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

doi:10.12114/j.issn.1007-9572.2020.00.356

Abstract

Abstract: Background　Simvastatin（SIM）is often clinically used for secondary prevention of cardio-cerebrovascular 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（0.312 5，0.625 0，1.250 0，2.500 0，5.000 0，10.000 0，20.000 0，40.000 0，and 80.000 0 μmol/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（200 μ l 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 receptor γ（PPARγ）and nuclear factor erythroid 2-related factor 2（NRF2）.Results　MTT assay found that SIM≥5 μmol/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，PPARγ and NRF2 in the model group were significantly up-regulated（P<0.05），and in SIM groups，the expression levels of CD36，PPARγ and 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.

Key words: Cerebral hemorrhage, Simvastatin, BV-2 microglia, Glial cell, Endogenous removal of hematoma, Cardiovascular diseases

摘要： 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 0 μmol/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（200 μl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≥5 μmol/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。

关键词: 脑出血, 辛伐他汀, BV-2小胶质细胞, 神经胶质细胞, 血肿内源性清除, 心血管疾病

WANG Ruonan,ZHAO Dexi,LIU Liming, et al. Efficacy and Mechanism of Simvastatin for Hematoma Absorption in an in Vitro Mouse Model of Intracerebral Hemorrhage[J]. Chinese General Practice, 2020, 23(23): 2883-2889. DOI: 10.12114/j.issn.1007-9572.2020.00.356.
王若男,赵德喜,刘立明等. 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[J]. 中国全科医学, 2020, 23(23): 2883-2889. DOI: 10.12114/j.issn.1007-9572.2020.00.356.

