[1]刘启明,张沂,徐中海,等.染料木素对骨关节炎软骨细胞凋亡的影响及其作用机制[J].中医正骨,2025,37(10):15-22.
　LIU Qiming,ZHANG Yi,XU Zhonghai,et al.Effects of genistein on chondrocyte apoptosis in osteoarthritis and its underlying mechanisms[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(10):15-22.
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染料木素对骨关节炎软骨细胞凋亡的影响及其作用机制()

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期数:
2025年10期
页码:
15-22
栏目:
基础研究
出版日期:
2025-10-20

文章信息/Info

Title:
Effects of genistein on chondrocyte apoptosis in osteoarthritis and its underlying mechanisms
作者:
刘启明; 张沂; 徐中海; 穆中杰; 柴君雷
杭州市富阳中医骨伤医院,浙江 杭州 311400
Author(s):
LIU Qiming; ZHANG Yi; XU Zhonghai; MU Zhongjie; CHAI Junlei
Hangzhou Fuyang Hospital of Orthopedics of Traditional Chinese Medicine,Hangzhou 311400,Zhejiang,China
关键词:
骨关节炎;  软骨细胞;  染料木黄酮;  细胞凋亡;  线粒体自噬;  Pink1-Parkin信号通路
Keywords:
osteoarthritis;  chondrocytes;  genistein;  apoptosis;  mitophagy;  Pink1-Parkin signaling pathway
摘要:
目的:探讨染料木素对骨关节炎(osteoarthritis,OA)软骨细胞凋亡的影响及其作用机制。方法:将处于对数生长期的人软骨细胞,分为对照组、模型组、染料木素组、染料木素联合G1组和染料木素联合G15组。模型组、染料木素组、染料木素联合G1组和染料木素联合G15组软骨细胞,加入终浓度为10 ng·mL-1的白细胞介素-1β诱导建立OA软骨细胞模型; 染料木素联合G1组和染料木素联合G15组软骨细胞,分别加入终浓度为1×108 mol·L-1的G蛋白偶联雌激素受体(G protein-coupled estrogen receptor,GPER)激动剂G1和抑制剂G15,培养48 h; 染料木素组、染料木素联合G1组和染料木素联合G15组软骨细胞,加入终浓度为100 μg·mL-1的染料木素,培养72 h。收集各组细胞,采用流式细胞仪检测各组软骨细胞凋亡率,采用溶酶体与线粒体共定位实验检测软骨细胞线粒体自噬情况,采用Western Blot法检测软骨细胞中Ⅱ型胶原、聚集蛋白聚糖、基质金属蛋白酶(matrix metalloproteinase,MMP)-13、半胱氨酸天冬氨酸蛋白酶(cysteine aspartic acid specific protease,Caspase)3、活化的半胱氨酸天冬氨酸蛋白酶(cleaved-cysteine aspartic acid specific protease,Cleaved-Caspase)3、GPER、Pink1和Parkin的蛋白表达水平。结果:①软骨细胞凋亡率检测结果。模型组软骨细胞凋亡率高于对照组(P=0.001),染料木素组和染料木素联合G1组软骨细胞凋亡率低于模型组(P=0.001,P=0.001),染料木素联合G15组软骨细胞凋亡率与模型组的差异无统计学意义(P=0.142)。②软骨细胞中细胞外基质代谢相关基因的蛋白表达水平检测结果。模型组软骨细胞中Ⅱ型胶原、聚集蛋白聚糖的蛋白相对表达量均低于对照组(P=0.000,P=0.000),MMP-13、Caspase3和Cleaved-Caspase3的蛋白相对表达量均高于对照组(P=0.000,P=0.000,P=0.000); 染料木素组和染料木素联合G1组软骨细胞中Ⅱ型胶原、聚集蛋白聚糖的蛋白相对表达量均高于模型组(P=0.001,P=0.001; P=0.001,P=0.001),MMP-13、Caspase3和Cleaved-Caspase3的蛋白相对表达量均低于模型组(P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000); 染料木素联合G15组软骨细胞中Ⅱ型胶原、聚集蛋白聚糖、MMP-13、Caspase3和Cleaved-Caspase3的蛋白相对表达量与模型组的差异均无统计学意义(P=0.965,P=0.999,P=0.999,P=0.999,P=0.999)。③软骨细胞中GPER蛋白表达水平检测结果。模型组软骨细胞中GPER的蛋白相对表达量低于对照组(P=0.001),染料木素组和染料木素联合G1组软骨细胞中GPER的蛋白相对表达量均高于模型组(P=0.001,P=0.001),染料木素联合G15组软骨细胞中GPER的蛋白相对表达量与模型组的差异无统计学意义(P=0.965)。④软骨细胞线粒体自噬情况检测结果。对照组有黄色荧光标记,模型组和染料木素联合G15组几乎无黄色荧光标记,染料木素组和染料木素联合G1组黄色荧光标记较模型组增多。⑤软骨细胞中线粒体自噬相关基因的蛋白表达水平检测结果。模型组软骨细胞中Pink1、Parkin的蛋白相对表达量均低于对照组(P=0.001,P=0.001),染料木素组和染料木素联合G1组软骨细胞中Pink1、Parkin的蛋白相对表达量均高于模型组(P=0.001,P=0.001; P=0.001,P=0.001),染料木素联合G15组软骨细胞中Pink1、Parkin的蛋白相对表达量与模型组的差异均无统计学意义(P=0.999,P=0.999)。结论:染料木素能够抑制OA软骨细胞凋亡,其作用机制可能与促进Ⅱ型胶原、聚集蛋白聚糖、GPER表达,抑制MMP-13、Caspase3和Cleaved-Caspase3表达及激活Pink1-Parkin信号通路促进线粒体自噬有关。
Abstract:
Objective:To investigate the effects of genistein on chondrocyte apoptosis in osteoarthritis(OA),and to explore its underlying mechanism.Methods:The human chondrocytes in the logarithmic growth phase were selected and divided into control group,model group,genistein group,genistein combined with G1 group,and genistein combined with G15 group.All the chondrocytes but the ones in control group were induced by interleukin-1β(IL-1β)at a final concentration of 10 ng/mL to establish OA chondrocyte models.Concurrently,the chondrocytes in genistein combined with G1 group and genistein combined with G15 group were further intervened with the G protein-coupled estrogen receptor(GPER)agonist G1 and antagonist G15,respectively,at a final concentration of 1×108 mol/L for consecutive 48 hours.Following the 48-hour