药学学报  2021, Vol. 56 Issue (3): 771-777     DOI: 10.16438/j.0513-4870.2020-1270   PDF    
黄芩素通过抑制JAK2/STAT1通路减轻Aβ25-35诱导的PC12细胞损伤
郑文鸽1,2, 周峰1,2, 高丽1, 秦雪梅1     
1. 山西大学中医药现代研究中心, 山西 太原 030006;
2. 山西大学化学化工学院, 山西 太原 030006
摘要: 本研究旨在探讨黄芩素对Aβ25-35诱导PC12细胞损伤的保护作用及机制。采用20 μmol·L-1 Aβ25-35损伤PC12细胞,通过细胞形态观察、Hoechst 33342染色和炎症因子检测考察黄芩素对Aβ25-35损伤PC12细胞的保护作用,采用Western blotting方法检测凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶3(cysteinyl aspartate specific proteinase 3,caspase-3)、活化型半胱氨酸天冬氨酸蛋白酶3(cleaved cysteinyl aspartate specific proteinase-3,cleaved caspase-3)、Janus激酶2/信号转导子和转录激活子1(Janus kinase 2/signal transducer and activator of transcription 1,JAK2/STAT1)通路相关蛋白及其下游一氧化氮合酶(inducible nitric oxide synthase,iNOS)和环氧合酶-2(cyclooxygenase-2,COX-2)炎症蛋白的表达,研究黄芩素的作用机制。结果显示,黄芩素(80 μmol·L-1)能显著抑制Aβ25-35诱导的PC12细胞凋亡和炎症因子IL-8和TNF-α的释放;Western blotting实验结果显示,黄芩素能抑制JAK2和STAT1的磷酸化,并抑制其下游蛋白iNOS和COX-2的表达。以上结果表明,黄芩素能有效抑制JAK2/STAT1信号通路,从而抑制Aβ25-35诱导的PC12细胞损伤。
关键词: 黄芩素    PC12细胞    JAK2/STAT1信号通路    炎症反应    
Baicalein reduces Aβ25-35-induced PC12 cell damage by inhibiting the JAK2/STAT1 pathway
ZHENG Wen-ge1,2, ZHOU Feng1,2, GAO Li1, QIN Xue-mei1     
1. Modern Research Center for Traditional Chinese Medicine, Shanxi University, Taiyuan 030006, China;
2. College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China
Abstract: This study investigated the mechanism by which baicalein protected PC12 cells from Aβ25-35-induced injury. PC12 cells were treated with Aβ25-35 (20 μmol·L-1) and the ability of baicalein to prevent apoptosis was investigated by monitoring changes in cell morphology, Hoechst 33342 staining, and measurement of inflammatory factors. Western blotting was used to detect the expression of the apoptosis-related proteins cysteinyl aspartate specific proteinase-3 (caspase-3), cleaved cysteinyl aspartate specific proteinase-3 (cleaved caspase-3), proteins involved in the Janus kinase 2/signal transducer and activator of transcription 1 (JAK2/STAT1) pathway, and downstream inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The results show that baicalein (80 μmol·L-1) can significantly inhibit apoptosis and the release of inflammatory factor IL-8 and TNF-α in Aβ25-35-treated PC12 cells. Western blotting results showed that baicalein can inhibit the phosphorylation of JAK2 and STAT1 and decrease the expression of downstream iNOS and COX-2, thereby inhibiting the JAK2/STAT1 signaling pathway and preventing Aβ25-35-induced PC12 cell damage.
Key words: baicalein    PC12 cell    JAK2/STAT1 pathway    inflammation    

阿尔茨海默症(Alzheimer's disease, AD) 是常见的中枢神经退行性疾病, 也是造成老年人认知功能障碍的主要原因。β-淀粉样蛋白(amyloid-protein, Aβ) 沉积和Tau蛋白异常磷酸化被认为是AD发生的重要特征。其中, Aβ假说得到了广泛的证据支持, 该假说认为由于Aβ “漏出”神经细胞, 引起周围的神经元和神经胶质细胞的细胞膜和线粒体膜损伤, 诱发炎症反应和氧化应激, 导致神经纤维缠结, 进而引发AD。Aβ由淀粉样前体蛋白(amyloid precursor protein, APP) 分解而来[1], 由无毒的Aβ单体、具有神经毒性的Aβ纤维和Aβ寡聚体聚合组成[2], Aβ的清除是通过非酶促和酶促途径, 如LDL受体相关蛋白1 (low density lipoprotein receptor-related protein 1, LRP1)、P-糖蛋白(permeability glycoprotein, P-gp) 和各种肽酶等。正常情况下, Aβ的产生和清除存在一个动态平衡[3]; 但是在衰老或病理情况下, 大脑中Aβ的产生和清除的稳态被打破[4], 导致Aβ积累和老年斑形成[5], 从而引发AD[6]

