单壁纳米碳管对小鼠中枢神经系统毒性的研究
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摘要
【目的】
纳米材料因其小尺寸而具有独特的理化和生物特性,越来越广泛地被应用于生产生活的各个领域。纳碳米管(Carbon nanotube, CNT)是纳米材料中当前研究热点之一,主要包括单壁纳米碳管(Single-walled carbon nanotube, SWCNT)和多壁纳米碳管(Muti-walled carbon nanotube, MWCNT)。SWCNT因其特殊的理化特性,使用范围包括空间结构、热控制装置、传感技术、能源、电子学和生物医药等多个领域。纳米材料(包括SWCNT)的应用带来了巨大的经济效益,但同时由于尺寸小及生物体暴露几率大大增加而产生的潜在的生物安全性与环境安全性等问题引起了世界范围的广泛关注。
已有研究表明空气中纳米粒子的浓度与呼吸道,心血管疾病等多种疾病的发病率和死亡率呈正相关,而SWCNT在体外试验中可引起细胞存活率下降、细胞凋亡、细胞周期改变等细胞毒性,体内试验发现SWCNT可经呼吸道、皮肤、消化道及注射等多种途径进入机体,并经血或淋巴循环分布到心、肝、脾、肾、胃、脑、皮肤、血液等多个组织器产生毒性效应。目前对SWCNT的呼吸系统毒性研究较多,结果发现SWCNT吸入后可引起肺炎性反应和氧化应激,产生肺组织纤维化和肉芽肿；并且进入肺间质部位后还可经多种途径转移到肺外组织从而分布到多个器官引起损伤,这使得人们更加重视SWCNT的肺外系统毒性。虽然相关研究较少,但已有研究发现SWCNT可引起主动脉线粒体损伤和氧化应激并可加速动脉粥样硬化的形成,并可跨越血脑屏障(blood brain barrier, BBB)进入脑内。
中枢神经系统(central nervous system, CNS)是神经系统的主要部分,大量的神经细胞聚集构成网络或回路,是学习、记忆、人类意识、心理、思维活动的基础,且神经组织的再生能力非常有限,其损伤多具有不可逆性。而BBB是存在于血液和脑组织之间的生物屏障,具选择透过性,对维持CNS的稳定有重要的作用。现有的研究表明,碳纳米材料可跨越BBB进入脑内,还可经鼻腔黏膜摄入至嗅球后经嗅神经转运或经感觉神经末梢直接转运至脑内并对CNS产生影响。虽然目前关于纳米材料的神经生物效应研究尚处于起步阶段,但已有研究报道显示进入CNS的纳米颗粒可引起一定的神经毒性效应,导致神经组织损伤。
鉴于SWCNT可经多种途径进入体内,对呼吸系统和心血管系统产生炎性损伤和氧化应激等毒性反应,同时还可跨越BBB在脑内分布,且CNS损伤多具有不可逆性,因此研究SWCNT对CNS的影响及其机制有积极的意义。本实验拟以小鼠为研究对象,探索SWCNT对BBB及CNS的损伤效应及其机制,通过组织病理学、神经损伤标志物、氧化损伤标志物和炎性因子等方面的改变,阐明SWCNT对不同脑区的损伤情况。
【材料和方法】
2.1实验动物
清洁级雄性ICR小鼠,体重22±2g,温度24±0.5℃,湿度55±5%,每日12h灯照,自由摄取食物和水,适应1周后开始实验。
2.2实验材料及配制
溶剂：生理盐水与吐温80(T80)配成0.1%T80生理盐水液,超声400 w,8 s×15次；
材料：单壁纳米碳管(SWCNT),先配成1 mg/ml的悬液,用细胞粉碎仪分散,超声功率400 w,8 s×15次。分散后立即按比例稀释成0.5、0.2、0.1 mg/ml不同浓度的悬液,稀释后立即超声(同前),现配现用。
2.3给药方法
实验分成两部分,一部分研究剂量效应关系,另一部分研究时间效应关系。
剂量效应研究,5组,包括1个对照组(0.1%T80生理盐水液)和4个实验组(2.5、5、12.5、25 mg/kg的SWCNT悬液),尾静脉注射0.5 ml/只,连续染毒5天,第5天给药结束后观察7天,共100只。
时间效应研究,8组,共设对照组(0.1%T80生理盐水液)和实验组(12.5 mg/kg的SWCNT悬液),尾静脉注射0.5 ml/只,连续染毒5天,第5天给药结束后观察1,7,14和28天四个时间点,共220只。
分组织病理学实验、抗氧化酶测定、western blotting、qPCR4部分实验,各部分各剂量组5只小鼠。开始给药前及到观察时间点时分别测定小鼠体重,用于组织病理学实验的小鼠经心脏灌流固定后取脑分小脑、纹状体、海马和皮质4个脑区,然后固定、包埋、切片后染色观察；其余3部分实验的小鼠颈椎脱臼处死取脑,于冰上进行分区(小脑、纹状体、海马和皮质),置于1.5 ml EP管中,于-80℃保存待用,qPCR实验的组织用无RNA酶和DNA酶的1.5 ml EP管置于-80℃保存待用。
2.4材料特性鉴定
称取2 mg的SWCNT,加4 ml含0.1%T80的ddH2O,超声400 w,8s×15次,配成0.5 mg/ml悬液,滴于铜网,立即用TEM观察材料特性。
2.5组织病理学检测
到观察时间点时,小鼠用戊巴比妥钠70 mg/kg腹腔注射麻醉后固定操作板,用生理盐水和4%多聚甲醛经升主动脉心脏灌流固定,至从右心房流出的灌流液无色再灌流至少30 min。取脑并分小脑,纹状体,海马和皮质4个脑区,固定于4%多聚甲醛,4℃1天后送至邵逸夫医院病理科,脱水包埋,切片,制片。常规HE染色,观察组织病理结构变化；免疫组化检测胶质纤维酸性蛋白(glial fibrillaryacidic protein, GFAP),紧密连接相关蛋白跨膜蛋白(zonula occludens-1, ZO-1)和胞浆附着蛋白(Claudin-1).
