芸香苷改善椎间盘退变大鼠髓核细胞焦亡的研究

Research of rutin on improving pyroptosis of nucleus pulposus cells in rats with intervertebral disc degeneration

目的从细胞水平探究芸香苷对椎间盘退变大鼠髓核细胞焦亡的影响及作用机制。方法将大鼠椎间盘髓核细胞随机分为对照组、模型组[白细胞介素-1β(IL-1β)诱导]、低剂量实验组(25μmol·L^(-1)芸香苷+10ng·mL^(-1)IL-1β)、中剂量实验组(50μmol·L^(-1)芸香苷+10ng·mL^(-1)IL-1β)、高剂量实验组(100μmol·L^(-1)芸香苷+10ng·mL^(-1)IL-1β)、高剂量+SIRT1激动药组[100μmol·L^(-1)芸香苷+10ng·mL^(-1)IL-1β+1μmol·L^(-1)沉默信息调节因子1(SIRT1)激动药SRT1720]。用实时定量聚合酶链反应(RT-qPCR)实验、蛋白质印迹法检测相关基因和蛋白的表达水平,用细胞计数试剂盒-8(CCK-8)实验检测细胞活力,用酶联免疫吸附试验(ELISA)法检测炎性因子的表达水平。结果对照组、模型组、高剂量实验组、高剂量+SIRT1激动药组的细胞活力分别为(100.00±3.56)%、(47.30±2.58)%、(76.78±2.83)%和(85.84±4.84)%,SIRT1mRNA表达水平分别为1.00±0.12、0.25±0.02、0.67±0.08和0.83±0.09,肿瘤坏死因子-α(TNF-α)表达水平分别为(15.03±1.21)、(126.50±8.84)、(53.88±3.43)和(28.19±4.02)pg·mL^(-1),PTEN诱导激酶1(PINK1)蛋白相对表达水平分别为0.30±0.03、0.26±0.04、0.89±0.12和1.28±0.08,N端-消皮素D(GSDMD-N)蛋白相对表达水平分别为0.38±0.02、1.20±0.08、0.53±0.05和0.40±0.03;高剂量实验组的上述指标与模型组比较,在统计学上差异均有统计学意义(均P<0.05);高剂量+SIRT1激动药组的上述指标与高剂量实验组比较,在统计学上差异均有统计学意义(均P<0.05)。结论芸香苷可通过激活SIRT1诱导线粒体自噬,抑制髓核细胞焦亡缓解大鼠椎间盘退变。

Objective To investigate the effect of rutin on pyroptosis of nucleus pulposus cells in rats with intervertebral disc degeneration and its mechanism at cellular level.Methods Rat interdisc nucleus pulposus cells were randomly divided into control group,model group[interleukin-1β(IL-1β)induction],experiment-L group(25μmol·L^(-1)rutin+10 ng·mL^(-1)IL-1β),experiment-M group(50μmol·L^(-1)rutin+10ng·mL^(-1)IL-1β),experiment-H group(100μmol·L^(-1)rutin+10 ng·mL^(-1)IL-1β),experiment-H+SRT1720 agonist group[100μmol·L^(-1)rutin+10 ng·mL^(-1)IL-1β+1μmol·L^(-1)silent information regulator 1(SIRT1)agonist SRT1720].Real-time quantitative polymerase chain reaction(RT-qPCR)assay and Western blot assay were used to detect the expression of related genes and proteins;cell count kit-8(CCK-8)assay and enzyme-linked immunosorbent assay were used to detect the expression of cell viability and inflammatory factors,respectively.Results The cell activities of control group,model group,experimental-H group and experimental-H+SIRT1 agonist group were(100.00±3.56)%,(47.30±2.58)%,(57.33±3.41)%,(66.15±4.85)%,(76.78±2.83)%and(85.84±4.84)%;SIRT1 mRNA expression levels were 1.00±0.12,0.25±0.02,0.39±0.04,0.52±0.03,0.67±0.08 and 0.83±0.09;tumor necrosis factor-α(TNF-α)expression level were(15.03±1.21),(126.50±8.84),(112.60±12.29),(83.71±6.85),(53.88±3.43)and(28.19±4.02)pg·mL^(-1);the relative expression levels of PTEN-induced putative kinase 1(PINK1)protein were 0.30±0.03,0.26±0.04,0.39±0.04,0.65±0.06,0.89±0.12 and 1.28±0.08;the relative expression levels of gasdermin D-N(CSDMD-N)were 0.38±0.02,1.20±0.08,0.89±0.06,0.69±0.07,0.53±0.05 and 0.40±0.03,respectively.Compared with the model group,the above indexes in the experimental-H group were statistically different(all P<0.05).There were statistically significant differences in the above indexes between the experimental-H+SIRT1 agonist group and the experimental-H group(all P<0.05).Conclusion Rutin can induce mitochondrial autophagy by activating SIRT1,inhibit pyroptosis of nucleus pulposus cells and alleviate intervertebral disc degeneration in rats.