References

［1］高利.高血压性脑出血急性期中西医结合诊疗专家共识［J］．中国全科医学，2016，19（30）：3641-3648．DOI：10.3969/j.issn.1007-9572.2016.30.001.
GAO L，Expert consensus on hypertensive intracerebral hemorrhage in acute stage in diagnosis and treatment combining traditional Chinese medicine and Western medicine［J］．Chinese General Practice，2016，19（30）：3641-3648．DOI：10.3969/j.issn.1007-9572.2016.30.001
［2］DUAN X C，WEN Z J，SHEN H T，et al．Intracerebral hemorrhage，oxidative stress，and antioxidant therapy［J］．Oxid Med Cell Longev，2016，2016：1203285．DOI：10.1155/2016/1203285.
［3］ARONOWSKI J，ZHAO X R.Molecular pathophysiology of cerebral hemorrhage：secondary brain injury［J］．Stroke，2011，42（6）：1781-1786．DOI：10.1161/STROKEAHA.110.596718.
［4］LECLERC J L，LAMPERT A S，LOYOLA AMADOR C，et al．The absence of the CD163 receptor has distinct temporal influences on intracerebral hemorrhage outcomes［J］．J Cereb Blood Flow Metab，2018，38（2）：262-273．DOI：10.1177/0271678X17701459.
［5］陈璟，熊小檍，王鹏飞，等．CD36促进脑出血患者血肿吸收及其临床意义［J］．第三军医大学学报，2015，37（8）：792-796．DOI：10.16016/j.1000-5404.201411044.
CHEN J，XIONG X Y，WANG P F，et al．CD36 promotes hematoma absorption in patients with intracerebral hemorrhage and its clinical significance［J］．Journal of Third Military Medical University，2015，37（8）：792-796．DOI：10.16016/j.1000-5404.201411044.
［6］FLORES J J，KLEBE D，ROLLAND W B，et al．PPARγ-induced upregulation of CD36 enhances hematoma resolution and attenuates long-term neurological deficits after germinal matrix hemorrhage in neonatal rats［J］．Neurobiol Dis，2016，87：124-133．DOI：10.1016/j.nbd.2015.12.015.
［7］ZHAO X R，GONZALES N，ARONOWSKI J.Pleiotropic role of PPARγ in intracerebral hemorrhage：an intricate system involving NRF2，RXR，and NF-κB［J］．CNS Neurosci Ther，2015，21（4）：357-366．DOI：10.1111/cns.12350.
［8］ZHAO X R，SUN G H，TING S M，et al．Cleaning up after ICH：the role of NRF2 in modulating microglia function and hematoma clearance［J］．J Neurochem，2015，133（1）：144-152．DOI：10.1111/jnc.12974.
［9］WANG Y C，WANG P F，FANG H，et al．Toll-like receptor 4 antagonist attenuates intracerebral hemorrhage-induced brain injury［J］．Stroke，2013，44（9）：2545-2552．DOI：10.1161/STROKEAHA.113.001038.
［10］FANG H，CHEN J，LIN S，et al．CD36-mediated hematoma absorption following intracerebral hemorrhage：negative regulation by TLR4 signaling［J］．J Immunol，2014，192（12）：5984-5992．DOI：10.4049/jimmunol.1400054.
［11］NI W，MAO S S，XI G H，et al．Role of erythrocyte CD47 in intracerebral hematoma clearance［J］．Stroke，2016，47（2）：505-511．DOI：10.1161/STROKEAHA.115.010920.
［12］KARKI K，KNIGHT R A，HAN Y X，et al．Simvastatin and atorvastatin improve neurological outcome after experimental intracerebral hemorrhage［J］．Stroke，2009，40（10）：3384-3389．DOI：10.1161/STROKEAHA.108.544395.
［13］YANG D M，KNIGHT R A，HAN Y X，et al．Vascular recovery promoted by atorvastatin and simvastatin after experimental intracerebral hemorrhage：magnetic resonance imaging and histological study［J］．J Neurosurg，2011，114（4）：1135-1142．DOI：10.3171/2010.7.JNS10163.
［14］HARA N，CHIJIIWA M，YARA M，et al．Metabolomic analyses of brain tissue in Sepsis induced by cecal ligation reveal specific redox alterations— protective effects of the oxygen radical scavenger edaravone［J］．Shock，2015，44（6）：578-584．DOI：10.1097/SHK.0000000000000465.
［15］ZHAO X，GROTTA J，GONZALES N，et al．Hematoma resolution as a therapeutic target：the role of microglia/macrophages［J］．Stroke，2009，40（3，Supplement 1）：S92-94．DOI：10.1161/strokeaha.108.533158.
