湿润烧伤膏对脂多糖致大鼠皮肤成纤维细胞损伤的改善作用及其机制研究

Study on Improvement Effect and Mechanism of MEBO on LPS-induced Injury of Rat Skin Fibroblasts

目的:研究湿润烧伤膏对脂多糖致大鼠皮肤成纤维细胞损伤的改善作用及其机制。方法:将大鼠皮肤成纤维细胞分为对照组、脂多糖组(5μg/mL)、康复新液组(阳性对照,5μg/mL脂多糖+1.25%康复新液)和湿润烧伤膏组(5μg/mL脂多糖+0.6 mg/mL湿润烧伤膏),每组设置6个复孔。以脂多糖诱导建立炎性损伤细胞模型(对照组除外)。培养一定时间后,检测各组细胞存活率和细胞迁移率,测定细胞上清液中肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)含量,检测细胞内IL-6蛋白定位和荧光强度,检测细胞中同源性磷酸酶张力蛋白(PTEN)、磷酸化p65(p-p65)、TNF-α、IL-6、磷脂酰肌醇3激酶(PI3K)和蛋白激酶B(Akt)蛋白的表达情况。结果:与对照组比较,脂多糖组细胞存活率显著升高、迁移率显著降低,细胞上清液中TNF-α、IL-6含量和PTEN、p-p65、TNF-α、IL-6和PI3K蛋白的相对表达量均显著升高(P<0.01),IL-6蛋白主要在细胞质中表达且荧光强度增强。与脂多糖组比较,康复新液组和湿润烧伤膏组细胞存活率均显著降低、迁移率均显著升高;TNF-α、IL-6含量和PTEN、p-p65、TNF-α、IL-6(除康复新液组外)、PI3K、Akt(除康复新液组外)蛋白的相对表达量均显著降低(P<0.05或P<0.01),IL-6蛋白荧光强度减弱,且湿润烧伤膏组细胞中TNF-α、IL-6、PI3K、Akt蛋白的相对表达量均显著低于康复新液组(P<0.05或P<0.01)。结论:湿润烧伤膏可抑制脂多糖诱导的大鼠皮肤成纤维细胞增殖,降低炎性因子水平和炎症反应强度,其机制可能与下调PTEN/核因子κB、PI3K/Akt信号通路有关。

OBJECTIVE:To study the improvement effect and mechanism of MEBO on lipopolysaccharide(LPS)-induced injury of rat skin fibroblasts.METHODS:Skin fibroblasts of rats were divided into control group,LPS group(5μg/mL),Kangfuxin solution group(positive control,5μg/mL LPS+1.25%Kangfuxin solution)and MEBO group(5μg/m L LPS+0.6 mg/mL MEBO),with 6 wells in each group.Inflammatory injury cell model was induced by LPS(except for control group).After a certain period of cultivation,the cell survival rate and cell migration rate were detected in each group.The contents of TNF-αand IL-6 in cell supernatant was detected.The localization and fluorescence intensity of IL-6 protein were detected.The protein expression of PTEN,p-p65,TNF-α,IL-6,PI3 K and Akt in the fibroblasts were also determined.RESULTS:Compared with control group,survival rate of the fibroblasts was increased significantly in LPS group,while cell migration was decreased significantly;the contents of TNF-αand IL-6 in cell supernatant as well as relative protein expression of PTEN,p-p65,TNF-α,IL-6 and PI3 K were increased significantly(P<0.05 or P<0.01);IL-6 protein mainly expressed in the cytoplasm,and the fluorescence intensity was enhanced.Compared with LPS group,survival rate of the fibroblasts was decreased significantly in Kangfuxin solution group and MEBO group,while migration rate was increased significantly;the contents of TNF-αand IL-6,relative protein expression of PTEN,p-p65,TNF-α,IL-6(except for Kangfuxin solution group),PI3 K and Akt(except for Kangfuxin solution group)were decreased significantly(P<0.05 or P<0.01),while fluorescence intensity of IL-6 protein decreased;relative protein expression of TNF-α,IL-6,PI3 K and Akt in MEBO group were significantly lower than Kangfuxin solution group(P<0.05 or P<0.01).CONCLUSIONS:MEBO can inhibit the proliferation of LPS-induced skin fibroblasts,reduce the level of inflammatory factors and the intensity of inflammatory reaction,which may be related to the down-regulation of PTEN/NF-κB,PI3 K/Akt signaling pathway.