左旋丁基苯酞抑制氧糖剥夺/复氧后小鼠脑胶质细胞内NF-κB的激活及机制
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摘要
背景:缺血性脑血管病是中枢神经系统(CNS)常见病、多发病,具有高致残率、高死亡率、高复发率,严重威胁人们生命健康,给社会和家庭带来沉重负担。国内外学者对该病作出了大量实验与临床研究,使人们对脑缺血损害及其相关预防、治疗、康复等的认识不断深化,针对该病不同病理生理环节进行了有目的、分阶段的联合治疗,使其预后有了较大改观。随着脑缺血治疗学研究的进展,现在逐步提出了脑缺血后全脑保护的概念,即就卒中后功能恢复而言,所有脑区、细胞类型和细胞成分均应受到保护。因此脑缺血后神经功能的最终恢复不仅有赖于对神经元和灰质的保护,而且也有赖于对白质的保护。而胶质细胞作为脑白质的主要组成细胞,深入研究其缺血性损伤的机制及抑制手段就显得尤为必要和重要。
胶质细胞对炎症、外伤等损伤具有反应性增生的能力,即胶质细胞数目增加,细胞增殖、肥大,较正常时出现更多的胶质丝和突起,代谢活动增强。增生的胶质细胞及其突起可以包围受损、变性的神经元,过度的胶质化可阻碍髓鞘和轴突的再生,影响周围神经组织的结构修复和功能恢复。增生胶质细胞形成的胶质瘢痕甚至还可能成为脑缺血后继发性癫痫病灶。
目前对脑缺血/再灌注损伤机制的研究,主要集中在细胞内钙超载、兴奋性毒性氨基酸、氧自由基损伤、炎症反应、细胞凋亡调控基因、胱冬酶家族基因、即早反应基因等方面。其中,缺血/再灌注后炎症反应促进了继发性脑损害,是脑缺血/再灌注损伤的主要原因之一。而胶质细胞的反应性增生也与炎症反应中分泌的细胞因子IL-1β、TGF、NGF等有关。因此,关注胶质细胞在脑缺血后的炎症反应及与之相关的抗炎、神经保护途径将对缺血性脑卒中的治疗和后期康复具有重要而现实的意义。
核因子-κB(nuclear factor-kappa B,NF-κB)是一种重要的细胞核内转录因子,其主导调节一系列与炎症相关的细胞因子、酶类等的转录和表达,介导或直接参与了脑缺血/再灌注后神经元的损伤、死亡。多数学者认为NF-κB在炎症反应和免疫应答中具有重要、关键的地位,是炎症反应的中心环节。它已成为干预脑缺血/再灌注损伤有前景的治疗靶点之一。
丁基苯酞(3-n-butylphthalide,NBP)是我国第3种化学合成类I类新药,亦是中国脑血管研究领域第1项拥有自主知识产权的国家I类新药。它主要从我国南方水芹籽中提取得到,包括三种光学异构体:左旋丁基苯酞(l-NBP)、右旋丁基苯酞(d-NBP)、消旋丁基苯酞(dl-NBP),其中l-NBP的药效学作用更强。经大量实验室研究证明丁基苯酞对缺血后神经元具有保护作用,多中心I-IV期临床实验研究也表明,l-NBP对急性缺血性脑卒中有明显疗效,具有良好的抗脑缺血活性。因此,我们以l-NBP为干预药物,进一步研究其对胶质细胞氧糖剥夺/复氧后炎性损伤的抑制作用及对胶质细胞反应性增生的抑制作用,并探讨其可能机制;明确l-NBP对缺血/再灌注后脑胶质细胞的保护作用;推测其可能对胶质瘢痕的形成具有一定的抑制作用;预测其可能对其它白质炎症相关性疾病(多发性硬化、CNS创伤、C NS感染等)具有一定的治疗或辅助治疗作用。
目的:(1)探讨小鼠脑胶质细胞(glial cell,GC)的培养方法并对之进行形态学观察,免疫细胞化学鉴定。
(2)通过体外细胞氧糖剥夺/复氧(oxygen glucose deprivation/ reoxygenation,OGD/R)模型研究GC在OGD/R不同时间段细胞数量、活性的变化情况及细胞内NF-κB p65蛋白表达、活化的规律。
(3)研究l-NBP对GC在OGD24h/R1d后细胞数量变化的影响。
(4)研究l-NBP对OGD24h/R1d后GC内IκB-α、NF-κB p65、IL-1β蛋白表达的影响。
方法:取昆明小鼠乳鼠脑组织,采用机械吹打分离、筛网过滤、离心等技术获取鼠脑胶质细胞并进行培养,通过倒置显微镜、免疫细胞化学进一步鉴定;采用LDH法、免疫细胞化学方法研究和评价OGD/R模型,用MTT法确定GC数量变化最大的OGD/R时间点;采用Western blot、ELISA方法研究药物l-NBP作用下OGD/R后GC内IκB-α、NF-κB p65以及其下游炎性细胞因子IL-1β蛋白表达情况。
结果:(1)经形态学、免疫细胞化学(GFAP、MAC-1、NSE)鉴定所培养的细胞为混合培养的星形胶质细胞和小胶质细胞。
(2) OGD前后细胞形态结构变化显著,LDH释放量增加,并与NC组相比有显著性差异(p<0.01)。
(3)免疫细胞化学显示OGD后细胞核内蛋白表达增加;OGD4h时开始明显增加,OGD12h时达到高峰,OGD24h时有所下降,但仍高于NC组;OGD12h与OGD24h相比无显著性差异(p>0.05);OGD各组与NC组相比均有显著性差异(p<0.01)。OGD前NF-κB p65弱阳性表达,多在胞浆中可见;OGD后NF-κB p65出现核转位,强阳性表达于胞核,细胞呈“实心”现象。
(4) MTT法测定发现OGD24h后细胞数量减少,与NC组和OGD24h/R各组相比均有显著性差异(p<0.05);OGD24h/R8h、OGD24h/R12h、OGD24h/R1d、OGD24h/R2d、OGD24h/R3d各时间段GC数量逐渐增加,OGD24h/R1d、OGD24h/R2d、OGD24h/R3d与NC组相比有显著性差异(p<0.05)。(5) MTT法测定发现正常培养时l-NBP各组(20μmol/L、100μmol/L、500μmol/L)细胞数量与NC组无统计学差异(p>0.05)。l-NBP 1mmol/L组细胞数量增多,与NC组相比有显著性差异(p<0.01)。PDTC 100μmol/L组细胞数量减少,与NC组相比有显著性差异(p<0.01)。OGD24h/R1d后细胞数量增加,l-NBP各组(20μmol/L、100μmol/L、500μmol/L)、PDTC 100μmol/L组细胞数量减少, PDTC 100μmol/L组与l-NBP各组相比均有显著性差异(p<0.05)。l-NBP 500μmol/L组与20μmol/L、100μmol/组组间比较差异显著(p<0.05)。
