益肾健骨丸对KOA软骨细胞AMPK/Cyclin Y/CDK16通路活性及细胞自噬的影响  

Effect of Yishen Jiangu Pill on AMPK/Cyclin Y/CDK 16 pathway activity and cellular autophagy in KOA chondrocytes

作　　者：柴喜平[1,2] 李盛华[2] 周明旺[2] 陈威[2] 朱保红 柴英英 CHAI Xi-ping;LI Sheng-hua;ZHOU Ming-wang;CHEN Wei;ZHU Bao-hong;CHAI Ying-ying(Gansu University of Chinese Medicine,Lanzhou 730000,China;Gansu Provincial Hospital of TCM,Lanzhou 730050,China)

机构地区：[1]甘肃中医药大学,甘肃兰州730000 [2]甘肃省中医院,甘肃兰州730050

出　　处：《时珍国医国药》2024年第1期60-65,共6页Lishizhen Medicine and Materia Medica Research

基　　金：甘肃省自然科学基金(22JR5RA634)。

摘　　要：目的 探讨益肾健骨丸对膝骨关节炎(KOA)软骨细胞AMPK/Cyclin Y/CDK16通路活性及自噬的影响。方法 在接受人工全膝关节置换术KOA患者软骨组织中提取软骨细胞,筛选最佳给药浓度及时间后,将其分成模型组(Modle)、对照组(Control)、AMPK激活剂组(AICAR)、AMPK抑制剂组(Compound C)、益肾健骨丸组(YSJGP)和AMPK抑制剂+益肾健骨丸组(Compound C+YSJGP),除模型组、对照组外,其余各组给予相对应的药物干预。使用CCK8法检测益肾健骨丸对软骨细胞活性的影响,qRT-PCR和Western blot检测软骨细胞中AMPK、Cyclin Y、CDK16 mRNA和蛋白表达以及ULK-1、Beclin 1、p62 mRNA和蛋白的表达及LC3Ⅱ/Ⅰ的比值,使用透射电镜观察自噬小体,并利用激光共聚焦观察LC3Ⅱ蛋白相对表达。结果 YSJGP可显著提升LPS诱导的软骨细胞中AMPK/Cyclin Y/CDK16通路活性,增加ULK-1、Beclin 1的活性及LC3Ⅱ/Ⅰ的比值,减少p62的表达,并可增加软骨细胞中自噬小体的数量及LC3Ⅱ的荧光强度,其对软骨细胞的影响与AICAR趋势一致,而Compound C干预则与之相反;此外,Compound C还显著抑制了YSJGP对AMPK/Cyclin Y/CDK16通路活性及ULK-1、Beclin 1、p62、LC3Ⅱ/Ⅰ的调节,逆转了YSJGP对软骨细胞自噬活性的调控作用。结论 益肾健骨丸能提升KOA软骨细胞自噬活性,其机制与上调AMPK/Cyclin Y/CDK16通路活性密切相关。Objective To explore the effect of Yishen Jiangu Pill on AMPK/CyclinY/CDK16 pathway activity and autophagy of chondrocytes in knee osteoarthritis(KOA).Methods Chondrocytes were extracted from the cartilage of KOA patients undergoing total knee arthroplasty.After screening the optimal concentration and time of administration,the chondrocytes were divided into model group(Modle),control group(Control),AMPK activator group(AICAR),AMPK inhibitor group(CompoundC),Yish⁃en Jiangu pill group(YSJGP)and AMPK inhibitor+Yishen Jiangu pill group(Compound C+YSJGP).Except the model group and control group,the other groups were treated with corresponding drugs.The effect of Yishen Jiangu Pill on the activity of chon⁃drocytes was detected by CCK8 method.The expression of AMPK,CyclinY,CDK16mRNA and protein,the expression of ULK-1,Beclin1,p62 mRNA and protein and the ratio of LC3 II/I in chondrocytes were detected by qRT-PCR and Westernblot.Au⁃tophagosomes were observed by transmission electron microscope,and the relative expression of LC3 II protein was observed by la⁃ser confocal.Results YSJGP could significantly increase the activity of AMPK/Cyclin Y/CDK16 pathway in chondrocytes induced by LPS,increase the activities of ULK-1 and Beclin 1 and the ratio of LC3Ⅱ/Ⅰ,decrease the expression of p62,and increase the number of autophagosomes and fluorescence intensity of LC3Ⅱin chondrocytes.Its effect on chondrocytes was consistent with the trend of AICAR,while the intervention of Compound C was on the contrary.In addition,Compound C significantly inhibited the regulation of AMPK/Cyclin Y/CDK16 pathway activity and ULK-1,Beclin-1,p62,LC3Ⅱ/I by YSJGP,and reversed the regulation of YSJGP on autophagy activity of chondrocytes.Conclusion Yishen Jiangu Pill can enhance the autophagy activity of KOA chondrocytes,and its mechanism is closely related to the up-regulation of AMPK/CyclinY/CDK16 pathway activity.

关 键 词：膝骨关节炎 软骨细胞 自噬 AMPK通路 益肾健骨丸 

分 类 号：R285.5[医药卫生—中药学]