独活寄生汤对白介素1β诱导髓核细胞凋亡和内质网应激的影响  

Effects of Duhuo Jisheng Decoction on Interleukin-1β-Induced Human Nucleus Pulposus Cell Apoptosis and Endoplasmic Reticulum Stress

作　　者：周鹃[1] 李艳丽 杜世阳 杨若鹏 夏平 刘伟[1] 冯晶[1] ZHOU Juan;LI Yanli;DU Shiyang;YANG Ruopeng;XIA Ping;LIU Wei;FENG Jing(Wuhan No.1 Hospital,Wuhan 430022,China;Wuhan No.4 Hospital,Wuhan 430033,China)

机构地区：[1]武汉市第一医院,武汉430022 [2]武汉市第四医院

出　　处：《中国中医骨伤科杂志》2024年第4期1-7,共7页Chinese Journal of Traditional Medical Traumatology & Orthopedics

基　　金：国家自然科学基金项目(82104899);武汉市知识创新专项项目(2022020801020531);武汉市卫计委项目(WX21M02,WZ21C04,WZ21C18)。

摘　　要：目的:探讨独活寄生汤含药血清对白介素1β诱导髓核细胞凋亡和内质网应激及其关键途径PERK/elF2α通路的影响,探究独活寄生汤对椎间盘退变的作用和分子机制。方法:将40只10周龄的SD大鼠以独活寄生汤灌胃,连续灌胃2周后经腹主动脉取血,获取含药血清。采用不同浓度独活寄生汤对髓核细胞进行不同时间的处理,细胞活力检测试剂盒(CCK-8)检测髓核细胞的活性。将髓核细胞随机分成3组:对照组(未经IL-1β和独活寄生汤含药血清处理)、IL-1β组(加入10 ng/mL IL-1β溶液干预24 h)和独活寄生汤组(加入10 ng/mL IL-1β溶液和独活寄生汤含药血清干预24 h)。TUNEL法检测分析髓核细胞凋亡率;免疫荧光法检测各组CHOP、GRP78的表达水平;qRT-PCR法检测各组CHOP、GRP78、Cleaved-caspase3、Bax、BCL-2和PERK、elF2α、ATF4通路mRNA的表达;Western Blot法检测CHOP、GRP78、Cleaved-caspase3、Bax、BCL-2、PERK、elF2α、p-PERK、p-elF2α、ATF4通路蛋白的表达。结果:独活寄生汤能够显著提高髓核细胞的活力,改善髓核细胞凋亡。与对照组相比,IL-1β组CHOP、GRP78、Cleaved-caspase3、Bax和PERK、elF2α、ATF4基因表达显著上升,BCL-2基因表达显著下降,差异有统计学意义(P<0.05);与IL-1β组相比,独活寄生汤组CHOP、GRP78、Cleaved-caspase3、Bax和PERK、elF2α、ATF4基因表达显著下降,BCL-2基因表达显著上升,差异有统计学意义(P<0.05)。与对照组相比,IL-1β组CHOP、GRP78、Cleaved-caspase3、Bax和PERK、elF2α、ATF4、p-PERK、p-elF2α蛋白表达显著上升,BCL-2蛋白显著下降,差异有统计学意义(P<0.05);与IL-1β组相比,独活寄生汤组CHOP、GRP78、Cleaved-caspase3、Bax和PERK、elF2α、ATF4、p-PERK、p-elF2α蛋白表达显著下降,BCL-2蛋白显著上升,差异有统计学意义(P<0.05)。结论:独活寄生汤含药血清能够抑制退变髓核细胞的内质网应激和凋亡,进而延缓椎间盘退变,作用途径可能与抑制PERK/elF2α通Objective:To study the effect of Duhuo Jisheng decoction drug-contained serum on interleukin 1β-induced human nucleus pulposus cell apoptosis and endoplasmic reticulum stress,and to explore the mechanism of Duhuo Jisheng decoction in intervening intervertebral disc degeneration.Methods:The 40 ten-week-old SD rats were gavaged with Duhuo Jisheng decoction,and the abdominal aorta blood after 2 weeks continuous gavage was collected to obtain drug-contained serum.The nucleus pulposus cells were treated by different concentrations of Duhuo Jisheng decoction for different times.The cell viability test kit(CCK-8)was used to detect the activity of nucleus pulposus cells.The nucleus pulposus cells were randomly divide into three groups:control group(without the treatment of IL-1βand Duhuo Jisheng decoction drug-contained serum),IL-1βgroup(adding 10 ng/mL IL-1βsolution for 24 h),Duhuo Jisheng decoction group(adding 10 ng/mL IL-1βsolution and Duhuo Jisheng decoction drug-contained serum for 24 h).The TUNEL method was used to analyze the apoptosis rate of nucleus pulposus cells.Immunofluorescence assay was used to detect the expressions of CHOP and GRP78 in each group;qRT-PCR was used to detect the mRNA expressions of CHOP,GRP78,Cleaved-caspase3,Bax,BCL-2,PERK,elF2αand ATF4 in each group;Western Blot was used to detect CHOP,GRP78,Cleaved-caspase3,Bax,BCL-2,PERK,elF2α,p-elF2α,p-PERK and ATF4 protein expressions.Results:Duhuo Jisheng decoction can significantly enhance the vitality and improve the apoptosis of nucleus pulposus cells.Compared with the control group,the mRNA expression of CHOP,GRP78,Cleaved-caspase3,Bax,PERK,elF2αand ATF4 in IL-1βgroup was significantly decreased;the mRNA expression of BCL-2 was increased,and the difference was statistically significant(P<0.05).Compared with the IL-1βgroup,the mRNA expression of CHOP,GRP78,Cleaved-caspase3,Bax,PERK,elF2αand ATF4 in Duhuo Jisheng decoction group was significantly increased;the mRNA expression of BCL-2 was decreased,and the difference was statistically

关 键 词：独活寄生汤 含药血清 椎间盘退变 内质网应激 

分 类 号：R-33[医药卫生]