| 刘启明,张沂,邵利芳,穆中杰,柴君雷.淫羊藿苷通过GPER介导Akt、ERK1/2表达保护骨关节炎软骨细胞[J].浙江中西医结合杂志,2026,36(4): |
| 淫羊藿苷通过GPER介导Akt、ERK1/2表达保护骨关节炎软骨细胞 |
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| 投稿时间：2025-04-22 修订日期：2026-03-17 |
| DOI： |
| 中文关键词: 淫羊藿苷 GPER Akt ERK1/2 软骨细胞 |
| 英文关键词:Icariin GPER Akt ERK1/2 Chondrocytes |
| 基金项目:浙江省中医药科技计划项目(2021ZQ079 );杭州市富阳区十四五时期“135”优秀中青年人才培养计划(富人社[2022]65号) |
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| 中文摘要: |
| 摘要：目的：探讨淫羊藿苷通过G蛋白偶联雌激素受体（GPER）介导蛋白激酶B（Akt）、细胞外信号调节激酶1/2(ERK1/2)表达保护骨关节炎软骨细胞的作用机制。方法：白细胞介素-1β(IL-1β)终浓度为10ng/mL,GPER选择性激动剂(G1)、GPER选择性拮抗剂(G15)处理浓度均为1×108mol/L。通过浓度为10ng/mL的IL-1β处理正常人软骨细胞建立骨关节炎软骨细胞模型。本试验将人软骨细胞分为：正常人软骨细胞组：正常组；正常人软骨细胞+IL-1β处理组：模型组；正常人软骨细胞+IL-1β处理+淫羊藿苷+G1组：淫羊藿苷联合G1组；正常人软骨细胞+IL-1β处理+淫羊藿苷+G15组：淫羊藿苷联合G15组；正常人软骨细胞+IL-1β处理+淫羊藿苷组：淫羊藿苷组；正常人软骨细胞+IL-1β处理+淫羊藿苷+TPA组:淫羊藿苷联合TPA组。采用蛋白免疫印迹法（Western blot）检测GPER、Akt、ERK1/2、Ⅱ型胶原（collagen Ⅱ）、聚集蛋白聚糖（Aggrecan）、基质金属蛋白酶-13（MMP-13）、半胱氨酸天冬氨酸蛋白酶3（Caspase-3）和活化型半胱氨酸天冬氨酸蛋白酶3（cleaved caspase3）蛋白表达量，流式细胞仪检测软骨细胞凋亡率、CCK-8法检测软骨细胞增殖活性，ELISA检测肿瘤坏死因子-α(TNF-α)含量。结果：与Control组相比，IL-1β处理后人软骨细胞GPER[(1.000±0.103)%比(0.305±0.030)%,P<0.01]、p-Akt/Akt[(0.610±0.062)%比(1.000±0.105)%]、collagen II[(0.472±0.048)%比(1.000±0.102),P<0.01]、AggrecanI[(0.562±0.053)%比(1.000±0.102),P<0.01]蛋白表达显著下降，MMP-13[(1.883±0.185)%比(1.000±0.102)%,P<0.01]、caspase3 [(4.035±0.410)%比(1.000±0.107)%,P<0.01]、cleaved caspase3[(8.200±0.828)%比(1.000±0.102)%, P<0.01]、p-ERK1/2/ERK1/2[(5.114±0.514)%(1.000±0.103)%,P<0.01]和TNF-α[(92.17±8.06)比(58.2±5.02)%,P<0.01]蛋白表达显著上升。软骨细胞凋亡率[(26.360±2.398)%比(5.070±0.592)%,P<0.01]明显上升，而软骨细胞增值活动[(0.64±0.05)%比(1.06±0.09)%,P<0.01]受到抑制。与IL-1β组相GPER [(0.586±0.057)%比(0.305±0.030)%,P<0.01]、p-Akt/Akt[(0.781±0.075)%比(0.610±0.062)%]、collagen II[(0.724±0.071)%比(0.472±0.048)%,P<0.01]、AggrecanI[(0.857±0.082±0.053)%比(0.562±0.053)%,P<0.01]蛋白表达显著下降，MMP-13[(1.903±0.182)%比(1.883±0.185)%,P<0.01]、caspase3[(5.073±0.492)%比(4.035±0.410)%,P<0.05，]、cleaved caspase3[(5.397±0.532)%比(1.000±0.102)%, P<0.01]、p-ERK1/2/ERK1/2[(10.959±1.065)%比(8.200±0.828)%,P<0.01]和TNF-α[(79.21±6.42)比(92.17±8.06)%,P<0.05]蛋白表达显著上升。软骨细胞凋亡率[(18.920±1.782)%比(26.360±2.398)%,P<0.01]明显下降，而软骨细胞增值活动[(0.74±0.07)%比(0.64±0.05)%,P<0.05]明显活跃。结论：淫羊藿苷可与GPER结合发挥雌激素效应抑制软骨细胞凋亡和炎症因子的释放，提高软骨细胞的增殖活性，促进collagen II、Aggrecan蛋白合成，其机制可能与调节Akt和ERK1/2蛋白表达相关。 |
| 英文摘要: |
| ABSTRACT Objective:This study explores the mechanism by which icariin protects osteoarthritis chondrocytes by mediating the expression of protein kinase B (Akt) and extracellular signal - regulated kinases 1/2 (ERK1/2) through G protein - coupled estrogen receptor (GPER).Methods:The final concentration of interleukin-1β(IL-1β) was 10ng/mL, and the treatment concentrations of the GPER agonist (G1) and GPER antagonist (G15) were both 1×108mol/L.A model of osteoarthritis chondrocytes was established by treating normal human chondrocytes with IL-1β at a concentration of 10 ng/mL.In this experiment, human chondrocytes were divided into the following groups: normal human chondrocytes group: control