(6) Western blot条带显示,OGD24h/R1d后核内NF-κB p65蛋白表达增加,与NC组相比有显著差异(p<0.01);l-NBP各组(20μmol/L、100μmol/L、500μmol/L)NF-κB p65蛋白表达减少,l-NBP浓度越高NF-κB p65蛋白表达越少,l-NBP 100μmol/L组、l-NBP 500μmol/L组与OGD24h/R1d组相比有显著差异(p<0.01)。PDTC 100μmol/L组NF-κB p65蛋白表达也减少,与OGD24h/R1d组比较有显著差异(p<0.01)。l-NBP 500μmol/L组与20μmol/L、100μmol/组组间比较差异显著(p<0.05)。
(7) Western Blot条带显示,OGD24h/R1d后胞浆内IκB-α蛋白表达减少,与NC组相比有显著差异(p<0.01);l-NBP各组(20μmol/L、100μmol/L、500μmol/L)IκB-α蛋白表达增加,l-NBP浓度越高IκB-α蛋白表达越多,l-NBP 100μmol/L、500μmol/L组与OGD24h/R1d组相比有显著差异(p<0.01)。PDTC 100μmol/L组IκB-α蛋白表达也增加,与OGD24h/R1d组比较有显著差异(p<0.01)。l-NBP 500μmol/L组与20μmol/L、100μmol/组组间比较差异显著(p<0.05)。
(8) ELISA测定发现,OGD24h/ R1d后IL-1β分泌量增加,与NC组相比有显著差异(p<0.05);l-NBP各组(20μmol/L、100μmol/L、500μmol/L)IL-1β表达减少,l-NBP浓度越高IL-1β表达越少,l-NBP 100μmol/L组、l-NBP 500μmol/L组与NC组比较无明显差异(p>0.05)。PDTC 100μmol/L组IL-1β表达也减少,与各组比较均有显著差异(p<0.01)。
结论:(1)成功进行了小鼠脑星形胶质细胞和小胶质细胞的混合原代培养及鉴定。
(2)对正常培养GC,l-NBP各浓度组(20μmol/L、100μmol/L、500μmol/L)无明显促进生长作用。l-NBP 1mmol/L组有促进细胞生长作用,PDTC 100μmol/L组有抑制细胞生长作用。表明l-NBP对GC没有毒副作用;PDTC对GC可能有一定毒性作用。
(3)体外细胞OGD/R模型简便有效,OGD后GC数量减少、活性减弱,NF-κB因子活化入核,OGD12h为其表达高峰。
(4) OGD24h/R1d后GC出现反应性增生。l-NBP和PDTC均能抑制细胞数量的增加,l-NBP 500μmol/L组抑制作用较其低浓度组(20μmol/L、100μmol/L)明显。
(5) OGD24h/R1d后GC内IκB-α蛋白降解,NF-κB p65活化入核,l-NBP能通过抑制IκB-α在胞浆的降解来减少NF-κB p65入核。l-NBP 500μmol/L组抑制作用较其低浓度组(20μmol/L、100μmol/L)明显。而且l-NBP 500μmol/L组比PDTC 100μmol/L组抑制作用更强。
(6) OGD24h/R1d后GC分泌IL-1β增多,l-NBP和PDTC均能抑制其分泌,l-NBP 100μmol/L、l-NBP 500μmol/L组抑制作用较其低浓度组(20μmol/L)更明显。
Background: Ischemic cerebrovascular disease is the common and multiple disease in central nervous system(CNS)diseases. It has high mutilation rates, mortality rate and relapse rate. It threatens people’s life and health severely, gives heavy burden to the society and families. Scholars did lots of experiments and clinical studies about ischemic cerebrovascular disease and the recognition of its damage, prophylaxis, therapy and restoration were improved. With the development of therapeutic study on cerebral ischemia, the concept of whole brain protection postischemia was proposed gradually. All encephalic region, cell types and cell composition should be protected for the functional recovery. Because the glial were the major component of white matter, it is very necessary and important to investigate the mechanism and inhibition means of its ischemic damage.
The glial reactive proliferate when subjected to inflammation, external injury et al. It includes cell number increasing, cell multiplication and hypertrophy, glia filament and protuberance increasing than normal, metabolic activity enhancement, expression of protein GFAP raise. The glial scar may be the focus of secondary epilepsy after brain ischemia. Inflammatory reaction after ischemic reperfusion enhance secondary brain damage, it is one cause of ischemic reperfusion damages. The reactive hyperplasia of glial has relationship to inflammation. So, paying close attention to the inflammation of glial after ischemic reperfusion and the anti-inflammation, methods of protecting nerves about the inflammation will have important and actual significance in the therapy and later stage convalescence of cerebral arterial thrombosis.
