[1]陈稼祥,徐诞,王其澜,等.基于电压门控钠离子通道NAV1.7探究运气方苁蓉牛膝汤对膝骨关节炎慢性疼痛的影响及作用机制[J].中医正骨,2025,37(09):7-15,25.
　CHEN Jiaxiang,XU Dan,WANG Qilan,et al.Investigating the effects and mechanisms of Congrong Niuxi Tang(苁蓉牛膝汤)from Yunqi Fang(运气方)on chronic pain in knee osteoarthritis based on the voltage-gated sodium channel NAV1.7:an experimental study[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2025,37(09):7-15,25.
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基于电压门控钠离子通道NAV1.7探究运气方苁蓉牛膝汤对膝骨关节炎慢性疼痛的影响及作用机制()

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第37卷
期数:
2025年09期
页码:
7-15,25
栏目:
膝骨关节炎
出版日期:
2025-09-20

文章信息/Info

Title:
Investigating the effects and mechanisms of Congrong Niuxi Tang(苁蓉牛膝汤)from Yunqi Fang(运气方)on chronic pain in knee osteoarthritis based on the voltage-gated sodium channel NAV1.7:an experimental study
作者:
陈稼祥1; 徐诞2; 王其澜1; 钱玉王璇3; 李林1; 胡雪琴1
(1.浙江中医药大学附属第一医院/浙江省中医院,浙江 杭州 310006; 2.浙江中医药大学公共卫生学院,浙江 杭州 310053; 3.浙江中医药大学第二临床医学院,浙江 杭州 310053)
Author(s):
CHEN Jiaxiang1; XU Dan2; WANG Qilan1; QIAN Yuwangxuan3; LI Lin1; HU Xueqin1
1.The First Affiliated Hospital of Zhejiang Chinese Medical University(Zhejiang Provincial Hospital of Chinese Medicine),Hangzhou 310006,Zhejiang,China 2.School of Public Health,Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China 3.The Second Clinical Medical College,Zhejiang Chinese Medical University,Hangzhou 310053,Zhejiang,China
关键词:
骨关节炎; 膝;  慢性疼痛;  苁蓉牛膝汤;  电压门控钠离子通道NAV1.7;  运气学说;  小鼠;  动物实验
Keywords:
osteoarthritis; knee;  chronic pain;  Congrong Niuxi Tang;  NAV1.7 voltage-gated sodium channel;  doctrine of five evolutive phases and six climatic factors;  mice;  animal experimentation
摘要:
目的:基于电压门控钠离子通道NAV1.7探究运气方苁蓉牛膝汤对膝骨关节炎(knee osteoarthritis,KOA)慢性疼痛的影响及作用机制。方法:将18只8周龄C57BL/6J雄性小鼠随机分为3组,每组6只。模型组和苁蓉牛膝汤组采用内侧半月板失稳术于小鼠右后肢构建KOA模型,假手术组暴露关节腔后缝合皮肤。造模手术后第2天起,苁蓉牛膝汤组按0.02 mL·g-1以苁蓉牛膝汤浓缩液(生药浓度1 g·mL-1)灌胃,模型组和假手术组以等量生理盐水灌胃,每天1次,连续灌胃12周。造模后6周、8周、10周、12周分别采用Von Frey测痛纤维丝和智能热板仪测定小鼠机械性疼痛阈值和热敏性疼痛阈值。造模后12周药物干预结束后,取小鼠右侧膝关节和L4～L6右侧背根神经节,采用Micro-CT进行膝关节骨微结构检测; 采用阿尔辛蓝-苏木素-橙黄G染色进行膝关节软骨组织病理学观察,并采用国际骨关节炎研究学会(Osteoarthritis Research Society International,OARSI)评分系统对膝关节软骨病理变化进行评价; 采用免疫组织化学染色检测膝关节软骨中Ⅱ型胶原、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)、白细胞介素(interleukin,IL)-1β、IL-18表达情况; 采用免疫荧光技术检测L4～L6背根神经节中NAV1.7和c-fos表达情况及膝关节软骨中NAV1.7表达情况。结果:①机械性疼痛阈值测定结果。模型组机械性疼痛阈值随时间变化不明显(F=1.764,P=0.186),假手术组和苁蓉牛膝汤组机械性疼痛阈值随时间变化均呈先升高后降低的趋势(F=6.764,P=0.002; F=4.188,P=0.019)。造模后6周、8周、10周、12周,模型组的机械性疼痛阈值均低于假手术组和苁蓉牛膝汤组(P=0.000,P=0.042; P=0.002,P=0.009; P=0.000,P=0.000; P=0.000,P=0.001)。②热敏性疼痛阈值测定结果。热敏性疼痛阈值随时间变化总体呈先升高后降低的趋势(F=14.383,P=0.000)。模型组的热敏性疼痛阈值低于假手术组和苁蓉牛膝汤组(P=0.000,P=0.000)。③膝关节骨微结构检测结果。Micro-CT检查结果显示,假手术组膝关节结构基本正常,模型组可见明显骨赘和软骨下骨钙化,苁蓉牛膝汤组可见轻微骨赘和软骨下骨钙化。