【摘要】 目的：探讨正丁酸钠（SB）对严重烧伤患者血管内皮细胞通透性和内皮糖萼层的影响以及初步分子机制。方法：将培养后的人脐静脉内皮细胞（HUVECs）分为正常血清组、烧伤血清组、低浓度SB组（烧伤血清+1 mmol/L SB）和高浓度SB组（烧伤血清+3 mmol/L SB）。正常血清组用健康人血清、烧伤血清组用严重烧伤患者血清、不同浓度SB组用严重烧伤患者血清及不同浓度SB共同培养12 h以后，采用蛋白质印迹法（Western blotting）检测细胞内带状闭合蛋白-1(ZO-1)的表达，酶联免疫吸附试验检测细胞上清液中血管内皮钙黏着蛋白（VE-cadherin）水平和多配体蛋白聚糖-1(SDC-1)脱落量，Transwell法检测单层内皮细胞通透系数（Pa），透射电镜下观察内皮细胞糖萼层的变化，探讨SB的最佳作用浓度和对血管内皮细胞通透性以及糖萼的影响。另将HUVECs复苏后分为正常血清组、烧伤血清组、烧伤血清+SB组（烧伤血清+3 mmol/L SB）和烧伤血清+SB+血管内皮细胞生长因子（VEGF）抑制剂（丹参酮ⅡA，使用浓度为20μg/mL）组，采用Western blotting检测细胞内低氧诱导因子-1α(HIF-1α)、VEGF与基质金属蛋白酶-9(MMP-9)蛋白的表达。结果：与正常血清组比较，烧伤血清组ZO-1蛋白表达显著降低，VE-cadherin蛋白表达、Pa与SDC-1脱落量均显著增加（P <0.001）；与烧伤血清组比较，低浓度SB组Pa、SDC-1脱落量显著降低（P <0.01），高浓度SB组ZO-1蛋白表达显著增加（P <0.05）而VE-cadherin蛋白水平显著降低（P <0.01）；与低浓度SB组比较，高浓度SB组VE-cadherin蛋白表达、Pa、SDC-1脱落量均显著降低（P <0.05）。正常血清组HUVECs的糖萼层相对浓密，结构比较完整；烧伤血清组糖萼层结构遭到破坏，已发生断裂、脱落；不同浓度SB组与烧伤血清组比较，糖萼层更浓密、结构的破坏程度较轻，其中以高浓度SB组最明显。烧伤血清组HIF-1α、VEGF、MMP-9蛋白表达均显著高于正常血清组（P <0.01）；烧伤血清+SB组HIF-1α、VEGF和MMP-9蛋白表达量均显著低于烧伤血清组（P <0.01），但均显著高于正常血清组（P <0.05）。烧伤血清+SB+VEGF抑制剂组HIF-1α、VEGF、MMP-9蛋白表达均显著低于烧伤血清组和烧伤血清+SB组（P <0.01）。结论：SB可能通过抑制HIF-1α-VEGFMMP-9信号通路的活化减轻严重烧伤后血管内皮细胞糖萼层的损伤，从而改善严重烧伤引起的血管内皮细胞通透性的增加。更多还原

【Abstract】 Objective: To investigate the effects of sodium butyrate(SB) on vascular permeability and endothelial glycocalyx in vascular endothelial cells after severe burn, and explore its molecular mechanisms. Methods: The cultured human umbilical vein vascular endothelial cells(HUVECs) were divided into the normal serum group, the burn serum group,the low-concentration(SB) group(burn serum +1 mmol/L SB), and the high-concentration SB group(burn serum +3 mmol/L SB). After 12 hours of co-culture, the normal serum group using the serum of healthy people, the burn serum group using the serum of patients with severe burns, and the different concentration SB groups using the serum of patients with severe burns and different concentrations of SB, the expression of protein zonula occludens-1(ZO-1) was detected by Western blotting. The level of vascular endothelial cadherin(VE-cadherin) and the shedding amount of Syndecan-1(SDC-1) in the cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA). The permeability coefficient(Pa) of monolayer endothelial cells was detected by Transwell assay. The changes of glycocalyx layer of endothelial cells were observed under transmission electron microscopy to explore the optimal concentration of SB and the effects on permeability of vascular endothelial cells and glycocalyx. After HUVECs resuscitation, they were divided into the normal serum group, the burn serum group, the burn serum + SB group(burn serum +3 mmol/L SB), and the burn serum + SB + vascular endothelial growth factor(VEGF)inhibitor group(tanshinone ⅡA, concentration: 20 μg/mL). The protein expressions of hypoxia-inducible factor 1α(HIF-1α), VEGF and matrix metalloproteinase-9(MMP-9) in cells were detected by Western blotting. Results: Compared with the normal serum group, the expression of ZO-1 protein in the burn serum group was significantly decreased, and the expression of VE-cadherin protein, as well as the shedding amount of Pa and SDC-1 were significantly increased(P < 0.001). Compared with the burn serum group, the shedding amount of Pa and SDC-1 in the low-concentration SB group were significantly decreased(P < 0.01); the expression of ZO-1 protein in the high-concentration SB group increased significantly(P < 0.05);the level of VE-cadherin protein in the high-concentration SB group decreased significantly(P < 0.01). Compared with the low-concentration SB group, the expression of VE-cadherin protein levels, the shedding amount of Pa and SDC-1 in the highconcentration SB group decreased significantly(P < 0.05). The endothelial glycocalyx of HUVECs in the normal serum group was relatively dense and structurally intact. The structure of the endothelial glycocalyx in the burn serum group was damaged and had been fractured and fell off. Compared to the burn serum group, the glycolycalyx layer was denser and the degree of structural damage was less in different concentration SB groups, among which the high concentration SB group was the most obvious. The protein expression of HIF-1α, VEGF and MMP-9 in the burn serum group were significantly higher than those in the normal serum group(P < 0.01). The expression of HIF-1α, VEGF and MMP-9 in the burn serum+SB group were significantly lower than those in the burn serum group(P < 0.01), but were still higher than those in the normal serum group(P < 0.05). The protein expression of HIF-1α, VEGF and MMP-9 in the burn serum + SB + VEGF inhibitor group were significantly lower than those in the burn serum group and burn serum + SB groups(P < 0.01). Conclusions: Sodium butyrate may alleviate the damage to the glycocalyx of vascular endothelial cells after severe burns by inhibiting the activation of the HIF-1α-VEGF-MMP-9 signal pathway, thereby improving the increase in vascular endothelial cell permeability caused by severe burns.更多还原