坏死状凋亡通路抑制剂在兔早期激素性股骨头坏死中的作用

Role of necroptosis and necrostatin-1 in early steroid-induced femoral head necrosis of rabbit model

目的探讨坏死状凋亡及其抑制剂Necrostatin-1(Nec-1)在兔早期激素性股骨头坏死中的作用机制,为股骨头坏死的研究提供新的理论依据。方法选取84只健康成年新西兰兔,雌雄不限,体重(2.9±0.3)kg,适应性喂养1周。选择60只采用激素联合脂多糖法构建早期激素性股骨头坏死模型,其余24只为空白对照模型。造模6周后MRI对动物模型进行筛选,50只发生早期股骨头坏死,选2只进行病理学观察,其余48只利用简单随机数法随机平均分为模型组和治疗组,治疗组给予每日1.65 mg/kg剂量静脉注射Nec-1,模型组和空白对照组按同剂量每日注射生理盐水,给药后分别于2、4、8周三个时间点处死动物并取双侧股骨头标本,通过大体观察、HE染色及透射电镜观察骨坏死情况;免疫组化及Western Blot检测受体相互作用蛋白1(RIP-1)的蛋白表达情况。利用单因素方差分析比较三组不同时间点空骨陷窝率,利用Quantity One半定量分析各组及同一组不同时间点蛋白表达情况,利用Image Pro Plus对免疫组化各组及同一组不同时间点平均蛋白吸光度行半定量分析,对半定量结果行单因素方差分析。结果兔早期股骨头坏死模型成模率83.3%。Nec-1治疗组在不同时间点的透射电镜下形态学改变及平均空骨陷窝率较模型组均改善(t值分别为2.41,2.43,4.13,P均小于0.05),但均劣于空白组(t值分别为8.42,8.80,11.38,P均小于0.05);免疫组化半定量分析示RIP-1表达治疗组与模型组相比在第2周,第4周,第8周均有统计学差异(t值分别为6.28,4.00,2.35;P均小于0.05)。Western Blot电泳结果提示RIP-1蛋白表达趋势与免疫组化结果相同,半定量分析示治疗组与模型组在第2周,第4周,第8周RIP-1表达有统计学差异(t值分别为6.89,3.69,2.50;P均小于0.05)。结论坏死状凋亡参与激素性股骨头坏死的发生,Nec-1通过对坏死状凋亡抑制从而对激素性股骨头坏死有一定的保护作用。

Objective To explore the necroptosis and the role of necrostatin-1 （Nec-1） in the early steroid-induced femoral head necrosis of the rabbit model, so as to provide a theoretical basis for further study of the femoral head necrosis. Methods Eighty-four adult New Zealand rabbits were selected, whether male or female, weighing （ 2.9 ± 0. 3 ） kg. Sixty rabbits were chosen to construct a model of steroid-induced necrosis of femoral head with hormone plus lipopolysaccharides and the other 24 rabbits were set as the control group. MRI was performed to screen the successfully-modeling animals after six weeks and 50 rabbits showed early stage bone necrosis, among which two rabbits were selected to obtainpathological observed model validation, and 48 rabbits were randomly divided into the model group （ 1.65 mg/kg intravenous saline daily） and the treatment group （ 1.65 mg/kg intravenous Nec-1 daily） （24 rabbits in each group） using simple random number method, while the control group injected with saline daily according to the same dose also. The animals were sacrificed in two, four and eight weeks and femoral heads specimens were observed by gross appearance, HE staining and transmission electron microscope ; receptor interacting protein-1 （ RIP-1 ） was verified by immunohistochemistry and Western Blot to discuss the variation of necroptosis. One way ANOVA was applied to compare the percentage of empty lacunae among the groups. ImageProPlus and Quantity One were used for semi-quantitative analysis of the images of immunohistochemical and Western Blot, and one way ANOVA was applied to analyzed the semi- quantitative data. Results The rate of steroid-induced necrosis of femoral head was 83.3%. The treatment group showed a significant improvement of morphology and percentage of empty lacunae when compared with model group （ t = 2.41, 2. 43, 4. 13, P 〈 0. 05 ）, but inferior to control group （ t equals 8.42, 8.80, 11.38, respectively, P 〈 0. 05 ）. The expression of RIP-1 in the treatment group was significantly lower than that in the model group at each time point which was proved by the semi- quantitative analysis of both immunohistochemistry （ t = 6. 28, 4. 00, 2. 35, P 〈 0. 05 ） and western bolt （t = 6. 89, 3.69, 2. 50, P 〈 0. 05 ）. Conclusion Necroptosis plays a role in steroid-induced femoral head osteonecrosis, and Nec-1 can inhibit the necroptosis pathway to protect against steroid-induced necrosis of femoral head.