基于Wnt/β-catenin信号通路的凉血散瘀法对糖尿病溃疡大鼠模型的干预作用

Research on Mechanism of Cooling-blood and Eliminating-stasis Therapy to Diabetic Ulcer Rat Models Based on Wnt/β-catenin Signaling Pathway

目的:观察凉血散瘀中药对糖尿病溃疡大鼠的干预作用,并从Wnt/β-catenin通路角度探讨其作用机制。方法:Wistar大鼠采用高脂高糖饲料联合小剂量链脲佐菌素(STZ)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大鼠制作溃疡模型,并分为溃疡对照(CU)组、糖尿病溃疡(DU)组、四妙勇安汤组(DU-四妙组)、愈疡灵组(DU-愈疡灵组)、四妙勇安汤+愈疡灵组(DU-四妙+愈疡灵组),每组各15只。观察造模后第3、7、14 d各组创面愈合情况和织形态结构的变化的变化,采用ELISA法、RT-PCR法检测创面组织β-catenin、糖原合成激酶-3β(GSK-3β)蛋白及m RNA的表达水平。结果:DU组大鼠与CU组大鼠相比,第3、7、14 d的创面愈合率明显降低[(5.21±0.94)%vs(20.87±1.39)%,(31.70±1.21)%vs (49.99±1.58)%,(62.20±1.37)%vs (91.60±1.58)%, P <0.05];创面组织中β-catenin蛋白表达[(5.14±0.56) vs (9.04±0.65),(5.83±0.43) vs (10.08±0.35),(6.55±0.58) vs (12.77±0.30), P <0.05]及mRNA表达[(4.40±0.35) vs (6.92±0.16),(5.20±0.49) vs (7.12±0.48),(6.06±0.47) vs (9.24±0.55), P <0.05]均较低,而GSK-3β蛋白表达[(15.52±1.00) vs (7.04±0.53),(14.58±0.45) vs (6.05±0.23),(13.37±0.62) vs (4.49±0.37), P <0.05]及mRNA表达[(7.07±0.29) vs (3.55±0.32),(6.64±0.47) vs (3.00±0.28),(5.68±0.46) vs (1.98±0.19), P <0.05]则较高。与DU组大鼠相比,各治疗组大鼠各时间点的创面愈合率、创面组织中β-catenin蛋白及mRNA的表达水平均升高,而GSK-3β蛋白及mRNA的表达水平则降低,且治疗组中,以DU-四妙+愈疡灵组改善最明显。结论:具有凉血散瘀作用的四妙勇安汤及愈疡灵软膏均可通过干预Wnt/β-catenint通路的表达促进DU大鼠创面愈合,两者同用的效果优于单用其中一种。

Objective To observe the effect of cooling-blood and eliminating-stasis therapy to the diabetic ulcer rats, and to explore the mechanism from Wnt/β -catenin signaling pathway. Methods Diabetic animal models were established in Wistar rats by intraperitoneal injection of low-dose Streptozotocin following high- fat diet feeding. A circular wound was made on the dorsum of rats in both control group and diabetic group. And then, the ulcer rats were divided into 5 groups： control ulcer group （CU group）, diabetic ulcer group （DU group）, diabetic ulcer with Si-Miao-Yong-An Decoction group （DU-Simiao group）, diabetic ulcer with Yu-Yang-Ling Ointment group （DU-Yu group）, diabetic ulcer with Si-Miao-Yong-An Decoction and Yu-Yang-Ling Ointment group （DU-Simiao±Yu group）. The condition of wound healing and the changes of structure of the wound tissues were observed in the 5 groups on the 3rd, 7th and 14th day after wounding. Also, the protein and mRNA expression of β -catenin and glycogen synthase kinase-3 β （GSK-3 β ） in wound tissues were detected by ELISA and RT-PCR. Results The wound healing rates of DU group at the 3rd, 7th and 14th time point were lower than those of CU group [（5.21 ± 0.94）% vs （20.87 ± 1.39）%, （31.70 ± 1.21）% vs （49.99 ± 1.58）%, （62.20 ± 1.37）% vs （91.60 ± 1.58）%, P 〈 0.05]; as well as the protein expression [（5.14 ± 0.56） vs （9.04 ± 0.65）, （5.83 ± 0.43） vs （10.08 ± 0.35）, （6.55 ± 0.58） vs （12.77 ± 0.30）,P 〈 0.05] and mRNA expression [（4.40 ± 0.35） vs （6.92 ± 0.16）, （5.20 ± 0.49） vs （7.12 ± 0.48）, （6.06 ± 0.47） vs （9.24 ± 0.55） ,P 〈 0.05] of [3 -catenin, the protein expression [（15.52 ± 1.00）vs （7.04 ± 0.53）,（14.58 ± 0.45） vs （6.05 ± 0.23）, （β.37 ± 0.62） vs （4,49 ± 0.37） , P 〈 0.05] and mRNA expression [（7.07 ± 0.29） vs （3.55±0.32）, （6.64±0.47） vs（3.00±0.28）, （5.68 ± 0.46） vs（1.98±0.19）,P 〈 0.05] of GSK-3β in DU group were higher than those of the CU group. Besides, the wound healing rate and the protein and mRNA expression of β -catenin in the treatment group were higer than those of the DU group, the protein expression of GSK-3 β was lower, and the DU-Simiao-Yu group was the lowest in the 3 treatment groups. Conclusion Si- Miao-Yong-An Decoction and Yu-Yang-Ling Ointment, the represent of cooling-blood and eliminating-stasis therapy, can promote the healing of diabetic ulcer by intervene the expression of Wnt/β -catenin signaling pathway, and the effect of combination is better than using one of them.