甲基莲心碱通过调节AMPK/mTOR/NLRP3信号通路减轻慢性皮肤溃疡大鼠的炎症反应

Neferine alleviates inflammation in rats with chronic skin ulcer by regulating AMPK/mTOR/NLRP3 signaling pathway

目的:探讨甲基莲心碱(Nef)调节AMP活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)/NOD样受体热蛋白结构域相关蛋白3(NLRP3)信号通路对慢性皮肤溃疡(CSU)大鼠炎症反应的影响。方法:将90只大鼠随机分为空白对照组(NC组)、CSU组、Nef组、AMPK抑制剂(Compound C)组、Nef+Compound C组,每组18只大鼠。除NC组外的其他各组大鼠通过剪开创口注射氢化可的松以及喷洒金黄色葡萄球菌构建CSU大鼠模型。造模完成后,Nef组和Compound C组分别将20%Nef、10μmol·L^(-1) Compound C与50 mL的20%高渗盐水凝胶混合敷在伤口处,Nef+Compound C组将20%Nef和10μmol·L^(-1) Compound C一起添加到20%高渗盐水凝胶中敷在伤口处,持续治疗2周,NC组、CSU组用等量盐水凝胶处理伤口。观察大鼠皮肤创面愈合情况;ELISA法检测血清白细胞介素(IL)-1β、IL-8、TNF-α水平;水解法检测创面肉芽组织中羟脯氨酸(HyP)水平;HE染色检测肉芽组织病理学变化;Western blotting检测CCL4、CCL2、CXCL12以及AMPK/mTOR/NLRP3信号通路蛋白表达水平。结果:CSU组大鼠可看到新生肉芽组织,并且有大量炎症细胞浸润现象,CSU组较NC组IL-1β、IL-8、TNF-α含量显著升高(P<0.05);与CSU组相比,Nef组创面愈合率、HyP含量、p-AMPK/AMPK蛋白水平显著增加(P<0.05),IL-1β、IL-8、TNF-α含量、CCL2、CXCL12、CCL4、mTOR、NLRP3蛋白水平显著下降(P<0.05),而Compound C组趋势相反(P<0.05);Compound C消除了Nef对CSU大鼠炎症反应的减轻作用。结论:Nef可能通过调控AMPK/mTOR/NLRP3信号通路减轻CSU大鼠炎症反应。

Objective:To investigate the effect of neferine(Nef)on inflammatory response in rats with chronic skin ulcer(CSU)by regulating AMP activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)/NOD-like receptor thermal protein domain associated protein 3(NLRP3)signaling pathway.Methods:90 rats were randomly divided into blank control group(NC group),CSU group,Nef group,AMPK inhibitor(Compound C)group,and Nef+Compound C group,with 18 rats in each group.Except the blank control group,rats in the other groups were injected with hydrocortisone and sprayed with Staphylococcus aureus to establish CSU rat model.After the modeling was completed,20%Nef and 10μmol·L^(-1 )Compound C were added to 20%hypertonic saline gel in the Nef group and Compound C group,respectively,and then applied to the wound.In the Nef+Compound C group,20%Nef and 10μmol·L^(-1) Compound C were added to 20%hypertonic saline gel and then applied to the wound.In the NC group and the CSU group,the same amount of saline gel was applied to the wound.All groups were treated for 2 weeks.The healing of skin wound in rats was observed.The levels of IL-1β,IL-8 and TNF-αin serum were detected by ELISA.The level of HyP in wound granulation tissue was detected by hydrolysis method.HE staining was used to detect pathological changes of granulation tissue.And the expression levels of CCL4,CCL2,CXCL12 and AMPK/mTOR/NLRP3 signaling pathway proteins were detected by Western blotting.Results:New granulation tissue and inflammatory cell infiltration were observed in the CSU group,the contents of IL-1β,IL-8 and TNF-αin CSU group were obviously higher than those in the NC group(P<0.05).Compared with the CSU group,the wound healing rate,content of HyP,and level of p-AMPK/AMPK protein in Nef group were obviously higher(P<0.05),the contents of IL-1β,IL-8,TNF-α,and the levels of CCL2,CXCL12,CCL4,mTOR,and NLRP3 proteins were obviously lower(P<0.05),while the trend in Compound C group was the opposite(P<0.05).Compound C eliminated the reducing effect of Nef on inflammatory reaction of CSU rats.Conclusion:Nef may reduce the inflammatory response of CSU rats by regulating AMPK/mTOR/NLRP3 signaling pathway.