incubation,the chondrocytes in genistein group,genistein combined with G1 group,and genistein combined with G15 group were further intervened with genistein at a final concentration of 100 μg/mL for another 72 hours.After the end of intervention,the incubated-chondrocytes were collected from each group.The apoptosis rate of chondrocytes in each group was determined by flow cytometry,the mitochondrial autophagy was evaluated by lysosomal and mitochondrial co-localization assays,and the protein expression levels of TypeⅡcollagen,aggrecan,matrix metalloproteinase(MMP)-13,cysteine aspartic acid specific protease(Caspase)-3,cleaved-cysteine aspartic acid specific protease(cleaved-Caspase)-3,GPER,Pink1,and Parkin in the incubated-chondrocytes were detected by Western Blot.Results:①The chondrocyte apoptosis rate.The apoptosis rate of chondrocytes was higher in model group compared to control group(P=0.001),conversely,it was lower in genistein group and genistein combined with G1 group compared to model group(P=0.001,P=0.001),but not different between genistein combined with G15 group and model group(P=0.142).②The protein expression levels of extracellular matrix metabolism-related genes in chondrocytes.The relative protein expression levels of TypeⅡcollagen and aggrecan in chondrocytes were lower,while those of MMP-13,Caspase-3,and cleaved-Caspase-3 were higher in model group compared to control group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000).The relative protein expression levels of TypeⅡcollagen and aggrecan in chondrocytes were higher,while those of MMP-13,Caspase-3,and cleaved-Caspase-3 were lower in genistein group and genistein combined with G1 group compared to model group(P=0.001,P=0.001; P=0.001,P=0.001; P=0.000,P=0.000,P=0.000; P=0.000,P=0.000,P=0.000),while all these markers showed no significant difference between genistein combined with G15 group and model group(P=0.965,P=0.999,P=0.999,P=0.999,P=0.999).③The protein expression level of GPER in chondrocytes.The relative protein expression level of GPER in chondrocytes was lower in model group compared to control group(P=0.001),conversely,it was higher in genistein group and genistein combined with G1 group compared to model group(P=0.001,P=0.001),but not different between genistein combined with G15 group and model group(P=0.965).④The mitochondrial autophagy in chondrocytes.The control group exhibited yellow fluorescence(colocalization signal),which was nearly absent in model group and genistein combined with G15 group,conversely,both genistein group and genistein combined with G1 group demonstrated increased yellow fluorescence compared to the model group.⑤The protein expression levels of mitochondrial autophagy-related genes in chondrocytes.The relative protein expression levels of Pink1 and Parkin in chondrocytes were lower in model group compared to control group(P=0.001,P=0.001),conversely,they were higher in genistein group and genistein combined with G1 group compared to model group(P=0.001,P=0.001; P=0.001,P=0.001),but not different between genistein combined with G15 group and model group(P=0.999,P=0.999).Conclusion:Genistein can inhibit chondrocyte apoptosis in OA.It may work by up-regulating the expressions of typeⅡcollagen,aggrecan,and GPER,suppressing the expressions of MMP-13,caspase-3,and cleaved caspase-3,as well as promoting the mitochondrial autophagy via activating the Pink-Parkin signaling pathway.

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备注/Memo

备注/Memo:
基金项目:浙江省卫生健康科技计划项目(2021KY971)
通讯作者:柴君雷 E-mail:286779764@qq.com
更新日期/Last Update: 1900-01-01