2.6抗氧化酶和脂质过氧化物检测组织称重,用生理盐水制成10%的组织后匀浆,4000 g离心10 min,取上清液备用,用考马斯亮兰法测定脑组织蛋白含量、硫代巴比妥酸法(TBA法)测定反应脂质过氧化情况的丙二醛(malondialdehyde, MDA)的含量、测抗氧化酶超氧化物岐化酶(superoxide dismutase, SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)活性。
2.7神经损伤相关蛋白
组织称重,按10 mg/150 ul的质量体积比加RIPA裂解液(含PMSF)提取总蛋白,用BCA法测定蛋白浓度,Western blotting法检测GFAP和NOS1蛋白表达水平的改变,实验结果用GFAP/β-actin和NOS1/β-actin光密度比值表示。
2.8炎性细胞因子
液氮研磨组织,用RNA pro plus (Takara)按步骤提取组织中的总RNA,测定RNA浓度,两步法RT-PCR试剂盒(Takara)检测炎症细胞因子IL-1β和TNF-αmRNA表达水平。
2.9统计分析
用SPSS统计软件对数据进行统计分析,结果用Mean±SD表示,采用t检验和单因素方差分析,LSD法用于比较两组间的差异,P<0.05视为有统计学差异。
【结果】
1.材料特性：
电镜下观察SWCNT呈分散的管状结构或团聚一起的块状结构,分散状态的纳米颗粒直径Φ≤100 nm,聚集的块状物粒径100 nm≤Φ≤500 nm,在放大10万倍条件下可见空心样的管状结构。
2.体重
SWCNT处理可抑制小鼠体重的增加。
剂量效应研究中,SWCNT可抑制小鼠体重增长,呈现为体重增加随剂量升高而减少的剂量-效应关系,最高剂量(25 mg/kg)抑制明显(P<0.05)。
时间效应研究中,第1,7,14,28天时都表现为处理组体重增加少于对照组的趋势但无统计学意义(P>0.05)。
3.组织病理学
HE染色结果显示：SWCNT处理引起组织结构的改变,纹状体、海马和皮质细胞排列紊乱。
免疫组化结果显示：小脑表现不明显,纹状体、海马和皮质三个脑区均显示SWCNT处理使GFAP表达先增加后减弱,ZO-1和Claudin-1表达呈先减少后增加的趋势,表明SWCNT处理对BBB紧密连接相关蛋白和神经损伤相关蛋白有影响。
GFAP表现为：剂量关系研究发现,小脑改变不明显,纹状体、海马和皮质脑区SWCNT处理组较高剂量组GFAP阳性表达升高,以12.5 mg/kg组最为显著；时间效应研究发现,小脑基本无变化,另三个脑区在第7和14天时SWCNT处理组阳性表达比对照组高,28天时趋向于与对照组在同水平。
紧密连接相关蛋白ZO-1和Claudin-1表现为：在剂量效应研究中,SWCNT高剂量组与对照相比损伤明显,以12.5 mg/kg组最为显著；时间效应研究发现纹状体和海马,在7和14天时实验组蛋白阳性表达比对照组明显减少,在第28天时,实验组和对照组又趋于平衡,小脑和皮质表现都不明显。Claudin-1总的变化趋势与ZO-1一致,但损伤表现不如ZO-1明显。
4.抗氧化酶和脂质过氧化物
SWCNT处理可使氧化系统的失衡,表现为抗氧化酶SOD和GSH-Px活性减弱,MDA含量增加,且对纹状体,海马和皮质三个脑区影响较大,小脑不明显。剂量效应研究发现纹状体、海马和皮质的抗氧化酶SOD和GSH-Px活性减弱和MDA含量增加,低浓度时(≤12.5 mg/kg)表现出随剂量增加损伤明显的剂量效应关系,12.5 mg/kg组损伤表现最明显(P<0.05),小脑略有变化但无差异(P>0.05)。时间效应研究发现在纹状体、海马和皮质抗氧化酶SOD和GSH-Px活性短期内先减弱后渐升高恢复,MDA含量先增加后逐渐减少恢复,在第7和14天时损伤明显(P<0.05),28 d时各指标逐渐恢复至与对照同水平,小脑未见明显变化(P>0.05)。
5.神经损伤相关蛋白
SWCNT处理后引起纹状体、海马和皮质的NOS1和GFAP蛋白表达水平增高,小脑变化不明显。剂量效应研究发现NOS1和GFAP在纹状体、海马和皮质脑区表达增高,在一定的浓度范围内(≤12.5 mg/kg)表现出随剂量增加损伤明显的剂量效应关系,12.5 mg/kg组损伤最明显(P<0.05),小脑有此趋势但无差异(P>0.05)。时间效应研究发现NOS1和GFAP水平在纹状体、海马和皮质脑区表达先增高后减低恢复至与对照水平,损伤效应在第7和14 d时明显(P<0.05),28天时逐渐恢复至与对照同水平(P>0.05),小脑变化不明显(P>0.05)。
6.炎症细胞因子
SWCNT处理后引起脑组织炎性反应,表现为细胞因子IL-1β和TNF-αmRNA表达水平升高。剂量效应研究发现SWCNT处理后小脑和皮质内12.5 mg/kg升高明显(P<0.05),纹状体和海马中TNF-α和IL-1β处理组高于对照组,12.5 mg/kg组升高最明显(P<0.05),皮质未见明显的改变。时间关系中,在四个脑区都表现为短期内明显升高,而后逐渐降低,TNF-a在小脑,纹状体和皮质在第1天明显升高后减低,在海马第7天时最高；IL-1β在小脑基本无变化,纹状体和海马7天时升高明显,皮质第1和28天明显升高。
【结论】
1.小鼠尾静脉注射SWCNT后可降低BBB紧密连接蛋白ZO-1和Claudin-1的表达,使抗氧化酶活性减弱、脂质过氧化物含量增加,促进炎症细胞因子表达和神经损伤相关蛋白的表达；
2. SWCNT处理对不同脑区的损伤效应不同,小脑损伤较小,纹状体和海马损伤较严重,皮质也有损伤表现；
3.在本实验条件下,SWCNT对中枢神经系统的损伤的部分因子变换呈现剂量效应和时间效应关系,且存在一定的可逆性；
4.本研究中,SWCNT神经系统毒性的具体机制可能涉及炎症反应和氧化应激。
[Objective]
Nanomaterial (NM) has unique physio-chemical properties because of the small size, and was produced for various applications. Carbon nanotube (CNT) are one of the remarkable NM of the world, including Single-walled carbon nanotube (SWCNT) and Muti-walled carbon nanotube (MWCNT). SWCNT has applications in space structures, thermal control, sensing technology, energy, electronic,biomedicine and pharmacy owing to the specific properties.The world widely use of NM (including SWCNT) generated enormous economic and scientific benefits, but at the same time, owing to the world widely utility and exposure, people pay close attention to the assessment of the potential impact on human health and environment suffers in recent years.
