CJI (Traditional Chinese Medicine)
活血化瘀合剂对膝骨性关节炎模型兔炎症因子及 TLR4/NF-κB 通路的影响
余毅,潘波,杨冬梅,祝家乐,赵洪庆,王芊芊,卢敏,余望贻
湖南中医药大学,湖南 长沙 410208
摘要:目的 观察活血化瘀合剂对膝骨性关节炎模型兔炎症因子及TLR4/NF-κB 通路的影响,探讨其关节保护作用机制。方法 将新西兰兔分为正常组、假手术组、模型组、中药组和阳性药组,每组8只。采用改良的 Hulth 造模法建立兔膝骨性关节炎模型,成模后中药组给予活血化瘀合剂(2.6 g/kg)灌胃,阳性药组给予
硫酸氨基葡萄糖溶液(15 mg/kg)灌胃,正常组、假手术组和模型组给予等体积蒸馏水灌胃,每日2次,连续
28 d。末次给药后,切除兔右后膝关节软骨用于组织病理分析,分离血清及关节液,检测肿瘤坏死因子-α
(TNF-α)、白细胞介素(IL)-1β、IL-6 含量,Western blot 检测关节软骨 Toll 样受体 4(TLR4)、核因子-κB (NF-κB)蛋白表达。结果 模型组兔关节软骨存在浅表层纤维化,软骨细胞成簇、肥大、排列紊乱、部分坏死,滑膜增厚,基质染色丢失等病理现象,血清和关节液中 TNF-α、IL-1β、IL-6 含量较正常组和假手术组均
显著升高(P<0.05,P<0.01),软骨 TLR4、NF-κB 蛋白表达显著升高(P<0.01);与模型组比较,中药组兔关节软骨病理损伤得以缓解,炎症因子 TNF-α、IL-1β、IL-6 含量均不同程度下降(P<0.05,P<0.01),TLR4、NF-κB 蛋白表达显著降低(P<0.05,P<0.01)。结论 活血化瘀合剂能缓解膝骨性关节炎模型兔关节软骨的损伤,其机制与抑制 TLR4/NF-κB 通路、降低炎症因子水平有关。
关键词:膝骨性关节炎;活血化瘀合剂;炎症因子;关节软骨;TLR4/NF-κB通路;兔
中图分类号:R285.5 文献标识码:A 文章编号:1005-5304(2020)03-0039-04
DOI:10.3969/j.issn.1005-5304.201907029 开放科学(资源服务)标识码(OSID):
Effects of Huoxue Huayu Mixture on Inflammatory Factors and TLR4/NF-κB Pathway in Rabbit Model of Knee Osteoarthritis
YU Yi, PAN Bo, YANG Dongmei, ZHU Jiale, ZHAO Hongqing, WANG Qianqian, LU Min, YU Wangyi
Hunan University of Chinese Medicine, Changsha 410208, China
Abstract: Objective To observe the effects of Huoxue Huayu Mixture on inflammatory factors and TLR4/NF-κB pathway in rabbit model of knee osteoarthritis; To discuss its protective mechanism for joints. Methods New Zealand rabbits were divided into normal group, sham-operation group, model group, TCM group and positive medicine group, with 8 rats in each group. A rabbit model of knee osteoarthritis was established by modified Hulth modeling method. After successful modeling, TCM group was given Huoxue Huayu Mixture (2.6 g/kg) for gavage, positive medicine group was given glucosamine sulfate solution (15 mg/kg) for gavage, and normal group, sham-operation group and model group were given equal volume of distilled water for gavage, twice a day, for 28 d After the last administration, the right posterior knee articular cartilage was removed for histopathological analysis, and serum and joint fluid were separated to detect the levels of inflammatory factors TNF-α, IL-1β and IL-6. Western blot was used to detect TLR4 and NF-κB protein levels in articular cartilage. Results Rabbits articular cartilage in model group had superficial fibrosis, chondrocyte clustering, hypertrophy, disordered arrangement, partial necrosis, thickening of synovial membrane, loss of matrix staining and other pathological phenomena. The levels of TNF-α, IL-1β and IL-6 in serum and joint fluid were significantly higher than those in normal group and sham-operation group (P<0.05,
基金项目:国家自然科学基金面上项目(81574004、81874476);湖南省教育厅一般项目(19C1420);湖南中医药大学基础医
学一流学科开放基金(2018JCYX04)
通讯作者:余望贻,E-mail:yuwangyi2006@163.com
P<0.01), and the expressions of TLR4 and NF-κB in cartilage increased (P<0.01). Compared with the model group, the pathological damage of rabbit articular cartilage of TCM group was alleviated, and the levels of TNF-α, IL-1β, and IL-6 decreased to different degrees (P<0.05, P<0.01). The protein expressions of TLR4 and NF-κB also decreased significantly (P<0.05, P<0.01). Conclusion Huoxue Huayu Mixture can alleviate the damage of articular cartilage in rabbit model with knee osteoarthritis, and its mechanism may be related to inhibiting TLR4/NF-κB pathway and reducing the level of inflammatory factors.