Aβ25-35引起PC12细胞损伤主要表现在引起细胞凋亡、氧化应激、炎症和神经毒性等方面。研究发现, Aβ25-35可引起PC12细胞c-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK) 磷酸化, 上调半胱氨酸天冬氨酸蛋白酶3 (cysteinyl aspartate specific proteinase 3, caspase-3) 和活化型半胱氨酸天冬氨酸蛋白酶3 (cleaved cysteinyl aspartate specific proteinase-3, cleaved caspase-3) 凋亡相关蛋白的水平[7], 并通过促进Fas途径来促进细胞凋亡[8]; Aβ25-35通过促进Kelch样环氧氯丙烷相关蛋白-1/核因子E2相关因子2/抗氧化反应元件-1 (kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2/hemo oxygenase-1, Keap1/Nrf2/HO-1) 信号通路引起PC12细胞内氧化应激[9], 并增加活性氧(reactive oxygen species, ROS)、乳酸脱氢酶(lactate dehydrogenase, LDH)、丙二醛(malondialdehyde, MDA) 和线粒体过氧化等氧化损伤[10]; Aβ25-35可激活Janus激酶/信号转导子和转录激活子(Janus kinase 2/signal transducer and activator of transcription 1, JAK2/STAT1)[11]信号通路引起PC12细胞内炎症水平升高, 增加白介素-1β (interleukin-1β, IL-1β)、白介素-8 (interleukin-8, IL-8) 和肿瘤坏死因子α (tumor necrosis factor α, TNF-α) 等促炎性细胞因子的分泌[12]; 同时Aβ25-35也可使PC12细胞内的钙调蛋白(calmodulin, CaM)、钙调蛋白依赖性蛋白激酶激酶(calmodulin dependent protein kinase kinase, CaMKK)、钙调蛋白依赖性蛋白激酶Ⅳ (calmodulin-dependent protein kinase Ⅳ, CaMKIV)、Tau蛋白表达升高, 引起神经毒性[13]。此外, Aβ25-35引起的PC12细胞损伤可通过其他途径消除。例如磷酸肌醇3-激酶/蛋白激酶B (phosphoinositide 3-kinase/protein kinase B, PI3K/Akt)[14]通路的激活可消除Aβ25-35引起的神经细胞凋亡; 酪氨酸转移RNA合成酶-多腺苷酸聚合酶的自动聚ADP核糖基化1-沉默信息调节因子1 [tyrosyl transfer-RNA synthetase/poly (adenosine diphosphate-ribose) polymerase 1/sirtuin 1, TyrRS-PARP1-SIRT1][15]信号通路的激活可诱导PC12细胞自噬, 减轻神经毒性。

基于天然产物发现改善学习记忆障碍的药物是当前研究的热点。传统中药黄芩最早收录于《神农本草经》, 药用价值高, 历史悠久[16]。黄芩素(baicalein) 是从中药黄芩中提取出来的黄酮类化合物, 具有抗氧化、抗炎、抗菌、抗病毒和清除自由基等作用[17]。研究发现, 在Aβ1-40[18, 19]和Aβ25-35[20]诱导的AD大鼠模型中, 黄芩素均能显著改善AD大鼠的遗忘和记忆障碍; 在Aβ25-35损伤的PC12细胞中, 黄芩素能够抑制氧化应激从而降低Aβ毒性反应[21], 表明黄芩素具有改善认知功能和学习记忆能力[22]

课题组前期发现200 mg·kg-1黄芩素能够改善D-半乳糖致衰老模型大鼠[23]和快速老化小鼠SAMP8的学习记忆障碍[24]。通过对SAMP8小鼠的大脑皮层进行转录组学研究, 结果表明黄芩素可能通过抑制JAK2/STAT1信号通路发挥神经保护作用[25]。JAK/STATs信号通路[26]是众多细胞因子信号转导的共同途径, 广泛参与细胞增殖、分化、凋亡以及炎症等过程, 研究表明[27]JAK/STATs信号通路激活会导致多种炎症反应。

PC12细胞为大鼠肾上腺嗜铬细胞瘤细胞系, 在结构、功能上与神经元有很多相似之处, 与神经元相比又相对容易培养。因此在本研究中, 以PC12细胞为研究对象, 以Aβ25-35损伤PC12细胞构建AD体外模型, 验证黄芩素的保护作用及对JAK2/STAT1信号通路的调节作用。

