中医正骨2025，Vol.37Issue(9)：7-15,25,10.
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
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
摘要
Abstract
Objective:To investigate the effects of Congrong Niuxi Tang(苁蓉牛膝汤,CRNXT)from Yunqi Fang(运气,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 equiva-lent 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 type Ⅱ collagen(COL2),matrix metalloproteinase-9(MMP-9),interleukin(IL)-1β,and IL-18 in the knee cartilage tissues were determined by using im-munohistochemical 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:① Mechanical 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 mechani-cal 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-1 β,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-1 β,and IL-18 compared to sham-opera-ted 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.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Key words
osteoarthritis,knee/chronic pain/Congrong Niuxi Tang/NAV1.7 voltage-gated sodium channel/doctrine of five evolutive pha-ses and six climatic factors/mice/animal experimentation引用本文复制引用
陈稼祥,徐诞,王其澜,钱玉王璇,李林,胡雪琴..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(9):7-15,25,10.基金项目
国家自然科学基金项目(82274679) （82274679）
