槐定碱对脂多糖诱导的肺泡上皮细胞损伤的影响

Effect of sophoridine on lipopolysaccharide-induced pulmonary alveolar epithelial cell damage

目的探讨槐定碱通过调节高迁移率族蛋白l(HMGB1)-晚期糖基化终产物受体(RAGE)信号通路对脂多糖(LPS)诱导的肺泡上皮细胞损伤的影响。方法将体外培养的A549和HAECⅡ细胞随机分为5组:对照组、LPS组、LPS+槐定碱组、LPS+空载组、LPS+槐定碱+HMGB1过表达组,除对照组外其余各组细胞以LPS诱导建立损伤模型,同时以槐定碱和质粒分组处理。以细胞计数试剂盒-8法、原位末端标记法检测各组细胞活力与凋亡率;以蛋白质印迹法检测各组细胞HMGB1和RAGE蛋白表达。结果对照组、LPS组、LPS+槐定碱组、LPS+空载组和LPS+槐定碱+HMGB1过表达组的A549细胞活力分别为(100.00±0.00)%、(56.73±8.31)%、(90.02±11.24)%、(53.26±9.15)%和(60.84±8.13)%,HAECⅡ细胞活力分别为(100.00±0.00)%、(50.86±7.56)%、(93.05±12.38)%、(54.10±8.42)%和(54.43±6.14)%,A549细胞凋亡率分别为(2.11±0.65)%、(42.46±5.20)%、(4.01±1.31)%、(44.74±4.93)%和(39.75±4.86)%,HAECⅡ细胞凋亡率分别为(2.30±0.72)%、(48.14±4.87)%、(3.83±1.23)%、(45.72±5.14)%和(44.81±5.25)%,A549细胞中HMGB1蛋白水平分别为0.16±0.02、0.87±0.13、0.19±0.04、0.89±0.11和0.84±0.12,RAGE蛋白水平分别为0.19±0.03、0.94±0.16、0.21±0.04、0.96±0.15和0.90±0.17,HAECⅡ细胞中HMGB1蛋白水平分别为0.13±0.04、0.79±0.10、0.15±0.04、0.80±0.14和0.75±0.12,RAGE蛋白水平分别为0.28±0.07、1.08±0.19、0.31±0.06、1.10±0.21和1.04±0.15。上述指标:LPS组与对照组比较,LPS+槐定碱组与LPS组比较,LPS+槐定碱+HMGB1过表达组与LPS+槐定碱组比较,在统计上差异均有统计学意义(均P<0.05)。结论槐定碱可通过降低HMGB1-RAGE信号活性而清除活性氧,减少炎症因子产生,增强抗氧化酶活性,进而抑制LPS诱导的肺泡上皮细胞炎症与氧化应激,最终减轻其细胞损伤。

Objective To investigate the effect of sophoridine on lipopolysaccharide(LPS)induced pulmonary alveolar epithelial cell damage by regulating the high-mobility group box 1(HMGB1)-receptor for advanced glycation end product(RAGE)signal pathway.Methods A549 and HAECⅡcells cultured in vitro were randomly divided into 5 groups:control group,LPS group,LPS+sophoridine group,LPS+empty group and LPS+sophoridine+HMGB1 overexpression group.Except for the control group,cells in other groups were induced by LPS to establish the injury models and treated with sophoridine and plasmids,respectively.Cell counting ki-—8(CCK-8)method and terminal-deoxynucleoitidyl transferase mediated nick end labeling staining were used to detect cell viability and apoptosis rate in each group;Western blotting was used to detect the expression of HMGB1 and RAGE proteins.Results The A549 cell viability of control group,LPS group,LPS+sophoridine group,LPS+empty group and LPS+sophoridine+HMGB1 overexpression group were(100.00±0.00)%,(56.73±8.31)%,(90.02±11.24)%,(53.26±9.15)%and(60.84±8.13)%;the cell viability of HAECⅡwere(100.00±0.00)%,(50.86±7.56)%,(93.05±12.38)%,(54.10±8.42)%and(54.43±6.14)%;the apoptosis rates of A549 cells were(2.11±0.65)%,(42.46±5.20)%,(4.01±1.31)%,(44.74±4.93)%and(39.75±4.86)%;the apoptosis rates of HAECⅡcells were(2.30±0.72)%,(48.14±4.87)%,(3.83±1.23)%,(45.72±5.14)%and(44.81±5.25)%;the HMGB1 protein levels in A549 cells were0.16±0.02,0.87±0.13,0.19±0.04,0.89±0.11 and 0.84±0.12;RAGE protein levels were 0.19±0.03,0.94±0.16,0.21±0.04,0.96±0,15 and 0.90±0.17;the HMGB1 protein levels in HAECⅡcells were0.13±0.04,0.79±0.10,0.15±0.04,0.80±0.14 and 0.75±0.12;RAGE protein levels were 0.28±0.07,1.08±0.19,0.31±0.06,1.10±0.21 and 1.04±0.15.The above indexes:there were statistically significant differences between LPS group and control group,LPS+sophoridine group and LPS group,LPS+sophoridine+HMGB1 overexpression group and LPS+sophoridine group(all P<0.05).Conclusion Sophoritine can clear reactive oxygen species,reduce the production of inflammatory factors,enhance antioxidant enzyme activity,and inhibit LPS induced inflammation and oxidative stress in alveolar epithelial cells by reducing HMGB1-RAGE signal activity,ultimately reducing cell damage.