group; normal human chondrocytes+IL-1β treatment group: model group; normal human chondrocytes + IL-1β treatment+icariin+G1 group: icariin combined with G1 group; normal human chondrocytes+IL-1β treatment+icariin+G15 group: icariin combined group; normal human chondrocytes+IL-1β treatment+icariin group: icariin group;normal human chondrocytes + IL-1β treatment + icariin + TPA group: icariin combined with TPA group.The protein expression levels of GPER, Akt, ERK1/2, collagen II, aggrecan, matrix metalloproteinase-13(MMP-13), caspase-3, and cleaved caspase-3 were detected by Western blot. The apoptosis rate of chondrocytes was detected by flow cytometry, the proliferative activity of chondrocytes was detected by CCK-8 assay, and the content of tumor necrosis factor-α(TNF-α) was detected by ELISA.Results:Compared with the Control group, the protein expression levels of GPER [(1.000±0.103)% vs (0.305±0.030)%, P<0.01], p-AKT/AKT [(0.610±0.062)% vs (1.000±0.105)%], collagen II [(0.472±0.048)% vs (1.000±0.102)%, P<0.01], and AggrecanI [(0.562±0.053)% vs (1.000±0.102)%, P<0.01] in human chondrocytes significantly decreased after IL-1β treatment, while the protein expression levels of MMP-13 [(1.883±0.185)% vs (1.000±0.102)%, P<0.01],caspase-3 [(4.035±0.410)% vs (1.000±0.107)%, P<0.01], cleaved caspase-3 [(8.200±0.828)% vs (1.000±0.102)%, P<0.01],p-ERK1/2/ERK1/2 [(5.114±0.514)% vs (1.000±0.103)%, P<0.01],and TNF-α[(92.17±8.06) vs(58.2±5.02)%,P<0.01] significantly increased. The apoptosis rate of chondrocytes [(26.360±2.398)% vs (5.070±0.592)%,P<0.01]significantly increased, while the proliferative activity of chondrocytes[(0.64±0.05)% vs(1.06±0.09)%,P<0.01] was inhibited. Compared with the IL-1β group, the protein expression levels of GPER [(0.586±0.057)% vs(0.305±0.030)%, P<0.01], p-AKT/AKT[(0.781±0.075)% vs (0.610±0.062)%], collagen II [(0.724±0.071)% vs (0.472±0.048)%, P<0.01],and AggrecanI [(0.857±0.082)% vs (0.562±0.053)%, P<0.01] significantly increased, while the protein expression levels of MMP-13 [(1.903±0.182)% vs (1.883±0.185)%, P<0.01],caspase-3 [(5.073±0.492)%vs(4.035±0.410)%, P<0.05], cleaved caspase-3 [(5.397±0.532)% vs (8.200±0.828)%, P<0.01], p-ERK1/2/ERK1/2 [(10.959±1.065)% vs (5.114±0.514)%, P<0.01], and TNF-α [(79.21±6.42) vs (92.17±8.06)%, P<0.05] significantly decreased. The apoptosis rate of chondrocytes [(18.920±1.782)% vs (26.360±2.398)%, P<0.01] significantly decreased, while the proliferative activity of chondrocytes [(0.74±0.07)% vs (0.64±0.05)%, P<0.05] significantly increased.Conclusion:Icariin can bind to GPER to exert estrogen-like effects,thereby inhibiting chondrocyte apoptosis and the release of inflammatory factors.It enhances the proliferative activity of chondrocytes and promotes the synthesis of collagen II and Aggrecan proteins. The mechanism underlying these effects is associated with the regulation of Akt and ERK1/2 protein expression. |
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