莲心碱通过抑制细胞自噬减轻体外胰腺腺泡细胞急性炎症反应
Liensinine alleviates acute inflammatory response of pancreatic acinar cells in vitro by inhibiting autophagy
惠博 1张健 1李韧 1周蕊 1杨正安1
作者信息
- 1. 西安交通大学第二附属医院干四病区,西安 710004
- 折叠
摘要
目的 探讨莲心碱(liensinine,LSN)对雨蛙素诱导的急性胰腺炎细胞模型的保护作用及其分子机制.方法 胰腺腺泡细胞AR42J分为对照组、模型组、20 μmol/L LSN组和40 μmol/L LSN组.模型组加入雨蛙素刺激造模;20 μmol/L LSN组和40 μmol/L LSN组加入LSN预处理12 h,再加入雨蛙素刺激造模.采用Western blot检测LC3、Beclin-1和p62蛋白表达水平,采用ELISA法检测炎症因子白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)和肿瘤坏死因子(tumor necrosis factor-α,TNF-α)分泌水平,采用淀粉酶活性检测试剂盒检测淀粉酶含量,采用Calcein AM细胞活性检测试剂盒检测细胞存活率,采用乳酸脱氢酶(lactic dehydrogenase,LDH)细胞毒性检测试剂盒检测细胞损伤,采用TUNEL法检测细胞凋亡.结果 与对照组比较,模型组LC3-Ⅱ/LC3-Ⅰ比率和Beclin-1蛋白水平显著升高(P＜0.01),而p62蛋白水平显著降低(P＜0.01);与模型组比较,20 μmol/L LSN组和40 μmol/L LSN组LC3-Ⅱ/LC3-I比率和Beclin-1蛋白水平显著降低(P＜0.05),而 p62 蛋白水平显著升高(P＜0.05);与 20 μmol/L LSN 组比较,40 μmol/L LSN 组 LC3-Ⅱ/LC3-Ⅰ 比率和 Beclin-1 蛋白水平显著减少(P＜0.05),而p62蛋白水平显著增加(P＜0.05).与对照组比较,模型组IL-1 β、IL-6和TNF-α分泌水平显著增加(P＜0.01),淀粉酶活性显著升高(P＜0.01),细胞存活率显著降低(P＜0.01),LDH活性显著升高(P＜0.01),细胞凋亡率显著增加(P＜0.01);与模型组比较,20 μmol/L LSN组和40 μmol/L LSN组IL-1 β、IL-6和TNF-α分泌水平显著降低(P＜0.05),淀粉酶活性显著下降(P＜0.05),细胞存活率显著升高(P＜0.05),LDH活性显著降低(P＜0.05),细胞凋亡率显著下降(P＜0.05);与20 μmol/L LSN组比较,40 μmol/L LSN组IL-1β、IL-6和TNF-α分泌水平显著减少(P＜0.05),淀粉酶活性显著降低(P＜0.05),细胞存活率显著提高(P＜0.05),LDH活性显著下降(P＜0.05),细胞凋亡率显著降低(P＜0.05).结论 莲心碱可有效保护雨蛙素诱导的胰腺腺泡细胞损伤,其作用机制可能与抑制细胞自噬相关.
Abstract
Objective To explore the protective role of liensinine(LSN)in a cellular model of cerulein-induced acute pancreatitis and its underlying mechanism.Methods Pancreatic acinar cells AR42J were divided into control group,model group,20 μmol/L LSN group,and 40 μmol/L LSN group.AR42J cells in model group were stimulated by cerulein,and AR42J cells in 20 μmol/L LSN group and 40 μmol/L LSN group were pretreated with LSN for 12 h followed by stimulation with cerulein.Protein levels of LC3,Beclin-1 and p62 were measured by Western blot.The levels of inflammatory cytokines including interleukin-1β(IL-1β),IL-6,and tumor necrosis factor-α(TNF-α)were assessed by ELISA.Amylase level was analyzed by amylase assay kit.Cell survival rate was measured by Calcein AM cell viability assay kit.Cell injury was determined by lactic dehydrogenase(LDH)cytotoxicity assay kit.Cell apoptosis was detected by TUNEL assay.Results Compared with control group,LC3-Ⅱ/LC3-Ⅰ ratio and Beclin-1 protein level were significantly increased(P＜0.01),while p62 protein level was significantly decreased in model group(P＜0.01).Compared with model group,LC3-Ⅱ/LC3-Ⅰ ratio and Beclin-1 protein level were significantly decreased(P＜0.05),while p62 protein level was significantly increased in 20 μmol/L LSN group and 40 μmol/L LSN group(P＜0.05).Compared with 20 μmol/L LSN group,LC3-Ⅱ/LC3-Iratio and Beclin-1 protein level were significantly reduced(P＜0.05),while p62 protein level was significantly upregulated in 40 μmol/L LSN group(P＜0.05).Compared with control group,the levels of IL-1β,IL-6 and TNF-α were significantly increased(P＜0.01),the amylase level was significantly increased(P＜0.01),the cell survival rate was significantly reduced(P＜0.01),LDH activity was significantly elevated(P＜0.01),and the cell apoptosis rate was increased in model group(P＜0.01).Compared with model group,the levels of IL-1 β,IL-6 and TNF-α were significantly decreased(P＜0.05),the amylase level was significantly decreased(P＜0.05),the cell survival rate was significantly improved(P＜0.05),LDH activity was significantly reduced(P＜0.05),and the cell apoptosis rate was significantly decreased in 20 μmol/L LSN group and 40 μmol/L LSN group(P＜0.05).Compared with 20 μmol/L LSN group,the levels of IL-1β,IL-6 and TNF-α were significantly decreased(P＜0.05),the amylase level was significantly reduced(P＜0.05),the cell survival rate was significantly increased(P＜0.05),LDH activity was significantly decreased(P＜0.05),and the cell apoptosis rate was significantly decreased in 40 μmol/L LSN group(P＜0.05).Conclusion LSN can effectively protect against cerulein-induced injury of pancreatic acinar cells possibly through the suppression of autophagy.
关键词
急性胰腺炎/胰腺腺泡细胞/细胞自噬/莲心碱/炎症反应/细胞损伤/细胞凋亡Key words
acute pancreatitis/pancreatic acinar cells/autophagy/liensinine/inflammatory response/cell injury/cell apoptosis引用本文复制引用
基金项目
陕西省社会发展科技攻关项目(2016SF-042)
出版年
2024
国家科技期刊平台
NSTL
万方数据