TLR4/ NF-κB 在酒精性股骨头坏死骨组织中的表达及意义

The expression of Toll-like receptor 4/ NF-κB in human non-traumatic osteonecrosis of the femoral head

目的通过研究人酒精性股骨头坏死(ONFH)股骨头骨组织中Toll样受体4/核因子κB(TLR4/NF-κB)的表达,初步探讨自身免疫在酒精性ONFH发病机制中的作用。方法收集酒精性ONFH组织标本16例。根据Ficat分类法分为FicatⅣ期组9例,平均年龄(65.761±8.316)岁,其中男8例,女1例;FicatⅢ期组7例,平均年龄(64.832±6.814)岁,其中男6例,女1例。取9例股骨颈骨折股骨头标本为对照组,平均年龄(64.515±7.834)岁,其中男7例,女2例。通过HE染色法观察组织病理学改变;实时定量PCR(RT-PCR)和Western-blot检测标本中TLR4、NF-κB mRNA和蛋白的相对表达量;免疫组化法检测组织标本中TLR4、NF-κB表达情况。计数指标进行卡方(χ~2)检验,三组间方差齐的资料组间比较采用单因素方差分析,方差不齐的组间比较采用Kruskal-Wallis检验。结果三组患者基本资料比较差异无统计学意义。组织病理学显示,对照组骨组织正常,坏死的股骨头骨组织内骨小梁减少、稀疏,甚至中断。TLR4、NF-κB在3种组织中都有不同程度的表达。免疫组织化学显示,两种蛋白在股骨头骨组织细胞胞质内表现为棕褐色,FicatⅣ期组表达最强;对照组两种蛋白的表达较弱。RT-PCR及Western-blot结果显示,FicatⅣ期组股骨头骨组织的TLR4、NF-κB mRNA及蛋白含量表达最高,对照组表达最低,差异有统计学意义。结论 TLR4/NF-κB在酒精性ONFH股骨头组织中表达明显上调,提示TLR4/NF-κB信号通路介导的自身免疫可能参与了酒精性ONFH的发病机制。

Objective To explore the expressions of Toll-like receptor4/nuclear factor kappa B (TLR4/NF-κB) in human alcohol-induced osteonecrosis of the femoral head (ONFH) so as to clarify the role of autoimmunity in pathogenesis of alcohol-induced ONFH. Methods Sixteen femoral head samples were collected from the patients with alcohol-induced osteonecrosis of the femoral head.According to Ficat classification methods,these was divided into 9 cases [M:F 8:1;mean age (65.761±8.316) y] of Ficat IV group and 7 cases [M:F 6:1;mean age (64.832±6.814)y] of Ficat Ⅲ group;9 [M:F 7:2;mean age (64.515±7.834) y].The femoral head samples from the patients with femoral neck fracture were treated as control group.The pathological changes in femoral heads were detected with hematoxylin-eosin (HE) staining method.The signaling molecules including TLR4 and NF-κB were detected by quantitative Real-time PCR and Western blot.The expressions of TLR4 and NF-κB in the samples were analyzed by immunohistochemical methods.The counting index was conducted as a chi-squared test.One-way ANOVA was used for comparison of homogeneity of variance among the three groups,while Kruskal-Wallis test was used for pairwise comparison among the groups with heterogeneity of variance. Results There was no significant difference in the baseline characteristics of the three groups.Histopathology showed that TLR4 and NF-κB were brown in the cytoplasm of bone tissue cells of the femoral head,bone tissue of Ficat Ⅳ group had the highest expressions of TLR4 and NF-κB,the group of normal femoral head had the least expression;the bone tissue of the control group was normal.RT-PCR and Western blot showed that the bone tissue of Ficat Ⅳ group had the highest expressions of the mRNA and protein in TLR4 and NF-κB,the second was the Ficat Ⅳ group,the group of normal femoral head bone tissue had the least expression.Statistical analysis suggested that the differences among the groups were significant ( P <0.05). Conclusion The expressions of TLR4 and NF-κB were upregulated significantly in the necrosis area of the femoral head,indicating that overactive TLR4/NF-κB signal pathway in alcohol-induced ONFH and downstream overexpressed NF-κB may be involved in the pathogenesis of alcohol-induced ONFH.