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

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.