黄芪甲苷调节PI3K/AKT/eNOS信号通路对糖尿病大鼠皮肤缺损的影响

Effects of Astragaloside Ⅳ Regulating PI3K/AKT/e NOS Signaling Pathway on Skin Defects in Diabetic Rats

目的:探究黄芪甲苷通过调控3-磷酸肌醇激酶(PI3K)/蛋白激酶B(AKT)/内皮型一氧化氮合酶(eNOS)信号通路对糖尿病大鼠皮肤损伤的影响。方法:36只雄性SD大鼠随机分为糖尿病组、皮肤损伤组、护理软膏组、黄芪甲苷高剂量组、黄芪甲苷低剂量组、黄芪甲苷+PI3K抑制剂组,每组6只。除糖尿病组外,其余各组均建立糖尿病大鼠皮肤损伤模型,各给药组分别给予相应干预,糖尿病组和皮肤损伤组不做处理。1次/d,持续用药21 d,记录各时间点(术后7、14、21 d)创面愈合情况;图片记录大鼠术后21 d创面血管生成情况,分析各组大鼠血管化面积比;HE染色和Masson染色观察皮肤病理学变化;检测PI3K/AKT/eNOS信号通路及VEGF/VEGFR信号通路相关基因的mRNA及蛋白的表达情况。结果:术后各时间点大鼠创面愈合率比较,差异有统计学意义(P<0.05),存在时间效应。5组大鼠创面愈合率总体比较,差异有统计学意义(P<0.05),存在分组效应。时间因素与分组因素对大鼠创面愈合率的影响存在交互作用(P<0.05)。与糖尿病组比较,皮肤损伤组大鼠创面组织坏死严重,创面血管化面积比明显降低(P<0.05),PI3K/AKT/eNOS通路相关基因mRNA、蛋白磷酸化表达及VEGF、VEGFR2蛋白表达水平均明显升高(P<0.05);与皮肤损伤组比较,黄芪甲苷高剂量组、黄芪甲苷低剂量组、护理软膏组大鼠创面组织逐渐恢复,创面血管化面积比明显升高(P<0.05),PI3K/AKT/eNOS通路相关基因mRNA、蛋白磷酸化表达及VEGF、VEGFR2蛋白表达水平均明显升高(P<0.05);与黄芪甲苷高剂量组比较,黄芪甲苷低剂量组、黄芪甲苷+PI3K抑制剂组大鼠创面组织恢复缓慢,创面血管化面积比明显降低(P<0.05),PI3K/AKT/eNOS信号蛋白磷酸化水平及VEGF、VEGFR2蛋白表达均明显降低(P<0.05);黄芪甲苷高剂量组各项指标与护理软膏组比较,差异均无统计学意义(P>0.05)。结论:黄芪甲苷可增强糖尿病皮肤缺损模型大鼠PI3K/AKT/eNOS通路激活水平,提高VEGF、VEGFR2蛋白的表达,加速创面组织的恢复和血管生成,缓解皮肤缺损。

Objective: To investigate the effect of astragaloside Ⅳ on skin damage in diabetic rats by regulating the 3-phosphoinositide kinase(PI3K) protein kinase B(AKT)/endothelial nitric oxide synthase(eNOS) signaling pathway. Methods: A total of 36 male SD rats were randomly divided into diabetes group, skin damage group,nursing ointment group, astragaloside Ⅳ high-dose group, astragaloside Ⅳ low-dose group, and astragaloside Ⅳ+PI3K inhibitor group, with 6 rats in each group. Except for the diabetes group, the rest of the groups were established with diabetic rat skin damage model. Each administration group was given corresponding intervention,and the diabetes group and the skin damage group were not treated. 1 time/d, continuous medication for 21 d.The wound healing at each time point(7, 14, 21 d after operation) was recorded. Pictures were recorded on the wound angiogenesis of rats on 21 d after operation, and the ratio of vascularized area in each group was analyzed.HE staining and Masson staining were used to observe skin pathological changes. The related genes mRNA and protein expressions of PI3K/AKT/eNOS signaling pathway and VEGF/VEGFR signaling pathway were detected.Results: The wound healing rate of rats at different time points after operation was compared, and the difference was statistically significant(P<0.05), and there was a time effect. The overall comparison of the wound healing rate of the five groups of rats showed a statistically significant difference(P<0.05), and there was a group effect.There was an interaction between time and grouping factors on the wound healing rate of rats(P<0.05). Compared with the diabetic group, the wound tissue of the rats in the skin damage group was severely necrotic, and the area ratio of wound vascularization was significantly reduced(P<0.05). Compared with the skin damage group, the wound tissue of the rats in astragaloside Ⅳ high-dose group, astragaloside Ⅳ low-dose group, and nursing ointment group gradually recovered, and the vascularized area ratio of the wound was significantly increased(P <0.05). The mRNA and protein phosphorylation levels of PI3K/AKT/eNOS pathway-related genes, and the expression levels of VEGF and VEGFR2 proteins were significantly increased(P<0.05). Compared with the astragaloside Ⅳ high-dose group, the wound tissue of rats in the astragaloside Ⅳ low-dose group and the astragaloside Ⅳ + PI3K inhibitor group recovered slowly, and the ratio of wound vascularization area was significantly decreased(P <0.05). The phosphorylation level of PI3K/AKT/eNOS signal protein and the expression of VEGF and VEGFR2 protein were significantly decreased(P<0.05). There was no significant difference in the indexes between the astragaloside Ⅳhigh-dose group and the nursing ointment group(P>0.05). Conclusion: Astragaloside Ⅳ can enhance the activation of PI3K/AKT/eNOS pathway in diabetic skin defect model rats, increase the expression of VEGF and VEGFR2 proteins,accelerate wound tissue recovery and angiogenesis, and relieve skin defects.