基于RIPK1/RIPK3/MLKL通路探讨补肾活血方对髓核细胞坏死性凋亡的影响  

Exploration on the Effect of Bushen Huoxue Prescription on Necroptosis in Human Nucleus Pulposus Cells Based on RIPK1/RIPK3/MLKL Pathway

作　　者：彭伟[1] 朱立国[2,3,4] 尹逊路 杨克新 罗杰[2] 冯敏山[2,3,4] 于杰[2] 李玲慧 展嘉文[2] 韩涛[2] 梁龙[2] 罗明义[1] 张典 PENG Wei;ZHU Liguo;YIN Xunlu;YANG Kexin;LUO Jie;FENG Minshan;YU Jie;LI Linghui;ZHAN Jiawen;HAN Tao;LIANG Long;LUO Mingyi;ZHANG Dian(Beijing University of Chinese Medicine,Beijing 100029,China;Wangjing Hospital,China Academy of Chinese Medical Sciences,Beijing 100102,China;Beijing Research Institute of Integrated Traditional Chinese and Western Medicine for Orthopedics and Traumatology,Beijing 100700,China;Beijing Key Laboratory of Traditional Chinese Medicine Orthopedic Techniques,Beijing 100700,China)

机构地区：[1]北京中医药大学,北京100029 [2]中国中医科学院望京医院,北京100102 [3]北京市中西医结合骨伤研究所,北京100700 [4]中医正骨技术北京市重点实验室,北京100700

出　　处：《中国中医药信息杂志》2025年第3期69-75,共7页Chinese Journal of Information on Traditional Chinese Medicine

基　　金：国家自然科学基金青年科学基金(82305280);首都卫生发展科研专项(首发2022-2-4163)。

摘　　要：目的观察补肾活血方对压力诱导的人髓核细胞坏死性凋亡及RIPK1/RIPK3/MLKL通路表达的影响,探讨其延缓椎间盘退变的可能机制。方法体外培养人髓核细胞,采用持续负载压力法建立髓核细胞退变模型,将造模后的髓核细胞分为模型组、补肾活血方组和抑制剂组,分别予空白血清、补肾活血方含药血清和坏死性凋亡抑制剂(Nec-1)干预,另设正常组髓核细胞常规培养。AO/EB荧光双染法检测细胞凋亡情况,流式细胞术检测细胞坏死性凋亡率,Western blot检测p-受体相互作用蛋白激酶(RIPK)1、p-RIPK3、p-混合谱系激酶结构域样蛋白(MLKL)蛋白表达,RT-qPCR检测RIPK1、RIPK3、MLKL mRNA表达。结果与正常组比较,模型组髓核细胞AO/EB染色下红色荧光较多,呈圆形、固缩状,髓核细胞坏死性凋亡率升高(P<0.05),p-RIPK1、p-RIPK3、p-MLKL蛋白表达升高(P<0.05,P<0.01),RIPK1 mRNA表达差异无统计学意义(P>0.05),RIPK3、MLKL mRNA表达升高(P<0.01);与模型组比较,补肾活血方组及抑制剂组髓核细胞红色固缩状染色质较少,补肾活血方组坏死性凋亡率降低(P<0.05),抑制剂组坏死性凋亡率有降低趋势(P>0.05),补肾活血方组及抑制剂组p-RIPK1、p-RIPK3、p-MLKL蛋白表达降低(P<0.05,P<0.01),RIPK1 mRNA表达差异无统计学意义(P>0.05),RIPK3、MLKL mRNA表达降低(P<0.01)。结论补肾活血方可减轻压力诱导的髓核细胞损伤,从而延缓椎间盘退变程度,其机制可能与抑制RIPK1/RIPK3/MLKL通路介导的坏死性凋亡有关。Objective To observe the effects of Bushen Huoxue Prescription on pressure-induced necroptosis in human nucleus pulposus cells and the expressions of RIPK1/RIPK3/MLKL pathway;To explore its potential mechanism in delaying intervertebral disc degeneration.Methods Human primary nucleus pulposus cells were cultured in vitro,and a model of nucleus pulposus cell degeneration was established using continuous load pressure method.After modeling,the nucleus pulposus cells were divided into model group,Bushen Huoxue Prescription group and inhibitor group,blank serum,Bushen Huoxue Prescription containing serum and necroptotic apoptosis inhibitor(Nec-1)intervention were administered,respectively.Normal group nucleus pulposus cells were cultured routinely.AO/EB fluorescence dual staining method was used for detecting cell apoptosis,flow cytometry was used to detect the necroptosis rate of nucleus pulposus cells,Western blot was used to detect the protein expressions of p-receptor interacting protein kinase(RIPK)1,p-RIPK3 and p-mixed lineage kinase domain like protein(MLKL),RT-qPCR was used to detect the mRNA expressions of RIPK1,RIPK3 and MLKL.Results Compared with the normal group,the model group showed more red fluorescence under AO/EB staining of nucleus pulposus cells,which were round and condensed,the necroptosis rate of nucleus pulposus cells increased(P<0.05),the protein expressions of p-RIPK1,p-RIPK3 and p-MLKL increased(P<0.05,P<0.01),while there was no significant difference in RIPK1 mRNA expression(P>0.05),and RIPK3 and MLKL mRNA expression increased(P<0.01).Compared with the model group,Bushen Huoxue Prescription group and the inhibitor group had less red condensed chromatin in the nucleus pulposus cells,Bushen Huoxue Prescription group had a lower rate of necroptosis(P<0.05),while the inhibitor group showed a decreasing trend in necroptosis rate(P>0.05),the protein expressions of p-RIPK1,p-RIPK3 and p-MLKL decreased in Bushen Huoxue Prescription group and the inhibitor group(P<0.05,P<0.01),while there was no sig

关 键 词：补肾活血方 髓核细胞 坏死性凋亡 椎间盘退变 RIPK1/RIPK3/MLKL通路 

分 类 号：R285.5[医药卫生—中药学]