[关键词]
[摘要]
目的 探讨高良姜素调节沉默信息调节因子 1(SIRT1)/增强腺苷酸活化蛋白激酶(AMPK)/哺乳动物雷帕 霉素靶蛋白(mTOR)信号通路对脂多糖(LPS)诱导的大鼠关节软骨细胞自噬和凋亡的影响。方法 将大鼠关节 软骨细胞分为对照组,模型组(1.0 μg·mL-1 LPS),高良姜素低(1.0 μg·mL-1 LPS+10 μmol·L-1 高良姜素)、 中(1.0 μg·mL-1 LPS+20 μmol·L-1高良姜素)、高(1.0 μg·mL-1 LPS+40 μmol·L-1高良姜素)剂量组,高良姜素高 剂量+EX527(SIRT1 抑制剂)组(1.0 μg·mL-1 LPS+40 μmol·L-1高良姜素+40 μmol·L-1 EX527)。用流式细胞术检 测关节软骨细胞凋亡;丹酰尸胺(MDC)染色观察关节软骨细胞自噬;酶联免疫吸附法检测细胞中白细胞介素 1β(IL-1β)、肿瘤坏死因子 α(TNF-α)、IL-6 水平;Western Blot 法检测苄氯素(Beclin)、微管相关蛋白 1 轻链 3(LC3)Ⅱ/Ⅰ、B 淋巴细胞瘤-2 相关 X 蛋白(Bax)、B 淋巴细胞瘤-2(Bcl-2)、SIRT1、AMPK、磷酸化 AMPK(p-AMPK)、mTOR、p-mTOR 表达情况。结果 与对照组比较,模型组 MDC 阳性细胞比例,Beclin、 LC3Ⅱ/Ⅰ、Bcl-2、SIRT1 蛋白表达及 p-AMPK/AMPK 比值均降低(P<0.05);细胞凋亡率、IL-1β、TNF-α、 IL-6 水平、Bax 蛋白表达、p-mTOR/mTOR 比值均升高(P<0.05)。与模型组比,高良姜素低、中、高剂量组 的 MDC 阳性细胞比例及 Beclin、LC3Ⅱ/Ⅰ、Bcl-2、SIRT1 蛋白表达及 p-AMPK/AMPK 比值均增加(P<0.05); 细胞凋亡率、IL-1β、TNF-α、IL-6 水平、Bax 蛋白表达及 p-mTOR/mTOR 比值均降低(P<0.05)。与高良姜素 高剂量组比较,高良姜素高剂量+EX527 组的 MDC 阳性细胞比例及 Beclin、LC3Ⅱ/Ⅰ、Bcl-2、SIRT1 蛋白表 达以及 p-AMPK/AMPK 比 值 明 显 降 低(P<0.05), 细 胞 凋 亡 率 IL-1β、 TNF- α、 IL-6 水 平 、 Bax 蛋 白 表达、p-mTOR/mTOR 比值明显升高(P<0.05)。结论 高良姜素能促进 LPS 诱导的关节软骨细胞自噬,抑制 细胞凋亡,可能是通过激活 SIRT1/AMPK/mTOR 信号通路发挥作用。
[Key word]
[Abstract]
Objective To investigate the effect of galangin on lipopolysaccharide (LPS)-induced autophagy and apoptosis of rat articular chondrocytes by regulating the silent information regulator 1(SIRT1)/adenosine 5’ -monophosphate-activated protein kinase (AMPK) /mammalian target of rapamycin (mTOR) signaling pathway. Methods Rat articular chondrocytes were grouped into control group, model group (1.0 μg·mL-1 LPS), low concentration galangin group (1.0 μg·mL-1 LPS + 10 μmol·L-1 galangin), medium concentration galangin group (1.0 μg·mL-1 LPS + 20 μmol·L-1 galangin),high concentration galangin group (1.0 μg·mL-1 LPS + 40 μmol·L-1 galangin),high concentration galangin+ EX527 (SIRT1 inhibitor) group (1.0 μg·mL-1 LPS + 40 μmol·L-1 galangin + 40 μmol·L-1 EX527). Apoptosis of articular chondrocyte was detected by flow cytometry. Monodansylcadaverine (MDC) staining was applied to observe autophagy of articular chondrocytes. Enzyme linked immunosorbent assay was applied to detect the levels of interleukin (IL) -1β,tumor necrosis factor α (TNF-α),and IL-6 in cells. Western Blot was applied to detect the expression of Beclin,microtubule-associated protein 1 light chain 3 (LC3) Ⅱ/Ⅰ , B lymphoblastoma-2-associated X protein (Bax), B lymphoblastoma-2 (Bcl-2), SIRT1, AMPK, phosphorylated (p) -AMPK,mTOR,and p-mTOR. Results Compared with the control group,the proportion of MDC positive cells,protein expression of Beclin,LC3 Ⅱ/Ⅰ,Bcl-2 and SIRT1,as well as p-AMPK/AMPK ratio in the model group decreased (P<0.05), while the apoptosis rate, levels of IL-1β, TNF- α and IL-6, Bax protein expression, and p-mTOR/mTOR ratio increased (P<0.05) . Compared with the model group, the proportion of MDC positive cells,protein expression of Beclin,LC3 Ⅱ/Ⅰ,Bcl-2 and SIRT1,as well as p-AMPK/ AMPK ratio in the low-,medium-,and high- concentration galangin groups all increased (P<0.05),but the apoptosis rate,levels of IL-1β,TNF-α and IL-6,Bax protein expression,and p-mTOR/mTOR ratio decreased (P<0.05) . Compared with the high concentration galangin group,the proportion of MDC positive cells,protein expression of Beclin,LC3 Ⅱ/Ⅰ,Bcl-2 and SIRT1,and p-AMPK/AMPK ratio in the high concentration galangin+ EX527 group reduced obviously (P<0.05),while the apoptosis rate,levels of IL-1β,TNF-α and IL-6,Bax protein expression,and p-mTOR/mTOR ratio increased obviously (P<0.05) . Conclusion Galangin promotes LPSinduced autophagy and inhibits apoptosis in articular chondrocytes,possibly by activating the SIRT1/AMPK/mTOR signaling pathway to exert its effect.
[中图分类号]
R285.5
[基金项目]
河南省医学科技攻关计划(联合共建)项目(LHGJ20230888)