西红花酸对AGEs诱导血管内皮细胞通透性增加的抑制作用

Inhibition of crocetin on AGEs-induced hyperpermeability in vascular endothelial cells

目的研究西红花酸对晚期糖基化终产物(advancedglycation end products,AGEs)诱导血管内皮细胞通透性增加的抑制作用并探讨其机制。方法用不同剂量西红花酸(0.01、0.1、1μmol.L-1)预孵牛主动脉血管内皮细胞(bo-vine vascular endothelial cells,BECs)12 h后,AGEs(100μg.L-1)刺激内皮细胞,以HRP作示踪剂检测内皮细胞单层通透性变化,ELISA法测定细胞上清MCP-1和TNF-α水平,罗丹明-鬼笔环肽荧光染色检测细胞骨架蛋白F-actin变化,Cell-based ELISA法和液闪法分别测定磷酸化p38 MAPK蛋白表达量和活性变化。同时设定正常对照组(control)、AGEs(100 mg.L-1)模型组和葛根素(1 g.L-1)阳性对照组。结果与AGEs模型照组相比,西红花酸(0.1、1μmol.L-1)预孵细胞后,F-actin骨架蛋白破环程度有所减轻,细胞单层通透性减小(P<0.01或0.05),TNF-α和MCP-1分泌降低,磷酸化p38MAPK的数量下降且活性被抑制(P<0.01或0.05)。结论西红花酸对AGEs诱导内皮细胞通透性增加有抑制作用,该作用可能与其抑制p38MAPK通路有关,这可能是其抗糖尿病血管病变的机制之一。

Aim To investigate the effects of crocetininduced by advanced glycation end products （AGEs） and its possible mechanisms. Methods Bovine vascular endothelial cells （ BECs ） were preincubated with crocetin （0. 01,0. 1,1 μmol · L-1 ） for 12 h,then exposed to AGEs （ 100 mg· L-1 ）, mono-layer perme- ability of endothelial cells was detected by methods of labeled compound by HRP. The MCP-1 and TNF-a in cell supernatant were measured by ELISA. Further- more, the treated cells were incubated with rhodaminephallodin to stain F-actin for visualizing the morphological changes of actin cytoskeleton protein. And cell- based ELISA and liquid scientillation method were adopted to analyze the expression of p-p38MAPK. In addition, control group, AGEs model group and Gegensu（ 1 mg ·L-l） positive group were also set in the study. Results Compared with AGEs group, crocetin （0. 1,1 Ixmol · L-l） was able to ameliorate disturbedon the hyperpermeability of vascular endothelial cellsramified rank of cytoskeletal protein （ F-actin ）, decrease mono-layer permeability of endothelial cells （P 〈 0. 05 or 0. 01 ） and also reduce levels of TNF-ct and MCP-1 in cell supernatant （P 〈 0.05 or 0. 01 ）. In ad- dition, results of cell-based ELISA and liquid scientil- lation methods demonstrated that the expression level and activity of p-p38MAPK were both inhibited by crocetin （ P 〈 0. 05 or 0. 01 ）. Conclusions Crocetin can inhibit AGEs-induced hyperpermeability in endothelial cells through attenuating p38MAPK phosphorylation signal pathway, which is one of the possible mechanisms for crocetin to attenuate diabetic vascular complications.