基于ERK信号通路研究补阳还五汤促糖尿病足溃疡愈合的作用机制

The mechanism of Buyang Huanwu Decoction promotion of diabetic foot ulcer healing based on analysis of the ERK signaling pathway

目的 基于细胞外调节激酶(ERK)信号通路研究补阳还五汤对人永生化表皮(HaCaT)细胞增殖和迁移的作用。方法 CCK-8法检测不同浓度补阳还五汤(0、5、10、15、25、50 mg/mL)对HaCaT细胞增殖的影响,Western blot检测不同浓度补阳还五汤作用下HaCaT细胞磷酸化细胞外信号调节激酶1/2(p-ERK1/2)蛋白的表达水平;划痕实验检测HaCaT细胞的迁移情况;CCK-8法检测适宜浓度补阳还五汤联合ERK抑制剂(U0126)作用下HaCaT细胞增殖的情况,Western blot法检测适宜浓度补阳还五汤以及ERK抑制剂(U0126)作用下HaCaT细胞p-ERK1/2蛋白的表达水平。结果 CCK-8法实验结果表明,5、10、15、25、50 mg/mL补阳还五汤细胞的吸光度值分别为(1.1704±0.076)、(1.2642±0.078)、(1.3845±0.060)、(1.6996±0.080)、(1.7211±0.088),与空白组(1.0514±0.080)比较,25 mg/mL时促增殖效果最佳(P<0.01)。Western blot实验结果表明,与空白组比较,5、15、25 mg/mL补阳还五汤浓度依赖性促进HaCaT细胞p-ERK1/2的表达[(0.545±0.005)、(1.038±0.126)、(2.414±0.456)比(0.260±0.092),P<0.01]。划痕实验结果表明,补阳还五汤能促进HaCaT细胞的迁移[12 h:(0.55±0.06)比(0.43±0.02),P<0.05;24 h:(0.84±0.06)比(0.58±0.06),P<0.01]。同时,CCK-8法检测结果显示,与ERK抑制剂(U0126)组比较,25 mg/mL补阳还五汤能明显促进ERK抑制剂(U0126)作用下HaCaT细胞的增殖[(1.3167±0.088)比(1.0474±0.056),P<0.01]。Western blot实验结果同样表明,与ERK抑制剂组比较,补阳还五汤促进ERK抑制剂(U0126)作用下p-ERK1/2蛋白的表达[(0.855±0.008)比(0.356±0.108),P<0.01]。结论 补阳还五汤可能通过激活ERK信号通路促进HaCaT细胞增殖、迁移,促进糖尿病足溃疡的愈合。

Objective To assess the effects of Buyang Huanwu Decoction(BYHWD) on human immortalized epidermal cells(HaCaT) proliferation and migration using the extracellular signal-regulated kinase(ERK) signaling ap proach.Methods HaCaT cells were grown and treated with BYHWD for different dosages(0,5,10,15,25,50 mg/mL)with or without an ERK inhibitor(U0126).Cell proliferation was used CCK-8 assay.Western blot was performed to determine the expression of phosphorylated extracellular signal-regulated kinases 1/2(p-ERK1/2) protein.Wound healing assay was conducted to evaluate the migration of HaCaT cells.Results The CCK-8 assay data showed that HaCaT cells treated with 5,10,15,25,50 mg/mL doses of BYHWD promoted cell viability(1.1704 ±0.076,1.2642±0.078,1.3845±0.060,1.6996±0.080,and 1.7211±0.088 vs.1.0514±0.080,respectively),especially with the25 mg/mL dosage(P0.01).Western blot data revealed that compared to the blank group,BYHWD at concentrations of 5,15,and 25 mg/mL significantly increased expression of p-ERK1/2 protein in HaCaT cells(0.545 ±0.005,1.038±0.126,and 2.414±0.456 vs.0.260±0.092,respectively;P0.01).The wound-healing assay demonstrated that BYHWD also enhanced HaCaT cell migration(0.55±0.06 vs.0.43±0.02 in 12 h;P0.05;0.84±0.06 vs.0.58±0.06 in24 h;P0.01).Furthermore,Compared to the ERK inhibitor(U0126) alone group,25 mg/mL BYHWD significantly promoted HaCaT cell viability after the addition of an ERK inhibitor U0126(1.3167±0.088 vs.1.0474±0.056;P0.01).Similarly,Western blot data showed that compared to that of the ERK inhibitor alone group,BYHWD plus U0126 still promoted level of p-ERK1/2 protein(0.855 ±0.008 vs.0.356 ±0.108;P 0.01).Conclusion BYHWD could promote HaCaT cell proliferation and migration to enhance healing of diabetic foot ulcers,which was possibly through activation of the ERK signaling.