Molecular mechanism of quercetin in the treatment of traumatic brain injury: its feasibility of clinical application

doi:10.3969/j.issn.2095-4344.1325

Abstract

Abstract:

BACKGROUND: Traumatic brain injury has pathological changes such as ischemia and hypoxia, which are similar to ischemic brain damage. Quercetin has been shown to hold significant effect in the treatment of traumatic brain injury.
OBJECTIVE: To review the research status and prospects of quercetin in the treatment of traumatic brain injury.
METHODS: Databases of WanFang, CNKI and PubMed were searched for the articles published from 1900 to December 2018. The keywords were “quercetin and brain injury; quercetin and traumatic brain injury; quercetin and oxidation reaction; quercetin and neurons apoptosis; quercetin and inflammatory factor; traumatic brain injury and oxidation reaction; traumatic brain injury and apoptosis; traumatic brain injury and inflammatory factor; quercetin and biosafety” in Chinese and English, respectively. The reports and reviews on the role of quercetin in traumatic brain injury were included.
RESULTS AND CONCLUSION: Quercetin can regulate antioxidant enzymes in oxidative stress to eliminate reactive oxygen species, improve the energy metabolism of mitochondria by promoting the biosynthesis of mitochondria themselves, and further protect mitochondria in neurons. Quercetin can also inhibit the activity and transport of phosphokinase C, antagonize the influx of sodium ions, thus inhibiting the release of histamine, prostaglandins and other inflammatory mediators, and alleviate inflammation. After treatment with quercetin, the autophagy and apoptosis of neurons can be weakened by activating PI3K/Akt signaling pathway and inhibiting ERK1/2 signaling pathway, and the neurological deficit and cognitive function can be improved. In animal safety evaluation test, the safety of quercetin can be considered to be high in a reasonable dose range. Quercetin has a significant therapeutic effect on traumatic brain injury, which is related to its scavenging oxygen free radicals, protecting mitochondria, antagonizing inflammatory reaction and reducing neuronal apoptosis. The potential molecular mechanism and clinical application of quercetin in the treatment of traumatic brain injury still need to be studied in depth.

Key words: quercetin, traumatic brain injury, mitochondria, oxidative stress, reactive oxygen species, inflammation reactions, neuroprotection, cell apoptosis

CLC Number:

R446
R318

Huang Chao, Huang Qinghua, You Di, Guo Wenlai, Qu Wenrui, Zhu Zhe, Li Rui . Molecular mechanism of quercetin in the treatment of traumatic brain injury: its feasibility of clinical application[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(23): 3760-3766.

