| 摘要: |
| 目的：通过观察Ras同源基因家族成员A/Rho相关卷曲螺旋蛋白激酶信号通路（RhoA/Rock signaling pathway）相关蛋白RhoA、ROCK1、细胞外信号调节激酶1（ERK1）、细胞外信号调节激酶2（ERK2）的表达以及血清基质金属蛋白酶-3（MMP-3）、基质金属蛋白酶-13（MMP-13）水平的变化，探讨温针灸延缓膝骨性关节炎（KOA）大鼠软骨退变的作用机制。方法：将30只SD大鼠随机分为空白组、模型组、温针灸组，每组各10只。其中，模型组与温针灸组大鼠在适应性喂养后的第1、4、7天，对其双膝关节注射4%木瓜蛋白酶0.9%氯化钠注射液0.2 mL复制KOA模型。正常饲养2周后，空白组、模型组大鼠与温针灸组同等同频抓取、捆绑固定，不做干预；温针灸组大鼠给予温针灸干预，选双膝内膝眼、犊鼻穴，进针约5 mm，留针20 min，每次每穴灸2壮，每天1次，干预6 d休息1 d，连续干预4周后取材。采用ELISA法检测大鼠血清MMP-3、MMP-13水平，番红O-固绿染色法检测大鼠膝关节软骨组织形态学并进行改良Mankin’s评分，免疫组化法检测大鼠膝关节软骨组织中RhoA、ROCK1、ERK1、ERK2的蛋白表达。结果：与空白组相比，模型组大鼠血清MMP-3、MMP-13水平显著上升，改良Mankin’s评分显著上升，RhoA、ROCK1、ERK1、ERK2蛋白表达显著增加，差异均具有统计学意义（P＜0.01）；番红O-固绿染色显示软骨组织表面有破损，部分存在裂隙可达移行层，软骨结构紊乱、潮线不清晰，染色程度减少，软骨细胞增多或簇集成团。与模型组相比，温针灸组大鼠血清MMP-3、MMP-13水平显著下降，改良Mankin’s评分显著下降，RhoA、ROCK1、ERK1、ERK2蛋白表达明显减少，差异均有统计学意义（P＜0.01），软骨总体状况较模型组有一定程度改善；软骨面较平整，四层结构较明确，染色程度介于模型组与空白组之间，软骨细胞数量相对增多，潮线较清楚。结论：温针灸可能通过下调KOA大鼠血清MMP-3、MMP-13水平，改善软骨病理形态，以及RhoA/ROCK信号通路相关蛋白RhoA、ROCK1、ERK1、ERK2表达水平以延缓软骨细胞退变，发挥对KOA的治疗作用。 |
| 关键词: 膝骨性关节炎 温针灸 RhoA/ROCK信号通路 ERK1 ERK2 软骨退变 |
| DOI： |
|
|
| Mechanism of action of warming acupuncture-moxibustion in delaying cartilage degeneration in a rat model of knee osteoarthritis |
| XIAO Yang,HONG Kunda |
| (Fujian University of Traditional Chinese Medicine，Fuzhou 350122，Fujian，China;The Second Affiliated Hospital of Fujian Medical University，Quanzhou 362000，Fujian，China) |
| Abstract: |
| Objective：To investigate the mechanism of action of warming acupuncture-moxibustion in delaying cartilage degeneration in a rat model of knee osteoarthritis (KOA) by observing the changes in the expression of ras homolog gene family，member A (RhoA)，Rho-associated coiled-coil kinase 1 (ROCK1)，extracellular signal-regulated kinase 1 (ERK1)，and extracellular signal-regulated kinase 2 (ERK2) associated with the RhoA/Rho-associated kinase (ROCK) signaling pathway and the serum levels of matrix metalloproteinase-3 (MMP-3) and matrix metalloproteinase-13 (MMP-13).Methods：A total of 30 Sprague-Dawley rats were randomly divided into blank group，model group，and warming acupuncture-moxibustion group，with 10 rats in each group.The rats in the model group and the warming acupuncture-moxibustion group were given injection of 4% papain normal saline solution (0.2 mL) into both knee joints on days 1，4，and 7 after adaptive feeding to establish a model of KOA.After normal feeding for 2 weeks，the rats in the blank group，the model group，and the warming acupuncture-moxibustion group were caught and fixed at the same frequency without any intervention，and the rats in the warming acupuncture-moxibustion group were given warming acupuncture-moxibustion at Neixiyan and Dubi points of both knee joints，with a depth of 5 mm for needle insertion，a needle retention time of 20 minutes，and 2 moxa cones for each acupoint each time，once a day for 6 consecutive days，followed by 1 day of rest.Samples were collected after intervention for 4 consecutive weeks.ELISA was used to measure the serum levels of MMP-3 and MMP-13；safranin O-fast green staining was used to observe the histomorphology of knee articular cartilage and determine modified Mankin’s score；immunohistochemistry was used to measure the protein expression levels of RhoA，ROCK1，ERK1，and ERK2 in knee articular cartilage.Results：Compared with the blank group，the model group had significant increases in the serum levels of MMP-3 and MMP-13，modified Mankin’s score，and the protein expression levels of RhoA，ROCK1，ERK1，and ERK2 (P＜0.01)，and safranin O-fast green staining showed ruptures on the surface of cartilage，fissures reaching the transitional layer，unclear structures of cartilage and tidal line，a reduction in the degree of staining，and an increase or aggregation of chondrocytes.Compared with the model group，the warming acupuncture-moxibustion group had significant reductions in the serum levels of MMP-3 and MMP-13，modified Mankin’s score，and the protein expression levels of RhoA，ROCK1，ERK1，and ERK2，as well as a certain degree of improvement in the overall condition of cartilage (P＜0.01)；with a relatively smooth surface of cartilage，a clear four-layer structure，a degree of staining between the model group and the blank group，an increase in the number of chondrocytes，and a relatively clear tidal line.Conclusion：Warming acupuncture-moxibustion can delay cartilage degeneration and exert a therapeutic effect on KOA by downregulating the serum levels of MMP-3 and MMP-13，improving the pathomorphology of cartilage，and reducing the protein expression levels of RhoA，ROCK1，ERK1，and ERK2 associated with the RhoA/ROCK signaling pathway. |
| Key words: knee osteoarthritis warming acupuncture-moxibustion ras homolog gene family,member A/Rho-associated kinase signaling pathway extracellular signal-regulated kinase 1 extracellular signal-regulated kinase 2 cartilage degeneration |
