摘要
目的探究A型肉毒毒素(BTX‑A)联合静态渐进性牵伸(SPS)治疗大鼠创伤性膝关节僵硬的效果及其机制。方法选取40只8周龄、体重220~300 g的健康雄性SD大鼠,按随机数字表法分为空白对照组(8只)和造模组(包括损伤组、BTX‑A组、SPS组和BTX‑A+SPS组,每组7只)。根据Hlidebrand的方法建立创伤性膝关节僵硬模型,主要步骤包括破坏关节囊、克氏针固定、关节打孔和4周后取出内固定。空白对照组不做任何处理,在笼中自由活动;损伤组在造模成功后自由活动;BTX‑A组在内固定取出当天于关节腔内注射BTX‑A;SPS组在内固定取出当天开始SPS治疗;BTX‑A+SPS组在内固定取出当天于关节腔内注射BTX‑A并开始SPS治疗。治疗周期为16 d。在治疗前及治疗后16 d检测关节活动度(ROM)和关节刚度;在治疗后16 d采集大鼠膝关节组织标本并处死大鼠,采用HE和Masson染色观察关节囊纤维组织增生情况,Western blot测定磷酸化(p)‑Smad2、Smad2、p‑Smad 3、Smad 3、Smad4、转化生长因子‑β1(TGF‑β1)、Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、α‑平滑肌动蛋白(α‑SMA)表达水平,再通过计算磷酸化蛋白与总蛋白的比值反映磷酸化水平。结果(1)ROM:治疗前,空白对照组ROM显著高于其余各组(P<0.05),其余各组间ROM差异均无统计学意义(P>0.05);治疗后16 d,损伤组、BTX‑A组、SPS组和BTX‑A+SPS组ROM均低于空白对照组(P<0.05);BTX‑A+SPS组ROM较损伤组、BTX‑A组和SPS组均增加(P<0.05)。治疗后16 d,空白对照组治疗前后ROM差异无统计学意义(P>0.05),其余各组ROM较治疗前均显著增加(P<0.01)。(2)关节刚度:治疗后16 d,损伤组、BTX‑A组和SPS组关节刚度分别为(0.95±0.24)N·cm/°、(0.86±0.22)N·cm/°、(0.65±0.09)N·cm/°,均低于空白对照组的(0.36±0.03)N·cm/°(P<0.05);BTX‑A+SPS组关节刚度为(0.49±0.04)N·cm/°,与空白对照组差异无统计学意义(P>0.05),但较损伤组、BTX‑A组和SPS组均降低(P<0.05)。(3)纤维组织增生情况:治疗后16 d,空白对照组关节囊结构完整、清晰,纤维排列有序,未见明显纤维组织增生;损伤组病理变化最严重,滑膜纤维组织大量增生,组织内血管明显增多,炎性细胞浸润;BTX‑A+SPS组较SPS组和BTX‑A组病变减轻,仅有轻度滑膜细胞增多,未见明显血管增生或淋巴细胞,总体病变最轻。(4)蛋白表达水平:治疗后16 d,损伤组、BTX‑A组和SPS组p‑Smad2/Smad2比值分别为1.552±0.234、1.328±0.272、1.194±0.277,均高于空白对照组的0.794±0.082(P<0.05);而BTX‑A+SPS组p‑Smad2/Smad2比值为1.013±0.123,与空白对照组、BTX‑A组和SPS组差异均无统计学意义(P>0.05),较损伤组降低(P<0.05)。治疗后16 d,损伤组、BTX‑A组、SPS组和BTX‑A+SPS组p‑Smad3/Smad3比值分别为2.272±0.309、1.664±0.285、1.381±0.276、1.003±0.060,均高于空白对照组的0.515±0.051(P<0.05);BTX‑A+SPS组p‑Smad3/Smad3比值较损伤组、BTX‑A组和SPS组均降低(P<0.05)。治疗后16 d,损伤组Smad4水平为1.001±0.015,高于空白对照组的0.294±0.076(P<0.05),而BTX‑A组(0.664±0.051)、SPS组(0.833±0.045)和BTX‑A+SPS组(0.467±0.068)与空白对照组差异均无统计学意义(P>0.05);BTX‑A+SPS组Smad4水平较损伤组、BTX‑A组和SPS组均降低(P<0.05)。治疗后16 d,损伤组TGF‑β1水平为1.004±0.407,高于空白对照组的0.269±0.122(P<0.05),而BTX‑A组(0.564±0.194)、SPS组(0.422±0.086)和BTX‑A+SPS组(0.347±0.161)与空白对照组差异均无统计学意义(P>0.05);BTX‑A+SPS组TGF‑β1水平较损伤组、BTX‑A组和SPS组均降低(P<0.05)。治疗后16 d,损伤组Ⅰ型胶原蛋白水平为0.999±0.170,高于空白对照组的0.299±0.139(P<0.05);而BTX‑A组(0.542±0.278)、SPS组(0.561±0.165)和BTX‑A+SPS组(0.537±0.045)与空白对照组差异均无统计学意义(P>0.05);BTX‑A+SPS组Ⅰ型胶原蛋白水平较损伤组、BTX‑A组和SPS组均降低(P<0.05)。治疗后16 d,损伤组Ⅲ型胶原蛋白水平为1.002±0.126,高于空白对照组的0.239±0.106(P<0.05),而BTX‑A组(0.661±0.062)、SPS组(0.595±0.062)和BTX‑A+SPS组(0.504±0.269)与空白对照组差异均无统计学意义(P>0.05);BTX‑A+SPS组Ⅲ型胶原蛋白水平较损伤组、BTX‑A组和SPS组均降低(P<0.05)。治疗后16 d,损伤组α‑SMA水平为0.998±0.074,高于空白对照组的0.130±0.023(P<0.05),而BTX‑A组(0.358±0.060)、SPS组(0.432±0.230)和BTX‑A+SPS组(0.293±0.135)与空白对照组差异均无统计学意义(P>0.05);BTX‑A+SPS组α‑SMA水平较损伤组、BTX‑A组和SPS组均降低(P<0.05)。结论与单一治疗相比,BTX‑A联合SPS治疗大鼠创伤性膝关节僵硬,在改善关节活动度和关节弹性、抑制关节囊纤维增生方面更具优势;其可能机制是通过进一步抑制关节囊TGF‑β1的表达来降低Smad2和Smad3的磷酸化水平,阻止其与Smad4受体结合,进而降低TGF‑β/Smad信号通路中下游蛋白质如Ⅰ型胶原蛋白、Ⅲ型胶原蛋白和α‑SMA的表达。
Objective To investigate the efficacy and mechanism of botulinum toxin type A(BTX‑A)combined with static progressive stretching(SPS)in the treatment of traumatic knee stiffness in rats.Methods Forty healthy male SD rats aged 8 weeks and weighing 220‑300 g,were randomly divided into blank control group(n=8)and model groups(n=28)(including injury group,BTX‑A group,SPS group and BTX‑A+SPS group,with 7 in each group).Hlidebrand′s method was used to construct a traumatic knee stiffness model,with the following main steps:destruction of the joint capsule,Kirschner wire fixation,joint drilling,and removal of the internal fixation at 4 weeks.The blank control group did not receive any treatment and could move freely in the cage.The injury group moved freely after successful modeling.On the day of internal fixation removal,BTX‑A was injected into the joint cavity in group BTX‑A,SPS treatment was started in the SPS