基于p38丝裂原活化蛋白激酶通路探讨瑶医除闷汤药浴治疗膝骨关节炎大鼠的作用机制

doi:10.20047/j.issn1673-7210.2023.31.02

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摘要目的基于p38丝裂原活化蛋白激酶（p38MAPK）通路探讨瑶医除闷汤药浴治疗膝骨关节炎（KOA）模型大鼠的效果与作用机制。方法在60只8周龄雄性SPF级SD大鼠（体重200～220 g）中随机选取10只作为空白组，在实验第1、4天，于空白组右膝关节腔内注射0.1 ml生理盐水，其余大鼠注射0.1 ml Ⅱ型胶原蛋白酶与弗氏完全佐剂的混合乳剂（质量浓度为0.5 mg/ml）。第7天根据大鼠膝关节情况评估造模结果，将50只造模成功的大鼠按照随机数字表法分为模型组、双氯芬酸钠组及除闷汤高、中、低剂量组，每组10只。实验第8天，对除闷汤高、中、低剂量组进行药浴干预（浓度分别为49.00、24.50、12.25 g/L），模型组进行温水泡浴，双氯芬酸钠组于造模部位予0.01 g的1%双氯芬酸钠凝胶，空白组常规饲养，连续干预4周，干预期间观察各组一般情况。干预后观察各组膝关节解剖学情况，测量膝关节直径，采用苏木精-伊红（HE）染色观察膝关节软骨组织形态学改变。采用酶联免疫吸附试验检测各组血清中白细胞介素-1β（IL-1β）、IL-6、IL-10含量；采用免疫组织化学染色检测各组软骨组织中p38MAPK、磷酸化p38MAPK（p-p38MAPK）、基质金属蛋白酶-13（MMP-13）的表达水平。结果药物干预期间，空白组一般情况正常；模型组强迫运动时跛行，伴膝关节屈伸功能障碍；除闷汤各剂量组膝关节屈伸功能与步态逐渐恢复，双足跛行状态减少。干预后，空白组膝关节解剖可见滑膜平滑半透明，软骨透明光滑，关节面完整，HE染色可见软骨细胞排列规则清晰；模型组膝关节解剖可见滑膜增厚糜烂，软骨表面粗糙磨损，色泽灰暗，HE染色可见软骨基质减少甚至消失，软骨细胞大量减少，形态不规则；除闷汤各剂量组膝关节解剖及HE染色可见膝关节软骨有不同程度改善。模型组膝关节直径大于空白组，血清IL-1β、IL-6及软骨p38MAPK、p-p38MAPK、MMP-13表达均高于空白组，血清IL-10低于对照组（P＜0.05）。除闷汤各剂量组膝关节直径小于模型组，血清IL-10高于模型组，软骨p-p38MAPK、MMP-13表达均低于模型组（P＜0.05）。除闷汤高、中剂量组血清IL-1β、IL-6及软骨p38MAPK低于模型组和除闷汤低剂量组，IL-10高于除闷汤低剂量组（P＜0.05）。除闷汤高剂量组膝关节直径小于除闷汤低、中剂量组，软骨p-p38MAPK、MMP-13表达低于除闷汤低剂量组，血清IL-10高于除闷汤低、中剂量组，软骨MMP-13表达低于除闷汤中剂量组（P＜0.05）。结论瑶医除闷汤可能通过靶向抑制p38MAPK通路表达，减轻KOA大鼠的炎症反应，改善软骨退变。

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	李彤1　　王艺锦1　　唐一洲1　　马艳2　　符标芳3　　闫国跃4

