黄芩清热除痹胶囊调控p53/p21信号通路延缓骨关节炎大鼠软骨细胞衰老的机制研究  

Mechanism of Huangqin Qingre Chubi Capsules regulating p53/p21 signaling pathway to delay chondrocyte senescence in osteoarthritic rats

作　　者：周巧[2,3] 刘健[1] 朱艳[2] 汪元[1] 王桂珍[1] 万磊[1] 黄旦 郭锦晨 齐亚军[3] 胡月迪 ZHOU Qiao;LIU Jian;ZHU Yan;WANG Yuan;WANG Gui-zhen;WAN Lei;HUANG Dan;GUO Jin-chen;QI Ya-jun;HU Yue-di(the First Affiliated Hospital,Anhui University of Chinese Medicine,Hefei 230031,China;the Second Affiliated Hospital,Anhui University of Chinese Medicine,Hefei 230061,China;Anhui University of Chinese Medicine,Hefei 230012,China)

机构地区：[1]安徽中医药大学第一附属医院,安徽合肥230031 [2]安徽中医药大学第二附属医院,安徽合肥230061 [3]安徽中医药大学,安徽合肥230012

出　　处：《中国中药杂志》2024年第12期3330-3339,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(82274310);安徽省名中医刘健工作室建设项目(中医药发展秘[2018] 11号);安徽省中医药领军人才项目(中医药发展秘[2018] 23号);高水平中医药重点学科建设项目(zyyzdxk-2023100);安徽现代中医内科应用基础与开发研究省级实验室项目(2016080503B041);安徽省高等学校科学研究(自然科学类)重点项目(2022AH050449);安徽省高校自然科学重大项目(2023AH040112);青年人才培养“杏林青秀培育计划”项目(0500-48-65)。

摘　　要：探讨黄芩清热除痹胶囊(HQC)调控肿瘤抑制蛋白53(tumor protein p53,p53)/细胞周期蛋白依赖性激酶抑制剂1A(cyclin-dependent kinase inhibitor 1A,p21)通路延缓骨关节炎(OA)大鼠软骨细胞衰老的机制。碘乙酸钠(MIA)联合外界风湿热环境刺激,诱导OA大鼠风湿热痹模型,分为正常(Con)组,OA模型(MIA)组,OA模型+风湿热刺激模型(MIA-M)组,MIA-M+HQC低(MIA-M+HQC-L)、中(MIA-M+HQC-M)、高(MIA-M+HQC-H)剂量组,MIA-M+氨基葡萄糖(MIA-M+GS)组,模型制备后灌胃30 d,苏木素-伊红(HE)和番红O固绿(SO)染色观察软骨病理变化,酶联免疫吸附(ELISA)法检测白细胞介素(IL)-1β和IL-6的表达,流式细胞术(FCM)检测细胞凋亡和周期,实时荧光定量PCR(RT-qPCR)检测基质降解酶13(MMP13)、聚蛋白多糖酶5(ADAMTS-5)、Ⅱ型胶原(COLⅡ)和转化生长因子-β(TGF-β)mRNA的表达,蛋白免疫印迹(WB)检测p53/p21通路、细胞周期蛋白依赖性激酶抑制剂2A(p16)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2关联X蛋白(Bax)蛋白的表达。Con组大鼠关节软骨表面光滑平整,潮线光滑平整;MIA组和MIA-M组软骨层破坏明显,软骨基质减少,MIA-M组情况更严重。HQC高剂量组和MIA-M+GS组软骨表面基本完整,分层清晰。与MIA-M+HQC-H组相比,低、中剂量Mankin′s评分较高,MIA-M+GS组变化不明显。与Con组相比,MIA和MIA-M组软骨细胞G_1的比例升高,S期、G_2期比例下降,细胞凋亡率增加;与MIA-M组相比,HQC各剂量组以浓度依赖的方式抑制细胞凋亡,促进增殖;与MIA-M+HQC-H组相比,高剂量较中、低剂量作用更显著,与MIA-M+GS组相比差异无统计学意义。与Con组相比,MIA和MIA-M组IL-1β和IL-6升高,MMP13和ADAMTS-5 mRNA水平升高,p53、p21、p16和Bax蛋白升高,COLⅡ、TGF-β mRNA水平下降;与MIA-M组相比,HQC和GS药物干预后IL-1β和IL-6下降,MMP13和ADAMTS-5 mRNA、p53、p21、Bax和p16蛋白水平下降,Bcl-2升高;与MIA-M+HQC-H组相比,这些指标改善优于HQC低、中剂量组,与MIA-M+GS组差异�This study aims to investigate the mechanism of Huangqin Qingre Chubi Capsules(HQC)in delaying chondrocyte senescence of osteoarthritic(OA)rats by regulating the p53/p21 signaling pathway.Rheumatic fever paralysis models of OA rats were induced based on monosodiun iodoacetate(MIA)combined with external rheumatic fever environmental stimuli and divided into normal(Con)group,OA model(MIA)group,OA model+rheumatic fever stimulation model(MIA-M)group,MIA-M+HQC low-dose(MIA-M+HQC-L)group,medium-dose(MIA-M+HQC-M)group,and high-dose(MIA-M+HQC-H)group,and MIA-M+glucosamine(MIA-M+GS)group.The models were successfully prepared and administered by gavage for 30 d.The pathological changes of cartilage were observed by hematoxylin-eosin(HE)and Senna O solid green(SO)staining.The expression of interleukin(IL)-1βand IL-6 was detected by enzyme-linked immunosorbent assay(ELISA).Flow cytometry(FCM)was used to detect apoptosis and cell cycle.The mRNA expression of MMP13,ADAMTS-5,COLⅡ,and TGF-βwas detected by RT-qPCR.The protein expression of p53/p21,p16,Bax,and Bcl-2 was detected by Western blot.The articular cartilage surface of rats in the Con group was smooth,and the tide line was smooth.The cartilage layer of MIA and MIA-M groups was obviously damaged,and the cartilage matrix was reduced.The above conditions were more severe in the MIA-M group.The cartilage surface of the HQC high-dose group and MIA-M+GS group was basically intact with clear delamination.Compared with the MIA-M+HQC-H group,Mankin's score was higher in the HQC low-dose and medium-dose groups,and the change was not obvious in the MIA-M+GS group.Compared with the Con group,the proportion of chondrocytes G1 was elevated in the MIA and MIA-M groups,and the proportion of the S phase and G2 phase was significantly decreased.In addition,the apoptosis rate was increased.Compared with MIA-M,HQC groups inhibited apoptosis and promoted cell proliferation in a concentration-dependent manner.Compared with the MIA-M+HQC-H group,the effect was more significant in the HQC h

关 键 词：黄芩清热除痹胶囊 骨关节炎 风湿热模型 细胞衰老 p53/p21 

分 类 号：R285.5[医药卫生—中药学]