膝痹宁通过CPT1调节大鼠滑膜氧化还原稳态缓解膝骨关节炎疼痛的效应机制  

Mitigation mechanism of Xibining decoction on pain of knee osteoarthritis by regulating redox homeostasis of synoviocytes through CPT1 enzyme

作　　者：廖太阳 张力[1,2,3] 丁亮[1,2] 李晓辰[1,2] 吴鹏[1,2] 梅伟 张农山[1,2] 王培民[1,2] 张立[1,2] Liao Tai-Yang;Zhang Li;Ding Liang;Li Xiao-Chen;Wu Peng;Mei Wei;Zhang Nong-Shan;Wang Pei-Min;Zhang Li(Department of Orthopedics,Affilliated Hospital of Nanjing University of Chinese Medicine,Nanjing,Jiangsu 210029,China;Department of Orthopedics,Jiangsu Province Hospital of Chinese Medicine,Nanjing,Jiangsu 210029,China;Key Laboratory for Metabolic Diseases in Chinese Medicine,Nanjing University of Chinese Medicine,Nanjing,Jiangsu 210023,China)

机构地区：[1]南京中医药大学附属医院骨伤科,江苏南京210029 [2]江苏省中医院骨伤科,江苏南京210029 [3]南京中医药大学代谢病中医研究重点实验室,江苏南京210023

出　　处：《解放军医学杂志》2023年第1期49-57,共9页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81904224);江苏省中医药领军人才项目(SLJ0207);江苏省研究生培养创新工程资助项目(KYCX21_1645,KYCX21_1675)。

摘　　要：目的探讨膝痹宁通过肉毒碱棕榈酰转移酶(CPT1)调节大鼠滑膜氧化还原稳态缓解膝骨关节炎(KOA)疼痛的效应机制。方法提取大鼠膝关节成纤维样滑膜细胞(FLS),采用CCK-8法筛选膝痹宁冻干粉干预FLS细胞的合适作用浓度。将FLS细胞分为对照组、KOA组、膝痹宁组,后两组采用5μg/ml脂多糖(LPS)诱导KOA炎症细胞模型,膝痹宁组加入100μg/ml膝痹宁,培养24 h。采用Real-time PCR和Western blotting检测CPT1、肉碱/有机阳离子转运体1(OCTN1)mRNA和蛋白的表达;采用试剂盒检测CPT1、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量;使用2?,7?-二氯荧光素二乙酸酯检测活性氧(ROS)水平。提取大鼠背根神经节(DRG)神经元细胞,采用βⅢ-微管蛋白(βⅢ-tubulin)与胶质纤维酸性蛋白(GFAP)免疫荧光染色进行鉴定。将收集的各组FLS上清加入DRG神经元中干预24 h,分别设为对照组、KOA组、膝痹宁组,采用Real-time PCR和Western blotting检测DRG神经元瞬时受体电位A1离子通道(TRPA1)mRNA和蛋白的表达,实时荧光钙成像观察DRG神经元中TRPA1开放后Ca^(2+)内流情况,ELISA法检测降钙素基因相关肽(CGRP)和P物质(SP)水平。结果选取膝痹宁冻干粉浓度100μg/ml进行实验。Real-time PCR和Western blotting检测结果显示,膝痹宁组CPT1、OCTN1 mRNA和蛋白表达水平高于KOA组(P<0.05)。与对照组比较,KOA组ROS水平[(26.46±2.07)AU vs.(5.52±0.78)AU]和MDA含量[(3.13±0.02)nmol/ml vs.(2.77±0.03)nmol/ml]明显升高(P<0.05),CPT1[(4.98±0.02)nmol/min vs.(11.50±0.21)nmol/min]和SOD[(11.38±0.05)U/ml vs.(17.6±0.07)U/ml]活性明显降低(P<0.05);与KOA组比较,膝痹宁组ROS水平[(14.07±1.41)AU]和MDA含量[(2.87±0.01)nmol/ml]明显降低(P<0.05),CPT1[(7.94±0.21)nmol/min]和SOD活性[(13.81±0.07)U/ml]明显升高(P<0.05)。与KOA组比较,膝痹宁组FLS上清干预可抑制DRG神经元中TRPA1 mRNA和蛋白的表达,以及TRPA1开放后Ca^(2+)的内流(P<0.05),降低CGRP和SP表达量[(19.93±1.2Objective To investigate the action mechanism of Xibining decoction on mitigation of pain of knee osteoarthritis(KOA)through regulation of redox homeostasis of synoviocytes by carnitine palmitoyl transferase 1(CPT1)enzyme.Methods Fibroblast-like synovial cells(FLS)were extracted from rats?knee joint and the optimal concentration of the freezedried powder of Xibining decoction on FLS was selected by CCK-8.Subsequently,the cells were divided into control group,KOA group and Xibining group.The inflammatory environment of KOA was induced by 5μg/ml lipopolysaccharide(LPS)in the last two groups,and in Xibining group,100μg/ml Xibining were added and cultured for 24 hours.The mRNA and protein expressions of CPT1 and carnitine/organic cation transporters 1(OCTN1)were detected by Real-time PCR and Western blotting.The activities of CPT1 and superoxide dismutase(SOD)and malondialdehyde(MDA)content were detected by assay kits.Reactive oxygen species(ROS)level was detected by a 2?,7?-dichlorofluorescein diacetate assay.The dorsal root ganglion(DRG)neurons were extracted from rats and identified byβⅢ-tubulin and glial fibrillary acidic protein(GFAP)immunofluorescence.The neurons were divided into control group,KOA group and Xibining group,the FLS supernatants of the three groups were added to the DRG for 24 hours.The mRNA and protein expression of transient receptor potential A1 ion channel(TRPA1)were detected by Real-time PCR and Western blotting.Ca^(2+)influx in DRG neurons after TRPA1 opening was observed by Real-time fluorescent calcium imaging.ELISA assay was used to detect the content of calcitonin gene related peptide(CGRP)and substance P(SP)in DRG supernatant.Results The ideal freeze-dried powder of Xibining decoction concentration was identified as 100μg/ml.Real-time PCR and Western blotting results showed that the expression levels of mRNA and protein in CPT1 and OCTN1 of Xibining group were significantly higher than those in KOA group(P<0.05).Compared with control group,the average fluorescence intensity and M

关 键 词：膝骨关节炎 肉毒碱棕榈酰转移酶 膝痹宁 背根神经节 疼痛 

分 类 号：R285.5[医药卫生—中药学]