摘要目的 观察17β-雌二醇对白细胞介素-1β(IL-1β)诱导的椎间盘细胞凋亡及基质降解的影响.方法 通过Ⅱ型胶原酶消化分离大鼠原代椎间盘细胞,并将椎间盘细胞分为5组,分别为正常组、IL-1β组、IL-1β ±0.1、1.0、10.0 μmol/L的17 β-雌二醇组.并检测各组细胞活力、凋亡率、含半胱氨酸的天冬氨酸蛋白水解酶-3(Caspase-3)活性、肿瘤坏死因子-α(TNF-α)、γ-干扰素(IFN-γ)、IL-6含量、B细胞淋巴瘤/白血病-2(bcl-2)、bcl-2相关X蛋白(bax)、基质金属蛋白酶(MMP)-3、MMP-13表达,组间比较采用t检验.结果 与正常组(0.48±0.04)比较,IL-1β组(0.35±0.03)中椎间盘细胞活力降低(t=5.896,P ＜0.01);与IL-1β组比较,0.1,1.0,10.0 μmol/L的17β-雌二醇组细胞活力提高(0.43 ±0.04比0.35 ±0.03,t=6.154,P＜ 0.01;0.51 ±0.05比0.35±0.03,t=6.127,P ＜0.01;0.62 ±0.06比0.35±0.03,t5.920,P＜0.01).与正常组比较,细胞早期凋亡率降低[(12.42±1.19)％比(18.52±1.64)％,t=6.295,P＜0.01;(7.56 ±0.76)％比(18.52±1.64)％,=6.127,P ＜0.01;(3.62±0.40)％比(18.52±1.64)％,=6.570,P ＜0.01];晚期凋亡率降低[(11.44±1.18)％比(17.39±1.74)％,t=6.236,P＜0.01;(8.24±0.82)％比(17.39±1.74)％,￡=5.907,P ＜0.01;(5.17 ±0.52)％比(17.39±1.74)％,t=6.575,P ＜0.01],Caspase-3活性降低(3.85±0.38比4.32±0.43,t=6.397,P＜0.01;2.91 ±0.33比4.32±0.43,t 5.685,P＜0.05;2.02 ±0.23比4.32±0.43,t=5.889,P＜0.01).与正常组比较,TNF-α含量降低[(6.50±0.65) ng/L比(8.74±0.89) ng/L,t=5.798,P＜0.05;(5.58 ±0.56) ng/L比(8.74±0.89) ng/L,t=6.701,P ＜0.01;(3.86 ±0.39) ng/L比(8.74 ±0.89) ng/L,t =6.352,P ＜0.01],IFN-γ含量降低[(15.28 ±1.53) ng/L比(17.34±1.73) ng/L,t =6.136,P＜0.01;(13.12±1.30) ng/L比(17.34±1.73) ng/L,t =6.520,P ＜0.05;(10.67±1.08) ng/L比(17.34±1.73) ng/L,t=6.735,P＜0.05]及IL-6含量降低[(2.23±0.22) ng/L比(2.78 ±0.28) ng/L,t=6.199,P＜0.01;(1.86 ±0.18) ng/L比(2.78 ±0.28) ng/L,t =5.989,P＜0.05;(1.57 ±0.16) ng/L比(2.78 ±0.28) ng/L,t =5.449,P＜0.05].与正常组比较,bax表达量下调[(0.52±0.05)μmol/L比(0.75±0.07) μmol/L,t=6.429,P ＜0.01;(0.33 ±0.03) μmol/L比(0.75 ±0.07) μmol/L,t =6.380,P ＜0.01;(0.31 ±0.30) μmol/L比(0.75 ±0.07) μmol/L,t=6.118,P＜0.01] 、MMP-3表达量下调[(0.70±0.07) μmol/L比(1.78±0.18) μmol/L,t =6.225,P ＜0.01;(0.40 ±0.04) μmol/L比(1.78±0.18) μmol/L,t =6.485,P＜0.01;(0.38 ±0.04) μmol/L比(1.78±0.18) μmol/L,t=6.106,P＜0.05],bcl-2表达量上调[(0.50±0.05) μmol/L比(0.30±0.03) μmol/L,t=6.120,P＜0.01;(0.95±0.09) μmol/L比(0.30±0.03) μmol/L,t=6.625,P＜0.01;(1.20±0.12) μmol/L比(0.30±0.03) μmol/L,t=6.460,P＜0.01].与正常组比较,1.0、10.0 μmol/L的17β-雌二醇组MMP4表达量下调[(0.25 ±0.03) μmol/L比(0.93 ±0.12) μmol/L,t =5.259,P ＜0.05;(0.32 ±0.09) μmol/L比(0.99 ±0.15) μmol/L,t =5.556,P ＜0.05];1.0、10.0 μmol/L的17β-雌二醇组MMP-13表达量下调[(0.20 ±0.02) μmol/L比(0.88±0.09) μmol/L,t=6.479,P＜0.01;(0.20 ±0.02) μmol/L比(0.88 ±0.09) μmol/L,t=6.732,P＜0.01].结论 17β-雌二醇能抑制IL-1β诱导的椎间盘细胞凋亡及细胞外基质降解.

