丹酚酸B-葛根素联用对氧糖剥夺/复氧损伤的SH-SY5Y神经细胞焦亡的影响  

Effect of Salvianolic Acid B Com bined with Puerarin on Pyroptosis of SH-SY5Y Cells Injured by Oxygen-glucose Deprivation/Reoxygenation

作　　者：涂舒欣 王艳[1] 贺晓丽[1] TU Shuxin;WANG Yan;HE Xiaoli(Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100193,China)

机构地区：[1]中国医学科学院,北京协和医学院,药用植物研究所,北京100193

出　　处：《中国实验方剂学杂志》2024年第3期54-63,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82274407)。

摘　　要：目的:基于细胞焦亡探讨丹酚酸B和葛根素合用对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)损伤的SH-SY5Y神经细胞的保护作用及机制。方法:利用SH-SY5Y细胞构建OGD/R损伤模型,设立正常对照组(control)、模型组(OGD/R)、10μmol·L^(-1)丹酚酸B组(salvianolic acid B,Sal B)、100μmol·L^(-1)葛根素组(puerarin,Pue)、10μmol·L^(-1)丹酚酸B和100μmol·L^(-1)葛根素合用组(SP)及10μmol·L^(-1)NOD样受体蛋白3(NLRP3)炎症小体抑制剂MCC950组。除正常组外,其余各组细胞在氧糖剥夺6 h后迅速复糖复氧12 h进行OGD/R造模。采噻唑蓝(MTT)法检测细胞存活率,光学显微镜下观测细胞形态,分光光度计法检测培养上清中乳酸脱氢酶(LDH)释放率,Hoechst/PI染色检测细胞膜损伤情况,实时荧光定量聚合酶链式反应(RT-qPCR)检测NLRP3、半胱氨酸蛋白酶1(caspase-1)、消皮素D(GSDMD)、凋亡斑点样蛋白(ASC)和白细胞介素-1β(IL-1β)的mRNA表达水平,免疫荧光检测NLRP3和caspase-1蛋白表达的变化,蛋白免疫印迹法(Western blot)检测IL-1β、ASC、NLRP3、caspase-1和活化半胱氨酸蛋白酶1(cleaved caspase-1)蛋白表达的变化。结果:与正常组比较,模型组SHSY5Y细胞存活率显著降低(P<0.01),细胞形态受损,培养上清中LDH渗漏率显著提高(P<0.01),细胞膜破损,细胞中NLRP3、caspase-1、GSDMD、ASC和IL-1β的mRNA表达水平显著提高(P<0.01),IL-1β、ASC、NLRP3、caspase-1和cleaved caspase-1蛋白的表达水平显著提高(P<0.01)。与模型组相比,丹酚酸B、葛根素与丹酚酸B和葛根素合用均能显著提高OGD/R损伤后SH-SY5Y细胞的存活率(P<0.01);与单用组相比,合用组具有更好的效果(P<0.01)。与模型组相比,丹酚酸B和葛根素合用及MCC950均能改善细胞形态,降低细胞上清液LDH的渗漏(P<0.05),减轻细胞膜损伤水平,降低SH-SY5Y细胞NLRP3、caspase-1、GSDMD、ASC和IL-1β的mRNA表达水平(P<0.05,P<0.01),降低IL-1β、ASC、NLRP3、caspase-1和cObjective:To investigate the effect and mechanism of salvianolic acid B and puerarin on SH-SY5Y cells damaged by oxygen glucose deprivation and reoxygenation(OGD/R)based on pyroptosis.Method:SH-SY5Y cells were used to construct the model of OGD/R damage,and the control group,OGD/R group,10μmol·L^(-1) salvianolic acid B group(Sal B),100μmol·L^(-1) puerarin group(Pue),10μmol·L^(-1) salvianolic acid B and 100μmol·L^(-1) puerarin group(SP)and 10μmol·L^(-1) NOD like receptor protein(NLRP3)inhibitor MCC950 group were set up.Except for the control group,other groups were were rapidly reoxygenated for12 h after 6 h OGD for modeling.Cell survival rate was detected by MTT assay,cell morphology was observed by optical microscope,lactic dehydrogenase(LDH)content in culture supernatant was detected,cell damage was measured by Hoechst/PI staining,mRNA expression levels of NLRP3,cysteinyl aspartate specific proteinase(caspase-1),gasdermin D(GSDMD),apoptosis-associated speck-like protein(ASC)and interleukin-1β(IL-1β)were determined by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).Caspase-1 and NLRP3 protein activation were detected by immunofluorescence.Protein expression levels of IL-1β、ASC、NLRP3、caspase-1 and cleaved caspase-1 were observed by western blot.Result:Compared with the control group,the survival rate was decreased remarkably(P<0.01),and the morphology was damaged,the leakage rate of LDH was increased(P<0.01),mRNA expression levels of NLRP3,caspase-1,GSDMD,ASC and IL-1βwere lifted(P<0.01),and also the activation of IL-1β、ASC、NLRP3、caspase-1 and cleaved caspase-1 protein were risen(P<0.01)in OGD/R group.Compared with OGD/R group,Salvianolic acid B,puerarin and salvianolic acid B combined with puerarin could improve cell survival rate(P<0.01),and the combined treatment group had better effect(P<0.01).Salvianolic acid B combined with puerarin and MCC950 can both improve cell morphology,reduce the leakage of LDH(P<0.05),decrease the level of cell damage,reduce the mRNA

关 键 词：缺血性脑卒中 氧糖剥夺/复氧模型 丹酚酸B 葛根素 细胞焦亡 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33