三叶青黄酮通过TLR4/NF-κB通路调控NLRP3小体激活改善大鼠心肌细胞缺氧再复氧损伤的作用及机制  

Effects and mechanisms of Radix Tetrastigma hemsleyani flavone on improving rat hypoxia/reoxygenation injury by regulating NLRP3 inflammasome activation via TLR4/NF-κB pathway

作　　者：沈盛晖[1] 叶建华[1] 吴相忠 张祺箐 彭鹏 吕珩 SHEN Shenghui;YE Jianhua;WU Xiangzhong;ZHANG Qiqing;PENG Peng;LYV Heng(Department of Cardiology,Tongde Hospital of Zhejiang Province,Hangzhou,Zhejiang 310012,China;Department of Psychiatric Geriatric Intensive Care Medicine,Tongde Hospital of Zhejiang Province,Hangzhou,Zhejiang 310012,China;Department of Oncology,Tongde Hospital of Zhejiang Province,Hangzhou,Zhejiang 310012,China;Department of Geriatric Medicine,Tongde Hospital of Zhejiang Province,Hangzhou,Zhejiang 310012,China)

机构地区：[1]浙江省立同德医院心血管科,杭州310012 [2]浙江省立同德医院精神老年重症医学科,杭州310012 [3]浙江省立同德医院肿瘤科,杭州310012 [4]浙江省立同德医院老年医学科,杭州310012

出　　处：《浙江中西医结合杂志》2024年第6期507-513,共7页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：浙江省中医药传承与创新“十百千”人才工程(杏林工程)省级中青年临床名中医项目(No.浙中医药[2017]27号);浙江省中医药科学研究基金项目(No.2022ZA036)。