Studies indicated that the concentration of the nanopartical in the air has the positive relationship to the respiratory and cardiovascular disease. Recent studies have already shown that SWCNT can decrease cell viability, induce apoptosis and change of cell cycles, also can get into the body via inhalation, digestive, skin and injection, then achieved the target organs through lymph and blood circulation induce biotoxicity. SWCNT could get into the lung and cause pulmonary inflammation, intersitital fibrosis and granulomas lesions via the pharyngeal aspiration which is the commonly way. Once nanoparticles distributed in the lung, it can transport from the pulmonary to the extra-pulmonary tissues, distributed to heart, liver, brain and so on then induce tissues damage. All these makes us pay more attention to the extra-pulmonary toxicity of SWCNT. Nowadays, it is evidenced that SWCNT can induce aorta mitochondria damage and oxidative stress, also accelerate atherosclerotic of. Recent studied show that the main machine processed of NM is inflammatory reaction and oxidative stress, SWCNT induce the body produce ROS cause oxidative stress, then lead to inflammatory cause tissue damage.
Central nervous system is the chief component element of nervous system, the network or loop are gathered with lots of neurone, is the basement of learning, memories, human consciousness, psychological and thinking, meanwhile regeneration capacity of the nervous tissue is extremely limited. The blood brain barrier (BBB) serves as a physical moshysical barrier and strictly filter the extra-substance into and out of the brain in order to maintain CNS homeostasis. Recent studies indicated that CNT can get into CNS via BBB, olfactory bulb and sensory nerve ending then induce CNS damage. Though the research of the neuro-bio reaction is still deficiency, some researches have already indicated that NP can induce neurotoxicity, cause nervous tissue lesion.
SWCNT can distributed in the whole body through different ways and induce inflammatory and oxidative stress in the espiratory and cardiovascular system, and can reach the brain via BBB, so it is impretive to know whether SWCNT effects the BBB and CNS and illustrate the machine. According to these, the objective of this study was to investigate the structure change of the BBB and CNS neurotoxicity, such as re-inflammatory cytokine expressions, ROS production, antioxidant enzyme levels and neurology index in different sub-brain, illustrate the relationship of NM and Central nervous system disease.
[Materials and methods] 1.Animals
Male ICR mice 22±2 g, housed in plastic cages under controlled environmental conditions, temperature 24±0.5℃, humidity 55±5%, and a 12h light/dark cycle was in effect, with food and water available ad libitum, adapted 1 week before experiment.
2.Single-walled Carbon Nanotube
Solvent:saline with 0.1% (v/v) polysorbate 80 and sonicated for 400w,8s×15 times;
SWCNT:The 1 mg/ml SWCNT were suspended in saline with 0.1% (v/v) polysorbate 80 and sonicated for 400w,8 s×15 times, then immediately diluted to 0.5、0.2.0.1 mg/ml. The suspensions used in each experiment were always prepared freshly.
3.Treatment
The experiment contains dose-dependent and time-dependent study.
Dose-dependent study, five groups,100 animals, one control (the saline contained 0.1% Tween80) and 4 treated groups (different concentrations,2.5、5、2.5、25 mg/kg), administered 0.5 ml per mouse for continuous 5 days through tail intravenous injection with the observation time points of 7 days.
Time-dependent study, eight groups,220animals, control group (the saline contained 0.1% Tween 80) and treated groups (concentrations,12.5mg/kg), administered 0.5ml per mouse for continuous five days through tail intravenous injection with the observation time points of 1,7,14 and 28 days.
The animals were divided randomly into 13 groups, including histopathology, antioxidant enzyme acticities and MDA levels, Western Blotting and PCR four examinattion, each 5 murine. Recorded the body weight before the the administration and the time point of the observation. At the post-administration time point, except the histopathology animals were treated with perfusion then make the slides, others were sacrificed directly, cerebellum, striatum, hippocampus and cerebral cortex were divided on the ice bath and kept in 1.5 ml EP tube under-80℃.
4.Nanomaterial character
SWCNT 2 mg were suspended in 4 ml saline with 0.1% (v/v) polysorbate 80 and sonicated for 400w,8s×15 times, then observed with TEM immediately.
5. Histopathology
The brain tissues were collected by brain perfusion in this study. Briefly, the mouse was anesthetized with sodium pentobarbital (70 mg/kg, i.p.), then perfused through heart with 20 ml saline first and 4% paraformaldehyde 30 ml afterwards. Immediately after perfusion, the sample of cerebellum, striatum, hippocampus and cortex were collected and stored in 4% paraformaldehyde at 4℃until the slides were done. The histopathological tests were performed as standard laboratory procedures. After that do the HE staining and IHC staining (ZO-1, Claudin-1, GFAP), the sections were observed and photos were taken by optical microscope.
