摘要
目的探讨天麻素(Gastrodin)、对BV-2小胶质细胞焦亡模型AKT/NLRP3通路的影响。方法BV-2小胶质细胞分为正常对照组、模型组(1μg·mL^(-1)LPS+10μmol·L^(-1)Nigericin)、天麻素组(10、100μg·mL^(-1)天麻素)、抑制剂对照组(10μmol·L^(-1)LY294002)、抑制剂组(100μg·mL^(-1)天麻素+10μmol·L^(-1)LY294002),采用1μg·mL^(-1)LPS联合10μmol·L^(-1)Nigericin共同干预,诱导BV-2小胶质细胞焦亡。CCK-8观察天麻素对细胞活性的影响,Western blot检测p-AKT、NLRP3、ASC、caspase-1、IL^(-1)β表达,免疫荧光检测NLRP3、p-AKT蛋白表达,Real-time PCR观察IL^(-1)βmRNA含量,分子对接确定天麻素与p-AKT1/2/3、NLRP3蛋白结合位点。结果与对照组(0μg·mL^(-1)天麻素)相比,0.1-1000μg·mL^(-1)天麻素对BV-2细胞活性无明显影响(P>0.05)。Western blot实验中,与正常组相比,模型组p-AKT、NLRP3、ASC、caspase-1、IL^(-1)β表达均明显上升(P<0.01);与模型组相比,天麻素组p-AKT、NLRP3、ASC、caspase-1、IL^(-1)β表达均下降(P<0.05或P<0.01)。免疫荧光实验中,与正常组相比,模型组NLRP3表达均明显上升(P<0.01);与模型组相比,天麻素组p-AKT、NLRP3表达明显下降(P<0.01);与天麻素组相比,抑制剂组p-AKT、NLRP3表达均下降(P<0.01)。Realtime PCR实验中,与正常组相比,模型组细胞IL^(-1)βmRNA含量均明显上升(P<0.01);与模型组相比,天麻素组细胞IL^(-1)βmRNA含量下降(P<0.01)。LDH活性检测实验中,与正常组相比,模型组细胞LDH活性明显上升(P<0.01);与模型组相比,100μg·mL^(-1)天麻素干预可降低焦亡模型细胞LDH活性(P<0.01)。分子对接中,天麻素与AKT1/2/3、NLRP3具有结合能力,结合能范围(-6.6)-(-6.8),结合方式包括氢键、范德华力等。结论天麻素通过抑制AKT/NLRP3通路改善BV-2小胶质细胞焦亡。
Objective To investigate the effects of Gastrodin on AKT/NLRP3 pathway in BV-2 microglial pyroptosis model.Methods BV-2 microglia were divided into normal control group and model group(1μg·mL^(-1)LPS+10μmol·L^(-1) Nigericin),Gastrodin group(10,100μg·mL^(-1)Gastrodin),inhibitor control group(10μmol·L^(-1) LY294002),inhibitor group(100μg·mL^(-1)Gastrodin+10μmol·L^(-1) LY294002),using 1μg·mL^(-1)LPS combination 10μmol·L^(-1) Nigericin co intervened to induce pyroptosis of BV-2 microglia.CCK-8 observed the effect of Gastrodin on cell activity,and western blot detected p-AKT,NLRP3,ASC,caspase-1,IL^(-1)βexpression,immunofluorescence detection of NLRP3,p-AKT protein expression,Realtime PCR observation of IL^(-1)βmRNA content.Molecular docking was used to determine the binding sites of Gastrodin to p-AKT1/2/3 and NLRP3 proteins.Results Compared with the control group(0μg·mL^(-1)Gastrodin),0.1-1000μg·mL^(-1)Gastrodin had no significant effect on BV-2 cell activity(P>0.05).In the western blot experiment,compared with the normal group,the expression p-AKT,NLRP3,ASC,caspase-1,IL^(-1)βin model group was significantly increased(P<0.01);Compared with the model group,the expression of p-AKT,NLRP3,ASC,caspase-1,IL^(-1)βin Gastrodin group decreased(P<0.05 or P<0.01).In immunofluorescence,the expression of NLRP3 in model group was significantly higher than that in normal group(P<0.01);Compared with the model group,the expression of p-AKT and NLRP3 in Gastrodin group decreased significantly(P<0.01);Compared with Gastrodin group,the expression of p-AKT and NLRP3 in the inhibitor group decreased(P<0.01).In the Real-time PCR experiment,compared with the normal group,IL^(-1)βmRNA content in model group cells increased significantly(P<0.01);Compared with the model group,IL^(-1)βmRNA content in Gastrodin group cells decreased(P<0.01).In the LDH content test,compared with the normal group,the LDH activity in the model group cells increased significantly(P<0.01);Compared with the model group,the intervention of 100
作者
刘泳
王宁宁
曾楚华
王雨
Liu Yong;Wang Ningning;Zeng Chuhua;Wang Yu(Qingdao hospital of Traditional Chinese Medicine(Qingdao Hiser hospital),Qingdao 266300,China;School of Basic Medicine,Yunnan University of Chinese Medicine,Kunming 650500,China;School of Basic Medical Sciences,Hubei University of Medicine,Shiyan 442000,China;Hubei Key Laboratory of Wudang Local Chinese Medicine Research,Hubei University of Medicine,Shiyan 442000,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2023年第10期3238-3245,共8页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
国家自然科学基金委员会地区项目(82060831):基于“微生物-肠-脑轴”理论探讨固本健脑法对AD的作用机制,负责人:曾楚华
湖北省科学技术厅湖北省自然科学基金青年项目(2023AFB275):天麻素通过SHP2/ANT1信号调节NLRP3炎症小体防治AD的机制研究,负责人:王雨
湖北省中医药管理局中医药科研项目(ZY2023Q048):天麻素通过AKT/mTOR/HIF-1通路调节糖酵解代谢防治抑郁的机制研究,负责人:王雨
