弱酸环境下食管内皮细胞活力与分泌因子的变化

Changes in the viability and secretion of esophageal endothelial cells under weak acidic culture condition

目的采用体外试验的方法,研究弱酸性培养对人正常食管内皮细胞(HEEC)活力的影响及潜在的调控机制。方法细胞培养液的pH值分别设为(6.0~6.5)和(7.0~7.4)。以中性培养组为对照。利用CCK8实验,检测弱酸培养条件下,不同时间点食管内皮细胞活力的变化。采用蛋白免疫印迹法检测p38和磷酸化p38(p-p38)的表达。利用酶联免疫吸附剂测定法检测培养基上清液中IL-1β和IL-8的表达水平,并检测加入p38激酶活性抑制剂SBS 203580后两者浓度的变化。结果弱酸环境下,细胞活力下降。培养1 h时,弱酸组细胞活力为(96.4±8.0)%,培养3 h和6 h时分别为(88.7±6.2)%和(87.7±7.4)%。细胞中p38的水平与培养基的pH值无关。弱酸培养可以促使细胞内p-p38的含量增加。基线时,弱酸组p-p38的灰度值比值为(0.37±0.02),在培养2 h和6 h时分别为(0.64±0.09)、(0.84±0.11),差异显著(P<0.01)。弱酸刺激诱导食管内皮细胞表达更多的IL-8和IL-1β。基线时弱酸组上清液中IL-8和IL-1β的浓度分别为(8.64±1.31)pg/mL,(3.35±0.49)pg/mL。培养6 h后,二者的浓度分别上升至(36.85±2.02)pg/mL和(19.19±1.60)pg/mL,差异显著(P<0.01)。加入SBS 203580后,IL-8和IL-1β的浓度明显下降(P<0.05)。结论弱酸刺激可以降低食管内皮细胞的活力。p38 MAPK可能通过调控IL-8和IL-1β的表达参与该调节过程。

Objective To study the effect of weak acidic culture on the viability of normal human esophageal endothelial cells(HEEC)and the potential regulatory mechanisms.Methods The pH values of cell culture medium were set at(6.0-6.5)and(7.0-7.4),respectively.The group in neutral medium was set as control.CCK8 experiment was used to detect the change of cell viability at different time points.The expressions of p38 and phosphorylated p38(p-p38)were detected by Western Blot experiment.Enzyme linked immunosorbent assay was used to detect IL-1βand IL-8 concentration in the medium supernatant before and after adding p38 activity inhibitor(SBS 203580).Results HEEC viability was decreased under weak acidic conditions.After 1 hour of cultivation,the HEEC viability was(96.4±8.0)%,after 3 and 6 hours,it decreased to(88.7±6.2)%and(87.7±7.4)%,respectively.The level of p38 in cells was independent of culture medium pH values.Weak acidic stimulation could promote an increase of p-p38 in HEEC.At baseline,the gray value ratio of p-p38 in the weak acidic group was(0.37±0.02),and after 2 and 6 hours of culturing,it increased to(0.64±0.09)and(0.84±0.11),respectively,which differences were significant(P0.01).More IL-8 and IL-1βwere expressed after weak acidic stimulation.At baseline,the concentrations of IL-8 and IL-1βin the medium supernatant of weak acidic group were(8.64±1.31)pg/mL and(3.35±0.49)pg/mL.After 6 hours of culturing,they increased significantly to(36.85±2.02)pg/m L and(19.19±1.60)pg/m L(P0.01),while the concentrations were decreased after adding SBS 203580(P0.05).Conclusions The HEEC viability was reduced by weak acidic stimulation,p38 MAPK may participate in the process by regulating the expression of IL-8 and IL-1β.