知母皂苷B对缺氧/复氧损伤星形胶质细胞的保护作用及其机制研究
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摘要
目的:探讨知母皂苷B对缺氧/复氧损伤星形胶质细胞的保护作用及其可能机制。方法:原代新生SD大鼠星形胶质细胞经培养、鉴定后,随机分为正常组、模型组、阳性对照组(尼莫地平,10μmol/L)以及知母皂苷B低、中、高剂量组(1、10、100μmol/L)。正常组和模型组均给予完全培养基1 000μL,给药组给予含相应药物的完全培养基1 000μL。除正常组外,其余各组细胞均采用氧糖剥夺/再灌注法复制缺氧/复氧损伤模型。复氧后,采用比色法检测细胞中乳酸脱氢酶(LDH)的相对释放率;采用四甲基偶氮唑盐法检测细胞相对活力;采用酶联免疫吸附测定法检测细胞中水通道蛋白4(AQP-4)、白细胞介素6(IL-6)、IL-1β、肿瘤坏死因子α(TNF-α)的含量。结果:与正常组比较,模型组细胞中LDH相对释放率以及AQP-4、IL-6、IL-1β、TNF-α的含量均显著升高,细胞相对活力显著降低(P<0.01)。与模型组比较,各给药组细胞中LDH相对释放率以及AQP-4、IL-6、IL-1β、TNF-α的含量均显著下降,细胞相对活力均显著升高(P<0.05或P<0.01)。结论:知母皂苷B可明显降低细胞损伤程度、增强细胞活力,对缺氧/复氧损伤星形胶质细胞具有一定的保护作用,且这种作用可能与其下调AQP-4和IL-6、IL-1β、TNF-α的分泌有关。
OBJECTIVE:To investigate the protective effects of anemarsaponin B on hypoxia/reoxygenation injury astrocytes and its possible mechanism. METHODS:The primary astrocytes of neonatal SD rats were cultured and identified,and then randomly divided into normal group,model group,positive control group (nimodipine,10 μmol/L),anemarsaponin B low-dose,medium-dose and high-dose groups (1,10,100 μmol/L),respectively. Normal group and model group were given complete medium 1 000 μL. Administration group was given complete medium with relevant medicine 1 000 μL. Except for normal group,hypoxia/reoxygenation injury model was established by oxygen-glucose deprivation/reperfusion in other groups. After reoxygenation,relative release rate of lactate dehydrogenase (LDH)in cell was detected by colorimetry. MTT assay was used to detect the relative viability of the cells. The contents of aquaporin 4 (AQP-4),IL-6,IL-1β and TNF-α in cell were measured by ELISA. RESULTS:Compared with normal group,relative release rate of LDH,the contents of AQP-4,IL-6,IL-1β and TNF-αin cell were increased significantly in model group,while relative viability of the cells were decreased significantly (P<0.01).Compared with model group,relative release rate of LDH,the contents of AQP-4,IL-6,IL-1β and TNF-α in cell were decreased significantly in administration groups,while relative viability of the cells were increased significantly (P<0.05 or P<0.01).CONCLUSIONS:Anemarsaponin B can significantly decrease cell injury degree,strengthen cell viability and protect hypoxia/reoxygenation injury astrocytes to certain extent. The effect may be related to the down-regulation of the secretion of AQP-4,IL-6,IL-1β and TNF-α.
OBJECTIVE:To investigate the protective effects of anemarsaponin B on hypoxia/reoxygenation injury astrocytes and its possible mechanism. METHODS:The primary astrocytes of neonatal SD rats were cultured and identified,and then randomly divided into normal group,model group,positive control group (nimodipine,10 μmol/L),anemarsaponin B low-dose,medium-dose and high-dose groups (1,10,100 μmol/L),respectively. Normal group and model group were given complete medium 1 000 μL. Administration group was given complete medium with relevant medicine 1 000 μL. Except for normal group,hypoxia/reoxygenation injury model was established by oxygen-glucose deprivation/reperfusion in other groups. After reoxygenation,relative release rate of lactate dehydrogenase (LDH)in cell was detected by colorimetry. MTT assay was used to detect the relative viability of the cells. The contents of aquaporin 4 (AQP-4),IL-6,IL-1β and TNF-α in cell were measured by ELISA. RESULTS:Compared with normal group,relative release rate of LDH,the contents of AQP-4,IL-6,IL-1β and TNF-αin cell were increased significantly in model group,while relative viability of the cells were decreased significantly (P<0.01).Compared with model group,relative release rate of LDH,the contents of AQP-4,IL-6,IL-1β and TNF-α in cell were decreased significantly in administration groups,while relative viability of the cells were increased significantly (P<0.05 or P<0.01).CONCLUSIONS:Anemarsaponin B can significantly decrease cell injury degree,strengthen cell viability and protect hypoxia/reoxygenation injury astrocytes to certain extent. The effect may be related to the down-regulation of the secretion of AQP-4,IL-6,IL-1β and TNF-α.