Figures/Tables 1

2.1 槲皮素在创伤性脑损伤中的作用机制槲皮素具有抗氧化、抗炎、抗肿瘤、抗病毒、抗凋亡与神经保护作用等药理作用，在脊髓损伤和缺血再灌注动物治疗中可以有效阻断继发性改变引起的病理环节，并拮抗神经元凋亡，而脑组织的局部缺血缺氧也是创伤性脑损伤继发病理损伤的主要表现形式，可见将槲皮素应用于创伤性脑损伤的治疗具有重要意义。 2.1.1 槲皮素减轻创伤性脑损伤后氧化应激反应并保护线粒体氧化应激是指体内氧化与抗氧化作用失衡，倾向于氧化，导致中性粒细胞炎性浸润，蛋白酶分泌增加，产生大量活性氧簇。活性氧簇是高活性自由基簇，主要由线粒体产生，包括羟自由基(•OH)，超氧阴离子自由基(•O?-)和非自由基，如过氧化氢(H?O?)和单线态氧等[14]。在正常的细胞中，当过量电子从电子传递链中逸出时，活性氧簇通常作为ATP生成过程中的副产物，多余的活性氧簇随后被抗氧化剂清除[15]。创伤性脑损伤后，线粒体受到刺激产生大量活性氧簇，抗氧化系统受到损伤导致正常的平衡被破坏，大量的自由基引起氧化应激反应，导致脂质，蛋白质和DNA损伤等，被认为是创伤性脑损伤后继发性损伤的起源[16-18]。超氧化物岐化酶、过氧化氢酶和谷胱甘肽过氧化物酶是体内重要的抗氧化剂。超氧化物岐化酶是体内天然存在的超氧自由基清除因子,可以把有害的O2-转化为H?O?，进一步被体内的过氧化氢酶和谷胱甘肽过氧化物酶分解为完全无害的水[19-20]。机体内过氧化氢酶的耗尽被认为是导致脑损伤的重要因素[21]。创伤性脑损伤后线粒体内超氧化物岐化酶、谷胱甘肽过氧化物酶和过氧化氢酶水平降低，增多的活性氧簇引起脂质过氧化，脑组织的丙二醛和硫代巴比妥酸反应物的水平升高[22-23]。使用槲皮素治疗后，可以显著提高超氧化物岐化酶、谷胱甘肽过氧化物酶和过氧化氢酶水平，降低丙二醛和硫代巴比妥酸反应物水平，所以槲皮素可以升高体内抗氧化酶清除活性氧簇。槲皮素在创伤性脑损伤后潜在的抗氧化机制是目前的研究热点，核因子E2相关因子2是一种防御性核转录因子，通过调控多种抗氧化酶参与细胞防御反应，使细胞免受氧化应激损伤。生理状态下，核因子E2相关因子2存在于胞质中，与胞浆蛋白伴侣分子(Keapl)结合而处于抑制状态[24]，当受到刺激时，核因子E2相关因子2与Keapl解离并进入细胞核，启动下游一系列抗氧化应激反应，增强细胞对外界刺激的抵抗力[25-26]。创伤性脑损伤后，核因子E2相关因子2通路被激活，可使神经胶质细胞活化，为脑损伤提供保护作用。李翔[27]将槲皮素用于治疗创伤性脑损伤小鼠，结果显示创伤性脑损伤和槲皮素是核因子E2相关因子2核转位的诱导因子，与空白治疗组相比，槲皮素治疗组的核因子E2相关因子2比率显着增加，细胞质核因子E2相关因子2比例显著降低，提示槲皮素可以促进核因子E2相关因子2核转位，通过核因子E2相关因子2-抗氧化反应元件途径激活下游基因分泌抗氧化酶从而发挥抗氧化功效。其更深入的作用机制目前仍不明确，有待进一步研究。除了核因子E2相关因子2，氧化应激还可以激活PGC-lα通路，进一步诱导UCP2和超氧化物岐化酶2 的上调，对过度氧化应激引起的损伤效应起着重要的中和作用[28-29]。PGC-lα已被证明可以诱导抗氧化酶基因的表达[30]，并参与促进线粒体的生物合成、调节葡萄糖代谢、脂肪酸氧化等过程[31-32]，是能量代谢的中央监管机构，在高代谢率的组织如横纹肌、肝脏和大脑中高表达[28-31]。PGC-1α作为共同激活因子PGC-1的家族成员，能使线粒体转录因子A(TFAM)的表达量增加，同时增加线粒体DNA含量和线粒体的数量[29]。