［16］WAN S，CHENG Y Y，JIN H，et al．Microglia activation and polarization after intracerebral hemorrhage in mice：the role of protease-activated receptor-1［J］．Transl Stroke Res，2016，7（6）：478-487．DOI：10.1007/s12975-016-0472-8.
［17］GONZALES N R，SHAH J，SANGHA N，et al．Design of a prospective，dose-escalation study evaluating the Safety of Pioglitazone for Hematoma Resolution in Intracerebral Hemorrhage（SHRINC）［J］．Int J Stroke，2013，8（5）：388-396．DOI：10.1111/j.1747-4949.2011.00761.x.
［18］YAO J C，ZHENG K B，ZHANG X.Rosiglitazone exerts neuroprotective effects via the suppression of neuronal autophagy and apoptosis in the cortex following traumatic brain injury［J］．Mol Med Rep，2015，12（5）：6591-6597．DOI：10.3892/mmr.2015.4292.
［19］ZHAO X R，SUN G H，ZHANG J，et al．Hematoma resolution as a target for intracerebral hemorrhage treatment：role for peroxisome proliferator-activated receptor Gamma in microglia/macrophages［J］．Ann Neurol，2007，61（4）：352-362．DOI：10.1002/ana.21097.
［20］DAS GUPTA S，SAE-TAN S，WAHLER J，et al．Dietary γ-tocopherol-rich mixture inhibits estrogen-induced mammary tumorigenesis by modulating estrogen metabolism，antioxidant response，and PPARγ［J］．Cancer Prev Res（Phila），2015，8（9）：807-816．DOI：10.1158/1940-6207.CAPR-15-0154.
［21］ZHANG M J，WANG S P，MAO L L，et al．Omega-3 fatty acids protect the brain against ischemic injury by activating NRF2 and upregulating heme oxygenase 1［J］．J Neurosci，2014，34（5）：1903-1915．DOI：10.1523/JNEUROSCI.4043-13.2014.
［22］KHANDELWAL S，VAN ROOIJEN N，SAXENA R K.Reduced expression of CD47 during murine red blood cell（RBC）senescence and its role in RBC clearance from the circulation［J］．Transfusion，2007，47（9）：1725-1732．DOI：10.1111/j.1537-2995.2007.01348.x.
［23］CAO S L，ZHENG M Z，HUA Y，et al．Hematoma changes during clot resolution after experimental intracerebral hemorrhage［J］．Stroke，2016，47（6）：1626-1631．DOI：10.1161/STROKEAHA.116.013146.
［24］MENG L L，ZHOU H X，YANG S S，et al．Intervention effect of simvastatin on TLR4 mediated inflammatory injury after cerebral hemorrhage in rats ［J］．Journal of Neuroanatomy，2013，29（5）：557-561.
［25］LI J F，LI W，SHI Z Y，et al．Study on the prevention and treatment of simvastatin in rats with cerebral hemorrhage with secondary inflammatory injury and its mechanism ［J］．Exploration of Rational Drug Use in China，2019，16（2）：
11-14.
［26］TIAN Q Y，YANG S S，TANG Z P，et al．Effects of simvastatin on TNF- alpha expression and apoptosis in experimental rats with cerebral hemorrhage ［J］．Journal of Neuroanatomy，2014，30（5）：587-591.
［27］ZHOU H X，GAO L H，MENG L L，et al．Preventive and therapeutic effect of simvastatin on secondary inflammatory damage of rats with cerebral hemorrhage［J］．Asian Pac J Trop Med，2017（2）．DOI：10.1016/j.apjtm.2017.01.003
［28］ZHANG W Q，ZHANG C，TIAN Z Y，et al．The protective effect and mechanism of simvastatin on cerebral hemorrhage in rats ［J］．Shandong Medicine，2015，55（38）：4-6.
［29］JIA W J.Effects of simvastatin on neurological function recovery in patients with cerebral hemorrhage ［J］．Journal of Practical Neurological Diseases，2015，18（12）：117-118.
［30］SANSING L H，HARRIS T H，WELSH F A，et al．Toll-like receptor 4 contributes to poor outcome after intracerebral hemorrhage［J］．Ann Neurol，2011，70（4）：646-656．DOI：10.1002/ana.22528.
［31］GUGLIANDOLO E，FUSCO R，GINESTRA G，et al．Involvement of TLR4 and PPAR-α receptors in host response and NLRP3 inflammasome activation，against pulmonary infection with Pseudomonas aeruginosa［J］．Shock，2019，51（2）：221-227．DOI：10.1097/SHK.0000000000001137

