SIRT1经p53和NF—κB的相关蛋白途径调控软骨细胞凋亡的体外研究

SIRT1 regulates in vitro apoptosis of chondrocytes via p53/bax and NF-κB/peroxisome proliferator-activated receptor-T coactivator-1α pathways

目的探讨S1RT1经p53／bax和NF-κB／过氧化物酶体增殖物受体1共激活因子-1α（PGC．1a）途径调控软骨细胞凋亡的作用机制。方法常规培养新西兰兔关节软骨细胞，分为3组（n：10）：SIRTI激活剂（白藜芦醇）组、对照组、SIRT1抑制剂（尼克酰胺）组，用硝普钠诱导细胞凋亡。采用噻唑蓝法俭测各组软骨细胞活性及增殖、4＇，6-二脒基-2-苯幕吲哚（DAPI）染色和流式细胞术检测各组细胞捌亡，采用WesternBlot技术检测各组细胞SIRT1、p53、NF-κB、bax、PGC-α的表达，逆转录-聚合酶链反应（RT-PCR）法检测各组细胞SIRTⅠ、Ⅱ型胶原蛋白、聚集蛋白聚糖mRNA的表达。结果SIRT1激活剂组、对照组、SIRT1抑制剂组OD值平均分别为0．139±0．016、0．098±0．006、0．079±0．002，细胞凋亡率平均分别为9．8％±0．7％、27．3％±1．6％、41．9％±2．0％，以上项目3组间两两比较差异均有统计学意义（P〈0。05）。SIRT1激活剂组细胞SIRT1、PGC-1α的表达较对照组增高，而SIRT1抑制剂组较对照组降低；SIRTI激活剂组p53、NF。KB、bax的表达较对照组降低，而SIRT1抑制剂组较对照组升高。SIRT1激活剂组细胞SIRTI、Ⅱ型胶原蛋白、聚集蛋白聚糖mRNA的表达最高，对照组次之，SIRT1抑制剂组最低。结论SIRT1可通过调控p53、NF-κB的表达改变其下游bax、PGC-1α的表达，从而调控软骨细胞的凋亡。

Objective To investigate the role and mechanism of SIRTI in regulating the apoptosis of chondrocytes in vitro. Methods The articular ehondrocytes from New Zealand rabhits were routinely cuhured and divided into 3 even groups（ n = 10）： SIRT1 activator （resveratrol） group, control group and SIRT1 inhibitor （nicotinamide） group. The apoptosis of chondrocytes was induced by sodium nitroprusside （SNP） . MTF assay was used to detect the activity of cartilage cells in each group. DAPI staiuing and flow cytometry were applied tu detect the apoptosis of chondrocytes. Western Blot was used to detect the expressions of SIRT1, p53, NF-κB. bax, PGC-1α in the cells in each group. RT-PCR method was applied to detect the mRNA expressions of SIRT1, type Ⅱ collagen and aggrecan in the eells in each group. Results The average OD value was 0. 139 ±0. 016 in the SIRT1 activator group, 0. 098 ±0. 006 in the control group and 0. 079 ± 0. 002 in the SIRTI inhibitor group. There was a significant difference among the 3 groups （ F = 51. 273, P = 0. 000） and between groups as well （ P 〈 0.05） . In terms of celhdar apoptosis, the SIRT1 inhibitor group is the highest, the control group lower and the SIRT1 activator group the lowest. Compared with the control gorp, the expressions of SIRT1 and PGC-1α were increased in the SIRT1 activator group but decreased in the SIRT1 inhibitor group. The expressions of p53, NF-KB and bax were lower in the SIRT1 activator group than in the control group while those were higher in the SIRT1 inhibitor group. The mRNA expressions of SIRT1, type Ⅱ collagen and aggrecan were the highest in the SIRT1 activator group, lower in the control group and the lowest in the SIRT1 inhibitor group. Conclusion SIRT1 may regulate the apoptosis of chondrocytes by regulating the expressions of p53 and NF-κB which in turn changes the expres- sions of downstream bax and PGC-1 α.