6. Lipid oxidation and antioxidant enzyme acticities detection
The samples were weighed and homogenized in saline, centrifuged at 4000×g for 10 min at 4℃, then the supernatants were used to measure total protein concentrations (Bradford's method), the content of malondialdehyde (MDA with thiobarbituric acid, TBA method), the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px).
7. Western blotting (Protein expression of neural lesion)
The frozen tissues were weighed and collected total protein using RIPA with 1 mmol/L PMSF, and determined the protein content using Bicinchoninic Acid (BCA) Protein Assay. Western Blotting determine GFAP and NOS1 protein levels in brain regions.
8. Inflammatory cytokine (qPCR)
The total RNA was collected with RNA pro plus (Takara) in liquid nitrogen, then determined the RNA viscosity and purity. Two-steps RT-PCR kits were used to detect the IL-1βand TNF-αmRNA levels.
9. Statistical analysis
Each experiment was conducted at least three times. Data were represented as mean±standard deviation (SD). Multi-group comparisons were evaluated using one-way-analysis of variance (ANOVA) followed by Least-significant difference (LSD) in post-hoc test for the experiment groups. Statistical probability of P<0.05 was considered significant.
Results
1. Nanomaterial character
The TEM result shows that the SWCNT dispersed like tubes (Φ≤100 nm) or agglomeration (100nm≤Φ≤500 nm). And also can found hollowness tubular shape.
2. Weight:
Both the dose and time dependent research shows that SWCNT treatement inhibit the the weight gain. In the dose-dependent study, the weight increased fewer in the high dose compared to control, and the 25 mg/kg treatment inhibited the weight gain significantly (P<0.05). However in the time-dependent study, the weight gain less in the treated group, but has no sinificant means (P>0.05).
3. Immunohistochemisty
HE staining:SWCNT affected the structure of the brain, cells in striatum, hippocampus and cortex disorganized.
The test indicated that SWCNT may increase the GFAP protein expression in the earlier time then decreased, but the ZO-1 and Claudin-1 protein expression first decreased then increased in striatum, hippocampus and cerebral cortex except the cerebellum. In the dose-dependent study, it is indicated that, except the cerebellum, GFAP, ZO-1 and Claudin-1 expression increased at the high concentration, especially at 12.5mg/kg. The time-dependent study shows that the GFAP level was higher in the treatment compared with the control at the time point of 7 and 14 days except the cerebellum, however ZO-1 and Claudin-1 levels only decreased in two sub-brain area (striatum and hippocampus).
4. anti-oxidant enzyme and lipid peroxidation
The test indicated that SWCNTs effects the anti-oxidant system balance, act as SOD and GSH-Px activity decreased and MDA level heightened in striatum, hippocampus and cerebral cortex. In the dose-dependent study, in striatum, hippocampus and cerebral cortex sub-brain areas, it is indicated that anti-oxidant enzyme SOD and GSH-Px activity attenuated and MDA level advanced in the treated group compared to control, and has a dose-dependent relationship in the low concentration, the 12.5 mg/kg was the peak (P<0.05). The time-dependent study shows that the SOD and GSH-Px activity attenuated first then recovered and MDA shows the same result compared with the control at the time point of 7 and 14 days in striatum, hippocampus and cerebral cortex sub-brain areas (P<0.05). Hower, no obvious alteration was observed in the cerebellum (P>0.05).
5. Protein expression of neural lesion
It was indicated that SWCNT upgrade NOS1 and GFAP protein levels in striatum, hippocampus and cerebral cortex, no obvious change was found in cerebellum. In the dose-dependent study, in striatum, hippocampus and cerebral cortex sub-brain areas, it is indicated that NOS1 and GFAP protein levels increased, and found a dose-dependent relationship in the low concentration, the 12.5mg/kg was the peak (P＜0.05), and found the same changes in cerebellum, but no difference (P>0.05). The time-dependent study shows that ompared with the control, the NOS1 and GFAP protein levels increased first then attenuated at the time point of 7 and 14 days in striatum, hippocampus and cerebral cortex sub-brain areas(P<0.05). However, no obvious alteration was observed in the cerebellum (P>0.05).
6.Inflammatory cytokine
The test indicated that SWCNT induce inflammation, cytokine such as IL-1βand TNF-αmRNA level increased in the brain. In the dose-dependent study, the TNF-α mRNA level upgraded in striatum and hippocampus with dose-dependent relationships (P＜0.05), obvious lesion was observed in cerebellum and cortex at the 12.5 mg/kg (P<0.05); IL-1βmRNA level increased in cerebellum in low dose,12.5 mg/kg treatment increased in striatum, IL-ip significantly increased SWCNT groups in hippocampus (P＜0.05), with no changes in cortex. The time-dependent study shows that TNF-a mRNA level increased first then decreased in the four sub-brain regions, in cerebellum, striatum, and coetex TNF-a level were the peak at the first day, at the time point of 7 days in n hippocampus; IL-1βmRNA level has no change in cerebellum, increased significantly at the time point of 7 days in striatum and hippocampus.
