芍药苷靶向钙激活氯通道TMEM16A减少NFκB活化治疗急性胰腺炎

Paeoniflorin targeting calcium-activated chloride channel TMEM16A to reduce the NFκB activation in the treatment of acute pancreatitis

目的探讨芍药苷通过抑制钙激活氯通道TMEM16A进而减少NFκB活性治疗急性胰腺炎作用。方法采用细胞培养、ELISA、Western blot、细胞转染、全细胞膜片钳技术及分子模拟对接等方法,观察腺泡细胞炎症时芍药苷对TMEM16A蛋白以及通道电流,及其对下游核转移因子NFκB活性和炎性细胞因子的作用,以及芍药苷与TMEM16A蛋白的结合情况。结果芍药苷能降低腺泡细胞中TMEM16A蛋白表达,减少腺泡细胞内NFκB的磷酸化程度,减少AR42J腺泡细胞炎症时细胞培养上清液中TNF-α和IL-6含量。此外芍药苷可降低转染TMEM16A质粒的HEK293细胞中TMEM16A钙激活氯电流作用。芍药苷分子与TMEM16A蛋白通过Arg373,Leu627和Asp812三个重要氨基酸残基紧密结合。结论芍药苷可靶向抑制钙激活氯通道TMEM16A减少NFκB磷酸化作用治疗急性胰腺炎。

Objective To investigate the effect of paeoniflorin in the treatment of acute pancreatitis by inhibiting the calcium-activated chloride channel TMEM16A and reducing the activity of NFκB.Methods Cell culture,ELISA,Western blot,cell transfection,whole-cell patch clamp technology and molecular simulation docking were used to observe the effects of paeoniflorin on TMEM16A protein and channel current,as well as its effect on the downstream nuclear transfer factor NFκB activity and inflammatory cytokines in the acinar cell inflammation,and the binding of paeoniflorin to TMEM16A protein.Results Paeoniflorin reduced the expression of TMEM16A protein in acinar cells,decreased the phosphorylation of NFκB in acinar cells,and reduced the contents of TNF-αand IL-6 in cell culture supernatant of AR42J acinar cells under inflammation.In addition,paeoniflorin reduced the activation of chlorine current by TMEM16A calcium in HEK293 cells transfected with TMEM16A plasmid.Paeoniflorin molecule was closely bound to TMEM16A protein through three important amino acid residues of Arg373,Leu627 and Asp812.Conclusion Paeoniflorin can target and inhibit the calcium-activated chloride channel TMEM16A to reduce NFκB phosphorylation in the treatment of acute pancreatitis.