group,BTX‑A was injected into the joint cavity and SPS treatment was started in the BTX‑A+SPS group.The treatments lasted 16 days.The range of motion(ROM)and joint stiffness were measured before treatment and at 16 days after treatment.At 16 days after treatment,knee joint tissue was collected and the rats were sacrificed,and the articular capsule fibrous tissue proliferation was observed by HE and Masson staining.The expression levels of phosphorylated(p)‑Smad2,Smad2,p‑Smad3,Smad3,Smad4,transforming growth factor‑β1(TGF‑β1),collagen type I,collagen type III,andα‑smooth actin(α‑SMA)were determined by Western blot.The ratio of phosphorylated protein to total protein was calculated to reflect the phosphorylation level.Results(1)ROM:Before treatment,the ROM in the blank control group was significantly higher than that in the other groups(P<0.05),with no significant difference in ROM among the other groups(P>0.05).At 16 days after treatment,ROM in the injury group,BTX‑A group,SPS group,and BTX‑A+SPS group was lower than that in the blank control group(P<0.05),among which ROM in the BTX‑A+SPS group was significantly higher than that in the injury group,BTX‑A group,and SPS group(P<0.05).At 16 days after treatment,there was no significant difference in ROM before and after treatment in the blank control group(P>0.05),and ROM in the other groups was significantly increased compared with that before treatment(P<0.01).(2)Joint stiffness:At 16 days after treatment,the joint stiffness levels in the injury group,the BTX‑A group,and the SPS group were(0.95±0.24)N·cm/°,(0.86±0.22)N·cm/°,and(0.65±0.09)N·cm/°respectively,which were significantly lower than(0.36±0.03)N·cm/°in the blank control group(P<0.05).The joint stiffness level of the BTX‑A+SPS group was(0.49±0.04)N·cm/°,which was not significantly different from that in the blank control group(P>0.05),but was significantly lower than those in the injury group,BTX‑A group,and SPS group(P<0.05).(3)Fibrous tissue proliferation:at 16 days after treatment,the joint capsular structure in the blank control group was complete and clear,the fibers were arranged in order,and there was no obvious fibrous tissue proliferation.The pathological changes in the injury group were the most serious,with a large number of synovial fibrous tissue proliferation,significantly increased blood vessels in the tissue,and inflammatory cell infiltration.Compared with the SPS group and BTX‑A group,the lesions in BTX‑A+SPS group were milder,with only slight increase in the number of synovial cells but no obvious vascular proliferation or lymphocytes,and the overall lesions were the least severe.(4)Protein expression:the ratios of p‑Smad2/Smad2 in the injury group,BTX‑A group and SPS group were 1.552±0.234,1.328±0.272 and 1.194±0.277 respectively,which were higher than 0.794±0.082 in the blank control group(P<0.05).The ratio of p‑Smad2/Smad2 in the BTX‑A+SPS group was 1.013±0.123,which was not significantly different from those in the blank control group,BTX‑A group or SPS group(P>0.05),but was lower than that in the injury group(P<0.05).At 16 days after treatment,the p‑Smad3/Smad3 ratios in the injury group,BTX‑A group,SPS group and BTX‑A+SPS group were 2.272±0.309,1.664±0.285,1.381±0.276 and 1.003±0.060 respectively,which were higher than 0.515±0.051 in the blank control group(P<0.05).The p‑Smad3/Smad3 ratio in the BTX‑A+SPS group was significantly lower than those in the injury group,BTX‑A group and SPS group(P<0.05).At 16 days after treatment,the level of Smad4 in the injury group(1.001±0.015)was higher than 0.294±0.076 in the