材料

细胞株 大鼠肾上腺嗜铬细胞瘤细胞(PC12细胞) 购自中国科学院细胞研究所(上海)。

药物与试剂 黄芩素(批号JZ20150711, 质量分数 > 98%) 购自南京景竹生物科技有限公司; Aβ25-35购自北京Solarbio公司; 高糖DMEM培养基、胰酶、胎牛血清、IL-8检测试剂盒、TNF-α检测试剂盒、细胞裂解液、BCA蛋白浓度检测试剂盒、ECL显影液、HRP羊抗兔二抗(货号: D110058) 购自上海生工生物工程有限公司; 二甲基亚砜(DMSO)、四甲基偶氮唑蓝(MTT) 购自Sigma公司; 抗体β-actin (货号: 20536-1-AP)、一氧化氮合酶(inducible nitric oxide synthase, iNOS) (货号: 18985-1-AP)、环氧合酶-2 (cyclooxygenase-2, COX-2) (货号: 12375-1-AP)、JAK2 (货号: 17670-1-AP)、STAT1 (货号: 10144-2-AP) 购自Proteintech公司; 抗体p-JAK2 (货号: 3776)、p-STAT1 (货号: 7649)、caspase-3 (货号: 9665)、cleaved caspase-3 (货号: 9664) 购自Cell Signaling Technology公司。

仪器 HF safe1800生物安全柜、HF 90 CO2培养箱、Neofuge 1600R台式高速离心机(Heal Force公司, 中国); Nikon Eclipse Ti-S荧光倒置显微镜(Nikon公司, 日本); M200多功能酶标仪(Tecan公司, 瑞士); 高速冷冻离心机(力新仪器上海有限公司, 中国); Mini-PROTEAN Tetra电泳仪、Mini-Trans-Bolt Cell半干转印槽、光成像系统ChemiDocTM XRS+ (Bio-Rad公司)。

PC12细胞的培养 将PC12细胞接种于25 cm2培养瓶, 置于37 ℃、5% CO2的培养箱中培养, 培养基为含10%胎牛血清的DMEM。每2~3天传代1次, 待细胞生长至对数生长期时进行后续实验。

分组与给药 将实验分为空白组、Aβ25-35组(20 μmol·L-1) 和黄芩素(40、60、80 μmol·L-1) 组, 部分实验选取黄芩素最佳剂量80 μmol·L-1展开。正常组更换DMEM无血清培养液, Aβ25-35组中加入含20 μmol·L-1 Aβ25-35的DMEM无血清培养液, 造模24 h; 黄芩素给药组先用不同浓度黄芩素预处理2 h, 再加入终浓度为20 μmol·L-1 Aβ25-35培养24 h, 每组设置3个复孔。

细胞形态学观察 以每毫升1×105个的浓度将细胞接种于6孔板中, 待细胞生长至对数生长期时, 按“分组与给药”的步骤处理细胞后, 在倒置显微镜下观察细胞形态并进行拍照。

细胞凋亡检测 将PC12细胞接种于6孔板中, 于37 ℃、5% CO2培养箱中培养24 h, 按“分组与给药”的步骤分组给药, 继续培养24 h。按照Hoechst染色试剂盒说明书进行染色, 每孔加入Hoechst 33342染色液500 μL, 室温避光染色5 min, PBS洗2次, 于倒置荧光显微镜下观察细胞核, 亮蓝色荧光即为凋亡细胞。

炎症因子检测 采用双抗体夹心酶联免疫吸附法测定黄芩素对IL-8含量的影响。收集培养上清液, 按试剂盒说明书操作, 用酶标仪在450 nm处测定各孔吸光值, 根据标准曲线得出相应IL-8含量, 每组设置3个复孔, 计算平均值。

Western blotting实验 按照上述分组分别作用PC12细胞24 h, 收集细胞, 并用PBS溶液清洗2~3次, 加入裂解液(含有1% PMSF和10% 10×磷酸酶抑制剂复合物), 冰上裂解30 min后, 14 000 r·min-1、4 ℃离心15 min, 上清液即为蛋白质提取物。BCA法测定蛋白含量, 100 ℃蛋白变性10 min。蛋白上样量为50 μg, 采用8% SDS-PAGE凝胶电泳分离蛋白, 转膜, 5%脱脂奶粉/5% BSA室温摇床封闭2 h, 加入一抗β-actin (1∶1 000)、caspase-3 (1∶800)、JAK2 (1∶600)、STAT1 (1∶800)、p-STAT1 (1∶1 000)、p-JAK2 (1∶1 000)、iNOS (1∶500)、COX-2 (1∶1 000), 4 ℃摇床孵育过夜, TBST洗膜3次, 每次10 min, 室温摇床孵育辣根过氧化物酶标记的二抗(1∶10 000), TBST洗膜3次, 每次10 min, 漂洗并滴加显影液后, 再使用ChemiDocTM XRS+凝胶成像仪对目的条带逐一进行扫描, 使用Quantity One软件对结果进行统计分析。