Nuclear factor-кB is an important intranuclear transcription factor. It regulates the transcription and expression of a series of cell factor and erzymes. NF-кB is the central link in inflammation course and it is one of perspective therapeutic targets in interfering in ischemic reperfusion damages.
3-n-butylphthalide(NBP)is a new drug which has the first national independence intellectual property rights in studies on cerebral vessels. It has three kinds of optical isomers: l-NBP, d-NBP, dl-NBP. l-NBP has the better pharmacodynamic action among them. l-NBP has curative effect obviously on therapy to the acute cerebral arterial thrombosis, it has better activity of anti-ischemic in brain. Therefore we studied on the inhibition of l-NBP to the inflammatory impair and reactive hyperplasia of glial was subjected to oxygen glucose deprivation/reoxygenation, studied the possible mechanism of it moreover. Study the protection of l-NBP to glial cell following oxygen glucose deprivation/reoxygenation. Presumed l-NBP may inhibit the form of glial scar. Predicted l-NBP may has therapy or adjunctive therapy effect on other inflammatory diseases in white matter(disseminated sclerosis, traumatic CNS injury, CNS infection).
Objective: (1) Investigate the cultivate method of mouse brain glial cells ( GC ) , observed by morphology and identify them by immunocytochemistry.
(2) Investigate GC cell population and activity in different time stages after oxygen glucose deprivation/ reoxygenation(OGD/R)by cell OGD/R model, study the dynamic rule of protein NF-κB p65 expression and activity in cell.
(3) Observe the effect of l-NBP to GC cell population after OGD24h/R1d.
(4) Observe the effect of l-NBP to expression of protein IκB-α, NF-κB p65 and IL-1βin GC cell after OGD24h/R1d.
Methods: Took suckling Kunming mouse brain tissue, mechanical blowing to demesh,filtering by grit, centrifugalization etc. technique to obtain mouse brain glial cell and to cultivate. Observed the cultivated cell by inverted microscope and identify them by immunocytochemistry. Evaluated OGD model by LDH determination and immunocytochemistry, determined the OGD/R time point whose chang was obvious in GC population. Investigated expression of protein of IκB-α, NF-κB p65 and IL-1βwhich was NF-κB downstream inflammation factor by Western blot and ELISA in GC interfering with l-NBP after subjected to OGD/R.