摘　　要：目的探究三叶青黄酮(RTHF)对大鼠心肌细胞缺氧再复氧(H/R)损伤的作用及其可能机制。方法以大鼠心肌细胞H9C2为研究对象,分为对照组、H/R组、H/R+低浓度RTHF(L-RTHF)组(25μg/mL)、H/R+中浓度RTHF(M-RTHF)组(50μg/mL)和H/R+高浓度RTHF(H-RTHF)组(100μg/mL)。通过CCK-8法检测各组细胞活力;试剂盒检测各组细胞上清液中乳酸脱氢酶(LDH)和超氧化物歧化酶(SOD)含量;DCFH-DA探针法检测细胞内活性氧(ROS)水平;实时荧光定量聚合酶链反应(qRT-PCR)和Western blot检测各组细胞中NOD样受体热蛋白结构域相关蛋白3(NLRP3)、半胱氨酸蛋白酶-1(Caspase-1)、凋亡相关斑点样蛋白(ASC)、消皮素D的N端切割产物(GSDMD-N)、Toll样受体4(TLR4)、磷酸化核因子κB p65(p-NF-κB p65)、核因子κB p65(NF-κB p65)的表达水平。结果与对照组比较,H/R组H9C2细胞活力[(71.33±1.70)%比(100.00±2.94)%,P<0.01]、SOD水平[(15.54±0.36)U/mg prot比(34.54±0.87)U/mg prot,P<0.01]降低,ROS相对DCF强度[(3.72±0.12)比(1.00±0.10),P<0.01]和LDH水平[(387.57±9.37)U/L比(182.80±11.53)U/L,P<0.01]升高,NLRP3[(2.75±0.04)比(1.00±0.02)、(4.71±0.18)比(1.00±0.11),P<0.01]、Caspase-1[(3.02±0.11)比(1.00±0.06)、(3.33±0.09)比(1.00±0.12),P<0.01]、ASC[(3.32±0.12)比(1.00±0.09)、(5.30±0.15)比(1.00±0.10),P<0.01]、GSDMD-N[(3.69±0.14)比(1.00±0.13)、(3.23±0.08)比(1.00±0.06),P<0.01]、TLR4[(4.00±0.12)比(1.00±0.09)、(5.68±0.20)比(1.00±0.10),P<0.01]、p-NF-κB p65[(6.81±0.16)比(1.00±0.10)、(3.25±0.07)比(1.00±0.05),P<0.01]相对mRNA和蛋白水平升高;与H/R组比较,H/R+M-RTHF组和H/R+H-RTHF组的细胞活力[(77.00±2.16)%、(82.00±2.16)%比(71.33±1.70)%,P<0.05或P<0.01]、SOD水平[(23.43±1.50)U/mg prot、(28.66±1.22)U/mg prot比(15.54±0.36)U/mg prot,P<0.05或P<0.01]升高,ROS[(3.24±0.05)、(2.57±0.04)比(3.72±0.12),P<0.05或P<0.01]、LDH[(332.77±5.76)U/L、(253.36±9.43)U/L比(387.57±9.37)U/L,P<0.05或P<0.01]水平降低,NLRP3[(1.86±0.06)、(1.56±0.09)比(2Objective To explore the effects and the underlying mechanisms of Radix Tetrastigma hemsleyani flavone(RTHF)on hypoxia/reoxygenation(H/R)injury in rat cardiomyocytes.Methods Using rat cardiomyocytes H9C2 as the research subjects,they were divided into control,H/R,H/R+low concentration RTHF(L-RTHF)(25μg/mL),H/R+medium concentration RTHF(M-RTHF)(50μg/mL),and H/R+high concentration RTHF(H-RTHF)(100μg/mL)groups.Cell viability was detected using CCK-8 in each group.The levels of lactate dehydrogenase(LDH)and superoxide dismutase(SOD)in the cell supernatant of each group were measured using commercial kits.The levels of intracellular reactive oxygen species(ROS)were assessed using DCFH-DA probe method.The expression levels of NOD-like receptor thermal protein domain associated protein 3(NLRP3),cysteine proteinase-1(Caspase-1),apoptosis-associated speck-like protein(ASC),N-terminal cleaved product of Gasdermin D(GSDMD-N),Toll-like receptor 4(TLR4),phosphorylated nuclear factorκB p65(p-NF-κB p65),and nuclear factorκB p65(NF-κB p65)in cells of each group using real-time fluorescence quantitative polymerase chain reaction and Western blotting.Results Compared with the control group,in the H/R group,the cell activity of H9C2 cells[(71.33±1.70)vs.(100.00±2.94)%,P<0.01],SOD level[(15.54±0.36)vs.(34.54±0.87)U/mg prot,P<0.01]were decreased,while the relative intensity of ROS to DCF(3.72±0.12 vs.1.00±0.10,P<0.01)and LDH level[(387.57±9.37)vs.(182.80±11.53)U/L,P<0.01]were increased.Additionally,the relative mRNA and protein levels of NLRP3(2.75±0.04 vs.1.00±0.02,4.71±0.18 vs.1.00±0.11,P<0.01),Caspase-1(3.02±0.11 vs.1.00±0.06,3.33±0.09 vs.1.00±0.12,P<0.01],ASC(3.32±0.12 vs.1.00±0.09,5.30±0.15 vs.1.00±0.10,P<0.01],GSDMD-N(3.69±0.14 vs.1.00±0.13,3.23±0.08 vs.1.00±0.06,P<0.01),TLR4(4.00±0.12 vs.1.00±0.09,5.68±0.20 vs.1.00±0.10,P<0.01),p-NF-κB p65(6.81±0.16 vs.1.00±0.10,3.25±0.07 vs.1.00±0.05,P<0.01)were increased.Compared with the H/R group,in the H/R+M-RTHF and H/R+RTHF groups,the cell viabili

关 键 词：大鼠 三叶青黄酮 心肌细胞 缺氧再复氧损伤 NLRP3小体 TLR4/NF-κB 

分 类 号：R285.5[医药卫生—中药学]