苍膝通痹胶囊调控p38 MAPK/NLRP3/Caspase-1通路介导膝关节骨性关节炎软骨细胞焦亡的机制  

Mechanism of Cangxitongbi Capsules in Mediating Chondrocyte Pyroptosis in Knee Osteoarthritis via Regulating p38 MAPK/NLRP3/Caspase-1 Pathway

作　　者：张志猛 袁道通 靳希民 巩瑞 宋振龙 张永奎[3] 王晓乐 毕荣修[3] 谢文鹏 ZHANG Zhimeng;YUAN Daotong;JIN Ximin;GONG Rui;SONG Zhenlong;ZHANG Yongkui;WANG Xiaole;BI Rongxiu;XIE Wenpeng(The First Clinical Medical College of Shandong University of Traditional Chinese Medicine(TCM),Jinan 250014,China;Shandong Sport University,Jinan 250102,China;Affiliated Hospital of Shandong University of TCM,Jinan 250014,China)

机构地区：[1]山东中医药大学第一临床医学院,济南250014 [2]山东体育学院,济南250102 [3]山东中医药大学附属医院,济南250014

出　　处：《中国实验方剂学杂志》2024年第22期61-68,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金青年科学基金项目(82104897);山东省自然科学基金青年项目(ZR2020QH312);美罗青年医师创新发展项目(GSKQNJJ-2023-006);山东省中医药科技青年项目(Q-2022063);山东省医药卫生科技项目(202304071516)。

摘　　要：目的:探究苍膝通痹胶囊(CXTB)调控p38丝裂原活化蛋白激酶(p38 MAPK)/NOD样受体蛋白3(NLRP3)/胱天蛋白酶-1(Caspase-1)信号通路抑制膝关节骨性关节炎(KOA)软骨细胞焦亡的机制。方法:60只雄性SD大鼠随机分为假手术组、模型组、CXTB低、中、高剂量组及阳性药组,每组10只。使用改良Hulth法构建膝关节骨性关节炎大鼠模型,根据分组给予苍膝通痹胶囊(0.25、0.5、1.0 g·kg^(-1))及塞来昔布(24 mg·kg^(-1))灌胃,假手术组与模型组给予等体积生理盐水,连续灌胃28 d,1次/d。微计算机断层扫描(Micro-CT)检测骨体积分数(BV/TV)、骨小梁分离度(Tb.Sp),苏木素-伊红(HE)染色、番红固绿(SO)染色及国际骨关节炎研究协会(OARSI)评分观察膝关节退变水平,蛋白免疫印迹法(Western blot)检测p38 MAPK、磷酸化p38 MAPK(p-p38 MAPK)、NLRP3、Caspase-1、消皮素D(GSDMD)蛋白表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测p38 MAPK、NLRP3、Caspase-1、GSDMD mRNA表达,酶联免疫吸附测定法(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)炎性因子的含量。膝关节置换术后的软骨组织以Western blot检测p38 MAPK、p-p38 MAPK、NLRP3、Caspase-1、GSDMD蛋白表达,Real-time PCR检测p38 MAPK、NLRP3、Caspase-1、GSDMD mRNA表达。结果:Micro-CT显示,与假手术组比较,KOA大鼠关节间隙明显狭窄并见骨赘增生,BV/TV值减少、Tb.Sp值增加(P<0.01),血清中TNF-α、IL-1β、IL-18含量升高(P<0.01),软骨中p-p38 MAPK、NLRP3、Caspase-1、GSDMD蛋白表达升高(P<0.01),p38 MAPK、NLRP3、Caspase-1、GSDMD mRNA表达增强(P<0.01),与膝关节置换术后的正常软骨组织相比,病变软骨中的p-p38 MAPK、NLRP3、Caspase-1、GSDMD蛋白表达升高(P<0.05),p38 MAPK、NLRP3、Caspase-1、GSDMD mRNA表达增强(P<0.01)。HE染色及SO染色中可见KOA大鼠关节面粗糙,软骨厚度变薄,细胞排列无序杂乱,同时OARSI评分增加(P<0.01);与模型组比Objective:To explore the mechanism of Cangxi Tongbi capsules(CXTB)in regulating the p38 mitogen-activated protein kinase(p38 MAPK)/NOD-like receptor protein 3(NLRP3)/cysteine protease-1(Caspase-1)signaling pathway to inhibit pyroptosis of cartilage cells in knee osteoarthritis(KOA).Method:Sixty male SD rats were randomly divided into a sham operation group,a model group,low,medium,and high dose CXTB groups,and a positive control group,with 10 rats per group.The modified Hulth method was employed to establish a rat model of KOA.According to their respective assignments,rats were administered CXTB(0.25,0.5,1.0 g·kg^(-1))and Celecoxib(24 mg·kg^(-1))by gavage.The sham operation and model groups were given an equivalent volume of physiological saline.Treatment was performed once daily for 28 days.Microcomputed tomography(Micro-CT)was used to assess bone volume/total volume(BV/TV)and trabecular separation(Tb.Sp).Joint degeneration was evaluated through hematoxylin-eosin(HE)staining,safranin-fast green(SO)staining,and Osteoarthritis Research Society International(OARSI)scoring.Western blot analysis was conducted to measure the expression levels of p38 MAPK,phosphorylated p38 MAPK(p-p38 MAPK),NLRP3,Caspase-1,and gasdermin D(GSDMD)proteins.Real-time PCR was used to assess mRNA expression levels of p38 MAPK,NLRP3,Caspase-1,and GSDMD genes.Enzyme-linked immunosorbent assay(ELISA)was used to measure serum concentrations of inflammatory cytokines including tumor necrosis factor-alpha(TNF-α),interleukin-1 beta(IL-1β),and interleukin-18(IL-18).After knee replacement surgery,cartilage tissue was analyzed using Western blot to assess the protein expression levels of p38 MAPK,p-p38 MAPK,NLRP3,Caspase-1,and GSDMD,and Real-time PCR was used to evaluate gene expression levels of p38 MAPK,NLRP3,Caspase-1,and GSDMD.Result:Micro-CT analysis revealed significant narrowing of the joint space and increased bone spur formation in KOA rats compared with the sham operation group,with a decrease in BV/TV ratio and an increase in Tb.Sp valu

关 键 词：信号通路 p38丝裂原活化蛋白激酶(p38 MAPK) NOD样受体蛋白3(NLRP3) 胱天蛋白酶-1(Caspase-1) 软骨细胞 细胞焦亡 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33