中医正骨2025，Vol.37Issue(1)：30-37,8.
龟鹿二仙胶含药血清对膝骨关节炎小鼠软骨细胞模型自噬和凋亡的影响及作用机制研究
Exploring the effects and mechanism of Guilu Erxian Jiao(龟鹿二仙胶)medicated serum on autophagy and apoptosis of knee osteoarthritis mice chondrocyte model:an experimental study
摘要
Abstract
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-Daw-ley(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-1 β at a final concen-tration 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 elec-tron microscopy.Furthermore,the mRNA and protein expression levels of adenosine monophosphate activated protein kinase(AMPK),mam-malian 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,respec-tively.Results:①The apoptosis rate of chondrocytes.The apoptosis rate of chondrocytes was higher in model group compared to blank ser-um group and GLEXJ medicated serum group(P=0.000;P=0.000).②The autophagosomes in chondrocytes.The spherical autophago-somes 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 ex-pression 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 ex-tracellular matrix degradation.It may exert the effects by activating the AMPK/mTOR/ULK1 signaling pathway.关键词
骨关节炎,膝/龟鹿二仙胶/软骨细胞/自吞噬/细胞凋亡/信号传导/TOR丝氨酸-苏氨酸蛋白激酶/小鼠Key words
osteoarthritis,knee/Gui Lu Er Xian Jiao/chondrocytes/autophagy/apoptosis/signal transduction/TOR serine-threonine kina-ses/mice引用本文复制引用
沈默金,郑珍萍,吴伟欣,杨美鑫,顾富城,耿秋东,王和鸣,李楠..龟鹿二仙胶含药血清对膝骨关节炎小鼠软骨细胞模型自噬和凋亡的影响及作用机制研究[J].中医正骨,2025,37(1):30-37,8.基金项目
国家自然科学基金项目(81973880) （81973880）
