透骨消痛胶囊调控Nav1.7减轻膝骨关节炎小鼠软骨细胞退变

doi:10.12122/j.issn.1673-4254.2024.11.03

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (11): 2074-2081.doi: 10.12122/j.issn.1673-4254.2024.11.03

• • 上一篇

透骨消痛胶囊调控Nav1.7减轻膝骨关节炎小鼠软骨细胞退变

付长龙¹^,²(), 林艳铭¹^,², 兰书洁¹^,², 陈悦³, 李超¹^,², 陆诗雨¹^,², 林晴³()

^1.福建中医药大学中西医结合学院（中西医结合研究院），福建福州 350122
^2.福建省中西医结合老年性疾病重点实验室，福建福州 350122
^3.福建中医药大学中医学院，福建福州 350122

收稿日期:2024-07-23 出版日期:2024-11-20 发布日期:2024-11-29
通讯作者: 林晴 E-mail:993001232@qq.com;707425971@qq.com
作者简介:付长龙，博士，副研究员，E-mail: 993001232@qq.com
基金资助:
国家自然科学基金(82474532);福建中医药大学校管课题(X2023024);福建中医药大学高层次人才科研启动资金项目（X2019011-人才）;陈可冀中西医结合发展基金项目(CKJ2021018)

Tougu Xiaotong Capsule alleviates cartilage degeneration in mice with knee osteoarthritis by modulating Nav1.7

Changlong FU¹^,²(), Yanming LIN¹^,², Shujie LAN¹^,², Yue CHEN³, Chao LI¹^,², Shiyu LU¹^,², Qing LIN³()

^1.College of Integrative Medicine (Academy of Integrative Medicine), Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China
^2.Fujian Key Laboratory of Integrative Medicine on Geriatrics, Fuzhou 350122, China
^3.College of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China

Received:2024-07-23 Online:2024-11-20 Published:2024-11-29
Contact: Qing LIN E-mail:993001232@qq.com;707425971@qq.com
Supported by:
National Natural Science Foundation of China(82474532)

摘要/Abstract

摘要：

目的从Nav1.7调控软骨细胞外基质稳态角度，研究透骨消痛胶囊对减轻膝骨关节炎（KOA）软骨细胞退变的作用机制。方法选择40只2月龄C57BL/6小鼠，通过随机数字表法将其分为空白组（n=10）和造模组（n=30）。造模组小鼠通过Hulth法手术建立KOA模型后，随机分为模型组（生理盐水灌胃）、透骨消痛胶囊组（368 mg/kg剂量灌胃）、阳性药物组（10 mg/kg双氯芬酸钠灌胃），10只/组；空白组为生理盐水灌胃，干预6次/周，共4周。干预后5%异氟醚麻醉处死，获得膝关节软骨组织。形态学染色观察软骨组织结构变化，实时荧光定量PCR检测Nav1.7 mRNA水平，Western blotting分析Nav1.7、MMP-3、ADAMTS-5、COX-2蛋白表达变化。荧光原位杂交检测软骨细胞中Nav1.7的表达量。此外，利用慢病毒载体敲减Nav1.7（sh-Nav1.7）转染软骨细胞，实时荧光定量PCR分析转染后sh-Nav1.7在软骨细胞中mRNA水平变化。Western blotting分析在sh-Nav1.7条件下，透骨消痛胶囊对KOA软骨细胞MMP-3、MMP-13、ADAMTS-4、ADAMTS-5、COX-2蛋白的调控。结果形态学结果显示，和模型组对比，透骨消痛胶囊组和阳性药物组软骨层结构更完整，关节结构破坏程度减轻。透骨消痛胶囊组和阳性药物组的Nav1.7蛋白与mRNA水平降低（P<0.05），MMP-3、ADAMTS-5、COX-2蛋白表达减少（P<0.05）。荧光原位杂交结果显示，透骨消痛胶囊能降低IL-1β诱导的Nav1.7的荧光强度。在Nav1.7敲减实验中，与IL-1β组相比，IL-1β+sh-Nav1.7组中Nav1.7水平下降（P<0.05）；与IL-1β+TGXTC组相比，IL-1β+sh-Nav1.7+TGXTC组中Nav1.7水平下降（P<0.05）。Western blotting结果表明，IL-1β组软骨细胞中升高的MMP-3、MMP-13、ADAMTS-4、ADAMTS-5、COX-2蛋白被透骨消痛胶囊干预后显著抑制（P<0.05），但在Nav1.7敲低后，透骨消痛胶囊对这些蛋白的调控作用减弱（P<0.05）。结论透骨消痛胶囊通过调控Nav1.7减轻KOA软骨细胞外基质代谢紊乱，从而发挥减轻软骨细胞退变的作用。

关键词: 透骨消痛胶囊, Nav1.7, 软骨退变, 膝骨关节炎

Abstract:

