N-乙酰半胱氨酸通过调控ADAM10表达抑制脓毒症血清诱导的血管内皮细胞高通透性

N-acetylcysteine inhibits sepsis-induced high permeability of vascular endothelial cells by regulating ADAM10 expression

目的:探讨N-乙酰半胱氨酸(NAC)对脓毒症血清诱导的血管内皮细胞高通透性的影响及其机制。方法:收集本院脓毒症患者和健康志愿者血清。体外培养人脐静脉内皮细胞EA.hy926,将细胞分为空白对照组(Blank)、实验对照组(CON)、脓毒症组(Sep)、NAC低剂量组(NAC-L)、NAC高剂量组(NAC-H)及高剂量NAC联合整合素金属蛋白酶10(ADAM10)抑制剂GI254023X组(NAC-H+GI254023X)。分组处理后,采用MTT法检测各组细胞的增殖活性;qRT-PCR法检测各组细胞ADAM10 mRNA表达水平;Transwell小室法检测各组单层细胞通透性;跨内皮细胞电阻法检测各组细胞跨膜电阻(TEER)值;Western blot法检测各组细胞ADAM10、Occludin、ZO-1及VE-cadherin蛋白表达水平。结果:与Blank组和CON组比较,Sep组细胞增殖活性、TEER值、VE-cadherin、Occludin及ZO-1蛋白表达水平明显降低,而ADAM10 mRNA和蛋白表达量、细胞通透性明显升高(P<0.05);与Sep组比较,NAC-H组细胞增殖活性、TEER值、VE-cadherin、Occludin及ZO-1蛋白表达水平明显升高,而ADAM10 mRNA和蛋白表达量、细胞通透性明显降低(P<0.05),而NAC-L组的差异无统计学意义(P>0.05);ADAM10抑制剂GI254023X能显著增强NAC对脓毒症血清诱导人脐静脉内皮细胞EA.hy926高通透性的抑制作用。结论:NAC通过下调AD⁃AM10的表达水平,抑制脓毒症血清诱导的人脐静脉内皮细胞高通透性。

Objective:To investigate the effect of N-acetylcysteine(NAC)on sepsis-induced high permeability of vascular en⁃dothelial cells,and its possible mechanism.Methods:Serum of sepsis patients and healthy volunteers in our hospital were collected.Human umbilical vein endothelial cells EA.hy926 were cultured in vitro and divided into blank control group(Blank),experimental control group(CON),sepsis experimental group(Sep),low-dose NAC group(NAC-L),high-dose NAC group(NAC-H)and highdose NAC combined with ADAM10 inhibitor GI254023X group(NAC-H+GI254023X).After the treatment,cell proliferative activity was detected by MTT.The mRNA expression level of ADAM10 was measured by qRT-PCR assay.The permeability and the transmem⁃brane resistance(TEER)of cells were detected by a Transwell method and epithelial voltohm meter.The protein expression levels of ADAM10,Occludin,ZO-1 and VE-cadherin were detected by Western blot.Results:Compared with the Blank and CON groups,the cell proliferative activity,TEER and the protein expression levels of VE-cadherin,Occludin and ZO-1 were significantly reduced in the Sep group(P<0.05),while the mRNA and protein expression of ADAM10 and the permeability of EA.hy926 cells were markedly increased(P<0.05).Compared with the Sep group,the cell proliferative activity,TEER and the protein expression levels of VE-cad⁃herin,Occludin and ZO-1 were significantly increased in the Sep group(P<0.05),while the mRNA and protein expression of AD⁃AM10 and the permeability of EA.hy926 cells were markedly suppressed(P<0.05),while the difference was not statistically signifi⁃cant in the NAC-L group(P>0.05).ADAM10 inhibitor GI254023X could significantly enhance the inhibitory effect of NAC on sepsisinduced high permeability in EA.hy926 cells.Conclusion:NAC inhibits the high permeability of sepsis-induced human umbilical vein endothelial cells by down-regulating the expression of ADAM10.