TFAM因子负责转录核编码的线粒体蛋白，包括结构蛋白以及线粒体DNA(mtDNA)的转录、翻译和修复[33-35]。PGC-lα对线粒体的保护作用已经在帕金森病和阿尔茨海默病等中枢神经系统疾病被报道[36]。缺少PGC-1α时会导致氧化磷酸化的基因表达减少，编码有关抑制活性氧簇蛋白的基因表达下调[37]。创伤性脑损伤后，大脑皮质神经元细胞核内PGC-1α蛋白水平升高，使用槲皮素治疗后，检测到细胞内PGC-1α总蛋白和核内PGC-1α蛋白均显着增加，表明槲皮素不仅促进了PGC-1α从细胞质向细胞核的转运，而且还促进了细胞内总PGC-1α蛋白质的表达[38]。在Li等[39]的研究中，除了上述PGC-1α改变，还观察到使用槲皮素后，促进细胞色素C从细胞质转入线粒体内以增强线粒体的生物合成，但这一过程是否与PGC-1α有关及PGC-1α易位的潜在分子机制仍需要进一步研究。所以，槲皮素可以通过调节氧化应激反应中的抗氧化酶实现自身对活性氧的清除，并促进线粒体自身的生物合成来改善线粒体的能量代谢，进一步保护神经元细胞内的线粒体。 2.1.2 槲皮素在创伤性脑损伤中的抗炎作用除了氧化应激反应，在创伤性脑损伤后，被激活的另一途径是损伤部位的炎症反应，其发生与氧化应激反应密切相关[40-41]。中枢神经系统损伤介导的炎症反应，包括巨噬细胞向损伤组织内的转移和促炎细胞因子，如肿瘤坏死因子α、白细胞介素1β和白细胞介素6等的表达，导致神经元细胞的损伤[42]。高迁移率族蛋白1是一种高度保存的核蛋白。它由被激活的巨噬细胞分泌，存在于细胞外，可以促进肿瘤坏死因子α、白细胞介素1β和其他单核细胞炎症介质的释放[43-44]。肿瘤坏死因子α是炎症反应中出现最早、最重要的炎性递质，能激活中性粒细胞和淋巴细胞，使血管内皮细胞通透性增加，促进其他细胞因子的合成和释放；白细胞介素1β主要由单核细胞和巨噬细胞分泌，参与机体的炎性反应；白细胞介素6能参与机体的免疫应答，是炎性反应的促发剂。槲皮素可以使高迁移率族蛋白1诱导的肿瘤坏死因子α和白细胞介素1β mRNA表达受到抑制，这表明槲皮素通过调节细胞信号传导，进而调节促炎细胞因子的表达。通过腹腔注射槲皮素(30 mg/kg)治疗创伤性脑损伤大鼠，ED-1染色显示使用槲皮素可以增强对损伤组织的保护[45]，减少巨噬细胞和活化的小胶质细胞进入损伤部位，限制他们分泌更多的促炎细胞因子和其他有害因素，导致中枢神经系统中损伤组织的扩散。同时，他们还发现，槲皮素能显着降低创伤性脑损伤后肿瘤坏死因子α、白细胞介素1β和白细胞介素6的表达，并上调抗炎因子白细胞介素10的表达，抑制巨噬细胞在促炎刺激后诱导发生的代谢程序改变[46]。研究表明，有丝分裂原激活蛋白激酶(MAPK)信号通路的激活是高迁移率族蛋白1诱导的基因表达过程中的重要步骤，其导致炎性细胞因子，肿瘤坏死因子α和白细胞介素1β在巨噬细胞、中性粒细胞和内皮细胞中释放。高迁移率族蛋白1或脂多糖在具有诱导巨噬细胞中p38，c-Jun N末端激酶和细胞外信号调节激酶的磷酸化。Degryse等[47]的研究显示槲皮素可以显着抑制由高迁移率族蛋白1或脂多糖诱导的各种激酶的磷酸化而发挥作用。中性粒细胞在机体早期的炎症反应中起重要的作用，处于炎症反应中核心位置。炎症部位老化的中性粒细胞通过自发性凋亡的形式逐渐丧失其生物学功能，最终被周围吞噬细胞吞噬清除掉。槲皮素对中性粒细胞自发性凋亡及其活性无明显的影响。然而预先用槲皮素处理中性粒细胞，则可以阻止脂多糖活化中性粒细胞并引起中性粒细胞自发性凋亡，同时也降低中性粒细胞对炎症因子的敏感性，阻止脂多糖诱导的中性粒细胞产生更多的促炎症细胞因子[48]。