Results: (1) The cultivated cell was identified by morphology and immuocytochemistry(GFAP, MAC-1, NSE), it was mixed culture of astrocyte and microglia.
(2) GC morphous changed obviously after OGD, LDH releasing increased and had significant difference from normal control group(p<0.01).
(3) Expression of protein NF-κB p65 in GC nuclear increased after OGD by immuocytochemistry. It began to increase in OGD4h, got to the peak in OGD12h and began to descent, the amount in OGD24h was still more than normal control group. There was no significant difference between expression level in OGD12h and in OGD24h(p>0.05). OGD groups had significant difference from normal control(p<0.01). NF-κB p65 weak positive expression in endochylema before OGD and strong positive expression in nuclear after OGD, cell showed“solid center”.
(4) GC population was obviously less than normal control group and OGD24h/R groups after OGD24h by MTT(p<0.05). GC population gradually increased after OGD24h/R8h, OGD24h/R12h, OGD24h/R1d, OGD24h/R2d, OGD24h/R3d. The quantity of OGD24h/R1d, OGD24h/R 2d, OGD24h/R3d were significantly more than normal control group(p<0.05).
(5) GC population of l-NBP groups ( 20μmol/L, 100μmol/L, 500μmol/L)had no significant difference from normal control group when normal cultured with l-NBP(p>0.05).GC population of l-NBP 500μmol/L group was significantly more than normal control group(p<0.01), but PDTC 100μmol/L group was significantly less than that(p<0.01). Population of GC in l-NBP group(s20μmol/L, 100μmol/L, 500μmol/L)and PDTC 100μmol/L group decreased after OGD24h/R1d while OGD24h/R1d group increased. PDTC 100μmol/L group had significant difference from other groups(p<0.05). l-NBP 500μmol/L group had significant difference from 20μmol/L and 100μmol/L group(p<0.05).
(6) Straps in Western blot indicated expression of proteim NF-κB p65 in nuclear of OGD24h/R1d group was obviously more than normal control group(p<0.05). Expression of protein NF-κB p65 of l-NBP groups(20μmol/L, 100μmol/L, 500μmol/L)decreased, the higher density of l-NBP the less protein NF-κB p65 expression. l-NBP 100μmol/L group and l-NBP 500μmol/L group had significant difference from OGD24h/R1d group(p<0.01). NF-κB p65 protein amount in PDTC 100μmol/L group was also obviously less than OGD24h/R1d group(p<0.01). L-NBP 500μmol/L group had significant difference from 20μmol/L and 100μmol/L group(p<0.05).
(7) Straps in Western blot indicated expression of proteim IκB-αin endochylema of OGD24h/R1d group was obviously less than normal control group(p<0.01). Expression of protein IκB-αof l-NBP groups(20μmol/L, 100μmol/L, 500μmol/L)increased, the higher density of l-NBP the more protein IκB-αexpression. l-NBP 100μmol/L group and l-NBP 500μmol/L group had significant difference from OGD24h/R1d group(p<0.01). IκB-αprotein amount in PDTC 100μmol/L group was also obviously more than OGD24h/R1d group(p<0.01).
(8) IL-1βsecretory volume of OGD24h/R1d group obviously more than normal control group by ELISA(p<0.05). IL-1βsecretory volume of l-NBP groups(20μmol/L, 100μmol/L, 500μmol/L)decreased, the higher density of l-NBP the less IL-1βsecretory. l-NBP 100μmol/L group and l-NBP 500μmol/L group had no significant difference from normal control group(p>0.05). IL-1βsecretory amount in PDTC 100μmol/L group was also obviously less than other groups(p<0.01).
Conclusions: (1) Done well in primary mixed culture of mouse brain astrocyte and microglia and in identifing them by immuocytochemistry.
(2) l-NBP groups(20μmol/L、100μmol/L、500μmol/L)had no the promoted action to the growth of GC when normal cultured. L-NBP 1mmol/L group promoted the growth of GC and PDTC 100μmol/L group inhibited the growth of GC when normal cultured. It indicated that l-NBP has no toxic side effect to GC but PDTC may has .
(3) Cell OGD/R model in vitro was convenient and utility, GC population decreased, cell activity attenuated and factor NF-κB was activated to get into the nulcear after OGD. OGD12h was the peak of expression of protein NF-κB.
(4) GC reactive hyperplasia after OGD24h/R1d, l-NBP and PDTC inhibited the increase of cell, the inhibition of l-NBP 500μmol/L group was more stronger than other groups of lower concentration(20μmol/L, 100μmol/L).
