内热针调节TGF-β1/Smads信号通路对大鼠肩袖损伤后早期愈合的影响

Effect of internal heat acupuncture regulating TGF-β1/Smads signaling pathway on early healing of rotator cuff injury in rats

目的探讨内热针对大鼠肩袖损伤后早期愈合及TGF-β1/Smads信号通路的影响。方法将72只8周龄(200±20)g SPF级SD雄性大鼠分为模型组(54只)及对照组(18只)。模型组大鼠通过离断大鼠右肩冈上肌腱止点构建肩袖损伤大鼠模型,对照组大鼠除不横切冈上肌腱外其余处理同模型组。造模成功的大鼠采用简单随机抽样法分为肩袖损伤组、内热针组、内热针+通路激活剂SRI-011381组(内热针+SRI-011381组),每组18只。内热针组给予内热针治疗,内热针+SRI-011381组在内热针治疗的同时腹腔注射7 mg/kg SRI-011381,对照组与肩袖损伤组给予内热针组同时间、同部位的普通针刺。测定各组大鼠右前足足底热缩足潜伏期(TWL);酶联免疫吸附试验法检测关节液白细胞介素(IL)-1β、IL-6、肿瘤坏死因子-α(TNF-α)水平;HE染色观察肩袖关节肌腱组织病理损伤;Masson染色观察肩袖关节肌腱组织纤维化程度;腱-骨界面生物力学检测肩袖的最大拉力负荷;免疫组织化学染色法检测肌腱组织相关蛋白表达;Western blot法检测TGF-β1/Smads信号通路相关蛋白表达。结果与对照组比较,肩袖损伤组TWL缩短,最大拉力负荷及Scx、TnC表达降低,IL-1β、IL-6、TNF-α水平及TGF-β1、p-Smad2/3/Smad2/3表达升高(P<0.05)。与肩袖损伤组比较,内热针组TWL延长,最大拉力负荷及Scx、TnC表达升高,IL-1β、IL-6、TNF-α水平及TGF-β1、p-Smad2/3/Smad2/3表达降低(P<0.05)。与内热针组比较,内热针+SRI-011381组TWL缩短,最大拉力负荷及Scx、TnC表达降低,IL-1β、IL-6、TNF-α水平及TGF-β1、p-Smad2/3/Smad2/3表达升高(P<0.05)。对照组肌腱纤维、软骨及骨组织各层次排列清晰,胶原纤维生成相对正常,存在蓝色胶原纤维沉积;肩袖损伤组组织细胞排列紊乱,炎症细胞浸润明显,胶原纤维生成相对减少,蓝色胶原纤维沉积减少;内热针组组织病理变化得到改善,细胞排列相对正常,炎症细胞浸润减少,有丰富的胶原纤维生成,蓝色胶原纤维沉积大面积增多,胶原纤维连续性、平行取向和密度增加;内热针+SRI-011381组组织病理损伤加重,组织细胞排列紊乱,炎症细胞浸润加剧,胶原纤维生成减少,蓝色胶原纤维沉积明显减少。结论内热针可促进大鼠肩袖损伤早期愈合,其作用机制可能与抑制TGF-β1/Smads信号通路有关。

Objective To investigate the effect of acupuncture regulating TGF-β1/Smads signaling pathway on early healing of rotator cuff injury in rats.Methods A total of 728-week-old(200±20)g SPF SD male rats were divided into model group(54 rats)and control group(18 rats).In the model group,the rotator cuff injury rat model was constructed by disconnecting the right supraspinatus tendon of the rat,and control group was treated the same as model group except that supraspinatus tendon was not transversed.The rats with successful modeling were divided into rotator cuff injury group,internal heat acupuncture group,and internal heat acupuncture+pathway activator SRI-011381 group(internal heat acupuncture+SRI-011381 group)by simple random sampling,with 18 rats in each group.The internal heat acupuncture group was treated with internal heat acupuncture,internal heat acupuncture+SRI-011381 group was treated with intraperitoneal injection of 7 mg/kg SRI-011381 at the same time of internal heat acupuncture,control group and rotator cuff injury group were treated with ordinary acupuncture at the same time and in the same place as internal heat acupuncture group.The thermal withdrawal latency(TWL)of the right anterior full floor of rats was measured;the levels of interleukin(IL)-1β,IL-6,and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay;HE staining was used to observe the pathological injury of rotator cuff tendon;Masson staining was used to observe the degree of rotator cuff joint tendon fibrosis;the maximum tensile load of rotator cuff was measured by tendine-bone interface biomechanics;the expression of tendon tissue related protein was detected by immunohistochemical staining;and the expression of TGF-β1/Smads signaling pathway was detected by Western blot.Results Compared with control group,TWL was shortened,maximum tensile load and the expression of Scx and TnC was decreased,the levels of IL-1β,IL-6,and TNF-α,and the expression of TGF-β1,p-Smad2/3/Smad2/3 was increased in rotator cuff injury group(P<0.05).Compared with rotator cuff injury group,TWL was longer,maximum tensile load and the expression of Scx and TnC was increased,the levels of IL-1β,IL-6,and TNF-α,and the expression of TGF-β1,p-Smad2/3/Smad2/3 was reduced in internal heat acupuncture group(P<0.05).Compared with internal heat acupuncture group,TWL was shortened,maximum tensile load and the expression of Scx and TnC was decreased,the levels of IL-1β,IL-6,and TNF-α,and the expression of TGF-β1,p-Smad2/3/Smad2/3 was increased in internal heat acupuncture+SRI-011381 group(P<0.05).In control group,all levels of tendon fiber,cartilage,and bone tissue were clearly arranged,collagen fiber generation was relatively normal,and blue collagen fiber deposition existed;in rotator cuff injury group,the tissue cell arrangement was disordered,inflammatory cell infiltration was obvious,collagen fiber production was relatively reduced,and blue collagen fiber deposition was reduced;in the internal heat acupuncture group,the histopathological changes were improved,cell arrangement was relatively normal,inflammatory cell infiltration was reduced,collagen fibers were abundant,blue collagen fiber deposition increased in a large area,and the continuity,parallel orientation,and density of collagen fibers increased;in internal heat acupuncture+SRI-011381 group,histopathological injury was aggravated,histopathic cell arrangement was disordered,inflammatory cell infiltration was aggravated,collagen fiber production was reduced,and blue collagen fiber deposition was significantly reduced.Conclusion Internal heat acupuncture can promote early healing of rotator cuff injury in rats,and its mechanism of action may be related to the inhibition of TGF-β1/Smads signaling pathway.