摘要
目的:探讨筋骨痛消丸含药血清通过腺苷一磷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)/自噬/核苷酸结合寡聚化结构域样受体蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)信号轴调控膝骨关节炎(knee osteoarthritis,KOA)软骨细胞退变的作用机制。方法:取培养至第2代的人软骨细胞分为5组:空白组、模型组、筋骨痛消丸组、AMPK抑制剂组、自噬抑制剂组。空白组加入10%空白血清,模型组加入10%空白血清和10μg·L^(−1)白细胞介素-1β(interleukin-1β,IL−1β),筋骨痛消丸组加入10%筋骨痛消丸含药血清和10μg·L^(−1)IL−1β,AMPK抑制剂组加入10%筋骨痛消丸含药血清、10μg·L^(−1)IL−1β及10μmol·L^(−1)AMPK抑制剂(Dorsomorphin),自噬抑制剂组加入10%筋骨痛消丸含药血清、10μg·L^(−1)IL−1β及5 mmol·L^(−1)自噬抑制剂(3-MA),继续培养24 h。采用CCK-8检测各组细胞增殖率,通过流式细胞术检测细胞凋亡率及自噬活性,采用实时定量PCR检测AMPK、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,m TOR)、微管相关蛋白1轻链3B(microtubule-associated protein 1 light chain 3B,LC3B)、Beclin-1、UNC-51样自噬激活激酶1(UNC-51 like autophagy activating kinase 1,ULK1)、NLRP3、凋亡相关斑点样蛋白(apoptosis-associated specklike protein containing a CARD,ASC)、胱天冬氨酸蛋白酶-1(cysteine-requiring aspartate protease-1,Caspase-1)、IL−1β、白细胞介素-18(interleukin-18,IL−18)的mRNA表达水平,通过Western blot检测磷酸化AMPK(phospho-AMPK,p-AMPK)、Beclin-1、ULK1、LC3B-Ⅰ、LC3B-Ⅱ、磷酸化mTOR(phospho-mTOR,p-mTOR)、NLRP3、Caspase-1、IL−1β的蛋白表达水平。结果:与空白组对比,模型组细胞增殖率、细胞自噬率显著降低(P<0.05),细胞凋亡率显著升高(P<0.05),AMPK、LC3B、Beclin-1、ULK1 mRNA和蛋白表达均显著下调(均P<0.05),mTOR、NLRP3炎症小体相关的mRNA和蛋白表达水平均显著升高(P<0.05);与模型组对比,筋骨痛消丸组细胞增殖率、细胞自噬率显著升高(P<0.05),细胞凋亡率显著降低(P<0.05),AMPK、LC3B、Beclin-1及ULK1 mRNA和蛋白表达均显著升高(均P<0.05),mTOR、NLRP3炎症小体相关的mRNA和蛋白表达水平均显著降低(P<0.05);与筋骨痛消丸组对比,AMPK抑制剂组和自噬抑制剂组的细胞增殖率、细胞自噬率显著降低(P<0.05),细胞凋亡率显著升高(P<0.05),AMPK抑制剂组和自噬抑制剂组AMPK、LC3B、Beclin-1、ULK1 mRNA表达均显著下调(均P<0.05),mTOR、NLRP3炎症小体相关的mRNA和蛋白表达水平均显著上调(P<0.05)。结论:筋骨痛消丸可能通过激活AMPK/自噬轴抑制NLRP3炎性小体活化,促进KOA软骨细胞增殖和自噬,抑制凋亡,调控KOA软骨细胞退变。
Objective:To explore the mechanism by which serum containing Jingu Tongxiao Pill regulates the degeneration of chondrocytes in knee osteoarthritis(KOA)through the adenosine monophosphate activated protein kinase(AMPK)/autophagy/NOD like receptor thermal protein domain associated protein 3(NLRP3)signaling axis.Methods:Human chondrocytes at passage 2 were divided into five groups:blank control group,model group,Jingu Tongxiao Pill group,AMPK inhibitor group,and autophagy inhibitor group.The blank control group was treated with 10%blank serum;the model group was treated with 10%blank serum and 10μg·L^(−1) interleukin-1β(IL−1β);the Jin Gu Tong Xiao Pills group was treated with 10%serum containing Jin Gu Tong Xiao Pills and 10μg·L^(−1) IL 1β;the AMPK inhibitor group was treated with 10%serum containing Jin Gu Tong Xiao Pills,10μg·L^(−1) IL 1β,and 10μmol·L^(−1) AMPK inhibitor(Dorsomorphin);the autophagy inhibitor group was treated with 10%serum containing Jingu Tongxiao Pill,10μg·L^(−1) IL−1β,and 5 mmol·L^(−1) autophagy inhibitor(3-MA).The cells were then cultured