苍术素调节腺苷酸活化蛋白激酶/沉默信息调节因子1信号通路对白细胞介素-1β诱导软骨细胞自噬和凋亡的影响

Effect of atractylodin on IL-1β-induced autophagy and apoptosis of chondrocytes by regulating AMPK/SIRT1 signaling pathway

目的探讨苍术素(ATR)通过调节腺苷酸活化蛋白激酶/沉默信息调节因子1(AMPK/SIRT1)信号通路对白细胞介素1β(IL-1β)诱导的乳鼠软骨细胞自噬与凋亡的影响。方法2022年1―8月从乳鼠中分离软骨细胞并鉴定;使用不同浓度苍术素(0、5.0、10.0、20.0、40.0、80.0μmol/L)预处理软骨细胞,再使用IL-1β诱导软骨细胞培养24 h,噻唑蓝(MTT)法检测增殖活性,筛选最佳药物浓度;将软骨细胞分为对照组、IL-1β组(10μg/L IL-1β)、苍术素组(10μg/L IL-1β+20μmol/L苍术素)、苍术素+AMPK抑制剂组(苍术素+compound C组,10μg/L IL-1β+20μmol/L苍术素+50μmol/L compound C);流式细胞术与TUNEL法检测软骨细胞凋亡,单丹磺酰尸胺(MDC)染色观察自噬小体;蛋白质印迹法检测自噬及AMPK/SIRT1通路相关蛋白表达。结果与对照组比较,IL-1β组软骨细胞增殖活性、自噬水平、微管相关蛋白轻链3Ⅱ/Ⅰ(LC3Ⅱ/LC3Ⅰ)[(0.47±0.08)比(1.23±0.14)]、Beclin-1[(0.67±0.09)比(1.45±0.19)]、自噬相关基因-5(Atg5)[(0.35±0.06)比(1.14±0.13)]、磷酸化AMPK(p-AMPK)[(0.37±0.05)比(0.91±0.05)]、SIRT1[(0.51±0.06)比(1.31±0.14)]、磷酸化Unc-51样自噬激活蛋白酶(p-ULK1)蛋白表达[(0.25±0.04)比(0.85±0.11)]降低,细胞凋亡率[(28.46±3.55)%比(2.54±0.82)%]升高(P<0.05);与IL-1β组比较,苍术素组细胞增殖活性、自噬水平、LC3Ⅱ/LC3Ⅰ、Beclin-1、Atg5、p-AMPK、SIRT1、p-ULK1蛋白表达升高,细胞凋亡率降低(P<0.05);compound C可逆转苍术素对IL-1β诱导的软骨细胞自噬激活作用。结论苍术素通过激活AMPK/SIRT1信号通路促进IL-1β诱导的大鼠软骨细胞自噬,抑制细胞凋亡。

Objective To investigate the impacts of atractylodin(ATR)on interleukin-1β(IL-1β)induced autophagy and apoptosis of neonatal rat chondrocytes by regulating AMP-activated protein kinase/silencing Message regulator 1(AMPK/SIRT1)signaling pathway.Methods Chondrocytes were isolated from suckling mice and identified from January to August 2022;different concentrations of atractylodin(0,5.0,10.0,20.0,40.0,80.0μmol/L)was used to pretreat chondrocytes,and then IL-1βwas used to induce chondrocytes to culture for 24 hours,MTT assay was used to detect the proliferation activity to screen the best drug concentration;chondrocytes were divided into Control group,IL-1βgroup(10μg/L IL-1β),ATR group(10μg/L IL-1β+20μmol/L ATR),and ATR+AMPK inhibitor group(ATR+compound C group,10μg/L IL-1β+20μmol/L ATR+50μmol/L compound C),flow cytometry and TUNEL were used to de⁃tect chondrocyte apoptosis,the autophagosomes were observed by MDC staining;Western blotting was used to detect the expression of autophagy and AMPK/SIRT1 pathway related proteins.Results Compared with the Control group,the proliferation activity of chondro⁃cytes,autophagy level,protein expression of Microtubule-associated protein light chain 3Ⅱ/Ⅰ(LC3Ⅱ/LC3Ⅰ)[(0.47±0.08)vs.(1.23±0.14)],Beclin-1[(0.67±0.09)vs.(1.45±0.19)],Autophagy-related gene(Atg5)[(0.35±0.06)vs.(1.14±0.13)],Phosphorylated AMPK(p-AMPK)[(0.37±0.05)vs.(0.91±0.05)],SIRT1[(0.51±0.06)vs.(1.31±0.14)],andPhosphorylated Autophagy Activating Kinase1(p-ULK1)[(0.25±0.04)vs.(0.85±0.11)]in IL-1βgroup decreased,and the apoptosis rate[(28.46±3.55)%vs.(2.54±0.82)%]increased(P<0.05);compared with IL-1βgroup,the proliferation activity of chondrocytes,autophagy level,protein expression of LC3Ⅱ/LC3Ⅰ,Beclin-1,Atg5,p-AMPK,SIRT1,and p-ULK1 in ATR group increased,and the apoptosis rate decreased(P<0.05);compound C was able to re⁃verse the activation of IL-1β-induced chondrocyte autophagy induced by atractylonin.Conclusion Atractylodin promotes IL-1β-in⁃duced autophagy of rat chondrocytes and inhibits apoptosis by activating AMPK/SIRT1 signaling pathway.