(5) The protein IκB-αdegradated and the activated protein NF-κB p65 entered the nucleus in GC after OGD24h/R1d. l-NBP prevented NF-κB p65 from entering the nucleus by reducing the degradation of IκB-α. The inhibition of l-NBP 500μmol/L group was more stronger than other groups of lower concentration(20μmol/L, 100μmol/L)and PDTC 100μmol/L group.
(6) The volume secretion of factor IL-1βincreased after OGD24h/R1d, l-NBP and PDTC inhibited the secretion of factor IL-1β, the inhibition of l-NBP 100μmol/L and 500μmol/L group was more stronger than l-NBP 20μmol/L group.
胶质细胞对炎症、外伤等损伤具有反应性增生的能力,即胶质细胞数目增加,细胞增殖、肥大,较正常时出现更多的胶质丝和突起,代谢活动增强。增生的胶质细胞及其突起可以包围受损、变性的神经元,过度的胶质化可阻碍髓鞘和轴突的再生,影响周围神经组织的结构修复和功能恢复。增生胶质细胞形成的胶质瘢痕甚至还可能成为脑缺血后继发性癫痫病灶。
目前对脑缺血/再灌注损伤机制的研究,主要集中在细胞内钙超载、兴奋性毒性氨基酸、氧自由基损伤、炎症反应、细胞凋亡调控基因、胱冬酶家族基因、即早反应基因等方面。其中,缺血/再灌注后炎症反应促进了继发性脑损害,是脑缺血/再灌注损伤的主要原因之一。而胶质细胞的反应性增生也与炎症反应中分泌的细胞因子IL-1β、TGF、NGF等有关。因此,关注胶质细胞在脑缺血后的炎症反应及与之相关的抗炎、神经保护途径将对缺血性脑卒中的治疗和后期康复具有重要而现实的意义。
核因子-κB(nuclear factor-kappa B,NF-κB)是一种重要的细胞核内转录因子,其主导调节一系列与炎症相关的细胞因子、酶类等的转录和表达,介导或直接参与了脑缺血/再灌注后神经元的损伤、死亡。多数学者认为NF-κB在炎症反应和免疫应答中具有重要、关键的地位,是炎症反应的中心环节。它已成为干预脑缺血/再灌注损伤有前景的治疗靶点之一。
丁基苯酞(3-n-butylphthalide,NBP)是我国第3种化学合成类I类新药,亦是中国脑血管研究领域第1项拥有自主知识产权的国家I类新药。它主要从我国南方水芹籽中提取得到,包括三种光学异构体:左旋丁基苯酞(l-NBP)、右旋丁基苯酞(d-NBP)、消旋丁基苯酞(dl-NBP),其中l-NBP的药效学作用更强。经大量实验室研究证明丁基苯酞对缺血后神经元具有保护作用,多中心I-IV期临床实验研究也表明,l-NBP对急性缺血性脑卒中有明显疗效,具有良好的抗脑缺血活性。因此,我们以l-NBP为干预药物,进一步研究其对胶质细胞氧糖剥夺/复氧后炎性损伤的抑制作用及对胶质细胞反应性增生的抑制作用,并探讨其可能机制;明确l-NBP对缺血/再灌注后脑胶质细胞的保护作用;推测其可能对胶质瘢痕的形成具有一定的抑制作用;预测其可能对其它白质炎症相关性疾病(多发性硬化、CNS创伤、C NS感染等)具有一定的治疗或辅助治疗作用。
目的:(1)探讨小鼠脑胶质细胞(glial cell,GC)的培养方法并对之进行形态学观察,免疫细胞化学鉴定。
(2)通过体外细胞氧糖剥夺/复氧(oxygen glucose deprivation/ reoxygenation,OGD/R)模型研究GC在OGD/R不同时间段细胞数量、活性的变化情况及细胞内NF-κB p65蛋白表达、活化的规律。
(3)研究l-NBP对GC在OGD24h/R1d后细胞数量变化的影响。
(4)研究l-NBP对OGD24h/R1d后GC内IκB-α、NF-κB p65、IL-1β蛋白表达的影响。
方法:取昆明小鼠乳鼠脑组织,采用机械吹打分离、筛网过滤、离心等技术获取鼠脑胶质细胞并进行培养,通过倒置显微镜、免疫细胞化学进一步鉴定;采用LDH法、免疫细胞化学方法研究和评价OGD/R模型,用MTT法确定GC数量变化最大的OGD/R时间点;采用Western blot、ELISA方法研究药物l-NBP作用下OGD/R后GC内IκB-α、NF-κB p65以及其下游炎性细胞因子IL-1β蛋白表达情况。
结果:(1)经形态学、免疫细胞化学(GFAP、MAC-1、NSE)鉴定所培养的细胞为混合培养的星形胶质细胞和小胶质细胞。
(2) OGD前后细胞形态结构变化显著,LDH释放量增加,并与NC组相比有显著性差异(p<0.01)。
(3)免疫细胞化学显示OGD后细胞核内蛋白表达增加;OGD4h时开始明显增加,OGD12h时达到高峰,OGD24h时有所下降,但仍高于NC组;OGD12h与OGD24h相比无显著性差异(p>0.05);OGD各组与NC组相比均有显著性差异(p<0.01)。OGD前NF-κB p65弱阳性表达,多在胞浆中可见;OGD后NF-κB p65出现核转位,强阳性表达于胞核,细胞呈“实心”现象。
(4) MTT法测定发现OGD24h后细胞数量减少,与NC组和OGD24h/R各组相比均有显著性差异(p<0.05);OGD24h/R8h、OGD24h/R12h、OGD24h/R1d、OGD24h/R2d、OGD24h/R3d各时间段GC数量逐渐增加,OGD24h/R1d、OGD24h/R2d、OGD24h/R3d与NC组相比有显著性差异(p<0.05)。(5) MTT法测定发现正常培养时l-NBP各组(20μmol/L、100μmol/L、500μmol/L)细胞数量与NC组无统计学差异(p>0.05)。l-NBP 1mmol/L组细胞数量增多,与NC组相比有显著性差异(p<0.01)。PDTC 100μmol/L组细胞数量减少,与NC组相比有显著性差异(p<0.01)。OGD24h/R1d后细胞数量增加,l-NBP各组(20μmol/L、100μmol/L、500μmol/L)、PDTC 100μmol/L组细胞数量减少, PDTC 100μmol/L组与l-NBP各组相比均有显著性差异(p<0.05)。l-NBP 500μmol/L组与20μmol/L、100μmol/组组间比较差异显著(p<0.05)。