Keywords: knee osteoarthritis; Huoxue Huayu Mixture; inflammatory factors; articular cartilage; TLR4/NF-κB pathway; rabbits
膝骨性关节炎是由于膝关节局部损伤、炎症或慢性劳损引起的软骨变性及活动障碍的慢性退行性骨关节病,常见于中老年人,我国 55~64 岁人群中患病率高达49%[1]。目前,膝骨性关节炎发病机制尚未完全阐明,通常认为炎症因子在其发生发展过程中起到关键作用。炎症因子过度表达能导致骨基质降解酶
的大量释放,引起软骨组织损伤[2-3]。TLR4/NF-κB 通路作为天然免疫模式识别,介导炎症因子的表达,在
多种免疫性疾病的发生发展过程中起着重要作用[4]。活血化瘀合剂由当归、赤芍、姜黄3味中药组成,前
期临床研究证实其治疗膝骨性关节炎疗效显著[5],但作用机制尚不明确。本研究采用新西兰兔建立膝骨性关节炎模型,观察活血化瘀合剂对软骨组织病理形态及炎症因子表达的影响,从免疫相关的 TLR4/NF-κB通路着手,明确其治疗膝骨性关节炎的具体分子机制,为其临床应用提供依据。材料与方法
动物
14358S),核因子-κB(NF-κB)一抗(美国 CST,
8242S),二抗(武汉博士德,BM3243)。切片机(德国 Leica,RM223),倒置光学显微镜(德国 Leica,
DMi1 ),凝胶成像仪(德国 Bio-Rad , ChemiDoc
XRS+),酶标仪(美国Thermo Fisher Scientific,MK3)。
1.4 分组、造模与给药
将 42只新西兰兔随机分为正常组(8只)、假手
术组(8 只)和造模组(26 只)。造模组均采用改良的 Hulth 造模法建立兔膝骨性关节炎模型[6]。兔耳缘静脉注射 20%乌拉坦(4 mL/kg)麻醉,固定,右侧后腿膝关节常规脱毛、消毒,使膝关节屈曲,沿兔膝关节内侧入路,使内侧副韧带及前交叉韧带完全离断。经前抽屉试验、内侧应力试验验证韧带完全离断后,充分冲洗创口,逐层缝合,术后连续3d注射青霉素。假手术组从膝关节内侧打开关节腔,但不破坏韧带,而后直接缝合伤口,注射青霉素。造模后4 周,行右后膝关节X线片证实模型是否成功。选取24 只膝骨性关节炎模型兔,分为模型组、中药组、阳性药组,每组8只。中药组给予临床等效剂量活血化瘀合
剂(2.6 g/kg),阳性药组给予临床等效剂量硫酸氨基
葡萄糖溶液(15 mg/kg),正常组、假手术组和模型组给予等体积蒸馏水灌胃。给药体积为5 mL/kg,每日
2次,连续28 d。末次给药后1h麻醉,取血,于右后膝后髌上囊处常规脱毛,作长约 0.5 cm 的纵行切口,用1 mL生理盐水灌洗关节腔3次后,收集关节液,而后切除右后膝关节软骨,置于 4%多聚甲醛中固定或保存于液氮中。
1.5 HE 染色取固定的兔关节软骨,常规石蜡包埋后切片,经脱蜡、水化后进行HE染色,脱水、透明、封片,装盒待用。镜下观察、拍照,分析病理损伤情况,进行Mankin 评分[7]。
1.6 炎症因子含量检测
收集兔血液或关节液,4 ℃、4000 r/min 离心
10 min,取上清液。严格按 ELISA 试剂盒说明书进行
正常组