益气活血脑泰方干预脑缺血后铁代谢相关机制研究

Researching on the Mechanism of Correlation between IL-6/STAT3 Pathway and Hepcidin in Cerebral Ischemia and Intervention of Naotaifang

目的:探讨炎症通路IL-6/stat3/Hepcidin调节铁代谢相关机制,研究益气活血脑泰方通过抑制炎症反应,调节铁代谢,减少细胞内铁聚集,为脑泰方防治脑缺血的临床应用提供重要实验依据。方法:细胞实验中,SH-SY5Y及CHME5细胞株模型干预24 h后,检测Fe^(2+)表达、Western Blotting检测白细胞介素-6(IL-6)、Stat3及Hepcidin表达。动物实验中,随机将SD大鼠分为对照组、模型组、脑泰方低、中、高剂量组(3、9、27 g/kg)、去铁酮组。各组大鼠预处理灌胃给药连续3 d,大脑中动脉栓塞(MCAO)模型制备术后灌胃给药1 d。术后1 d取材,Western Blotting检测IL-6、Stat3及Hepcidin、Fpn的表达。结果:细胞实验结果显示:SH-SY5Y及CHME5细胞株模型组较正常组,细胞质内Fe^(2+)表达明显增加(P<0.01),IL-6、Stat3蛋白表达水平增高(P<0.05),Hepcidin表达明显增高(P<0.05)。动物实验结果显示:与模型组比较,大鼠局灶性脑缺血后脑泰方高剂量组及去铁酮组,大脑皮质及髓质IL-6和皮质stat3、Hepcidin的表达明显减少(P<0.01),脑泰方高剂量组髓质Hepcidin的表达明显减少(P<0.01),去铁酮组髓质Hepcidin的表达降低(P<0.05);脑泰方高、去铁酮组大脑皮质及髓质Fpn的表达较模型组明显增加(P<0.01)。结论:脑泰方通过抑制IL-6/stat3信号通路,抑制脑缺血后炎症反应,减少Hepcidin分泌进而促进Fpn的表达,促进铁离子外排,对脑缺血导致的铁超载损伤有一定保护作用。

Objective:To explore the mechanism of IL-6/stat3/Hepcidin in the inflammatory pathway to regulate iron metabolism,and to study the effect of Yiqi Huoxue Naotai Formula on inhibiting inflammation,regulating iron metabolism,and reducing intracellular iron accumulation,providing important experimental basis of Naotai Formula in preventing and treating cerebral ischemia.Methods:In vitro experiment:SH-SY5Y and CHME5 cell lines culture for 24 h under OGD environment.The Fe^(2+) of the 2 cell lines were detected by kit of Metallofluor ferhonox-1 iron(Ⅱ)living cell imaging probe,and the expression of inflammatory and iron metabolism related proteins were detected by western-blot.In animal experiments,The rats were randomly divided into 5 groups:a normal group,a Sham surgery group,a model group,a low dose of NTF group(3 g/kg),a middle dose of NTF group(9 g/kg),a high dose of NTF group(27 g/kg),a Defriprone group.The expression of inflammatory and iron metabolism related proteins,IL-6/STAT3,hepcidin and Fpn1,were detected by wstern-blot.Results:Cell experiment results showed that compared with the normal group,the expression of Fe ^(2+) in the cytoplasm of the SH-SY5Y and CHME5 cell lines in the OGD group was significantly increased(P<0.01),the expression of IL-6 and Stat3 protein was increased(P<0.05),and the expression of Hepcidin was significantly increased(P<0.05).The results of animal experiments showed that compared with the model group,the expression of IL-6 in the cerebral cortex and medulla,and the expression of stat3 and Hepcidin in the cerebral cortex and medulla in the high-dose Naotaifang group and the deferiprone group were significantly reduced after focal cerebral ischemia in rats(P<0.01),the expression of Hepcidin in the medulla of the Naotaifang high-dose group was significantly reduced(P<0.01),and the expression of Hepcidin in the medulla of the deferiprone group was decreased(P<0.05);The expression of medullary Fpn was significantly higher than that of the model group(P<0.01).Conclusion:Naotai Formula can inhibits the IL-6/stat3 signaling pathway,the inflammatory response after cerebral ischemia,reduce the secretion of Hepcidin and promote the expression of Fpn,promote the efflux of iron ions,and has a certain protective effect on iron overload damage caused by cerebral ischemia.