体外胰腺腺泡细胞炎性反应中ZFP580与内质网应激的相互作用

 目的 探讨锌指蛋白ZFP580在体外蛙皮素诱导的胰腺腺泡细胞炎性反应过程中通过内质网应激通路发挥作用的机制,为急性胰腺炎(acute pancreatitis, AP)发病机制提供新的认识。方法 用蛙皮素体外刺激AR42J细胞,模拟AP过程中胰腺腺泡细胞的炎性反应状态,用MTT法测定细胞活力以确定蛙皮素的最佳诱导时间;分别用RT-PCR和Western Blot检测细胞中内质网应激相关分子和ZFP580的mRNA和蛋白表达;应用IRE1抑制剂MKC-3946干预AR42J细胞,观察ZFP580表达变化;最后用慢病毒转染方法沉默ZFP580后,用Hochest33342染色和RT-PCT、Western Blot评价其对细胞凋亡-坏死的影响。结果 以蛙皮素刺激后诱导AR42J细胞发生内质网应激,ZFP580表达在刺激8 h后开始升高,呈时间依赖性上升;应用MKC-3946后,ZFP580的表达显著降低;沉默ZFP580后,XBP-1s表达下降,同时沉默ZFP580组的Chop表达量要显著高于对照组,而Caspase-3的表达显著低于普通AP组;Hochest染色观察发现沉默ZFP580组的坏死细胞比例要显著高于普通AP组。结论 ZFP580在胰腺腺泡细胞内质网应激信号通路中的位置可能位于IRE1与XBP-1s之间,其可能通过改善凋亡-坏死比例,对减轻AP炎性反应有一定作用。

Role of ZFP580 in acute pancreatitis induced by caerulein through endoplasmic reticulum stress pathway

Objective To investigate the role of zinc finger protein 580 (ZFP580) in acute pancreatitis (AP) induced by caerulein through endoplasmic reticulum (ER) stress pathway, and provide a new therapeutic target for AP treatment.Methods MTT method was used to detect cell viability. RT-PCR and Western Blot were used to detect endoplasmic reticulum stress-related mRNA and the protein expression of ZFP580 in AP model. IRE1 inhibitor (MKC-3946) was used to observe the position of ZFP580 in endoplasmic reticulum stress related signaling pathway. After Silencing ZFP580, Hochest33342 staining and WB, RT-PCT method were used to investigate the effect of ZFP580 on the apoptosis-necrosis process.Results After using cerulein to stimulate acute pancreatitis in AR42J cells, the endoplasmic reticulum stress occurred. 8 hours later, the expression of ZFP580 started to increase (showing a time-dependent increase). After using IRE1 inhibitor (MKC-3946), the expression of ZFP580 was significantly lower than that of the group without MKC-3946 treatment (P<0.05). After silencing ZFP580, the expression of XBP-1s significantly decreased. At the same time, RT-PCR and Western blot results showed that the expression of Chop in the si-ZFP580 group was significantly higher than that in the normal AP group, while the expression of Caspase-3 was lower than that in the normal AP group. The result of Hochest staining revealed that the proportion of necrotic cells in the si-ZFP580 group was significantly higher than that in the normal AP group.Conclusions ZFP580 which may be located between IRE1 and XBP-1s may play a protective role in regulating the severity of AP by improving the apoptosis-necrosis ratio.