-1 group. Granular cells successfully isolated from ovarian tissue of rats in the normal control group were taken as the control group. Enzyme-linked immunosorbent assay (ELISA) was applied to detect levels of follicle-stimulating hormone (FSH), testosterone (T) and luteinizing hormone (LH) in supernatant of ovarian granulosa cells. CCK-8 method and EdU staining were applied to detect granulosa cell proliferation. Flow cytometry was applied to detect granulosa cell apoptosis. Western blot assay was applied to detect the expression of Bcl-2 associated X (Bax), Cleaved aspartate-specific cysteine protease-3 (Cleaved Caspase-3), SDF-1, and CXCR4 proteins in granulosa cells. Results Compared with the control group, the FSH level, OD₄₅₀ value and EdU positive cell rate were decreased in the PCOS group, and T and LH levels, apoptosis rate, Bax, Cleaved Caspase-3, SDF-1 and CXCR4 protein expression increased (P<0.05). Compared with PCOS group, the FSH level, OD₄₅₀ value and EdU positive cell rate were increased sequentially in the icariin low, medium and high dose groups, T and LH levels, apoptosis rate, Bax, Cleaved Caspase-3, SDF-1 and CXCR4 protein expression levels were decreased sequentially, and change trends of corresponding indicators were opposite to the above in the Rr-SDF-1 group (P<0.05). Compared with the high-dose icariin group, FSH level, OD₄₅₀ value and EdU positive cell rate were decreased in the high-dose icariin+Rr-SDF-1 group, and T and LH levels, apoptosis rate, Bax, Cleaved Caspase-3, SDF-1 and CXCR4 protein expression levels were increased (P<0.05). Conclusion Icariin may inhibit the apoptosis of ovarian granulosa cells in PCOS rats by inhibiting the SDF-1/CXCR4 signaling pathway.

Key words: icariin, stromal cell-derived factor-1/CXC chemokine receptor-4 signaling pathway, polycystic ovarian syndrome, granular cells, proliferation, apoptosis

中图分类号:

R285.5

图/表 8

图1 颗粒细胞的形态观察（×100）

Fig.1 Morphological observation of granular cells (×100)

图2 免疫荧光鉴定细胞中FSHR表达（×400）

Fig.2 Immunofluorescence identification of FSHR expression in cells (×400)

表1 各组卵巢颗粒细胞上清液中FSH、T、LH水平比较

Tab.1 Comparison of FSH, T and LH levels in supernatant of ovarian granulosa cells between the seven groups

组别	FSH/（U/L）	T/（nmol/L）	LH/（U/L）
对照组	72.25±3.11	1.01±0.08	58.65±2.33
PCOS组	26.69±1.25^a	2.46±0.12^a	84.47±3.13^a
淫羊藿苷低剂量组	31.17±1.36^b	2.04±0.11^b	78.56±3.08^b
淫羊藿苷中剂量组	40.55±1.87^bc	1.58±0.09^bc	72.86±3.01^bc
淫羊藿苷高剂量组	63.35±2.81^bcd	1.11±0.07^bcd	64.43±2.85^bcd
Rr-SDF-1组	19.96±0.87^b	2.87±0.14^b	89.96±2.77^b
淫羊藿苷高剂量+ Rr-SDF-1组	35.67±1.62^e	1.63±0.09^e	71.88±2.93^e
F	564.801^＊＊	267.652^＊＊	86.309^＊＊

表2 各组卵巢颗粒细胞OD450值、EdU阳性细胞率及凋亡率比较（n=6, $\bar{x}±s$）

Tab.2 Comparison of OD450 value, EdU positive cell rate and apoptosis rate of ovarian granulosa cells between the seven groups

组别	OD₄₅₀值	EdU阳性细胞率/%	凋亡率/%
对照组	1.08±0.10	57.94±2.68	8.78±0.31
PCOS组	0.37±0.03^a	12.96±0.51^a	27.75±1.27^a
淫羊藿苷低剂量组	0.49±0.04^b	20.33±0.86^b	22.15±1.33^b
淫羊藿苷中剂量组	0.62±0.05^bc	29.95±1.37^bc	18.06±0.71^bc
淫羊藿苷高剂量组	0.87±0.08^bcd	43.36±2.45^bcd	11.53±0.62^bcd
Rr-SDF-1组	0.22±0.01^b	7.35±0.21^b	32.34±1.58^b
淫羊藿苷高剂量+ Rr-SDF-1组	0.53±0.04^e	24.56±1.08^e	19.94±0.87^e
F	156.952^＊＊	753.575^＊＊	384.293^＊＊

图3 EdU染色检测卵巢颗粒细胞增殖（×200） A：对照组；B：PCOS组；C：淫羊藿苷低剂量组；D：淫羊藿苷中剂量组；E：淫羊藿苷高剂量组；F：Rr-SDF-1组；G：淫羊藿苷高剂量+Rr-SDF-1组。

Fig.3 EdU staining for detection of ovarian granulosa cell proliferation (×200)

图4 流式细胞术检测卵巢颗粒细胞凋亡

Fig.4 Detection of ovarian granulosa cell apoptosis by flow cytometry

表3 各组卵巢颗粒细胞中Bax、Cleaved Caspase-3、SDF-1、CXCR4蛋白表达水平比较(n=6, $\bar{x}±s$)

Tab.3 Comparison of protein expression changes of Bax, Cleaved Caspase-3, SDF-1 and CXCR4 in ovarian granulosa cells between the seven groups

组别	Bax	Cleaved Caspase-3	SDF-1	CXCR4
对照组	0.56±0.04	0.36±0.04	0.39±0.02	0.26±0.02
PCOS组	1.37±0.11^a	0.89±0.07^a	1.68±0.13^a	1.01±0.10^a
淫羊藿苷低剂量组	1.12±0.09^b	0.73±0.06^b	1.25±0.11^b	0.86±0.07^b
淫羊藿苷中剂量组	0.95±0.08^bc	0.61±0.05^bc	0.78±0.06^bc	0.49±0.05^bc
淫羊藿苷高剂量组	0.70±0.05^bcd	0.43±0.03^bcd	0.56±0.04^bcd	0.37±0.04^bcd
Rr-SDF-1组	1.69±0.12^b	1.05±0.08^b	2.21±0.13^b	1.33±0.09^b
淫羊藿苷高剂量+Rr-SDF-1组	0.97±0.08^e	0.66±0.05^e	1.03±0.11^e	0.68±0.07^e
F	121.771^＊＊	110.491^＊＊	273.868^＊＊	186.704^＊＊

图5 Western blot检测颗粒细胞中Bax、Cleaved Caspase-3、SDF-1、CXCR4蛋白表达 A：对照组；B：PCOS组；C：淫羊藿苷低剂量组；D：淫羊藿苷中剂量组；E：淫羊藿苷高剂量组；F：Rr-SDF-1组；G：淫羊藿苷高剂量+Rr-SDF-1组。

Fig.5 Western blot detection of Bax, Cleaved Caspase-3, SDF-1 and CXCR4 protein expression in granulosa cells

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