for another 24 hours.Cell proliferation rates in each group were detected using CCK 8,cell apoptosis and autophagy activity were detected by flow cytometry,and real time quantitative PCR was used to detect the mRNA expression levels of AMPK,mammalian target of rapamycin(mTOR),microtubule-associated protein 1 light chain 3B(LC3B),Beclin-1,UNC-51 like autophagy activating kinase 1(ULK1),NLRP3,apoptosis associated speck like protein containing a CARD(ASC),cysteine requiring Aspartate Protease-1(Caspase−1),IL-1β,and interleukin 18(IL-18).Western blot was used to detect the protein expression levels of phospho AMPK(p-AMPK),Beclin−1,ULK1,LC3B-I,LC3B-II,phospho-mTOR(p-mTOR),NLRP3,Caspase−1,and IL−1β.Results:Compared with the blank control group,the proliferation rate and autophagy rate of cells in the model group were significantly decreased(P<0.05),while the apoptosis rate was significantly increased(P<0.05).The mRNA and protein expressions of AMPK,LC3B,Beclin-1,and ULK1 were all significantly downregulated(all P<0.05),whereas the mRNA and protein expression levels related to mTOR and NLRP3 inflammasome were significantly elevated(P<0.05).Compared with the model group,the cell proliferation rate and autophagy rate in the Jin Gu Tong Xiao Wan group were significantly increased(P<0.05),and the apoptosis rate was significantly decreased(P<0.05).The mRNA and protein expressions of AMPK,LC3B,Beclin-1,and ULK1 were all significantly upregulated(all P<0.05),while the mRNA and protein expression levels related to mTOR and NLRP3 inflammasome were significantly reduced(P<0.05).Compared with the Jingu Tongxiao Pill group,the AMPK inhibitor group and autophagy inhibitor group showed significantly decreased cell proliferation and autophagy rates(P<0.05)and significantly increased apoptosis rates(P<0.05).In both inhibitor groups,the mRNA expression of AMPK,LC3B,Beclin-1,and ULK1 was significantly downregulated(all P<0.05),while the mRNA and protein expression levels related to mTOR and NLRP3 inflammasome were significantly upregulated(P<0.05).Conclusion:Jingu Tongxiao Pill may inhibit NLRP3 inflammasome activation by activating the AMPK/autophagy axis,thereby promoting proliferation and autophagy of KOA chondrocytes,inhibiting apoptosis,and regulating KOA chondrocyte degeneration.
作者
王泽宇
申晟
卢岩岩
张甜甜
朱俊博
段卫锋
WANG Zeyu;SHEN Sheng;LU Yanyan;ZHANG Tiantian;ZHU Junbo;DUAN Weifeng(School of Orthopedics,Henan University of Chinese Medicine,Zhengzhou Henan China 450046;Luoyang Orthopedic Hospital of Henan Province(Henan Orthopedic Hospital),Zhengzhou Henan China 450006)
出处
《中医学报》
2026年第1期195-202,共8页
Acta Chinese Medicine
基金
河南省中医药科学研究专项项目(2024ZY1019)。