Conclusion
1. SWCNT with tail vein injection may decrease the expression of BBB tight junction protein ZO-1 and Claudin-1, disrupt the balance of antioxidant system, promote the expression of inflammatory cytokines and neural lesion,
2. SWCNT has different toxicity in each brain region, striatum and hippocampus were more sensitive than cerebellum and cortex,
3. Under these conditions, SWCNT demonstrated a dose-dependent and time-dependent relationship damage in finite concentration, morever the damge is reversible,
4. In this research, the machanism of SWCNT may the inflammatory reaction and oxidative stress.
纳米材料因其小尺寸而具有独特的理化和生物特性,越来越广泛地被应用于生产生活的各个领域。纳碳米管(Carbon nanotube, CNT)是纳米材料中当前研究热点之一,主要包括单壁纳米碳管(Single-walled carbon nanotube, SWCNT)和多壁纳米碳管(Muti-walled carbon nanotube, MWCNT)。SWCNT因其特殊的理化特性,使用范围包括空间结构、热控制装置、传感技术、能源、电子学和生物医药等多个领域。纳米材料(包括SWCNT)的应用带来了巨大的经济效益,但同时由于尺寸小及生物体暴露几率大大增加而产生的潜在的生物安全性与环境安全性等问题引起了世界范围的广泛关注。
已有研究表明空气中纳米粒子的浓度与呼吸道,心血管疾病等多种疾病的发病率和死亡率呈正相关,而SWCNT在体外试验中可引起细胞存活率下降、细胞凋亡、细胞周期改变等细胞毒性,体内试验发现SWCNT可经呼吸道、皮肤、消化道及注射等多种途径进入机体,并经血或淋巴循环分布到心、肝、脾、肾、胃、脑、皮肤、血液等多个组织器产生毒性效应。目前对SWCNT的呼吸系统毒性研究较多,结果发现SWCNT吸入后可引起肺炎性反应和氧化应激,产生肺组织纤维化和肉芽肿；并且进入肺间质部位后还可经多种途径转移到肺外组织从而分布到多个器官引起损伤,这使得人们更加重视SWCNT的肺外系统毒性。虽然相关研究较少,但已有研究发现SWCNT可引起主动脉线粒体损伤和氧化应激并可加速动脉粥样硬化的形成,并可跨越血脑屏障(blood brain barrier, BBB)进入脑内。
中枢神经系统(central nervous system, CNS)是神经系统的主要部分,大量的神经细胞聚集构成网络或回路,是学习、记忆、人类意识、心理、思维活动的基础,且神经组织的再生能力非常有限,其损伤多具有不可逆性。而BBB是存在于血液和脑组织之间的生物屏障,具选择透过性,对维持CNS的稳定有重要的作用。现有的研究表明,碳纳米材料可跨越BBB进入脑内,还可经鼻腔黏膜摄入至嗅球后经嗅神经转运或经感觉神经末梢直接转运至脑内并对CNS产生影响。虽然目前关于纳米材料的神经生物效应研究尚处于起步阶段,但已有研究报道显示进入CNS的纳米颗粒可引起一定的神经毒性效应,导致神经组织损伤。
鉴于SWCNT可经多种途径进入体内,对呼吸系统和心血管系统产生炎性损伤和氧化应激等毒性反应,同时还可跨越BBB在脑内分布,且CNS损伤多具有不可逆性,因此研究SWCNT对CNS的影响及其机制有积极的意义。本实验拟以小鼠为研究对象,探索SWCNT对BBB及CNS的损伤效应及其机制,通过组织病理学、神经损伤标志物、氧化损伤标志物和炎性因子等方面的改变,阐明SWCNT对不同脑区的损伤情况。
【材料和方法】
2.1实验动物
清洁级雄性ICR小鼠,体重22±2g,温度24±0.5℃,湿度55±5%,每日12h灯照,自由摄取食物和水,适应1周后开始实验。
2.2实验材料及配制
溶剂：生理盐水与吐温80(T80)配成0.1%T80生理盐水液,超声400 w,8 s×15次；
材料：单壁纳米碳管(SWCNT),先配成1 mg/ml的悬液,用细胞粉碎仪分散,超声功率400 w,8 s×15次。分散后立即按比例稀释成0.5、0.2、0.1 mg/ml不同浓度的悬液,稀释后立即超声(同前),现配现用。
2.3给药方法
实验分成两部分,一部分研究剂量效应关系,另一部分研究时间效应关系。
剂量效应研究,5组,包括1个对照组(0.1%T80生理盐水液)和4个实验组(2.5、5、12.5、25 mg/kg的SWCNT悬液),尾静脉注射0.5 ml/只,连续染毒5天,第5天给药结束后观察7天,共100只。
时间效应研究,8组,共设对照组(0.1%T80生理盐水液)和实验组(12.5 mg/kg的SWCNT悬液),尾静脉注射0.5 ml/只,连续染毒5天,第5天给药结束后观察1,7,14和28天四个时间点,共220只。
分组织病理学实验、抗氧化酶测定、western blotting、qPCR4部分实验,各部分各剂量组5只小鼠。开始给药前及到观察时间点时分别测定小鼠体重,用于组织病理学实验的小鼠经心脏灌流固定后取脑分小脑、纹状体、海马和皮质4个脑区,然后固定、包埋、切片后染色观察；其余3部分实验的小鼠颈椎脱臼处死取脑,于冰上进行分区(小脑、纹状体、海马和皮质),置于1.5 ml EP管中,于-80℃保存待用,qPCR实验的组织用无RNA酶和DNA酶的1.5 ml EP管置于-80℃保存待用。
2.4材料特性鉴定
称取2 mg的SWCNT,加4 ml含0.1%T80的ddH2O,超声400 w,8s×15次,配成0.5 mg/ml悬液,滴于铜网,立即用TEM观察材料特性。
2.5组织病理学检测
到观察时间点时,小鼠用戊巴比妥钠70 mg/kg腹腔注射麻醉后固定操作板,用生理盐水和4%多聚甲醛经升主动脉心脏灌流固定,至从右心房流出的灌流液无色再灌流至少30 min。取脑并分小脑,纹状体,海马和皮质4个脑区,固定于4%多聚甲醛,4℃1天后送至邵逸夫医院病理科,脱水包埋,切片,制片。常规HE染色,观察组织病理结构变化；免疫组化检测胶质纤维酸性蛋白(glial fibrillaryacidic protein, GFAP),紧密连接相关蛋白跨膜蛋白(zonula occludens-1, ZO-1)和胞浆附着蛋白(Claudin-1).