abstractsObjective To explore effect of 17β-estradiol on apoptosis and matrix degradation of intervertebral disc cells induced by interleukin (IL)-1β.Methods Primary rat intervertebral disc cells were isolated by type Ⅱ collagenase digestion.The cells were divided into 5 groups:normal group,IL-1 β group,IL-1β + 0.1,1.0,10.0 μmol/L 17β-estradiol group.Cell viability,cell apoptotic rate,Caspase-3 activity,the content of tumor necrosis factor alpha (TNF-α),interferon-γ (IFN-γ) and IL-6,the expression of B cell lymphoma/lewkmia-2 (bcl-2),bcl-2 related X protein (bax),matrix metalloproteinase-3 (MMP-3) and MMP-13 was detected.Results Compared with the normal group (0.48 ±0.04),the activity of intervertebral disc cells was decreased in the IL-1β group (0.35 ±0.03)(t =5.896).P ＜ 0.01);Compared with the IL-1 β group,the cell activity of the 17 betaestradiol group was improved (0.43 ±0.04 vs.0.35 ±0.03,t=6.154,P＜0.01;0.51 ±0.05 vs.0.35 ±0.03,t=6.127,P＜0.01;0.62 ±0.06 vs.0.35 ±0.03,t=5.920,P＜0.01).Compared with the normal group,the rate of early apoptosis was reduced [(12.42 ± 1.19)％ vs.(18.52 ± 1.64)％,t =6.295,P＜0.01;(7.56 ±0.76)％ vs.(18.52 ± 1.64)％,t=6.127,P＜0.01;(3.62 ±0.40)％ vs.(18.52 ± 1.64) ％,t =6.570,P ＜ 0.01].The rate of late apoptosis was decreased [(11.44 ± 1.18) ％ vs.(17.39±1.74,t=6.236,P＜0.01;(8.24±0.82)％ vs.(17.39±1.74)％,t=5.907,P＜0.01;(5.17 ± 0.52) ％ vs.(17.39 ± 1.74) ％,t =6.575,P ＜ 0.01] ％,and the activity of Caspase-3 was decreased [3.85 ± 0.38 vs.4.32 ± 0.43,t =6.397,P ＜ 0.01;2.91 ± 0.33 vs.4.32 ± 0.43,t =5.685,P＜0.05;2.02 ±0.23 vs.4.32 ±0.43,t=5.889,P＜0.01].Compared with the normal group,tnf-alpha content decreased [(6.50 ± 0.65) ng/L vs.(8.74 ± 0.89) ng/L,t =5.798,P ＜0.05;(5.58 ±0.56) ng/Lvs.(8.74±0.89) ng/L,t=6.701,P＜0.01;(3.86 ±0.39) ng/L vs.(8.74 ± 0.89) ng/L,t =6.352,P ＜ 0.01],and ifn-gamma content decreased [(15.28 ± 1.53) ng/L vs.(17.34±1.73) ng/L,t=6.136].P＜0.01;(13.12±1.30) ng/Lvs.(17.34±1.73) ng/L,t=6.520,P ＜ 0.05;(10.67 ± 1.08) ng/L vs.(17.34 ± 1.73) ng/L,t =6.735,P ＜ 0.0];and IL-6 content decreased [2.23 ± 0.22) ng/L vs.(2.78 ± 0.28) ng/L,t =6.199,P ＜ 0.01;(1.86 ± 0.18) ng/Lvs.(2.78 ±0.28) ng/L,t=5.989,P＜0.05;(1.57 ±0.16) ng/L vs.(2.78 ±0.28) ng/L,P ＜ 0.05].t =5.449,P ＜ 0.05].Compared with the normal group,bax expression was down-regulated [0.52 ± 0.07) μmol/L vs.(0.75 ± 0.07) μmol/L,t =6.429,P ＜ 0.01;(0.33 ±0.03) μmol/L vs.(0.75 ±0.07) μmol/L,t =6.380,P ＜0.01;(0.31 ±0.30) μmol/L vs.(0.75 ±0.07) μmol/L,t =6.118,P＜ 0.01],and MMP-3 expression was down-regulated [0.70 ±0.07) μmol/L vs.(1.78 ± 0.18) μmol/L,t =6.225].P ＜ 0.01;0.40 ± 0.04) μmol/L vs.(1.78 ±0.18) μmol/L,t =6.485,P ＜ 0.01;(0.38 ± 0.04) μmol/L vs.(1.78 ± 0.18) μmol/L,t =6.120 ±0.03,t=6.120,P＜0.01;(0.95±0.09) μmol/Lvs.(0.30±0.03) μmoL/L,t=6.625,P＜0.01;(1.20 ± 0.12) μmol/L vs.(0.30 ± 0.03) μmol/L,P ＜ 0.01;t =6.460,P ＜ 0.01].Compared with the normal group,the expression of MMP-3 in the 17 betaestradiol group was decreased by 1.0,10.0 μmoL/L [0.25 ± 0.03) μmol/L vs.(0.93 ± 0.12) μmol/L,t =5.259,P ＜ 0.05;(0.32 ± 0.09) μmol/L vs.(0.99 ±0.15) μmoL/L,t =5.556,P＜0.05].The expression level of MMP-13 was decreased in the 17 betaestradiol group [(0.20 ± 0.02) μmol/L vs.(0.88 ± 0.09) μmol/L,t =6.479,P ＜ 0.01;(0.20±0.02) μmol/Lvs.(0.88±0.09) μmol/L,t=6.732,P＜0.01).Conclusion These results suggested nicotine could inhibit inflammatory response of chondrocytes induced by IL-1β via inhibition of NF-κB signal pathway.