摘要
目的:探讨龟鹿二仙胶含药血清对白细胞介素-1β诱导的膝骨关节炎(knee osteoarthritis,KOA)小鼠软骨细胞模型自噬和凋亡的影响及作用机制。方法:将10只SD大鼠随机分为龟鹿二仙胶含药血清制备组和空白血清制备组,分别给予龟鹿二仙胶药液和生理盐水灌胃,连续灌胃1周后,采集腹主动脉血,制备龟鹿二仙胶含药血清和空白血清。取培养至第2代的C57BL/6小鼠膝关节软骨细胞,分为空白血清组、模型组、龟鹿二仙胶含药血清组。模型组、龟鹿二仙胶含药血清组加入终浓度为10 ng·mL-1的白细胞介素-1β,诱导培养24 h后,空白血清组、模型组加入10%空白血清,龟鹿二仙胶含药血清组加入10%龟鹿二仙胶含药血清,继续培养24 h。采用流式细胞分析仪检测各组软骨细胞凋亡率,采用电子显微镜观察各组软骨细胞自噬小体,分别采用实时定量PCR和Western Blot法检测腺苷一磷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、UNC-51样自噬激活激酶1(UNC-51 like autophagy activating kinase 1,ULK1)、自噬相关蛋白5(autophagy related protein 5,ATG5)、微管相关蛋白1轻链3B(microtubule associated protein 1 light chain 3B,LC3B)、B淋巴细胞瘤-2相关X蛋白(Bcl-2 associated X protein,Bax)、基质金属蛋白酶(matrix metalloproteinase,MMP)-13、趋化因子配体2(chemokine ligand 2,CCL2)的mRNA和蛋白表达水平。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空白血清组(P=0.000),龟鹿二仙胶含药血清组软骨细胞凋亡率低于模型组(P=0.000)。②软骨细胞自噬小体观察结果。空白血清组可见双层膜封闭的球状自噬小体,模型组自噬小体数量较空白血清组减少,龟鹿二仙胶含药血清组自噬小体数量较模型组增加。③软骨细胞自噬和凋亡相关基因mRNA和蛋白表达水平检测结果。模型组软骨细胞AMPK、ULK-1、ATG5、LC3B、Bcl-2的mRNA和蛋白表达水平均低于空白血清组(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000;P=0.002,P=0.000,P=0.000,P=0.000,P=0.000),mTOR、Bax、MMP-13、CCL2的mRNA和蛋白表达水平均高于空白血清组(P=0.002,P=0.000,P=0.000,P=0.000;P=0.004,P=0.000,P=0.000,P=0.000);龟鹿二仙胶含药血清组软骨细胞AMPK、ULK1、ATG5、LC3B、Bcl-2的mRNA和蛋白表达水平均高于模型组(P=0.000,P=0.003,P=0.001,P=0.001,P=0.001;P=0.020,P=0.002,P=0.000,P=0.008,P=0.004),mTOR、Bax、MMP-13、CCL2的mRNA和蛋白表达水平均低于模型组(P=0.008,P=0.001,P=0.000,P=0.000;P=0.040,P=0.000,P=0.001,P=0.000)。结论:龟鹿二仙胶含药血清能够增强KOA软骨细胞自噬,抑制其凋亡与细胞外基质降解,且龟鹿二仙胶含药血清可能通过激活AMPK/mTOR/ULK1信号通路发挥作用。
Objective:To explore the effects and underlying mechanism of Guilu Erxian Jiao(龟鹿二仙胶,GLEXJ)medicated serum on autophagy and apoptosis of interleukin(IL)-1β-induced knee osteoarthritis(KOA)mice chondrocyte model.Methods:Ten Sprague-Dawley(SD)rats were randomized into GLEXJ medicated serum preparation group and blank serum preparation group,with 5 ones in each group.The rats in the 2 groups were intervened by intragastric administration with GLEXJ solution and normal saline,respectively,twice a day for consecutive 7 days.Two hours after the end of the last intervention,the blood was drawn from the abdominal aorta of rats in each group for making GLEXJ medicated serum and blank serum.The chondrocytes extracted from the knee cartilage tissues of C57BL/6 mice were cultured,and the second-generation ones were collected and divided into blank serum group,model group and GLEXJ medicated serum group.The second-generation chondrocytes in model group and GLEXJ medicated serum group were intervened by IL-1βat a final concentration of 10 ng/mL.After 24-hour culture,the chondrocytes in blank serum group and model group were further intervened by 10%blank serum,whereas,the ones in GLEXJ medicated serum group by 10%GLEXJ medicated serum.After 24-hour culture,the apoptosis rate of chondrocytes in each group was detected by flow cytometry,and the autophagosomes in chondrocytes in each group were observed by electron microscopy.Furthermore,the mRNA and protein expression levels of adenosine monophosphate activated protein kinase(AMPK),mammalian target of rapamycin(mTOR),UNC-51 like autophagy activating kinase 1(ULK1),autophagy related protein 5(ATG5),microtubule associated protein 1 light chain 3B(LC3B),B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),matrix metalloproteinase(MMP)-13,and chemokine ligand 2(CCL2)were detected by using real-time quantitative PCR(RT-qPCR)and Western blotting,respectively.Results:①The apoptosis rate of chondrocytes.The apoptosis rate of chondrocytes was higher in model group compared to blank serum group and GLEXJ medicated serum group(P=0.000;P=0.000).②The autophagosomes in chondrocytes.The spherical autophagosomes enclosed by double membrane were observed in chondrocytes of blank serum group,besides,the number of autophagosomes decreased in model group compared to blank serum group,while that increased in GLEXJ medicated serum group compared to model group.③The mRNA and protein expression levels of autophagy and apoptosis-related genes in chondrocytes.The mRNA and protein expression levels of AMPK,ULK1,ATG5,LC3B and Bcl-2 in chondrocytes were lower,while,that of mTOR,Bax,MMP-13 and CCL2 were higher in model group compared to blank serum group(P=0.000,P=0.000,P=0.000,P=0.000,P=0.000;P=0.002,P=0.000,P=0.000,P=0.000,P=0.000;P=0.002,P=0.000,P=0.000,P=0.000;P=0.004,P=0.000,P=0.000,P=0.000).The mRNA and protein expression levels of AMPK,ULK1,ATG5,LC3B and Bcl-2 in chondrocytes were higher,while,that of mTOR,Bax,MMP-13 and CCL2 were lower in GLEXJ medicated serum group compared to model group(P=0.000,P=0.003,P=0.001,P=0.001,P=0.001;P=0.020,P=0.002,P=0.000,P=0.008,P=0.004;P=0.008,P=0.001,P=0.000,P=0.000;P=0.040,P=0.000,P=0.001,P=0.000).Conclusion:GLEXJ medicated serum can enhance the autophagy of KOA chondrocytes,thereby inhibiting their apoptosis and extracellular matrix degradation.It may exert the effects by activating the AMPK/mTOR/ULK1 signaling pathway.
作者
沈默金
郑珍萍
吴伟欣
杨美鑫
顾富城
耿秋东
王和鸣
李楠
SHEN Mojin;ZHENG Zhenping;WU Weixin;YANG Meixin;GU Fucheng;GENG Qiudong;WANG Heming;LI Nan(School of Traditional Chinese Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,Fujian,China;Key Laboratory of Orthopedics&Traumatology and Rehabilitation of Traditional Chinese Medicine of Ministry of Education,Fuzhou 350122,Fujian,China)
出处
《中医正骨》
2025年第1期30-37,共8页
The Journal of Traditional Chinese Orthopedics and Traumatology
基金
国家自然科学基金项目(81973880)。