(6) Western blot条带显示,OGD24h/R1d后核内NF-κB p65蛋白表达增加,与NC组相比有显著差异(p<0.01);l-NBP各组(20μmol/L、100μmol/L、500μmol/L)NF-κB p65蛋白表达减少,l-NBP浓度越高NF-κB p65蛋白表达越少,l-NBP 100μmol/L组、l-NBP 500μmol/L组与OGD24h/R1d组相比有显著差异(p<0.01)。PDTC 100μmol/L组NF-κB p65蛋白表达也减少,与OGD24h/R1d组比较有显著差异(p<0.01)。l-NBP 500μmol/L组与20μmol/L、100μmol/组组间比较差异显著(p<0.05)。
(7) Western Blot条带显示,OGD24h/R1d后胞浆内IκB-α蛋白表达减少,与NC组相比有显著差异(p<0.01);l-NBP各组(20μmol/L、100μmol/L、500μmol/L)IκB-α蛋白表达增加,l-NBP浓度越高IκB-α蛋白表达越多,l-NBP 100μmol/L、500μmol/L组与OGD24h/R1d组相比有显著差异(p<0.01)。PDTC 100μmol/L组IκB-α蛋白表达也增加,与OGD24h/R1d组比较有显著差异(p<0.01)。l-NBP 500μmol/L组与20μmol/L、100μmol/组组间比较差异显著(p<0.05)。
(8) ELISA测定发现,OGD24h/ R1d后IL-1β分泌量增加,与NC组相比有显著差异(p<0.05);l-NBP各组(20μmol/L、100μmol/L、500μmol/L)IL-1β表达减少,l-NBP浓度越高IL-1β表达越少,l-NBP 100μmol/L组、l-NBP 500μmol/L组与NC组比较无明显差异(p>0.05)。PDTC 100μmol/L组IL-1β表达也减少,与各组比较均有显著差异(p<0.01)。
结论:(1)成功进行了小鼠脑星形胶质细胞和小胶质细胞的混合原代培养及鉴定。
(2)对正常培养GC,l-NBP各浓度组(20μmol/L、100μmol/L、500μmol/L)无明显促进生长作用。l-NBP 1mmol/L组有促进细胞生长作用,PDTC 100μmol/L组有抑制细胞生长作用。表明l-NBP对GC没有毒副作用;PDTC对GC可能有一定毒性作用。
(3)体外细胞OGD/R模型简便有效,OGD后GC数量减少、活性减弱,NF-κB因子活化入核,OGD12h为其表达高峰。
(4) OGD24h/R1d后GC出现反应性增生。l-NBP和PDTC均能抑制细胞数量的增加,l-NBP 500μmol/L组抑制作用较其低浓度组(20μmol/L、100μmol/L)明显。
(5) OGD24h/R1d后GC内IκB-α蛋白降解,NF-κB p65活化入核,l-NBP能通过抑制IκB-α在胞浆的降解来减少NF-κB p65入核。l-NBP 500μmol/L组抑制作用较其低浓度组(20μmol/L、100μmol/L)明显。而且l-NBP 500μmol/L组比PDTC 100μmol/L组抑制作用更强。
(6) OGD24h/R1d后GC分泌IL-1β增多,l-NBP和PDTC均能抑制其分泌,l-NBP 100μmol/L、l-NBP 500μmol/L组抑制作用较其低浓度组(20μmol/L)更明显。
Background: Ischemic cerebrovascular disease is the common and multiple disease in central nervous system(CNS)diseases. It has high mutilation rates, mortality rate and relapse rate. It threatens people’s life and health severely, gives heavy burden to the society and families. Scholars did lots of experiments and clinical studies about ischemic cerebrovascular disease and the recognition of its damage, prophylaxis, therapy and restoration were improved. With the development of therapeutic study on cerebral ischemia, the concept of whole brain protection postischemia was proposed gradually. All encephalic region, cell types and cell composition should be protected for the functional recovery. Because the glial were the major component of white matter, it is very necessary and important to investigate the mechanism and inhibition means of its ischemic damage.