关键词 ：瑶医, 除闷汤, 膝骨关节炎, p38丝裂原活化蛋白激酶通路, 炎症因子, 作用机制

Abstract：Objective To investigate the effect and mechanism of medicinal bath of Yaoyi Chumen Decoction in the treatment of knee osteoarthritis (KOA) model rats based on p38 mitogen-activated protein kinase (p38MAPK) pathway. Methods Ten of 60 8-week-old male SPF SD rats (weight 200-220 g) were randomly selected as blank group, 0.1 ml of physiological saline was injected into the right knee cavity of the blank group on the 1st and 4th days of the experiment. The remaining rats were injected with a mixed emulsion of 0.1 ml type Ⅱ collagenic proteinase and complete Freund’s adjuvant (mass concentration was 0.5 mg/ml). On the 7th day, the modeling results were evaluated according to the knee joint conditions of the rats, 50 rats successfully modeled were divided into model group, Diclofenac Sodium group, and Chumen Decoction high, medium, low dose groups with ten rats in each group according to random number table method. On the 8th day of the experiment, the Chumen Decoction high, medium, low dose groups were given medicinal bath intervention (the concentration was 49.00, 24.50, 12.25 g/L, respectively), the model group was given warm water bath, the Diclofenac Sodium group was given 0.01 g of 1% Diclofenac Sodium Gel at the modeling site, and the blank group was fed routinely, the intervention lasted for four weeks in each group. The general condition of each group was observed during the intervention. After intervention, the anatomy of knee joint in each group was observed, the diameter of knee joint was measured, and the histological changes of knee joint cartilage were observed by hematoxylin-eosin (HE) staining. The serum levels of interleukin-1β(IL-1β), IL-6, and IL-10 were detected by enzyme-linked immunosorbent assay. Immunohistochemical staining was used to detect the location and expression levels of p38MAPK, phosphorylated p38MAPK (p-p38MAPK), and matrix metalloproteinase-13 (MMP-13) in cartilage tissue of each group. Results During the drug intervention, the general condition of the blank group was normal, the model group limp during forced exercise with knee flexion and extension dysfunction, the flexion and extension function of knee joint and gait of Chumen Decoction dose groups were gradually recovered, and the lameness of both feet was reduced. After the intervention, the knee joint anatomy of the blank group showed smooth and translucent synovium, transparent, and smooth cartilage, and complete articular surface, HE staining showed that the chondrocytes were arranged regularly and clearly. The knee joint anatomy of the model group showed synovial thickening and erosion, cartilage surface roughness, and wear, and gray color, HE staining showed that cartilage matrix was reduced or even disappeared, chondrocytes were reduced in large numbers, and the shape was irregular. The knee joint anatomy and HE staining showed that the knee joint cartilage of Chumen Decoction dose groups were improved to varying degrees. The knee diameter of model group was higher than that of blank group, the expressions of serum IL-1β, IL-6, and cartilage p38MAPK, p-p38MAPK, MMP-13 were higher than those of blank group, and serum IL-10 was lower than that of control group (P＜0.05). The knee diameter of each Chumen Decoction dose groups were lower than that of model group, the serum IL-10 was higher than that of model group, and the expressions of cartilage p-p38MAPK and MMP-13 were lower than those of model group (P＜0.05). Serum IL-1β, IL-6, and cartilage p38 MAPK in Chumen Decoction high and medium dose groups were lower than those in model group and Chumen Decoction low dose group, and serum IL-10 was higher than that in Chumen Decoction low dose group (P＜0.05). The knee diameter of Chumen Decoction high dose group was lower than those of Chumen Decoction low and medium dose groups, the expressions of cartilage p-p38MAPK and MMP-13 were lower than those of Chumen Decoction low dose group, the serum IL-10 was higher than that of Chumen Decoction low dose group, and the expression of cartilage MMP-13 was lower than that of Chumen Decoction medium group (P＜0.05). Conclusion Yaoyi Chumen Decoction may reduce the inflammatory response and improve cartilage degeneration in rats with KOA through targeted inhibition of p38MAPK pathway expression.

Key words： Yaoyi Chumen Decoction Knee osteoarthritis p38 mitogen-activated protein kinase pathway Inflammatory cytokines Mechanism of action

基金资助:广西壮族自治区科技攻关计划项目（桂科攻1598012-49）；
广西中医药大学研究生教育创新计划项目（YCSZ2022012）；
广西中医药大学博士科研启动基金项目（2020BS 022）；
广西中医药大学“桂派杏林青年英才”项目（2022C036）；
广西中医药重点学科建设项目（GZXK-Z-20-63）。

通讯作者: 符标芳（1985-），女，硕士；研究方向：中医经典及民族医药疾病防治。闫国跃（1985-），男，医学博士，副教授；研究方向：民族医药教育及瑶医药疾病防治。

作者简介: 李彤（1966-），男，主任医师；研究方向：瑶医药基础理论及疾病防治。

引用本文:

李彤1　　王艺锦1　　唐一洲1　　马艳2　　符标芳3　　闫国跃4. 基于p38丝裂原活化蛋白激酶通路探讨瑶医除闷汤药浴治疗膝骨关节炎大鼠的作用机制[J]. 中国医药导报, 2023, 20(31): 12-19.
LI Tong1 WANG Yijin1 TANG Yizhou1 MA Yan2 FU Biaofang3 YAN Guoyue4. Study on the mechanism of medicinal bath of Yaoyi Chumen Decoction in the treatment of rats with knee osteoarthritis based on p38MAPK pathway. 中国医药导报, 2023, 20(31): 12-19.

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https://www.yiyaodaobao.com.cn/CN/10.20047/j.issn1673-7210.2023.31.02 或 https://www.yiyaodaobao.com.cn/CN/Y2023/V20/I31/12

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