李昕等[49]的研究发现槲皮素(40 μmol/L)处理中性粒细胞半小时后，脂多糖虽能诱导其表达白细胞介素6 mRNA，但白细胞介素6的合成和分泌过程会被抑制。蒋飞等[50]的研究发现槲皮素还通过活化核因子κB，在A549细胞中抑制白细胞介素1β诱导表达细胞间黏附分子1。岳扬等[51]通过研究发现槲皮素可以拮抗脂多糖促进中性粒细胞黏附分子，如CD62L、CD11b/CD18的表达,进一步抑制脂多糖诱导的中性粒细胞的活化，从而阻止中性粒细胞与血管内皮细胞的黏附,使炎症细胞向炎症局灶的浸润减少，这可能是槲皮素发挥抗炎作用的一个重要机制。槲皮素还可通过抑制磷酸激酶C(PKC)的活性及转运，拮抗钠离子内流，从而抑制组胺、前列腺素等炎症递质的释放，缓解炎症[52]。 2.1.3 槲皮素在创伤性脑损伤中抑制神经元细胞凋亡和自噬发挥神经保护作用槲皮素已被证明可以减轻脑卒中后的继发性损伤对神经元的损害和在缺血性脑损伤动物模型中具有明显的神经保护作用[53-54]。近年来，研究槲皮素在创伤性脑损伤中神经保护作用成为研究的热点[55]。自噬和凋亡均参与了创伤性脑损伤后神经元细胞的死亡和功能丧失[56-57]。自噬是真核生物对细胞内物质进行周转的重要过程，导致细胞中蛋白质和细胞器的降解。微管相关蛋白1轻链3(LC3)是自噬标志物，当自噬激活时，胞浆型微管相关蛋白1轻链3(LC3-I)会酶解掉一小段多肽，转变为膜型微管相关蛋白1轻链3(LC3-II)。LC3-II含量的增加被认为是自噬发生的生化证据，抑制LC3-I到LC3-II的转化有助于减轻神经损伤和功能缺陷[46，58-59]。创伤性脑损伤后检测到增加的微管相关蛋白1轻链3免疫染色主要见于神经元细胞。Yousuf等[60]提出创伤性脑损伤通过激活自噬造成脑损伤，其可能在6 h或更早的时间内开始，并且在海马中长期保持高水平达48 h。Lipinski等[61]认为自噬的增加和/或恢复可能为治疗创伤性脑损伤提供潜在的治疗靶点。Du等[62]首次报道槲皮素可以降低创伤性脑损伤后大鼠脑海马组织中微管相关蛋白1轻链3的蛋白表达。除自噬外，凋亡是另一种重要的程序性细胞死亡类型[63]，细胞凋亡是多基因严格控制的过程。这些基因在种属之间非常保守，如Bcl-2家族、caspase家族等。Bax与Bcl-2同属于Bcl-2蛋白家族，Bcl-2是促进细胞存活的因子，能够阻止细胞色素C从线粒体释放到细胞质，从而抑制了细胞凋亡。Bax是促进细胞凋亡因子基因，Bax的过度表达可拮抗Bcl-2的保护效应而使细胞趋于死亡。caspase-3在caspase家族中参与执行细胞凋亡，是细胞凋亡过程中最主要的终末剪切酶，导致DNA修复的抑制并启动DNA的降解。Marone等[64]的研究显示抑制caspase或者增加Bcl-2的表达对神经元损伤具有保护作用。创伤性脑损伤后Bax和caspase-3水平增加，而Bcl-2水平降低，使用槲皮素治疗后，观察到TUNEL阳性细胞数量减少，Bax和caspase-3蛋白表达下调，而Bcl-2的蛋白水平上调，水迷宫检测也显示槲皮素可以显著改善创伤性脑损伤诱导的认知功能障碍[65]。关于其具体的分子作用机制，Yao等[66]证明了槲皮素通过激活PI3K/Akt通路减少缺血诱导的神经元细胞凋亡。许多报道也表明PI3K/Akt信号通路具有抑制细胞凋亡的功能[67-68]。Akt，也称为蛋白激酶B，在急性脑损伤中可以促进细胞存活[69-70]。为了确定Akt活化是否也是槲皮素在创伤性脑损伤诱导的神经元自噬中的作用机制，Du等[62]通过Western印迹和免疫荧光研究了创伤性脑损伤后Akt的磷酸化，观察到与假手术组相比，创伤性脑损伤组脑组织中磷酸化Akt(p-Akt)水平在48 h内增加，表明创伤性脑损伤后激活了Akt通路，与创伤性脑损伤组相比，用槲皮素治疗显着增加了p-Akt的蛋白质表达。