从糖原合成酶激酶-3β干预炎症反应角度探讨四妙勇安汤对糖尿病溃疡大鼠的促愈机制

Promoting healing mechanism of Simiao Yong’an Decoction in diabetic ulcer rats from the perspective of glycogen synthase kinase-3βintervention in inflammatory response

目的:观察四妙勇安汤对糖尿病溃疡大鼠巨噬细胞浸润状态、糖原合成酶激酶-3β(GSK-3β)及炎症因子白介素(IL)-6、IL-10表达的影响。方法:雄性Wistar大鼠采用高脂高糖饲料联合小剂量链脲佐菌素液腹腔注射法制备糖尿病模型。分别取正常对照组与糖尿病组制作溃疡模型,并分为对照组、模型组、四妙组、氯化锂组,每组20只。四妙组给予四妙勇安汤水煎液灌胃,氯化锂组给予氯化锂水溶液灌胃,余给予0.9%氯化钠溶液灌胃。观察创面造模后第3、7、14、21天各组创面愈合情况的变化,并采用CD68免疫组化染色法检测创面巨噬细胞浸润状态的变化,采用ELISA法、Q-PCR法检测创面组织中GSK-3β、IL-6、IL-10蛋白及mRNA的变化。结果:模型组大鼠创面巨噬细胞较对照组大鼠呈现慢进慢出的状态,而四妙组改善了巨噬细胞的浸润状态。与对照组比较,模型组、四妙组、氯化锂组大鼠的创面愈合率显著降低(P<0.05),GSK-3β、IL-6蛋白及mRNA水平显著升高(P<0.05),IL-10水平显著降低(P<0.05)。与模型组比较,四妙组大鼠的创面愈合率及GSK-3β、IL-6、IL-10水平均得到显著改善(P<0.05),同时,四妙组对GSK-3β的影响弱于氯化锂组,对IL-10水平的影响优于氯化锂组。另外,四妙组使IL-6的高峰时间前移。结论:GSK-3β可通过影响创面巨噬细胞浸润状态及炎症因子的水平而延缓糖尿病溃疡大鼠的创面愈合,而四妙勇安汤通过改善GSK-3β及炎症反应达到促进愈合的目的。

Objective: To observe the effects of the Simiao Yong’an Decoction on the state of macrophage infiltration,the level of glycogen synthase kinase-3β(GSK-3β),interleukin(IL)-6 and IL-10 in diabetic ulcer rats. Mehtods: Diabetic models were established in the male Wistar rats by intraperitoneal injection of low-dose Streptozotocin following high-fat diet feeding.A circular wound was made on the dorsum of the rats in both control group and diabetic group. And then,the ulcer rats were divided into 4 groups: the control ulcer group,the model group,the Simiao group,and LiCl group,20 rats in each group. The rats in Simiao group were given Simiao Yong’an Decoction by intragastric administration,while the rats in LiCl group were given LiCl solution,and others were given 0.9% sodium chloride solution. The condition of wound healing was recorded and the state of macrophage infiltration was observed by CD68 immunohistochemical staining in the four groups at 3rd,7th,14th and 21st day after wounding. Also,the protein and mRNA expression of GSK-3β,IL-6,IL-10 in wound tissues were detected by ELISA and Q-PCR. Results: Compared with the control group,the wound macrophages in the model group showed a state of slow entry and slow exit,while Simiao group could improve the state. Compared with the control group,the protein and mRNA expression of GSK-3β and IL-6 in wound tissues in model group,Simiao group and LiCl group were increased(P<0.05),while the wound healing rate and the expression of IL-10 were decreased(P<0.05). Compared with model group,the wound healing rate and the levels of GSK-3β,IL-6 and IL-10 in Simiao group were improved(P<0.05),but the effect of Simiao group on GSK-3β was weaker than that of LiCl group,and the effect on IL-10 level was better than that of LiCl group. Meanwhile,Simiao group moves the peak time of IL-6 forward. Conclusion: GSK-3β can delay wound healing in diabetic ulcer rats by affecting macrophage infiltration and inflammatory factors,however Simiao Yong’an Decoction can promote healing by improving GSK-3β and inflammatory response.