基于CCL2/CCR2/NF-κB通路探讨活膝汤含药血清对软骨细胞焦亡的影响  

Effect of Huoxi Decoction(活膝汤)Drug-Containing Serum on Chondrocyte Pyroptosis Based on CCL2/CCR2/NF-κB Pathway

作　　者：谢芳 简功辉 戴斌玉 邓志[4] 邵先舫 陈竹明 陈绍军 XIE Fang;JIAN Gonghui;DAI Binyu;DENG Zhi;SHAO Xianfang;CHEN Zhuming;CHEN Shaojun(Affiliated Hospital of Hunan Academy of Traditional Chinese Medicine,Changsha 410006;Hunan University of Traditional Chinese Medicine,Changsha 410006;Changde First Traditional Chinese Medicine Hospital,Changde 415000;The Second Affiliated Hospital of Hunan University of Traditional Chinese Medicine,Changsha 410005)

机构地区：[1]湖南省中医药研究院附属医院,长沙410006 [2]湖南中医药大学,长沙410006 [3]常德市第一中医医院,常德415000 [4]湖南中医药大学第二附属医院,长沙410005

出　　处：《中药药理与临床》2024年第5期30-36,共7页Pharmacology and Clinics of Chinese Materia Medica

基　　金：湖南省中医药科研计划重点项目(编号:2021048);湖南省教育厅科学研究项目(编号:22C0193);湖南省常德市科技创新发展项目(编号:2020S045)。

摘　　要：目的:探讨活膝汤含药血清对脂多糖诱导的软骨细胞焦亡的保护作用。方法:构建CCL2过表达质粒,制备活膝汤含药血清,CCK8法检测血清干预软骨细胞的最佳浓度及最佳时间。将培养的软骨细胞分为:空白对照组、模型对照组、12.81 g/kg活膝汤20%含药血清组、核因子κB(NF-κB)抑制组、CCL2过表达组、CCL2过表达+NF-κB抑制组、CCL2过表达+12.81 g/kg活膝汤20%含药血清组;应用ELISA法检测细胞上清中白介素-18(IL-18)、IL-1β的含量;RT-PCR法检测半胱天冬酶-1(Caspase-1)、削皮素D(Gsdmd)、NOD样受体热蛋白结构域相关蛋白3(Nlrp3)mRNA的表达;Western blot法检测半胱天冬酶剪切体-1(Cleaved-Caspase-1)、GSDMD、NLRP3、磷酸化P65(Ser536)(p-P65(Ser536))蛋白的表达;免疫荧光双染检测Caspase-1介导的软骨细胞焦亡水平。结果:与空白对照组比较,模型对照组软骨细胞IL-18、IL-1β含量显著升高,NLRP3、GSDMD、Caspase-1、Cleaved-Caspase-1、p-P65(Ser536)表达显著上调(P<0.01),软骨细胞焦亡增多;与模型对照组比较,12.81 g/kg活膝汤20%含药血清组及NF-κB抑制组软骨细胞IL-18、IL-1β含量显著降低,NLRP3、GSDMD、Caspase-1、Cleaved-Caspase-1、p-P65(Ser536)表达显著下调(P<0.01),软骨细胞焦亡减少;CCL2过表达组软骨细胞IL-18、IL-1β含量显著升高,NLRP3、GSDMD、Caspase-1、Cleaved-Caspase-1、p-P65(Ser536)表达显著上调(P<0.01),软骨细胞焦亡增多。结论:活膝汤含药血清可能通过抑制CCL2/CCR2/NF-κB信号轴下调Caspase-1介导的软骨细胞焦亡。Objective:To investigate the protective effect of Huoxi Decoction(HXD)(活膝汤)drug-containing serum on lipopolysaccharide-induced chondrocyte pyroptosis.Method:CCL2 over-expression plasmid was constructed,HXD drug-containing serum was prepared,and the optimal concentration and intervention time on chondrocytes were determined through CCK8 test.The cultured chondrocytes were divided into 7 groups:blank control group,model control group,HXD 12.81 g/kg group,nuclear factorκB(NF-κB)inhibition group,CCL2 over-expression group,CCL2 over-expression+NF-κB inhibition group,and CCL2 over-expression+HXD 12.81 g/kg group.The expressions of interleukin-18(IL-18)and interleukin-1β(IL-1β)in the supernatant were detected by ELISA.The mRNA expressions of caspase-1,gasdermin-D(Gsdmd)and NOD-like receptor pyrin domain-containing protein 3(Nlrp3)were detected by RT-PCR.Western blot was conducted to detect the expressions of cleaved-caspase-1,GSDMD,NLRP3,and phosphorylated p65(Ser536)(p-p65(Ser536))proteins.The level of caspase-1 mediated chondrocyte pyroptosis was detected by immunofluorescence double staining.Result:Compared with the blank control group,the contents of IL-18 and IL-1βwere increased in the model control group.Moreover,the expressions of NLRP3,GSDMD,caspase-1,cleaved-caspase-1,p-p65(Ser536)were significantly up-regulated(P<0.01),accompanied by increased chondrocyte pyroptosis.Compared with the model control group,the HXD 12.81 g/kg group and NF-κB inhibition group showed a significant reduction in the contents of IL-18 and IL-1βin chondrocytes.The expressions of NLRP3,GSDMD,caspase-1,cleaved-caspase-1,p-p65(Ser536)were significantly down-regulated(P<0.01),and chondrocyte pyroptosis was decreased.CCL2 over-expression group exhibited significantly elevated IL-18 and IL-1βcontents,significantly up-regulated levels of NLRP3,GSDMD,caspase-1,cleaved-caspase-1 and p-p65(Ser536)(P<0.01),as well as boosted chondrocyte pyroptosis.Conclusion:HXD drug-containing serum may down-regulate caspase-1-mediated chondrocyte

关 键 词：活膝汤 软骨细胞 细胞焦亡 CC趋化因子配体2 核因子ΚB 

分 类 号：R285[医药卫生—中药学]