基于PI3K/AKT信号通路研究沙棘籽油乳膏促进皮肤创伤修复的作用机制

Effects of Sea Buckthorn Seed Oil Cream on Skin Wound Repair Based on PI3K/Akt Signal Pathway

目的基于PI3K/AKT信号通路研究沙棘籽油乳膏对皮肤创伤的修复作用。方法SPF级BALB/c小鼠18只,采用皮肤环钻制成圆形全层皮肤缺损创面,将小鼠随机分成空白对照组、橄榄油组和沙棘籽油组,空白对照组不予处理,橄榄油组和沙棘籽油组分别采用30 mg橄榄油和沙棘籽油乳膏涂抹创面。在造模的第0、3、5、7、10 d,测量小鼠伤口面积。体外实验采用人永生化角质形成细胞(HaCaT),与梯度浓度的沙棘油(0、50、100、200、400 mg/L)共同孵育24 h,通过细胞增殖和细胞凋亡实验研究沙棘籽油对细胞活性的影响,采用Western Blot技术检测P-AKT、AKT、P-PI3K和PI3K的表达。采用LPS诱导HaCaT细胞炎症模型,以100、200 mg/L的沙棘籽油干预,采用ELISA方法检测培养上清液IL-1β、IL-6、TNF-α的水平。结果与空白对照组和橄榄油干预组比较,沙棘籽油干预组小鼠的创面愈合率显著提高(P<0.01)。HaCaT细胞经梯度浓度的沙棘籽油处理24 h,与空白对照组比较,100、200、400 mg/L的沙棘籽油均可明显促进HaCaT细胞的增殖(P<0.05);100、200 mg/L对HaCaT细胞的凋亡无明显影响(P>0.05);400 mg/L的沙棘籽油均可显著提高HaCaT细胞的凋亡率(P<0.01)。100、200 mg/L的沙棘籽油可显著降低LPS诱导的HaCaT细胞IL-1β和TNF-α的水平(P<0.01),200 mg/L的沙棘籽油可显著降低IL-6的水平(P<0.01)。100、200 mg/L的沙棘籽油可显著上调P-AKT和P-PI3K蛋白的表达(P<0.01)。结论沙棘籽油可能通过PI3K/AKT信号通路促进皮肤创伤的修复。

Objective To study the effects of sea buckthorn seed oil cream on skin wound repair based on phosphoinositide-3 kinase(PI3K)/protein kinase B(Akt)signal pathway.Methods Eighteen SPF BALB/c mice were made round full-thickness wounds in the skin with skin biopsy punches and then the mice were randomly divided into blank control group,olive oil group and sea buckthorn seed oil group.The olive oil group and seabuckthorn seed oil group were smeared with 30 mg olive oil and sea buckthorn seed oil cream respectively.The wound area of mice was measured on 0,3,5,7 d and 10 d.Human immortalized keratinocytes(HaCaT)cells were incubated with gradient concentrations of sea buckthorn seed oil(0,50,100,200,400 mg/L)for 24 h in vitro.The effect of sea buckthorn seed oil on cell activity was studied by cell proliferation and apoptosis assay.The expressions of phosphorylated AKT(P-AKT),AKT,phosphorylated inositol 3 kinase(P-PI3K)and phosphate inositol 3 stimulation(PI3K)were detected by western blot.The HaCaT cells were induced by lipopolysaccharide(LPS),intervened with seabuckthorn seed oil(100 and 200 mg/L).ELISA assay was adopted to detect the levels of interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α)in the culture supernatant.Results Compared with those of the blank control group and the olive oil group,the wound healing rate of the sea buckthorn seed oil group was significantly higher(P<0.01).HaCaT cells were treated with gradient concentrations of sea buckthorn seed oil for 24 hours.Compared with those of the blank control group,sea buckthorn seed oil with the concentrations of 100,200 mg/L and 400 mg/L could significantly promote the proliferation of HaCaT cells(P<0.05);sea buckthorn seed oil with the concentrations of 100 and 200 mg/L had no significant effect on HaCaT cell apoptosis(P>0.05);sea buckthorn seed oil with the concentration of 400 mg/L could significantly increase the apoptosis rate of HaCaT cells(P<0.01).Sea buckthorn seed oil with the concentrations of 100 and 200 mg/L significantly reduced the levels of IL-1β and TNF-α in LPS-induced HaCaT cells.Sea buckthorn seed oil with the concentrations of 200 mg/L decreased the level of IL-6(P<0.01).Sea buckthorn seed oil with the concentrations of 100 and 200 mg/L could significantly up regulate the expressions of P-AKT and P-PI3K protein(P<0.01).Conclusion Sea buckthorn seed oil may promote the skin wound repair through PI3K/AKT signal pathway.