[1]	LI Yuan, MU Yanling, XUE Mengzhou. Mechanism of Artesunate Regulating NLRP3/ASC/Caspase-1 Signaling Pathway to Reduce Inflammation and Protect Neurological Function in Mice with Intracerebral Hemorrhage [J]. Chinese General Practice, 2023, 26(33): 4194-4202.
[2]	YU Xinyan, ZHAO Jun, ZHAO Xiaoye, JIANG Qingru, CHEN Yatian, WANG Yan, ZHANG Haicheng. Application of Mobile Smart Healthcare in the Prevention and Control of Cardiovascular Diseases in Elderly Patients with Chronic Diseases in Primary Care [J]. Chinese General Practice, 2023, 26(33): 4167-4172.
[3]	PAN Yaojia, WANG Weiqiang, YI Weizhuo, GAO Bing, FU Fanglin, HAN Zheng, SUN Meng, DONG Yaqin, GU Huaicong. Relationship between Triglyceride-glucose Index and Risk of Cardiovascular Diseases in Middle-aged Obese Residents of Different Genders [J]. Chinese General Practice, 2023, 26(29): 3628-3635.
[4]	LIU Taotao, LI Tianrong, WANG Xue, CHEN Jiameng, SHUAI Zhiqin, LI Lisheng, XU Shangfu. Nanoparticle- and Exosome-based Targeted Drug Delivery Systems Used in the Diagnosis and Treatment of Atherosclerosis: Opportunities and Challenges [J]. Chinese General Practice, 2023, 26(08): 903-910.
[5]	ZHANG Haicheng, YU Xinyan, WANG Hongyu, XUE Tao, LIAO Xiaoyang, FAN Yongmei, ZHANG Qinghong. Discussion on the Difficulties and Bottlenecks in the Management of Remote ECG-based Screening for Cardiovascular Disease Risk in Hierarchical Diagnosis and Treatment [J]. Chinese General Practice, 2023, 26(05): 525-531.
[6]	WU Changyong, BAO Suli, XU Fei, PENG Yunzhu. Latest Advances in Exercise-induced Autophagy in Improving Cardiovascular Disease Prognosis [J]. Chinese General Practice, 2023, 26(05): 629-634.
[7]	WANG Zhe, ZHAO Haibin, WANG Guoliang, MA Xiaojuan, YIN Huijun. Complete Revascularization for Acute Myocardial Infarction and Multivessel Disease: an Overview of Systematic Reviews [J]. Chinese General Practice, 2023, 26(02): 142-153.
[8]	TANG Zhaoyun, HE Yang, CHEN Cong, YANG Xiaodan, WU Xinhua, LIU Hong. Risk Assessment of Ischemic Cardiovascular Disease in Rural Naxi Population in Low-to-high Altitudes [J]. Chinese General Practice, 2022, 25(36): 4522-4527.
[9]	SUN Zhenzhen, LIU Huanhuan, CHEN Kaining, LOU Qingqing. New Advances in the Value of the Normal Urinary Albumin as a Predictor of Multiple Diseases [J]. Chinese General Practice, 2022, 25(33): 4191-4195.
[10]	LIAO Jia, WEI Shuangyi, LIU Gang. Overview and Trends of Premature Deaths Due to Four Major Noncommunicable Diseases among Shenzhen Permanent Residents, 2014-2020 [J]. Chinese General Practice, 2022, 25(32): 4085-4090.
[11]	Fang HE, Fulan WANG, Liling XIE, Shuya LING, Mingchao XIAO. Application of TeamSTEPPS in the Management of Out-of-hospital Medication Safety for Patients with Chronic Heart Failure [J]. Chinese General Practice, 2022, 25(30): 3768-3774.
[12]	Xu CHEN, Hanxiong LIU, Xiuqiong YU, Lianchao CHENG, Lingyao QI, Siqi YANG, Yan LUO, Lin CAI. Total Occlusion of the Culprit Artery in Non-ST-segment Elevation Acute Coronary Syndrome: an Analysis of the Influencing Factors and Long-term Outcome Following Percutaneous Coronary Intervention [J]. Chinese General Practice, 2022, 25(27): 3365-3372.
[13]	Ziao RUI, Dongpu DAI, Yingying GUO, Shuo PANG, Yuanhang ZHOU, Yang DU, Xiaoyan ZHAO, Jianzeng DONG. Efficacy and Related Inflencing Factors of Intra-aortic Balloon Counterpulsation Combined with Extracorporeal Membrane Oxygenation in Patients with Acute Myocardial Infarction and Cardiogenic Shock [J]. Chinese General Practice, 2022, 25(21): 2597-2604.
[14]	Siqi YANG, Hanxiong LIU, Xiuqiong YU, Lin TONG, Jin LI, Lianchao CHENG, Xu CHEN, Lingyao QI, Yan LUO, Lin CAI. Sex-specific Analysis of the Management and Outcome of Elderly Patients with Acute Myocardial Infarction [J]. Chinese General Practice, 2022, 25(14): 1694-1699,1706.
[15]	Lida YIN, Jiasen WANG, Yongchao HE, Aiqun LI. A Meta-analysis of the Effect of Unprofessional Early Defibrillation on the Survival Rate of Patients with Out-of-hospital Cardiac Arrest [J]. Chinese General Practice, 2022, 25(14): 1757-1764.