槲皮素对白细胞介素-1β诱导的软骨细胞凋亡的影响及机制

Effect of quercetin on chondrocyte apoptosis induced by interleukin-1βand underlying mechanism

目的探讨槲皮素对白细胞介素-1β(IL-1β)诱导的软骨细胞凋亡的影响及其机制。方法酶消化法获得SD大鼠软骨细胞, 并将细胞分为5组, 对照组(无任何刺激);IL-1β组(10 ng/ml的IL-1β);槲皮素低剂量组(10 ng/ml的IL-1β+100 μmol/L槲皮素);槲皮素中剂量组(10 ng/ml的IL-1β+200 μmol/L槲皮素);槲皮素高剂量组(10 ng/ml的IL-1β+400 μmol/L槲皮素)。采用噻唑蓝(MTT)法检测细胞活力、膜联蛋白V-碘化丙啶(Annexin V-PI)流式双染法检测细胞凋亡、蛋白质印迹法(Western blot)检测细胞中B淋巴细胞瘤-2(bcl-2)、bcl-2相关X蛋白(bax)、剪切的半胱氨酸蛋白酶3(clveaved Caspase-3)、基质金属蛋白酶-1(MMP-1)及MMP-13表达, 核因子-κB抑制因子α(IκBα)及核因子-κB p65(NF-κB p65)磷酸化水平, 采用LSD-t检验进行数据分析。结果槲皮素低、中、高剂量组胞活力[(0.43±0.03)、(0.47±0.02)、(0.53±0.04)比(0.36±0.03), t=5.421、6.345、6.521, P<0.01]及bcl-2表达量[(0.45±0.03)、(0.46±0.03)、(0.57±0.04)比(0.22±0.01), t=6.021、6.239、5.984, P<0.01]高于IL-1β组。槲皮素低、中、高剂量组细胞早期凋亡率[(17.73±1.34)%、(11.68±0.90)%、(6.48±0.42)%比(21.46±2.28)%, t=5.421、6.345、6.521, P<0.01]、细胞晚期凋亡率[(9.46±0.36)%、(6.26±0.52)%、(3.87±0.40)%比(15.53±1.25)%, t=5.873、6.126、6.436, P<0.01], bax[(0.50±0.04)、(0.41±0.03)、(0.19±0.01)比(1.24±0.09), t=6.325、6.984、6.324, P<0.01]、cleaved Caspase-3[(0.43±0.03)、(0.31±0.02)、(0.23±0.0)比(1.02±0.05), t=5.687、5.214、5.983, P<0.01]、MMP-1[(0.43±0.03)、(0.42±0.02)、(0.57±0.04)比(1.03±0.08), t=6.418、5.647、6.942, P<0.01]、MMP-13[(0.98±0.06)、(0.47±0.03)、(0.41±0.02)比(1.24±0.08), t=6.017、5.283、6.216, P<0.01]、p-IκBα[(0.95±0.06)、(0.48±0.03)、(0.33±0.02)比(1.32±0.11), t=5.487、5.328、5.987, P<0.05]及p-NF-κB p65[(0.98±0.08)、(0.35±0.02)、(0.25±0.01)比(1.14±0.08), t=6.328、6.457、5.984, P<0.05]表达量低于IL-1β组。结论槲皮素能显著的抑制IL-1β诱导的软骨细胞凋亡, 与阻断NF-κB信号通路有关。

Objective To explore the effect of quercetin on chondrocyte apoptosis induced by interleukin-1β(IL-1β)and underlying mechanism.Methods The chondrocytes from SD rats were ob-tained by enzyme digestion and divided into 5 groups:control group(without any stimulation);IL-1βgroup(10 ng/ml IL-1β);low-dose quercetin group(10 ng/ml IL-1β+100μmol/L quercetin),medium-dose quercetin group(10 ng/ml IL-1β+200μmol/L quercetin),high-dose quercetin group(10 ng/ml IL-1β+400μmol/L quercetin).Cell viability was detect by methylthiazol tetrazolium(MTT)assay.Cell apoptosis was detected by Annexin V-propidium iodide(PI)flow cytometry.The expression of B-cell lym-phoma-2(bcl-2),bcl-2-associated X protein(bax),clevead cysteinyl aspartate specific proteinase 3(clev-ead Caspase-3),matrix metalloproteinase-3(MMP-3),MMP-13 and phosphorylation of inhibitor of NF-kB(IkBα)and nuclear factor kappa-B(NF-kB)p65 was detected by Western blotting.Data were analyzed by LSD-t test.Results Cell viability[(0.43±0.03),(0.47±0.02),(0.53±0.04)vs.(0.36±0.03),t=5.421,6.345,6.521,P<0.01]and the expression of bcl-2[(0.45±0.03),(0.46±0.03),(0.57±0.04)vs.(0.22±0.01),t=6.021,6.239,5.984,P<0.01]in low-,medium-and high-dose quercetin groups was higher than those in IL-1βgroup.Cell early apoptotic rate[(17.73±1.34)%,(11.68±0.90)%,(6.48±0.42)%vs.(21.46±2.28)%,t=5.421,6.345,6.521,P<0.01],cell late apoptotic rate[(9.46±0.36)%,(6.26±0.52)%,(3.87±0.40)%vs.(15.53±1.25)%,t=5.873,6.126,6.436,P<0.01],the expression of bax[(0.50±0.04),(0.41±0.03),(0.19±0.01)vs.(1.24±0.09),t=6.325,6.984,6.324,P<0.01],cleaved Caspase-3[(0.43±0.03),(0.31±0.02),(0.23±0.0)vs.(1.02±0.05),t=5.687,5.214,5.983,P<0.01],MMP-1[(0.43±0.03),(0.42±0.02),(0.57±0.04)vs.(1.03±0.08),t=6.418,5.647,6.942,P<0.01],MMP-13[(0.98±0.06),(0.47±0.03),(0.41±0.02)vs.(1.24±0.08),t=6.017,5.283,6.216,P<0.01],p-IkBα[(0.95±0.06),(0.48±0.03),(0.33±0.02)vs.(1.32±0.11),t=5.487,5.328,5.987,P<0.05]and p-NF-kB p65[(0.98±0.08),(0.35±0.02),(0.25±0.01)vs.(1.14±0.08),t=6.328,6.457,5.984,P<0.05]in low-,medium-and high-quercetin groups were lower than those in IL-1βgroup.Conclusion Quercetin could significantly inhibit IL-1β-induced chondrocyte apoptosis probably by inhibiting the NF-kB signal pathway.