2.6抗氧化酶和脂质过氧化物检测组织称重,用生理盐水制成10%的组织后匀浆,4000 g离心10 min,取上清液备用,用考马斯亮兰法测定脑组织蛋白含量、硫代巴比妥酸法(TBA法)测定反应脂质过氧化情况的丙二醛(malondialdehyde, MDA)的含量、测抗氧化酶超氧化物岐化酶(superoxide dismutase, SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)活性。
2.7神经损伤相关蛋白
组织称重,按10 mg/150 ul的质量体积比加RIPA裂解液(含PMSF)提取总蛋白,用BCA法测定蛋白浓度,Western blotting法检测GFAP和NOS1蛋白表达水平的改变,实验结果用GFAP/β-actin和NOS1/β-actin光密度比值表示。
2.8炎性细胞因子
液氮研磨组织,用RNA pro plus (Takara)按步骤提取组织中的总RNA,测定RNA浓度,两步法RT-PCR试剂盒(Takara)检测炎症细胞因子IL-1β和TNF-αmRNA表达水平。
2.9统计分析
用SPSS统计软件对数据进行统计分析,结果用Mean±SD表示,采用t检验和单因素方差分析,LSD法用于比较两组间的差异,P<0.05视为有统计学差异。
【结果】
1.材料特性：
电镜下观察SWCNT呈分散的管状结构或团聚一起的块状结构,分散状态的纳米颗粒直径Φ≤100 nm,聚集的块状物粒径100 nm≤Φ≤500 nm,在放大10万倍条件下可见空心样的管状结构。
2.体重
SWCNT处理可抑制小鼠体重的增加。
剂量效应研究中,SWCNT可抑制小鼠体重增长,呈现为体重增加随剂量升高而减少的剂量-效应关系,最高剂量(25 mg/kg)抑制明显(P<0.05)。
时间效应研究中,第1,7,14,28天时都表现为处理组体重增加少于对照组的趋势但无统计学意义(P>0.05)。
3.组织病理学
HE染色结果显示：SWCNT处理引起组织结构的改变,纹状体、海马和皮质细胞排列紊乱。
免疫组化结果显示：小脑表现不明显,纹状体、海马和皮质三个脑区均显示SWCNT处理使GFAP表达先增加后减弱,ZO-1和Claudin-1表达呈先减少后增加的趋势,表明SWCNT处理对BBB紧密连接相关蛋白和神经损伤相关蛋白有影响。
GFAP表现为：剂量关系研究发现,小脑改变不明显,纹状体、海马和皮质脑区SWCNT处理组较高剂量组GFAP阳性表达升高,以12.5 mg/kg组最为显著；时间效应研究发现,小脑基本无变化,另三个脑区在第7和14天时SWCNT处理组阳性表达比对照组高,28天时趋向于与对照组在同水平。
紧密连接相关蛋白ZO-1和Claudin-1表现为：在剂量效应研究中,SWCNT高剂量组与对照相比损伤明显,以12.5 mg/kg组最为显著；时间效应研究发现纹状体和海马,在7和14天时实验组蛋白阳性表达比对照组明显减少,在第28天时,实验组和对照组又趋于平衡,小脑和皮质表现都不明显。Claudin-1总的变化趋势与ZO-1一致,但损伤表现不如ZO-1明显。
4.抗氧化酶和脂质过氧化物
SWCNT处理可使氧化系统的失衡,表现为抗氧化酶SOD和GSH-Px活性减弱,MDA含量增加,且对纹状体,海马和皮质三个脑区影响较大,小脑不明显。剂量效应研究发现纹状体、海马和皮质的抗氧化酶SOD和GSH-Px活性减弱和MDA含量增加,低浓度时(≤12.5 mg/kg)表现出随剂量增加损伤明显的剂量效应关系,12.5 mg/kg组损伤表现最明显(P<0.05),小脑略有变化但无差异(P>0.05)。时间效应研究发现在纹状体、海马和皮质抗氧化酶SOD和GSH-Px活性短期内先减弱后渐升高恢复,MDA含量先增加后逐渐减少恢复,在第7和14天时损伤明显(P<0.05),28 d时各指标逐渐恢复至与对照同水平,小脑未见明显变化(P>0.05)。
5.神经损伤相关蛋白
SWCNT处理后引起纹状体、海马和皮质的NOS1和GFAP蛋白表达水平增高,小脑变化不明显。剂量效应研究发现NOS1和GFAP在纹状体、海马和皮质脑区表达增高,在一定的浓度范围内(≤12.5 mg/kg)表现出随剂量增加损伤明显的剂量效应关系,12.5 mg/kg组损伤最明显(P<0.05),小脑有此趋势但无差异(P>0.05)。时间效应研究发现NOS1和GFAP水平在纹状体、海马和皮质脑区表达先增高后减低恢复至与对照水平,损伤效应在第7和14 d时明显(P<0.05),28天时逐渐恢复至与对照同水平(P>0.05),小脑变化不明显(P>0.05)。
6.炎症细胞因子
SWCNT处理后引起脑组织炎性反应,表现为细胞因子IL-1β和TNF-αmRNA表达水平升高。剂量效应研究发现SWCNT处理后小脑和皮质内12.5 mg/kg升高明显(P<0.05),纹状体和海马中TNF-α和IL-1β处理组高于对照组,12.5 mg/kg组升高最明显(P<0.05),皮质未见明显的改变。时间关系中,在四个脑区都表现为短期内明显升高,而后逐渐降低,TNF-a在小脑,纹状体和皮质在第1天明显升高后减低,在海马第7天时最高；IL-1β在小脑基本无变化,纹状体和海马7天时升高明显,皮质第1和28天明显升高。
【结论】
1.小鼠尾静脉注射SWCNT后可降低BBB紧密连接蛋白ZO-1和Claudin-1的表达,使抗氧化酶活性减弱、脂质过氧化物含量增加,促进炎症细胞因子表达和神经损伤相关蛋白的表达；
2. SWCNT处理对不同脑区的损伤效应不同,小脑损伤较小,纹状体和海马损伤较严重,皮质也有损伤表现；
3.在本实验条件下,SWCNT对中枢神经系统的损伤的部分因子变换呈现剂量效应和时间效应关系,且存在一定的可逆性；
4.本研究中,SWCNT神经系统毒性的具体机制可能涉及炎症反应和氧化应激。
[Objective]
Nanomaterial (NM) has unique physio-chemical properties because of the small size, and was produced for various applications. Carbon nanotube (CNT) are one of the remarkable NM of the world, including Single-walled carbon nanotube (SWCNT) and Muti-walled carbon nanotube (MWCNT). SWCNT has applications in space structures, thermal control, sensing technology, energy, electronic,biomedicine and pharmacy owing to the specific properties.The world widely use of NM (including SWCNT) generated enormous economic and scientific benefits, but at the same time, owing to the world widely utility and exposure, people pay close attention to the assessment of the potential impact on human health and environment suffers in recent years.
