推拿对肩关节周围炎模型兔滑膜炎症及关节囊纤维化因子的影响

Effect of Massage on Synovial Inflammation and Joint Capsule Fibrosis Factors in Model Rabbits with Periarthritis of Shoulder Joint

目的:观察推拿对肩关节周围炎模型兔关节囊及外周血清中炎症因子和纤维化因子的影响,探讨推拿治疗肩关节周围炎可能的分子机制。方法:18只新西兰兔按随机数字表法分为空白组、模型组和干预组,每组6只。模型组及干预组采用持续劳损加冰敷方式制备肩关节周围炎兔模型。造模成功后,干预组进行推拿干预,每日1次,共干预3周。干预结束后取材,采用苏木精-伊红染色(HE染色)观察肩关节滑膜及关节囊病理形态,采用蛋白质印迹法(Western blotting)、免疫组织化学染色法(IHC)检测兔肩关节囊组织中转化生长因子-β1(TGF-β1)、基质金属蛋白酶-1(MMP-1)、基质金属蛋白酶-9(MMP-9)、基质金属蛋白酶抑制因子-1(TIMP-1)、Ⅰ型胶原蛋白(COL-Ⅰ)、Ⅲ型胶原蛋白(COL-Ⅲ)表达水平,酶联免疫吸附测定(ELISA)法检测各组兔外周血清中白细胞介素-1β(IL-1β)和前列腺素E2(PGE2)的水平。结果:HE染色可见,模型组滑膜细胞异常增生、排列紊乱,炎症细胞浸润,滑膜深层毛细血管增生、红细胞浸润;干预组滑膜细胞排列基本正常,未见明显炎症细胞浸润。模型组关节囊内胶原蛋白纤维束致密、排列方向紊乱,胶原蛋白异常堆积,切片中呈现强嗜酸性的深染区域;干预组胶原蛋白纤维束排列稍紊乱,无异常深染。与空白组比较,模型组关节囊组织中TGF-β1、MMP-1、MMP-9、TIMP-1、COL-Ⅰ、COL-Ⅲ表达均升高,MMP-1/TIMP-1、MMP-9/TIMP-1比值降低(P<0.05或P<0.01),外周血清中IL-1β和PGE2的水平升高(P<0.01);与模型组比较,干预组关节囊中TGF-β1、TIMP-1、COL-Ⅰ、COL-Ⅲ表达水平降低,MMP-1、MMP-9表达水平升高,差异具有统计学意义(P<0.05),且MMP-1/TIMP-1、MMP-9/TIMP-1比值均升高(P<0.05),外周血清中IL-1β和PGE2水平下降(P<0.01)。结论:推拿可能通过降低IL-1β、PGE2水平,减轻滑膜炎症反应,缓解肩周疼痛,下调关节囊中TGF-β1、TIMP-1表达,升高MMP-1、MMP-9表达,调节MMP-1/TIMP-1、MMP-9/TIMP-1比值平衡,重塑细胞外基质,抑制COL-Ⅰ和COL-Ⅲ异常堆积,拮抗关节囊纤维化,改善肩关节活动度。

Objective:To observe the effect of massage on inflammatory factors and fibrosis factors in joint capsule and peripheral serum in model rabbits with periarthritis of shoulder joint,and to explore the possible molecular mechanism of massage in the treatment of periarthritis of shoulder joint.Methods:According to the random number table method,18 New Zealand rabbits were divided into the blank group,the model group and the intervention group,with 6 rabbits in each group.Rabbit models with periarthritis of shoulder joint in the model group and the intervention group were established by continuous strain and ice application.After successful modeling,the intervention group received massage intervention,once a day,for 3 weeks.After the intervention,samples were collected and the pathological morphologies of shoulder synovium and joint capsule were observed by hematoxylin-eosin staining(HE staining).Western blotting and immunohistochemical staining(IHC)were used to detect the expression levels of transforming growth factor-β1(TGF-β1),matrix metalloproteinase-1(MMP-1),matrix metalloproteinase-9(MMP-9),inhibitor of matrix metalloproteinase-1(TIMP-1),typeⅠcollagen(COL-Ⅰ)and typeⅢcollagen(COL-Ⅲ)in the shoulder joint capsule of rabbits.The concent-rations of interleukin-1β(IL-1β)and prostaglandin E2(PGE2)in peripheral serum were measured by enzyme-linked immunosorbent assay(ELISA).Results:HE staining showed that synovial cells in the model group had abnormal proliferation,disordered arrangement,inflammatory cell infiltration,capillary proliferation and red blood cell infiltration in the deep synovial membrane.The arrangements of synovial cells in the intervention group were basically normal,and there was no obvious inflammatory cell infiltration.In the model group,the collagen fiber bundles in the joint capsule were dense and arranged disorderly,the collagen was abnormally accumulated,and the sections showed strongly eosinophilic dark staining areas.In the intervention group,the collagen fiber bundles were arranged slightly disorderly without abnormal dark staining.Compared with the blank group,the expression of TGF-β1,MMP-1,MMP-9,TIMP-1,COL-Ⅰand COL-Ⅲwere significantly increased,and the ratios of MMP-1/TIMP-1 and MMP-9/TIMP-1 were significantly decreased in the joint capsule of the model group(P<0.05 or P<0.01).The levels of IL-1βand PGE2 in peripheral blood were significantly increased(P<0.01).Compared with the model group,the expression levels of TGF-β1,TIMP-1,COL-Ⅰand COL-Ⅲin the joint capsule of the intervention group were decreased,and the expression levels of MMP-1 and MMP-9 were increased,the difference was statistically significant(P<0.05).The MMP-1/TIMP-1 and MMP-9/TIMP-1 ratios were increased(P<0.05),and the levels of IL-1βand PGE2 in peripheral blood were decreased(P<0.01).Conclusions:Massage may reduce the content of IL-1βand PGE2,alleviate synovial inflammation,relieve shoulder pain,down-regulate the expression of TGF-β1 and TIMP-1 in shoulder capsule,increase the expression of MMP-1 and MMP-9,regulate the ratio balance of MMP-1/TIMP-1,MMP-9/TIMP-1,reshape extracellular matrix,inhibit abnormal accumulation of COL-Ⅰand COL-Ⅲ,antagonize joint cystic fibrosis,improve the range of motion of shoulder joint.