大黄提取物对脂多糖诱导的内皮细胞的保护作用研究

Effect of Rhubarb Extract on the Protection of Endothelial Cells after Lipopolysaccharide-induced Injury

目的以脂多糖(LPS)诱导的人脐静脉内皮细胞(HUVEC)作为脓毒症内皮损伤的模型,研究大黄提取物对内皮细胞的保护作用。方法使用大黄提取物(1、2.5、5、10、20、40μg/mL)干预1μg/mL LPS处理的HUVEC细胞,实验分为空白对照组、LPS刺激组、大黄提取物组;CCK-8法检测HUVEC增殖;ELISA法检测HUVEC的血管内皮生长因子(VEGF)和血管生成素(Ang)的分泌变化;WB法研究大黄提取物对NF-κB和ERK信号通路激活的作用。结果 HUVEC用1μg/mL的LPS处理后,细胞增殖吸光度值与空白对照组相比有明显下降(0.36±0.02比0.65±0.02,P<0.01);使用1μg/mL、2.5μg/mL、5μg/mL、10μg/mL、20μg/mL和40μg/mL大黄提取物干预LPS诱导的HUVEC,细胞增殖吸光度分别为(0.40±0.03)、(0.44±0.03)、(0.52±0.03)、(0.55±0.04)、(0.57±0.03)和(0.57±0.02),其中浓度>5μg/mL的大黄提取物组与LPS处理组相比,差异有统计学意义(P均<0.01)。5μg/mL大黄提取物处理组与单纯LPS处理组相比,VEGF分泌下降[(240.92±3.09)pg/mL比(283.59±1.38)pg/mL,P<0.01],Ang1分泌上升[(115.64±0.61)pg/mL比(104.30±2.38)pg/mL,P<0.05],Ang2分泌下降[(105.40±1.40)pg/mL比(118.79±6.81)pg/mL,P<0.05]。同样,5μg/mL大黄提取物处理组与单纯LPS处理组相比,Ang1的基因表达上升(0.77±0.10比0.35±0.04,P<0.01),Ang2的基因表达下降(1.76±0.09比2.30±0.37,P<0.05)。5μg/mL大黄提取物处理后,pERK和p-p65明显下降。结论大黄提取物对脂多糖诱导的内皮细胞损伤和功能异常具有保护作用。

Objective To investigate the protective effect of rhubarb extract on lipopolysaccharide（LPS）-induced endothelial cells in the model of endothelial injury in sepsis. Methods Human umbilical vein endothelial cells（HUVEC） cells treated by 1μg/m L LPS were treated with rhubarb extract（1, 2.5, 5, 10, 20, 40μg/mL）. HUVEC were divided in blank, LPS treatment, and rhubarb extract groups. Proliferation of HUVEC was detected by CCK（Cell Counting Kit）-8 after LPS treatment. The changes of vascular endothelial growth factor（VEGF） and angiopoietin（Ang） secretion in HUVEC were detected by ELISA（enzyme linked immunosorbent assay）. Western Blot was used to determine the activation of NF-κB and ERK signal pathway. Results After treatment with 1μg/m L LPS, the absorbance measured by MTT decreased significantly（0.36±0.02 vs 0.65±0.02, P0.01）; LPS-induced HUVEC were treated with 1, 2.5, 5, 10, 20, and 40μg/m L rhubarb extracts showed absorbance of 0.40 ±0.03, 0.44 ±0.03, 0.52±0.03, 0.55±0.04, 00.57±0.03 and 0.57±0.02, respectively; the absorbance of groups with the concentration more than 5μg/mL of rhubarb extract were significantly different to that of LPS treatment group（all P0.05）.Compared with LPS treatment group, 5μg/mL of rhubarb extract group had decreased VEGF secretion（240.92 ±3.09 pg/mL vs 283.59±1.38 pg/mL, P0.01）, increased Ang1 secretion（115.64±0.61 pg/mL vs 104.30±2.38 pg/mL, P0.05）, and decreased Ang2 secretion（105.40±1.40 pg/mL vs 118.79±6.81 pg/mL,P0.05）. Meanwhile, the expression of Ang1 gene was increased（0.77 ±0.10 vs. 0.35 ±0.04, P 0.01） and the expression of Ang2 gene was decreased（1.76±0.09 vs. 2.30±0.37, P0.05） in the 5μg/m L rhubarb extract treatment group compared with LPS treatment group. After 5μg/mL rhubarb extract treatment, p-ERK and p-p65 decreased significantly. Conclusion Rhubarb extract can protect the endothelial cell injury and dysfunction induced by LPS.