Studies indicated that the concentration of the nanopartical in the air has the positive relationship to the respiratory and cardiovascular disease. Recent studies have already shown that SWCNT can decrease cell viability, induce apoptosis and change of cell cycles, also can get into the body via inhalation, digestive, skin and injection, then achieved the target organs through lymph and blood circulation induce biotoxicity. SWCNT could get into the lung and cause pulmonary inflammation, intersitital fibrosis and granulomas lesions via the pharyngeal aspiration which is the commonly way. Once nanoparticles distributed in the lung, it can transport from the pulmonary to the extra-pulmonary tissues, distributed to heart, liver, brain and so on then induce tissues damage. All these makes us pay more attention to the extra-pulmonary toxicity of SWCNT. Nowadays, it is evidenced that SWCNT can induce aorta mitochondria damage and oxidative stress, also accelerate atherosclerotic of. Recent studied show that the main machine processed of NM is inflammatory reaction and oxidative stress, SWCNT induce the body produce ROS cause oxidative stress, then lead to inflammatory cause tissue damage.
Central nervous system is the chief component element of nervous system, the network or loop are gathered with lots of neurone, is the basement of learning, memories, human consciousness, psychological and thinking, meanwhile regeneration capacity of the nervous tissue is extremely limited. The blood brain barrier (BBB) serves as a physical moshysical barrier and strictly filter the extra-substance into and out of the brain in order to maintain CNS homeostasis. Recent studies indicated that CNT can get into CNS via BBB, olfactory bulb and sensory nerve ending then induce CNS damage. Though the research of the neuro-bio reaction is still deficiency, some researches have already indicated that NP can induce neurotoxicity, cause nervous tissue lesion.
SWCNT can distributed in the whole body through different ways and induce inflammatory and oxidative stress in the espiratory and cardiovascular system, and can reach the brain via BBB, so it is impretive to know whether SWCNT effects the BBB and CNS and illustrate the machine. According to these, the objective of this study was to investigate the structure change of the BBB and CNS neurotoxicity, such as re-inflammatory cytokine expressions, ROS production, antioxidant enzyme levels and neurology index in different sub-brain, illustrate the relationship of NM and Central nervous system disease.
[Materials and methods] 1.Animals
Male ICR mice 22±2 g, housed in plastic cages under controlled environmental conditions, temperature 24±0.5℃, humidity 55±5%, and a 12h light/dark cycle was in effect, with food and water available ad libitum, adapted 1 week before experiment.
2.Single-walled Carbon Nanotube
Solvent:saline with 0.1% (v/v) polysorbate 80 and sonicated for 400w,8s×15 times;
SWCNT:The 1 mg/ml SWCNT were suspended in saline with 0.1% (v/v) polysorbate 80 and sonicated for 400w,8 s×15 times, then immediately diluted to 0.5、0.2.0.1 mg/ml. The suspensions used in each experiment were always prepared freshly.
3.Treatment
The experiment contains dose-dependent and time-dependent study.
Dose-dependent study, five groups,100 animals, one control (the saline contained 0.1% Tween80) and 4 treated groups (different concentrations,2.5、5、2.5、25 mg/kg), administered 0.5 ml per mouse for continuous 5 days through tail intravenous injection with the observation time points of 7 days.
Time-dependent study, eight groups,220animals, control group (the saline contained 0.1% Tween 80) and treated groups (concentrations,12.5mg/kg), administered 0.5ml per mouse for continuous five days through tail intravenous injection with the observation time points of 1,7,14 and 28 days.
The animals were divided randomly into 13 groups, including histopathology, antioxidant enzyme acticities and MDA levels, Western Blotting and PCR four examinattion, each 5 murine. Recorded the body weight before the the administration and the time point of the observation. At the post-administration time point, except the histopathology animals were treated with perfusion then make the slides, others were sacrificed directly, cerebellum, striatum, hippocampus and cerebral cortex were divided on the ice bath and kept in 1.5 ml EP tube under-80℃.
4.Nanomaterial character
SWCNT 2 mg were suspended in 4 ml saline with 0.1% (v/v) polysorbate 80 and sonicated for 400w,8s×15 times, then observed with TEM immediately.
5. Histopathology
The brain tissues were collected by brain perfusion in this study. Briefly, the mouse was anesthetized with sodium pentobarbital (70 mg/kg, i.p.), then perfused through heart with 20 ml saline first and 4% paraformaldehyde 30 ml afterwards. Immediately after perfusion, the sample of cerebellum, striatum, hippocampus and cortex were collected and stored in 4% paraformaldehyde at 4℃until the slides were done. The histopathological tests were performed as standard laboratory procedures. After that do the HE staining and IHC staining (ZO-1, Claudin-1, GFAP), the sections were observed and photos were taken by optical microscope.
