解毒生肌膏调控GSK-3β/β-catenin减轻炎症反应促糖尿病SD大鼠慢性创面修复的影响

Jiedu-Shengji ointment regulates GSK-3β/β-catenin to reduce inflammatory response to promote chronic wound healing in diabetic SD rats

摘要:

目的 从GSK-3β/β-catenin角度探讨炎症反应对糖尿病SD大鼠慢性创面修复的影响。方法 使用高脂饲料喂养联合腹腔注射链脲霉素（Streptozocin，STZ）及手术制备全层皮肤缺损的方法建立糖尿病SD大鼠创面模型，成模后随机分为模型组（n=15），磺胺嘧啶银组（n=15）及解毒生肌膏组（n=15），并设立正常创面组（n=15）。磺胺嘧啶银组创面和解毒生肌膏组创面予以磺胺嘧啶银软膏和解毒生肌膏外敷，每日1次，模型组与正常创面组不予干预。给药后第3天、7天和14天肉眼观察各组大鼠的创面愈合情况，计算创面愈合率；HE观察组织基本形态；Masson检测胶原生成情况；超微电镜观察内质网结构差异；免疫组织化学法检测肿瘤坏死因子-α（Tumor necrosis factor-α，TNF-α）和白细胞介素-6（Interleukin-6，IL-6）表达；蛋白质免疫印迹法检测糖原合成酶激酶-3β（glycogen synthase kinase-3β，GSK-3β）蛋白和β-连环蛋白（β-catenin）蛋白表达。结果 给药后3天解毒生肌膏组创面愈合率高于模型组（P<0.05）；给药后14天解毒生肌膏组创面愈合率高于磺胺嘧啶银组（P<0.05）。HE结果 给药后3、7天解毒生肌膏组创面较其他组肿胀程度较小，毛细血管数量丰富；14天，解毒生肌膏组和正常创面组出现皮肤附属器新生，组织结构完整度好。Masson结果 解毒生肌膏组较其他各组胶原纤维分布均匀，排列结构较为整齐，新生胶原纤维更为粗壮，分布均匀，排列整齐。电镜结果：解毒生肌膏组内质网丰富，数量较多，形态结构趋于正常。免疫组织化学法结果：与模型组相比，各组TNF-α、IL-6表达水平明显降低；解毒生肌膏组TNF-α表达水平低于正常创面组；解毒生肌膏组TNF-α、IL-6表达低于磺胺嘧啶银组。Western blot法结果显示：与正常创面组相比，各组β-catenin表达量均上调（P<0.05），解毒生肌膏组上调表达量最少；与正常创面组相比，各组GSK-3β蛋白表达含量明显下调（P<0.05）；给药后7天，磺胺嘧啶银组和解毒生肌膏组GSK-3β蛋白表达含量上调（P<0.05），但仍低于正常创面组。结论 解毒生肌膏作用糖尿病大鼠创面修复机制通过调控GSK-3β和β-catenin表达，使炎症因子IL-6和TNF-α表达下调，最终达到糖尿病大鼠慢性创面愈合。

Abstract:

Objective To investigate the effects of inflammatory response on the repair of chronic wounds in Sprague-Dawley（SD） rats with diabetes from the perspective of glycogen synthase kinase-3β（GSK-3β）/β-catenin.Methods A wound model in diabetic SD rats was established by high-fat feeding combined with intraperitoneal injection of streptozocin and surgical preparation of full-thickness skin defects. The model rats were randomly divided into model group（n=15），silver sulfadiazine group（n=15），and Jiedu-Shengji ointment group（n=15），with normal wound group（n=15） set as a control. The wounds in the silver sulfadiazine group and Jiedu-Shengji ointment group were externally treated with silver sulfadiazine ointment and Jiedu-Shengji ointment，respectively，once daily，with no intervention in the model group and normal wound group. On the 3rd，7th，and 14th days after ointment administration，the wound healing status of each group was examined to calculate the wound healing rate. The basic tissue morphology was examined with hematoxylin-eosin（HE） staining. Collagen production was detected by Masson. The structure of endoplasmic reticulum was observed using a transmission electron microscope. The expression of tumor necrosis factor-α（TNF-α） and interleukin-6（IL-6） was measured by immunohistochemistry. The protein expression of GSK-3β and β-catenin was determined by Western blot.Results On day 3，the wound healing rate of the Jiedu-Shengji ointment group was significantly higher than that of the model group（P<0.05）. On day 14，the wound healing rate of the Jiedu-Shengji ointment group was significantly higher than that of the silver sulfadiazine group（P<0.05）. HE staining revealed less swelling and more abundant capillaries in the wounds of the Jiedu-Shengji ointment group as compared with the other groups on days 3 and 7 and new skin appendages and intact tissue structure in the wounds of the Jiedu-Shengji ointment group and normal wound group on day 14. Masson staining revealed evenly distributed and orderly organized collagen fibers as well as stronger，evenly distributed，and orderly arranged new collagen fibers in the Jiedu-Shengji ointment group，as compared with the other groups. Under the electron microscope，the Jiedu-Shengji ointment group had abundant endoplasmic reticulum with nearly normal morphological structure. Immunohistochemical results showed that the expression levels of TNF-α and IL-6 were significantly higher in the model group than in the other groups； the Jiedu-Shengji ointment group had a significantly lower TNF-α level than the normal wound group and significantly lower TNF-α and IL-6 levels than the silver sulfadiazine group. Western blot results showed that compared with that in the normal wound group，the expression of β-catenin was significantly up-regulated in the other groups（P<0.05），to the least extent in the Jiedu-Shengji ointment group；compared with the normal wound group，the other groups had significantly down-regulated GSK-3β protein expression（P<0.05）；on day 7，GSK-3β expression was significantly increased in the silver sulfadiazine group and Jiedu-Shengji ointment group（P<0.05），but still lower than that in the normal wound group.Conclusion Jiedu-Shengji ointment can regulate the expression of GSK-3β and β-catenin to down-regulate the expression of the inflammatory factors IL-6 and TNF-α，eventually achieving the healing of chronic wounds in diabetic rats.