白细胞介素1β调控信号素3A表达诱发椎间盘退变的机制  

Mechanism by which interleukin-1beta regulates the expression of Semaphorin 3A to induce intervertebral disc degeneration

作　　者：黄杰 蒋强[2] 韩嘉恒 刘江 张燕[2] 卢正操[2] 丁宇 Huang Jie;Jiang Qiang;Han Jiaheng;Liu Jiang;Zhang Yan;Lu Zhencao;Ding Yu(Department of Orthopedics,School of Medicine,South China University of Technology,Guangzhou 510006,Guangdong Province,China;Orthopedics of TCM Senior Department,The Sixth Medical Center of PLA General Hospital,Beijing 100048,China)

机构地区：[1]华南理工大学医学院,广东省广州市510006 [2]解放军总医院第六医学中心中医医学部骨伤科,北京市100048

出　　处：《中国组织工程研究》2024年第23期3680-3685,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(82274637),项目负责人:丁宇。

摘　　要：背景:信号素3A是重要的神经血管生长抑制因子,目前尚不清楚信号素3A是如何参与盘源性腰痛发病的,研究信号素3A在椎间盘退变中的潜在机制可为防治盘源性腰痛提供新的靶点和理论依据。目的:通过激活核因子κB信号通路影响信号素3A的表达,探讨白细胞介素1β诱导大鼠椎间盘退变的机制。方法:采用RT-qPCR检测人未退变与退变髓核组织内的信号素3A mRNA表达。分离培养SD大鼠髓核细胞,传代至第3代时分3组培养:空白对照组常规培养48 h,退变组加入10 ng/mL白细胞介素1β干预48 h,退变+抑制剂组加入5μmol/L核因子κB信号通路特异性抑制剂BAY11-7082干预1 h后加入白细胞介素1β干预48 h。干预结束后,采用CCK-8法检测细胞活力,Annexin V/FITC染色法检测细胞凋亡,RT-qPCR检测细胞基质、血管、神经标志物及信号素3A的mRNA表达,Western blot检测标志蛋白、核因子κB信号通路蛋白p65及p-p65的蛋白表达。结果与结论:①RT-qPCR检测显示,人退变髓核组织内的信号素3A mRNA表达低于未退变髓核组织(P<0.05);②CCK-8检测与Annexin V/FITC染色显示,与空白对照组比较,退变组髓核细胞活力降低、凋亡率增加(P<0.05);与退变组比较,退变+抑制剂组髓核细胞活力升高、凋亡率降低(P<0.05);③RT-qPCR检测显示,与空白对照组比较,退变组Ⅱ型胶原蛋白、聚蛋白多糖、信号素3A的mRNA表达降低(P<0.05),CD31、神经丝蛋白200的mRNA表达升高(P<0.05);与退变组比较,退变+抑制剂组Ⅱ型胶原蛋白、聚蛋白多糖、信号素3A的mRNA表达升高(P<0.05),CD31、神经丝蛋白200的mRNA表达降低(P<0.05);④Western blot检测显示,与空白对照组比较,退变组Ⅱ型胶原蛋白、聚蛋白多糖、信号素3A的蛋白表达降低(P<0.05),CD31、神经丝蛋白200、p65及p-p65的蛋白表达升高(P<0.05);与退变组比较,退变+抑制剂组Ⅱ型胶原蛋白、聚蛋白多糖、信号素3A的蛋白表达升高(P<0.05),CD31BACKGROUND:Semaphone 3A(Sema3A)is an important neurovascular growth inhibitor.It is not clear how Sema3A is involved in the pathogenesis of discogenic low back pain.Exploring the potential mechanism of Sema3A in intervertebral disc degeneration can provide a new target and theoretical basis for the prevention and treatment of discogenic low back pain.OBJECTIVE:To explore the mechanism of interleukin-1βinhibiting the expression of Sema3A by activating the nuclear factor-κB signaling pathway to induce intervertebral disc degeneration in rats.METHODS:RT-qPCR was used to detect the expression of Sema3A mRNA in normal and degenerative human nucleus pulposus tissues.Nucleus pulposus cells of Sprague-Dawley rats were isolated,cultured,and passaged to the 3rd generation.Then,passage 3 cells were divided into three groups:the blank control group was routinely cultured for 48 hours,the degeneration group was intervened with 10 ng/mL interleukin 1βfor 48 hours,and the degeneration+inhibitor group was treated by 5μmol/L nuclear factor-κB signaling pathway-specific inhibitor BAY11-7082 for 1 hour,followed by interleukin-1βfor 48 hours.At the end of the intervention,cell viability was detected by cell counting kit-8,cell apoptosis was detected by Annexin V/FITC staining,mRNA expression of cellular matrix,vascular and neural markers and Sema3A was detected by RT-qPCR,and protein expression of marker proteins,p65 and p-p65 was detected by western blot.RESULTS AND CONCLUSION:RT-qPCR assay showed that the expression of Sema3A mRNA was lower in degenerative human nucleus pulposus tissue than in normal human nucleus pulposus tissue(P<0.05).Compared with the blank control group,the nucleus pulposus cell viability decreased and the apoptotic rate increased in the degeneration group(P<0.05);compared with the degeneration group,the nucleus pulposus cell viability increased and the apoptotic rate decreased in the degeneration+inhibitor group(P<0.05).Compared with the blank control group,mRNA expression of type II collagen,polyprote

关 键 词：白细胞介素1Β 核因子ΚB信号通路 信号素3A 椎间盘退变 盘源性腰痛 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R331.1