Objective To investigate the mechanism by which Tougu Xiaotong Capsule (TGXTC) alleviates chondrocyte degeneration in knee osteoarthritis (KOA). Methods Thirty 2-month-old C57BL/6 mouse models of KOA established using the Hulth method were randomized into model group, TGXTC group, and diclofenac sodium group and received treatment with saline, TGXTC (368 mg/kg), and diclofenac sodium (10 mg/kg) by gavage, respectively, with another 10 untreated mice as the blank control group. All interventions were administered 6 times a week for 4 weeks. After the treatments, structural changes in the cartilage tissue were observed with morphological staining, and Nav1.7 mRNA expression and the protein expression levels of Nav1.7, MMP-3, ADAMTS-5, and COX-2 were detected using RT-qPCR and Western blotting. Fluorescence in situ hybridization (FISH) was used to detect Nav1.7 expression in the chondrocytes. In cultured KOA chondrocytes, the effect of TGXTC and lentivirus-mediated Nav1.7 knockdown on MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, and COX-2 protein expressions were assessed with Western blotting. Results In KOA mice treatments with TGXTC and diclofenac sodium both significantly alleviated structural damage of the cartilage layer, reduced Nav1.7 protein and mRNA expressions and lowered the expressions of MMP-3, ADAMTS-5, and COX-2 proteins in the cartilage tissues. FISH results indicated that TGXTC treatment significantly reduced IL-1β-induced Nav1.7 expression in the chondrocytes. In Nav1.7 knockdown experiment, Nav1.7 levels were significantly lower in IL-1β+sh-Nav1.7 group than in IL-1β group, and also lower in IL-1β+TGXTC group than in IL-1β+sh-Nav1.7+TGXTC group. TGXTC treatment significantly inhibited IL-1β-induced elevation of MMP-3, MMP-13, ADAMTS-4, ADAMTS-5 and COX-2 protein expressions in the chondrocytes, but its effects were strongly weakened by Nav1.7 knockdown. Conclusion TGXTC alleviates extracellular matrix metabolic disorder in KOA chondrocytes by regulating Nav1.7, thereby mitigating chondrocyte degeneration in KOA mice.

Key words: Tougu Xiaotong Capsule, Nav1.7, chondrocyte degeneration, knee osteoarthritis

付长龙, 林艳铭, 兰书洁, 陈悦, 李超, 陆诗雨, 林晴. 透骨消痛胶囊调控Nav1.7减轻膝骨关节炎小鼠软骨细胞退变[J]. 南方医科大学学报, 2024, 44(11): 2074-2081.

Changlong FU, Yanming LIN, Shujie LAN, Yue CHEN, Chao LI, Shiyu LU, Qing LIN. Tougu Xiaotong Capsule alleviates cartilage degeneration in mice with knee osteoarthritis by modulating Nav1.7[J]. Journal of Southern Medical University, 2024, 44(11): 2074-2081.

图/表 5

图1 各组关节软骨HE、番红O-固绿和甲苯胺蓝染色图

Fig.1 HE, safranin O and toluidine blue staining of the articular cartilage in different groups (Original magnification: ×100).

图2 软骨组织中Nav1.7、MMP-3、ADAMTS-5、COX-2蛋白表达

Fig.2 Nav1.7, MMP-3, ADAMTS-5, and COX-2 protein expressions in the cartilage tissue in different groups. A-E: Western blotting and quantitative data of Nav1.7, MMP-3, ADAMTS-5, and COX-2 expressions in each group (n=3). *P<0.05 vs Sham group, #P<0.05 vs Model group.

图3 Nav1.7转染小鼠软骨细胞的MOI

Fig.3 Multiplicity of infection conditions of Nav1.7 transfection into the chondrocytes.

图4 荧光原位杂交检测结果

Fig.4 Fluorescence in situ hybridization reveals Nav1.7 expression in the cytoplasm of the chondrocytes (×100). *P<0.05 vs Sham group; #P<0.05 vs IL-1β group.

图5 MMP-3、MMP-13、ADAMTS-4、ADAMTS-5、COX-2蛋白表达

Fig.5 MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, and COX-2 proteins expressions in the chondrocytes with TGXTC treatment and lentivirus-mediated Nav1.7 knockdown. A-F: Western blotting and quantitative data of MMP-3, MMP-13, ADAMTS-4, ADAMTS-5 and COX-2 expressions in each group (n=3). *P<0.05 vs Sham group; #P<0.05 vs IL-1β group; &P<0.05 vs IL-1β+sh-Nav1.7 group; △P<0.05 vs IL-1β+TGXTC group.

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透骨消痛胶囊调控Nav1.7减轻膝骨关节炎小鼠软骨细胞退变

Tougu Xiaotong Capsule alleviates cartilage degeneration in mice with knee osteoarthritis by modulating Nav1.7

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