异钩藤碱对大鼠急性胰腺炎细胞的炎症和凋亡改善及ERK信号通路调控作用

Effects of isorhynchophylline on inflammation,apoptosis and ERK signaling pathway of acute pancreatitis cells in rats

目的观察异钩藤碱(LSO)对大鼠急性胰腺炎(AP)细胞炎症和凋亡改善及ERK信号通路调控作用。方法将AR42J细胞分为6组,对照组正常培养,模型组、LSO组、抑制剂组、LSO+抑制剂组、LSO+激活剂组加入100 nmol/L雨蛙素制备AP细胞模型,LSO组制模后加入40μmol/L LSO,抑制剂组制模后加入10μmol/L U0126,LSO+抑制剂组制模后加入40μmol/L LSO和10μmol/L U0126,LSO+激活剂组制模后加入40μmol/L LSO和0.1μmol/L C16-PAF。采用ELISA法、EdU法及Hoechst 33258染色法分别测定细胞培养液中的炎症因子(TNF-α、IL-6、IL-8、IL-1β)、细胞增殖率和凋亡率,qPCR法测定细胞中NLRP3、斑点样蛋白(ASC)、半胱氨酸蛋白酶-1(Caspase-1)mRNA,WB法测定细胞中ERK 1/2、p-ERK 1/2、NLRP3、ASC、Caspase-1蛋白。结果与对照组比较,模型组细胞TNF-α、IL-6、IL-8、IL-1β、凋亡率、p-ERK 1/2蛋白及NLRP3、ASC、Caspase-1 mRNA和蛋白升高,增殖率降低(P均<0.05);与模型组比较,LSO组、抑制剂组细胞炎症因子(TNF-α、IL-6、IL-8、IL-1β)、凋亡率、p-ERK 1/2蛋白及NLRP3、ASC、Caspase-1 mRNA和蛋白降低,增殖率升高(P均<0.05);与LSO组比较,LSO+抑制剂组中U0126增强了LSO对细胞上述指标趋势的作用,而LSO+激活剂组中C16-PAF则逆转了LSO对细胞上述指标趋势的作用(P均<0.05)。结论LSO对大鼠急性胰腺炎细胞的炎症和凋亡有改善作用,可能通过调控ERK信号通路来实现。

Objective To observe the effects of isorhynchophylline(LSO)on inflammation,apoptosis and ERK signaling pathway of acute pancreatitis(AP)cells in rats.Methods AR42J cells were divided into six groups.Cells in the control group were cultured normally;cells in the model group,LSO group,inhibitor group,LSO+inhibitor group and LSO+activator group were added with 100 nmol/L cerulein to prepare AP cell models,and cells in the LSO group were added with 40μmol/L LSO after modeling,10μmol/L U0126 was added to the inhibitor group,40μmol/L LSO and 10μmol/L U0126 were added to the LSO+inhibitor group,and 40μmol/L LSO and 0.1μmol/L C16-PAF were added to the LSO+activator group.Inflammatory factors(TNF-α,IL-6,IL-8,IL-1β),proliferation rate,and apoptosis rate in cell culture medium were determined by ELISA,EdU method and Hoechst 33258 staining,respectively.The mRNA levels of nucleotide oligomerization domain-like receptor protein 3(NLRP3),speckle-like protein(ASC)and Caspase-1 in the cells were determined by qPCR,and the protein levels of ERK 1/2,p-ERK 1/2,NLRP3,ASC and Caspase-1 were determined by WB.Results Compared with the control group,TNF-α,IL-6,IL-8,IL-1β,apoptosis rate,p-ERK 1/2 protein,NLRP3,ASC,Caspase-1 mRNA and protein increased,and the proliferation rate decreased in the model group(all P<0.05).Compared with the model group,inflammatory cytokines(TNF-α,IL-6,IL-8,IL-1β),apoptosis rate,p-ERK 1/2 protein,NLRP3,ASC,Caspase-1 mRNA and protein decreased,and proliferation rate increased in the LSO group and inhibitor group(all P<0.05).Compared with the LSO group,U0126 in the LSO+inhibitor group enhanced the effect of LSO on the above trend of cells,while C16-PAF in the LSO+activator group reversed the effect of LSO on the above trend of cells(all P<0.05).Conclusion LSO can inhibit the inflammation and apoptosis of AP cells probably by regulating ERK signaling pathway.