6. Lipid oxidation and antioxidant enzyme acticities detection
The samples were weighed and homogenized in saline, centrifuged at 4000×g for 10 min at 4℃, then the supernatants were used to measure total protein concentrations (Bradford's method), the content of malondialdehyde (MDA with thiobarbituric acid, TBA method), the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px).
7. Western blotting (Protein expression of neural lesion)
The frozen tissues were weighed and collected total protein using RIPA with 1 mmol/L PMSF, and determined the protein content using Bicinchoninic Acid (BCA) Protein Assay. Western Blotting determine GFAP and NOS1 protein levels in brain regions.
8. Inflammatory cytokine (qPCR)
The total RNA was collected with RNA pro plus (Takara) in liquid nitrogen, then determined the RNA viscosity and purity. Two-steps RT-PCR kits were used to detect the IL-1βand TNF-αmRNA levels.
9. Statistical analysis
Each experiment was conducted at least three times. Data were represented as mean±standard deviation (SD). Multi-group comparisons were evaluated using one-way-analysis of variance (ANOVA) followed by Least-significant difference (LSD) in post-hoc test for the experiment groups. Statistical probability of P<0.05 was considered significant.
Results
1. Nanomaterial character
The TEM result shows that the SWCNT dispersed like tubes (Φ≤100 nm) or agglomeration (100nm≤Φ≤500 nm). And also can found hollowness tubular shape.
2. Weight:
Both the dose and time dependent research shows that SWCNT treatement inhibit the the weight gain. In the dose-dependent study, the weight increased fewer in the high dose compared to control, and the 25 mg/kg treatment inhibited the weight gain significantly (P<0.05). However in the time-dependent study, the weight gain less in the treated group, but has no sinificant means (P>0.05).
3. Immunohistochemisty
HE staining:SWCNT affected the structure of the brain, cells in striatum, hippocampus and cortex disorganized.
The test indicated that SWCNT may increase the GFAP protein expression in the earlier time then decreased, but the ZO-1 and Claudin-1 protein expression first decreased then increased in striatum, hippocampus and cerebral cortex except the cerebellum. In the dose-dependent study, it is indicated that, except the cerebellum, GFAP, ZO-1 and Claudin-1 expression increased at the high concentration, especially at 12.5mg/kg. The time-dependent study shows that the GFAP level was higher in the treatment compared with the control at the time point of 7 and 14 days except the cerebellum, however ZO-1 and Claudin-1 levels only decreased in two sub-brain area (striatum and hippocampus).
4. anti-oxidant enzyme and lipid peroxidation
The test indicated that SWCNTs effects the anti-oxidant system balance, act as SOD and GSH-Px activity decreased and MDA level heightened in striatum, hippocampus and cerebral cortex. In the dose-dependent study, in striatum, hippocampus and cerebral cortex sub-brain areas, it is indicated that anti-oxidant enzyme SOD and GSH-Px activity attenuated and MDA level advanced in the treated group compared to control, and has a dose-dependent relationship in the low concentration, the 12.5 mg/kg was the peak (P<0.05). The time-dependent study shows that the SOD and GSH-Px activity attenuated first then recovered and MDA shows the same result compared with the control at the time point of 7 and 14 days in striatum, hippocampus and cerebral cortex sub-brain areas (P<0.05). Hower, no obvious alteration was observed in the cerebellum (P>0.05).
5. Protein expression of neural lesion
It was indicated that SWCNT upgrade NOS1 and GFAP protein levels in striatum, hippocampus and cerebral cortex, no obvious change was found in cerebellum. In the dose-dependent study, in striatum, hippocampus and cerebral cortex sub-brain areas, it is indicated that NOS1 and GFAP protein levels increased, and found a dose-dependent relationship in the low concentration, the 12.5mg/kg was the peak (P＜0.05), and found the same changes in cerebellum, but no difference (P>0.05). The time-dependent study shows that ompared with the control, the NOS1 and GFAP protein levels increased first then attenuated at the time point of 7 and 14 days in striatum, hippocampus and cerebral cortex sub-brain areas(P<0.05). However, no obvious alteration was observed in the cerebellum (P>0.05).
6.Inflammatory cytokine
The test indicated that SWCNT induce inflammation, cytokine such as IL-1βand TNF-αmRNA level increased in the brain. In the dose-dependent study, the TNF-α mRNA level upgraded in striatum and hippocampus with dose-dependent relationships (P＜0.05), obvious lesion was observed in cerebellum and cortex at the 12.5 mg/kg (P<0.05); IL-1βmRNA level increased in cerebellum in low dose,12.5 mg/kg treatment increased in striatum, IL-ip significantly increased SWCNT groups in hippocampus (P＜0.05), with no changes in cortex. The time-dependent study shows that TNF-a mRNA level increased first then decreased in the four sub-brain regions, in cerebellum, striatum, and coetex TNF-a level were the peak at the first day, at the time point of 7 days in n hippocampus; IL-1βmRNA level has no change in cerebellum, increased significantly at the time point of 7 days in striatum and hippocampus.
Conclusion
1. SWCNT with tail vein injection may decrease the expression of BBB tight junction protein ZO-1 and Claudin-1, disrupt the balance of antioxidant system, promote the expression of inflammatory cytokines and neural lesion,
2. SWCNT has different toxicity in each brain region, striatum and hippocampus were more sensitive than cerebellum and cortex,
3. Under these conditions, SWCNT demonstrated a dose-dependent and time-dependent relationship damage in finite concentration, morever the damge is reversible,
4. In this research, the machanism of SWCNT may the inflammatory reaction and oxidative stress.
引文
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