The glial reactive proliferate when subjected to inflammation, external injury et al. It includes cell number increasing, cell multiplication and hypertrophy, glia filament and protuberance increasing than normal, metabolic activity enhancement, expression of protein GFAP raise. The glial scar may be the focus of secondary epilepsy after brain ischemia. Inflammatory reaction after ischemic reperfusion enhance secondary brain damage, it is one cause of ischemic reperfusion damages. The reactive hyperplasia of glial has relationship to inflammation. So, paying close attention to the inflammation of glial after ischemic reperfusion and the anti-inflammation, methods of protecting nerves about the inflammation will have important and actual significance in the therapy and later stage convalescence of cerebral arterial thrombosis.
Nuclear factor-кB is an important intranuclear transcription factor. It regulates the transcription and expression of a series of cell factor and erzymes. NF-кB is the central link in inflammation course and it is one of perspective therapeutic targets in interfering in ischemic reperfusion damages.
3-n-butylphthalide(NBP)is a new drug which has the first national independence intellectual property rights in studies on cerebral vessels. It has three kinds of optical isomers: l-NBP, d-NBP, dl-NBP. l-NBP has the better pharmacodynamic action among them. l-NBP has curative effect obviously on therapy to the acute cerebral arterial thrombosis, it has better activity of anti-ischemic in brain. Therefore we studied on the inhibition of l-NBP to the inflammatory impair and reactive hyperplasia of glial was subjected to oxygen glucose deprivation/reoxygenation, studied the possible mechanism of it moreover. Study the protection of l-NBP to glial cell following oxygen glucose deprivation/reoxygenation. Presumed l-NBP may inhibit the form of glial scar. Predicted l-NBP may has therapy or adjunctive therapy effect on other inflammatory diseases in white matter(disseminated sclerosis, traumatic CNS injury, CNS infection).
Objective: (1) Investigate the cultivate method of mouse brain glial cells ( GC ) , observed by morphology and identify them by immunocytochemistry.
(2) Investigate GC cell population and activity in different time stages after oxygen glucose deprivation/ reoxygenation(OGD/R)by cell OGD/R model, study the dynamic rule of protein NF-κB p65 expression and activity in cell.
(3) Observe the effect of l-NBP to GC cell population after OGD24h/R1d.
(4) Observe the effect of l-NBP to expression of protein IκB-α, NF-κB p65 and IL-1βin GC cell after OGD24h/R1d.
Methods: Took suckling Kunming mouse brain tissue, mechanical blowing to demesh,filtering by grit, centrifugalization etc. technique to obtain mouse brain glial cell and to cultivate. Observed the cultivated cell by inverted microscope and identify them by immunocytochemistry. Evaluated OGD model by LDH determination and immunocytochemistry, determined the OGD/R time point whose chang was obvious in GC population. Investigated expression of protein of IκB-α, NF-κB p65 and IL-1βwhich was NF-κB downstream inflammation factor by Western blot and ELISA in GC interfering with l-NBP after subjected to OGD/R.
Results: (1) The cultivated cell was identified by morphology and immuocytochemistry(GFAP, MAC-1, NSE), it was mixed culture of astrocyte and microglia.
(2) GC morphous changed obviously after OGD, LDH releasing increased and had significant difference from normal control group(p<0.01).
(3) Expression of protein NF-κB p65 in GC nuclear increased after OGD by immuocytochemistry. It began to increase in OGD4h, got to the peak in OGD12h and began to descent, the amount in OGD24h was still more than normal control group. There was no significant difference between expression level in OGD12h and in OGD24h(p>0.05). OGD groups had significant difference from normal control(p<0.01). NF-κB p65 weak positive expression in endochylema before OGD and strong positive expression in nuclear after OGD, cell showed“solid center”.