引文
[1]张亚洲,何前松,胡斐然,等.锦鸡儿总黄酮预处理对局灶性脑缺血再灌注损伤模型大鼠血脑屏障通透性的影响及机制研究[J].中国药房,2018,29(13):1793-1797.
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[14]丁娟,丁敬宾,马小虎,等.一种改进的大鼠大脑皮质星形胶质细胞的培养方法[J].神经解剖学杂志,2017,33(3):349-353.
[15]隋海娟,马瑞国,刘佳颖,等.知母皂苷对Aβ1-42诱导的皮层神经元突触损伤的保护作用[J].中药药理与临床,2015,31(6):27-30.
[16]谢娜,杨建军,杨芳,等.CM-DiI应用于标记大鼠牙髓干细胞的实验研究[J].山西医科大学学报,2018,49(6):627-631.
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[19]HAN D,SUN M,HE PP,et al.Ischemic postconditioning alleviates brain edema after focal cerebral ischemia reperfusion in rats through down-regulation of aquaporin-4[J].J Mol Neurosci,2015,56(3):722-729.
[20]李小亮,刘文进,田书娟,等.脑出血大鼠血清炎性因子水平与脑水肿的相关性探讨[J].临床合理用药杂志,2016,9(3A):108-122.
[21]VESPA P,MCARTHUR DL,STEIN N,et al.Tight glycemic control in creases metabolic distress in traumatic brain injury:a randomized controlled within-subjects trial[J].Crit Care Med,2012,40(6):1923-1929.
[22]李国龙.伴生物质对知母皂苷类成分体内过程的影响研究[D].咸阳:陕西中医药大学,2015.
[23]吴非,钟雷,程玉芳,等.知母皂苷对脑缺血再灌注损伤的保护机制[J].中国临床康复,2006,10(31):22-24.
[24]陈丹丹,谢晓芳,万峰,等.氢溴酸樟柳碱对急性脑缺血再灌注损伤模型大鼠脑组织细胞凋亡及ERK1/2磷酸化水平的影响[J].中国药房,2017,28(28):3907-3910.
[25]丁玲,胡毅,刘洁,等.知母皂苷对大鼠局灶性脑缺血/再灌注损伤的保护作用[J].中国现代应用药学杂志,2018,35(12):1821-1825.
[26]王宇红,谭小雯,曾嵘,等.左归降糖解郁方对糖尿病并发抑郁症大鼠海马血脑屏障精细结构AQP4、Cx43的影响[J].中成药,2015,37(10):2109-2114.
[2]陈朝,刘泰,黄一挚,等.疏血通脉胶囊对脑缺血再灌注损伤模型大鼠的神经保护作用研究[J].中国药房,2018,29(16):2184-2188.
[3]MOZAFFARIAN D,BENJAMIN EJ,GO AS,et al.Heart disease and stroke statistics:2015 update:a report from the American Heart Association[J].Circulation,2015,131(4):e29-e322.
[4]ZHENG H,CHEN C,ZHANG J,et al.Mechanism and therapy of brain edema after intracerebral hemorrhage[J].Cerebrovasc Dis,2016,42(3/4):155-169.
[5]COLANGELO AM,ALBER GHINA L,PAPA M.Astrogliosis as a therapeutic target for neurodegenerative diseases[J].Neurosci Lett,2014.DOI:10.1016/j.neulet.2014.01.014.
[6]YAO XM,DERUGIN N,MANLEY GT,et al.Reduced brain edema and infarct volume in aquaporin-4 deficient mice after transient focal cerebral ischemia[J].Neurosci Lett,2015.DOI:10.1016/j.neulet.2014.10.040.
