辛伐他汀对脑出血体外模型血肿清除的影响及作用机制研究

Efficacy and Mechanism of Simvastatin for Hematoma Absorption in an in Vitro Mouse Model of Intracerebral Hemorrhage

背景辛伐他汀(SIM)在临床多用于心脑血管疾病的二级预防,但其对脑出血(ICH)后血肿内源性吸收的影响目前尚无明确结论。目的研究SIM对红细胞(RBC)诱导的小鼠神经胶质细胞BV-2的ICH模型内源性血肿清除的影响及其机制。方法 2017年12月-2018年12月,选取生长良好的BV-2细胞制成单细胞悬液,根据加入试剂不同分为空白组、不同浓度SIM组(0.312 5、0.625 0、1.250 0、2.500 0、5.000 0、10.000 0、20.000 0、40.000 0及80.000 0μmol/L),采用MTT比色法检测不同浓度SIM对于BV-2细胞存活率的影响,采用免疫荧光法观察BV-2细胞吞噬RBC的ICH体外模型。建模后,分为空白组(200μl的MEM完全培养基)、模型组(空白组+RBC)、SIM组(模型组+SIM),采用流式细胞术检测SIM对BV-2细胞清除RBC的影响,蛋白免疫印迹法(Western blot)检测SIM对BV-2细胞内相关蛋白CD47、CD36、Toll样受体(TLR4)、过氧化物酶体增殖物激活受体γ(PPARγ)、核转录因子2(NRF2)的表达。结果 MTT比色法结果显示,SIM≥5μmol/L时对BV-2细胞具有明显的抑制作用(P<0.05)。免疫荧光法观察到BV-2细胞吞噬了RBC,显示ICH体外模型成立。流式细胞检测显示,与模型组相比,SIM组小胶质细胞(MG)吞噬RBC数量上调(P<0.05)。Western blot显示,与空白组比较,模型组CD47、CD36、TLR4、PPARγ、NRF2蛋白的表达上调(P<0.05);与模型组相比,SIM组CD47、TLR4蛋白的表达下调(P<0.05),CD36、PPARγ、NRF2蛋白的表达上调(P<0.05)。结论 SIM可以有效促进RBC诱导的BV-2细胞ICH体外模型血肿的内源性清除。其作用机制可能与通过抑制CD47、TLR4的表达,上调CD36、TLR4、PPARγ、NRF2蛋白的表达相关。

Background Simvastatin(SIM)is often clinically used for secondary prevention of cardiocerebrovascular disease,but its effect on endogenous absorption of hematoma after intracerebral hemorrhage is still unclear.Objective To study the efficacy and mechanism of SIM for endogenous absorption of hematoma in a mousse model of BV-2 encephalorrhagia inducted by red blood cells(RBCs).Methods This experiment was implemented from December 2017 to December 2018.Single-cell suspensions of well-cultured mouse BV-2 cells were prepared,and were divided into blank control group A,model group,SIM groups(0.3125,0.6250,1.2500,2.5000,5.0000,10.0000,20.0000,40.0000,and 80.0000μmol/L)according to the reagents added.MTT colorimetry was used to detect the effect of different concentrations of SIM on the survival rate of BV-2 cells.Immunofluorescence technique was used to observe the in vitro intracerebral hemorrhage model of RBCs phagocytosed by BV-2 cells.After modeling,it was divided into blank group(200μl MEM complete medium),model group(blank group+RBC),SIM group(model group+SIM).Flow cytometry was used to examine the effect of BV-2 cells on removing RBCs.Western blot was used to measure the effect of SIM on the expression levels of CD47,CD36,toll-like receptor 4(TLR4),peroxisome proliferator-activated receptorγ(PPARγ)and nuclear factor erythroid 2-related factor 2(NRF2).Results MTT assay found that SIM≥5μmol/L had a significant inhibitory effect on BV-2 cells(P<0.05).Immunofluorescence microscopy showed that RBCs were phagocytosed by BV-2 cells in all groups except the blank control group,indicating that the in vitro mouse model of intracerebral hemorrhage was successfully developed.Flow cytometry revealed that SIM groups had more RBCs phagocytosed by microglia cells compared with the model group(P<0.05).Western blot found that compared with the blank group,the expression levels of CD47,CD36,TLR4,PPARγand NRF2 in the model group were significantly up-regulated(P<0.05),and in SIM groups,the expression levels of CD36,PPARγand NRF2 were also significantly up-regulated,but those of CD47 and TLR4 were down-regulated significantly(P<0.05).Conclusion SIM may effectively promote the endogenous removal of the hematoma in a mousse model of BV-2 cells encephalorrhagia inducted by RBCs.The action mechanism may be related to the restrain of the expression of CD47 and TLR4 proteins,and the upregulation of the expression of CD36,PPARγand NRF2 proteins.