(4) GC population was obviously less than normal control group and OGD24h/R groups after OGD24h by MTT(p<0.05). GC population gradually increased after OGD24h/R8h, OGD24h/R12h, OGD24h/R1d, OGD24h/R2d, OGD24h/R3d. The quantity of OGD24h/R1d, OGD24h/R 2d, OGD24h/R3d were significantly more than normal control group(p<0.05).
(5) GC population of l-NBP groups ( 20μmol/L, 100μmol/L, 500μmol/L)had no significant difference from normal control group when normal cultured with l-NBP(p>0.05).GC population of l-NBP 500μmol/L group was significantly more than normal control group(p<0.01), but PDTC 100μmol/L group was significantly less than that(p<0.01). Population of GC in l-NBP group(s20μmol/L, 100μmol/L, 500μmol/L)and PDTC 100μmol/L group decreased after OGD24h/R1d while OGD24h/R1d group increased. PDTC 100μmol/L group had significant difference from other groups(p<0.05). l-NBP 500μmol/L group had significant difference from 20μmol/L and 100μmol/L group(p<0.05).
(6) Straps in Western blot indicated expression of proteim NF-κB p65 in nuclear of OGD24h/R1d group was obviously more than normal control group(p<0.05). Expression of protein NF-κB p65 of l-NBP groups(20μmol/L, 100μmol/L, 500μmol/L)decreased, the higher density of l-NBP the less protein NF-κB p65 expression. l-NBP 100μmol/L group and l-NBP 500μmol/L group had significant difference from OGD24h/R1d group(p<0.01). NF-κB p65 protein amount in PDTC 100μmol/L group was also obviously less than OGD24h/R1d group(p<0.01). L-NBP 500μmol/L group had significant difference from 20μmol/L and 100μmol/L group(p<0.05).
(7) Straps in Western blot indicated expression of proteim IκB-αin endochylema of OGD24h/R1d group was obviously less than normal control group(p<0.01). Expression of protein IκB-αof l-NBP groups(20μmol/L, 100μmol/L, 500μmol/L)increased, the higher density of l-NBP the more protein IκB-αexpression. l-NBP 100μmol/L group and l-NBP 500μmol/L group had significant difference from OGD24h/R1d group(p<0.01). IκB-αprotein amount in PDTC 100μmol/L group was also obviously more than OGD24h/R1d group(p<0.01).
(8) IL-1βsecretory volume of OGD24h/R1d group obviously more than normal control group by ELISA(p<0.05). IL-1βsecretory volume of l-NBP groups(20μmol/L, 100μmol/L, 500μmol/L)decreased, the higher density of l-NBP the less IL-1βsecretory. l-NBP 100μmol/L group and l-NBP 500μmol/L group had no significant difference from normal control group(p>0.05). IL-1βsecretory amount in PDTC 100μmol/L group was also obviously less than other groups(p<0.01).
Conclusions: (1) Done well in primary mixed culture of mouse brain astrocyte and microglia and in identifing them by immuocytochemistry.
(2) l-NBP groups(20μmol/L、100μmol/L、500μmol/L)had no the promoted action to the growth of GC when normal cultured. L-NBP 1mmol/L group promoted the growth of GC and PDTC 100μmol/L group inhibited the growth of GC when normal cultured. It indicated that l-NBP has no toxic side effect to GC but PDTC may has .
(3) Cell OGD/R model in vitro was convenient and utility, GC population decreased, cell activity attenuated and factor NF-κB was activated to get into the nulcear after OGD. OGD12h was the peak of expression of protein NF-κB.
(4) GC reactive hyperplasia after OGD24h/R1d, l-NBP and PDTC inhibited the increase of cell, the inhibition of l-NBP 500μmol/L group was more stronger than other groups of lower concentration(20μmol/L, 100μmol/L).
(5) The protein IκB-αdegradated and the activated protein NF-κB p65 entered the nucleus in GC after OGD24h/R1d. l-NBP prevented NF-κB p65 from entering the nucleus by reducing the degradation of IκB-α. The inhibition of l-NBP 500μmol/L group was more stronger than other groups of lower concentration(20μmol/L, 100μmol/L)and PDTC 100μmol/L group.
(6) The volume secretion of factor IL-1βincreased after OGD24h/R1d, l-NBP and PDTC inhibited the secretion of factor IL-1β, the inhibition of l-NBP 100μmol/L and 500μmol/L group was more stronger than l-NBP 20μmol/L group.
引文
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