[7]DENG J,ZHAO F,YU X,et al.Expression of aquaporin4 and breakdown of the blood-brain barrier after hypoglycemia-induced brain edema in rats[J].PLoS One,2014.DOI:10.1371/journal.pone.0107022.
[8]RAO KV,VERKMAN AS,CURTIS KM,et al.Aquaporin-4 deletion in mice reduces encephalopathy and brain edema in experimental acute liver failure[J].Neurobiol Dis,2014.DOI:10.1016/j.nbd.2013.11.018.
[9]FAZZINA G,AMORINI AM,MARMAROU CR,et al.The protein kinase C activator phorbol myristate acetate decreases brain edema by aquaporin 4 downregulation after middle cerebral artery occlusion in the rat[J].J Neurotrauma,2013,27(2):453-461.
[10]YANG B,ZADOR Z,VERKMAN AS.Glial cell aquaporin-4 overexpression in transgenic mice accelerates cytotoxic brain swelling[J].J Biol Chem,2012,283(22):15280-15286.
[11]ZHANG X,LI H,HU S,et al.Brain edema after intracerebral hemorrhage in rats:the role of infiammation[J].Neurol India,2013,54(4):402-407.
[12]DING Z,ZHANG J,XU J,et al.Propofol administration modulates AQP-4 expression and brain edema after traumatic brain injury[J].Cell Biochem Biophys,2013,67(2):615-622.
[13]冯菲.知母的药理作用研究进展[J].中医临床研究,2017,9(12):133-137.
[14]丁娟,丁敬宾,马小虎,等.一种改进的大鼠大脑皮质星形胶质细胞的培养方法[J].神经解剖学杂志,2017,33(3):349-353.
[15]隋海娟,马瑞国,刘佳颖,等.知母皂苷对Aβ1-42诱导的皮层神经元突触损伤的保护作用[J].中药药理与临床,2015,31(6):27-30.
[16]谢娜,杨建军,杨芳,等.CM-DiI应用于标记大鼠牙髓干细胞的实验研究[J].山西医科大学学报,2018,49(6):627-631.
[17]POTOKAR M,JORGA?EVSKI J,ZOREC R.Astrocyte aquaporin dynamics in health and disease[J].Int J Mol Sci,2016.DOI:10.3390/ijms17071121.
[18]IKESHIMA-KATAOKA H.Neuroimmunological implications of AQP4 in astrocytes[J].Int J Mol Sci,2016.DOI:10.3390/ijms17081306.
[19]HAN D,SUN M,HE PP,et al.Ischemic postconditioning alleviates brain edema after focal cerebral ischemia reperfusion in rats through down-regulation of aquaporin-4[J].J Mol Neurosci,2015,56(3):722-729.
[20]李小亮,刘文进,田书娟,等.脑出血大鼠血清炎性因子水平与脑水肿的相关性探讨[J].临床合理用药杂志,2016,9(3A):108-122.
[21]VESPA P,MCARTHUR DL,STEIN N,et al.Tight glycemic control in creases metabolic distress in traumatic brain injury:a randomized controlled within-subjects trial[J].Crit Care Med,2012,40(6):1923-1929.
[22]李国龙.伴生物质对知母皂苷类成分体内过程的影响研究[D].咸阳:陕西中医药大学,2015.
[23]吴非,钟雷,程玉芳,等.知母皂苷对脑缺血再灌注损伤的保护机制[J].中国临床康复,2006,10(31):22-24.
[24]陈丹丹,谢晓芳,万峰,等.氢溴酸樟柳碱对急性脑缺血再灌注损伤模型大鼠脑组织细胞凋亡及ERK1/2磷酸化水平的影响[J].中国药房,2017,28(28):3907-3910.
[25]丁玲,胡毅,刘洁,等.知母皂苷对大鼠局灶性脑缺血/再灌注损伤的保护作用[J].中国现代应用药学杂志,2018,35(12):1821-1825.
[26]王宇红,谭小雯,曾嵘,等.左归降糖解郁方对糖尿病并发抑郁症大鼠海马血脑屏障精细结构AQP4、Cx43的影响[J].中成药,2015,37(10):2109-2114.