blank control group(P<0.05).However,there was no significant difference between the BTX‑A group(0.664±0.051),SPS group(0.833±0.045),BTX‑A+SPS group(0.467±0.068)or the blank control group(P>0.05).The level of Smad4 in the BTX‑A+SPS group was significantly lower than those in the injury group,BTX‑A group and SPS group(P<0.05).At 16 days after treatment,the level of TGF‑β1 in the injury group(1.004±0.407)was higher than 0.269±0.122 in the blank control group(P<0.05),while there was no significant difference between the BTX‑A group(0.564±0.194),SPS group(0.422±0.086)and BTX‑A+SPS group(0.347±0.161)and the blank control group(P>0.05).The level of TGF‑β1 in the BTX‑A+SPS group was significantly lower than those in the injury group,BTX‑A group and SPS group(P<0.05).At 16 days after treatment,the level of type I collagen in the injury group was 0.999±0.170,higher than 0.299±0.139 in the blank control group(P<0.05),while there was no significant difference between the BTX‑A group(0.542±0.278),SPS group(0.561±0.165),and BTX‑A+SPS group(0.537±0.045)and the blank control group(P>0.05).The level of collagen type I in the BTX‑A+SPS group was significantly lower than those in the injury group,BTX‑A group,and SPS group(P<0.05).At 16 days after treatment,the level of type III collagen in the injury group was 1.002±0.126,higher than 0.239±0.106 in the blank control group(P<0.05),while there was no significant difference between the BTX-A group(0.661±0.062),SPS group(0.595±0.062),and BTX-A+SPS group(0.504±0.269)and the blank control group(P>0.05).The level of collagen type III in the BTX-A+SPS group was significantly lower than those in the injury group,BTX-A group,and SPS group(P<0.05).At 16 days after treatment,the level ofα-SMA in the injury group was 0.998±0.074,higher than 0.130±0.023 in the blank control group(P<0.05),while there was no significant difference between the BTX-A group(0.358±0.060),SPS group(0.432±0.230),and BTX-A+SPS group(0.293±0.135)and the blank control group(P>0.05).The level ofα-SMA in the BTX-A+SPS group was significantly lower than those in the injury group,BTX-A group and SPS group(P<0.05).Conclusions Compared with single treatment,the combination of BTX-A and SPS demonstrates significantly greater efficacy in the treatment of traumatic knee stiffness in rats.This combined approach not only enhances joint mobility and elasticity but also effectively inhibits joint capsule fibrosis.The underlying mechanism may involve the further suppression of TGF-β1 expression in the joint capsule,leading to reduced phosphorylation levels of Smad2 and Smad3.This,in turn,inhibits the binding of Smad2 and Smad3 to the Smad4 receptor,ultimately downregulating the expression of the downstream proteins of the TGF-β/Smad signaling pathway,such as collagen type I,collagen type III andα-SMA.
作者
陈科
张鑫
任凯
廖莹莹
何鑫
李肖菊
Chen Ke;Zhang Xin;Ren Kai;Liao Yingying;He Xin;Li Xiaoju(Department of Rehabilitation,Sichuan Orthopedic Hospital(National Traditional Chinese Medicine Rehabilitation Center Construction Unit,Sichuan Traditional Chinese Medicine Rehabilitation Hospital),Chengdu 610041,China;Department of Rehabilitation Medicine,Third People′s Hospital of Chengdu,Chengdu 610014,China;Department of Rehabilitation,Sichuan Electric Power Hospital,Chengdu 610065,China)
出处
《中华创伤杂志》
北大核心
2025年第2期201-211,共11页
Chinese Journal of Trauma
基金
四川省中医药管理局中医药基础研究课题(2020JC0098)
四川省科学技术厅科技计划项目(2021YFS0067)
四川省骨科医院院内科研项目(PY202405)。
关键词
肉毒毒素类
膝损伤
纤维化
静态牵伸
Botulinum toxins
Knee injuries
Fibrosis
Static stretching Fund programs:Basic Research Project of Traditional Chinese Medicine of