同时，他们发现p-Akt阳性细胞在创伤性脑损伤后主要与阳性神经元共同定位，而槲皮素显着增强与神经元共同标记的p-Akt阳性细胞。除激活PI3K/Akt通路外，槲皮素还抑制了创伤性脑损伤诱导的细胞外信号调节激酶1/2(ERK1/2)信号通路[71]。ERK1/2是广泛存在于真核细胞内的一类丝氨酸/苏氨酸蛋白激酶, 属丝裂原活化蛋白激酶(MAPK)家族成员。细胞内ERK1/2信号通路激活会引起线粒体外膜通透性增大、诱导死亡信号复合物形成、DNA断裂、细胞色素C(Cytc)释放和caspase3激活[72-73]。1994年, Wieloch首次提出在缺血性脑损伤中有ERK1/2活化[74]。使用控制性皮质冲击伤模型研究ERK1/2在体内和体外被激活后的表达，结果显示创伤性脑损伤后ERK1/2活性显著增高。Du等[62]通过Western印迹分析，观察到创伤性脑损伤后 24 h，与假手术组相比，创伤性脑损伤后p-ERK1/2水平显着增加；而槲皮素治疗组与空白治疗组相比，槲皮素的治疗产生了p-ERK1/2水平的显着减弱。陈万欣等[75]的研究发现大鼠在重度创伤性脑损伤后,大脑皮质损伤中心p-ERK1/2呈明显持续激活状态,高峰表达持续至伤后 24 h，在相邻切片的p-ERK1/2和caspase-3双重标记，发现p-ERK1/2的分布情况与caspase-3标记有很大程度的重叠性，且两者的阳性细胞形态出现极大的相似性, 提示创伤性脑损伤后ERK1/2通路的激活参与了线粒体受损启动的内源性细胞凋亡通路。Nowak[76]的研究发现,使用MEK抑制剂PD98059抑制ERK1/2活性的同时caspase-3的表达也显著下降,说明ERK1/2可直接调控caspase-3的活性。Fredrik等[77]应用激光共聚焦显微镜观测到损伤中心及周边区p-ERK1/2与末端脱氧核苷酸转移酶介导的dUTP 缺口末端标记测定法(TUNEL)阳性细胞共同定位,认为闭合性颅脑损伤后，ERK1/2活化后通过核转录机制,激活立早基因(IEGs)c-fos、c-jun促进了凋亡晚期基因caspase-3的表达，最终导致凋亡。总之，创伤性脑损伤后抑制了PI3K/Akt信号通路，激活了ERK1/2信号通路造成神经元细胞的损伤，使用槲皮素治疗后可以通过激活PI3K/Akt通路并抑制ERK1/2信号通路从而减弱神经元细胞的自噬和凋亡，达到改善神经功能缺损和认知功能的作用。 2.2 槲皮素的安全性槲皮素的安全性目前仍存在争议，但多数观点支持槲皮素属于无毒物质，安全性较高。冯香安等[78]根据急性毒性试验中槲皮素对小鼠的半数致死量在10 g/kg以上，将其判定为无毒物质，同时多个致突变试验结果均为阴性。陈鸿雨等[79]对槲皮素提取液也进行了急性毒性试验，结果显示槲皮素剂量达到6 g/kg的情况下，大鼠无任何异常表现，无一死亡，剖检内脏也无明显变化，同时在亚急性毒性试验中,各组试验大鼠一般状况良好，血常规、生化指标及组织病理学检查也未见明显异常。Utesch等[80]在使用啮齿目动物进行的长期研究显示，槲皮素没有致癌性，在给予大鼠2 000 mg/kg剂量的槲皮素后，并未出现遗传毒性。Harwood等[81]对槲皮素的安全性进行了全面综述，认为人对槲皮素具有良好的耐受性。所以，基于现今的动物安全性评价试验，在合理剂量范围内，可以认为槲皮素的安全性较高。 "

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