模型组的骨体积分数、骨小梁厚度均高于假手术组和苁蓉牛膝汤组(P=0.000,P=0.012; P=0.000,P=0.000),骨小梁分离度低于假手术组和苁蓉牛膝汤组(P=0.000,P=0.027)。④膝关节软骨组织病理学观察结果。模型组的膝关节软骨OARSI评分高于假手术组和苁蓉牛膝汤组(P=0.000,P=0.004)。⑤膝关节软骨中Ⅱ型胶原、MMP-9、IL-1β、IL-18表达情况检测结果。模型组的Ⅱ型胶原阳性表达面积小于假手术组和苁蓉牛膝汤组(P=0.000,P=0.000),MMP-9、IL-1β、IL-18阳性表达面积均大于假手术组和苁蓉牛膝汤组(P=0.000,P=0.001; P=0.000,P=0.002; P=0.000,P=0.000)。⑥L4～L6背根神经节中NAV1.7和c-fos表达情况检测结果。模型组L4～L6背根神经节中NAV1.7和c-fos阳性表达面积均大于假手术组(NAV1.7:P=0.001,P=0.000,P=0.000; c-fos:P=0.001,P=0.000,P=0.000)和苁蓉牛膝汤组(NAV1.7:P=0.006,P=0.013,P=0.003; c-fos:P=0.019,P=0.018,P=0.006)。⑦膝关节软骨中NAV1.7表达情况检测结果。模型组膝关节软骨中NAV1.7阳性表达面积大于假手术组和苁蓉牛膝汤组(P=0.005,P=0.006)。结论:运气方苁蓉牛膝汤可改善KOA模型小鼠的慢性疼痛症状,其机制可能与下调NAV1.7表达水平,以及减轻炎症反应和延缓软骨退变有关。
Abstract:
Objective:To investigate the effects of Congrong Niuxi Tang(苁蓉牛膝汤,CRNXT)from Yunqi Fang(运气方,YQF)on chronic pain in knee osteoarthritis(KOA)via the voltage-gated sodium channel subtype NAV1.7,and to explore its underlying mechanisms.Methods:Eighteen 8-week-old male C57BL/6J mice were randomly divided into 3 groups,6 cases in each group.All mice but the ones in sham-operated group were subjected to destabilization of the medial meniscus(DMM)on the right hind limbs to build KOA models,while the ones in sham-operated group were merely incised the skin to expose the knee joint cavity at the corresponding site and then sutured.From day 2 after the modeling surgery,the mice in CRNXT group were intervened by intragastric administration with CRNXT concentrate at a dose of 0.02 mL/g(the crude drug concentration was 1 g/mL),while the ones in model group and sham-operated group with the equivalent volume of normal saline,once a day for consecutive 12 weeks.At 6,8,10,and 12 weeks post-modeling,the mechanical allodynia and thermal hyperalgesia thresholds were assessed using Von Frey filaments and a hot plate test,respectively.At 12 weeks post-modeling(after the end of drug intervention),the mice were euthanized,and the right knee joints and ipsilateral L4-L6 dorsal root ganglia(DRGs)were harvested,then the knee joint specimens were scanned by Micro-CT for analyzing the bone microarchitecture such as bone volume/tissue volume(BV/TV),trabecular thickness(Tb.Th),and trabecular separation(Tb.Sp).After that,the knee joint cartilage tissues were stained with alcian blue-hematoxylin-orange G(ABH-OG)for observing the pathological changes,and the severity of cartilage degeneration was scored using the Osteoarthritis Research Society International(OARSI)grading system.Furthermore,the expression of typeⅡcollagen(COL2),matrix metalloproteinase-9(MMP-9),interleukin(IL)-1β,and IL-18 in the knee cartilage tissues were determined by using immunohistochemical staining,and the expression of NAV1.7 and c-fos in the L4-L6 DRGs,as well as the expression