淫羊藿苷-透明质酸对兔肩袖腱骨愈合的影响及机制研究

Effect of epimedium-hyaluronic acid on the healing of rotator cuff tendon bone in rabbits and its mechanism

目的 探讨淫羊藿苷-透明质酸结合物对兔肩袖损伤术后腱骨界面愈合的影响及其作用机制。方法 选取30只新西兰雄性大白兔,随机分为假手术组、模型组、淫羊藿苷组、透明质酸钠组、淫羊藿苷-透明质酸组,每组6只。假手术组只进行肩部皮肤切开缝合,其余组兔建立右侧肩袖损伤模型,并在造模1周后进行肩袖重建手术,术后进行关节内注射。假手术组、模型组注射生理盐水,淫羊藿苷组注射生理盐水溶解的淫羊藿苷含药血清冻干粉溶液,透明质酸钠组注射透明质酸钠注射液,淫羊藿苷-透明质酸组注射淫羊藿苷-透明质酸结合物,每周1次,连续注射5周。注射5周后处死各组兔,采用ELISA法检测肩关节液中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量,HE染色进行腱骨界面组织病理观察,免疫组化检测腱骨界面组织中血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)阳性表达情况。结果 淫羊藿苷组、透明质酸钠组、淫羊藿苷-透明质酸组IL-1β、IL-6、TNF-α含量均明显低于模型组(P均<0.05),且淫羊藿苷-透明质酸组IL-1β、IL-6、TNF-α含量均明显低于淫羊藿苷组和透明质酸钠组(P均<0.05)。假手术组肌腱与软骨紧密结合,正常腱骨界面固有结构;模型组腱骨界面散在炎性细胞和少量成纤维细胞,连接较疏松;淫羊藿苷组、透明质酸钠组腱骨界面软骨、肌腱胶原纤维连接较紧密,填充较多类圆形软骨细胞及成纤维细胞,部分新生血管生成;淫羊藿苷-透明质酸组腱骨界面软骨、肌腱胶原纤维紧密连接,有大量类圆形软骨细胞及新生血管生成。模型组VEGF和bFGF阳性表达平均光密度值均明显高于假手术组(P均<0.05);淫羊藿苷组、透明质酸钠组、淫羊藿苷-透明质酸组VEGF和bFGF阳性表达平均光密度值均明显高于模型组(P均<0.05),且淫羊藿苷-透明质酸组均明显高于淫羊藿苷组和透明质酸钠组(P均<0.05)。结论 淫羊藿苷-透明质酸能够促进肩袖损伤后肩袖腱骨愈合,其作用机制可能与减轻局部炎症反应、促进新生血管形成以及软骨细胞和成纤维细胞的增殖分化有关。

Objective It is to investigate the effect and mechanism of epimedium-hyaluronic acid conjugate on tendon bone interface healing after rotator cuff injury in rabbits.Methods Thirty male New Zealand white rabbits were randomly divided into 5 groups:sham operation group,model group,epimedium group,sodium hyaluronate group,and epimedium-hyaluronic acid group,with 6 rabbits in each group.The rabbits in the sham operation group only underwent shoulder skin incision and suture,and the rabbits in the other groups were used to establish models of right rotator cuff injury,and underwent rotator cuff reconstruction surgery one week after modeling,followed by intra articular injection.The sham operation group and model group were injected with normal saline,the epimedium group was injected with epimedium medicated serum lyophilized powder solution dissolved by normal saline,the sodium hyaluronate group was injected with sodium hyaluronate injection,and epimedium-hyaluronic acid group was injected with epimedium-hyaluronic acid conjugate,once per week,continuously injected for 5 weeks.After 5 weeks of administration,the rabbits were sacrificed,and the concentrations of interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)in shoulder joint fluid were detected by ELISA,the histopathological changes of the tendon-bone interface were observed by HE staining,and the positive expression of vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(bFGF)in the tendon-bone interface tissue was detected by immunohistochemistry.Results The contents of IL-1β,IL-6 and TNF-αin the epimedium group,sodium hyaluronate group and epimedium-hyaluronic acid group were significantly lower than those in the model group(all P<0.05),and the contents of IL-1β,IL-6 and TNF-αin the epimedium-hyaluronic acid group were significantly lower than those in the epimedium group and sodium hyaluronate group(all P<0.05).The tendon and cartilage were closely combined,and the natural structure of the tendine-bone interface was normal in the sham operation group;the tendine-bone interface was scattered with inflammatory cells and a few fibroblasts,and the connection was loose in the model group;the cartilage,tendon collagen fibers were less closely connected in the tendon bone interface,filled with more round-like chondrocytes and fibroblasts,and some neovascularization occurred in the epimedium group and sodium hyaluronate group;the cartilage,tendon collagen fibers were more closely connected in the tendon bone interface,with a large number of round-like chondrocytes and neovascularization in the epimedium-hyaluronic acid group.The mean optical density values of VEGF and bFGF positive expression in the model group were significantly higher than those in the sham operation group(all P<0.05);the mean optical density values of VEGF and bFGF positive expression in the epimedium group,sodium hyaluronate group,and epimedium-hyaluronic acid group were significantly higher than those in the model group(all P<0.05),and the epimedium-hyaluronic acid group were significantly higher than the epimedium group and sodium hyaluronate group(both P<0.05).Conclusion Epimedium-hyaluronic acid can promote rotator cuff tendon bone healing after rotator cuff injury,the mechanism of action may be related to reducing the local inflammatory response,promoting neovascularization and the proliferation and differentiation of chondrocytes and fibroblasts.