of NAV1.7 in the knee joint cartilage tissues were detected by using immunofluorescence staining.Results:①Mechanical pain threshold.The mechanical pain threshold in model group remained relatively stable,showing no significant change over time(F=1.764,P=0.186),whereas the mechanical pain thresholds in both sham-operated group and CRNXT group presented a biphasic trajectory of initial increase followed by decrease over time(F=6.764,P=0.002; F=4.188,P=0.019).Furthermore,the mechanical pain threshold was lower in model group compared to sham-operated group and CRNXT group at 6,8,10,and 12 weeks post-modeling(P=0.000,P=0.042; P=0.002,P=0.009; P=0.000,P=0.000; P=0.000,P=0.001).②Thermal pain threshold.The thermal pain threshold exhibited a biphasic trajectory of initial increase followed by subsequent decrease over time in general(F=14.383,P=0.000),and it was lower in model group compared to sham-operated group and CRNXT group(P=0.000,P=0.000).③Bone microarchitecture of the knee joints.Micro-CT examination revealed that the knee joint structure of mice in sham-operated group remained basically normal,while,in mice of model group,the obvious osteophytes and subchondral bone calcification were observed,which were mild in the CRNXT group.The BV/TV and Tb.Th were higher and the Tb.Sp was lower in model group compared to sham-operated group and CRNXT group(P=0.000,P=0.012; P=0.000,P=0.000; P=0.000,P=0.027).④Histopathological analysis of knee joint cartilage tissues.The OARSI score was higher in model group compared to sham-operated group and CRNXT group(P=0.000,P=0.004).⑤Expression of COL2,MMP-9,IL-1β,and IL-18 in knee joint cartilage.The model group exhibited a smaller positive area for COL2,but larger positive areas for MMP-9,IL-1β,and IL-18 compared to sham-operated group and CRNXT group(P=0.000,P=0.000; P=0.000,P=0.001; P=0.000,P=0.002; P=0.000,P=0.000).⑥Expression of NAV1.7 and c-fos in the L4-L6 DRGs.The positive expression areas of NAV1.7 and c-fos in the L4-L6 DRGs were larger in model group compared to sham-operated group and CRNXT group(NAV1.7:P=0.001,P=0.000,P=0.000; P=0.006,P=0.013,P=0.003; c-fos:P=0.001,P=0.000,P=0.000; P=0.019,P=0.018,P=0.006).⑦Expression of NAV1.7 in the knee joint cartilage.The NAV1.7-positive expression area in the knee joint cartilage were larger in model group compared to sham-operated group and CRNXT group(P=0.005,P=0.006).Conclusion:CRNXT from YQF can alleviate the chronic pain symptoms of KOA model mice.It may work by down-regulating the expression level of NAV1.7,reducing inflammatory response and delaying cartilage degeneration.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金项目(82274679)
通讯作者:胡雪琴 E-mail:20151095@zcmu.edu.cn
更新日期/Last Update: 1900-01-01