统计学分析 实验数据以x ± s表示, 采用GraphPad Prism 6.0对实验数据进行统计学分析, 组间比较采用单因素方差分析结合Dunnett's检验, P < 0.05认为有统计学意义。

结果 1 黄芩素对Aβ25-35诱导PC12细胞存活的影响

课题组前期研究[28]结果表明, Aβ25-35能显著性降低PC12细胞存活, 其中20 μmol·L-1 Aβ25-35与PC12细胞孵育24 h后, 细胞存活率显著下降至空白组的57.57%; 用不同浓度黄芩素预处理后均能提高细胞存活, 其中80 μmol·L-1黄芩素使PC12细胞存活显著提高了20.75%。在此基础上, 观察黄芩素对Aβ25-35损伤PC12细胞形态的影响。结果表明, 与空白组相比, Aβ25-35组(20 μmol·L-1) 细胞数量减少, 且细胞皱缩、变圆, 部分细胞悬浮于培养液中; 而80 μmol·L-1黄芩素作用24 h后, 能明显增加细胞数量并改善细胞形态, 使其接近于空白组细胞形态(图 1)。

Figure 1 Effects of baicalein on the morphology of PC12 cells injured by Aβ25-35 (200×)
2 黄芩素抑制Aβ25-35诱导PC12细胞凋亡

Hoechst 33342染色结果显示, 空白组细胞核呈现弥散均匀微弱的蓝色荧光, 表明其凋亡细胞少。Aβ25-35组较多细胞呈现出较强的蓝白色荧光, 细胞核变小, 呈现不规则形状, 有些细胞内可见较强的颗粒状荧光, 表明细胞凋亡数量明显增多, 而给予黄芩素(80 μmol·L-1) 后, 可见荧光减弱, 表明黄芩素能够抑制Aβ25-35诱导的细胞凋亡(图 2A)。与空白组比较, Aβ25-35使caspase-3和cleaved caspase-3水平显著增加; 而给予黄芩素能显著降低caspase-3和cleaved caspase-3水平, 说明黄芩素能够抑制caspase-3介导的PC12细胞凋亡(图 2BC)。

Figure 2 Effects of baicalein on apoptosis of PC12 cells injured by Aβ25-35. A: Hoechst 33342 staining (×200); B: Cysteinyl aspartate specific proteinase-3 (caspase-3) expression; C: Cleaved cysteinyl aspartate specific proteinase-3 (cleaved caspase-3) expression. n = 3, x ± s. **P < 0.01 vs control group; ##P < 0.01 vs Aβ25-35 group
3 黄芩素抑制Aβ25-35诱导PC12细胞炎症因子IL-8和TNF-α的释放

为了考察黄芩素对Aβ25-35诱导的PC12细胞炎症因子的抑制作用, 选取促炎因子IL-8和TNF-α作为检测指标。结果显示, 20 μmol·L-1 Aβ25-35可显著增加IL-8和TNF-α的释放, 而黄芩素能显著抑制Aβ25-35诱导的IL-8和TNF-α的释放(图 3)。

Figure 3 The effects of baicalein on inflammatory factors induced by Aβ25-35 in PC12 cells. A: Interleukin-8 (IL-8) level; B: Tumor necrosis factor-α (TNF-α) level. n = 3, x ± s. **P < 0.01 vs control group; ##P < 0.01 vs Aβ25-35 group
4 黄芩素阻断Aβ25-35诱导PC12细胞JAK2/STAT1的活化

为了考察黄芩素对Aβ25-35诱导PC12细胞JAK2/STAT1通路的作用, 进行了Western blotting检测。结果显示, Aβ25-35刺激PC12细胞24 h后, JAK2、STAT1的磷酸化显著增加, 而黄芩素(80 μmol·L-1) 能显著抑制JAK2和STAT1的磷酸化(图 4), 表明黄芩素可以显著抑制JAK2/STAT1信号通路的活化。

Figure 4 Effects of baicalein on the phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 1 (STAT1) in PC12 cells induced by Aβ25-35. n = 3, x ± s. *P < 0.05, **P < 0.01 vs control group; #P < 0.05 vs Aβ25-35 group
5 黄芩素抑制Aβ25-35诱导PC12细胞炎症相关蛋白iNOS和COX-2的表达

为了验证黄芩素是否可以抑制JAK/STAT通路相关炎症蛋白的表达, 采用Western blotting检测黄芩素对iNOS (图 5A) 和COX-2蛋白表达的影响(图 5B)。结果显示, Aβ25-35能显著增加iNOS和COX-2蛋白表达, 而黄芩素(80 μmol·L-1) 可显著抑制iNOS和COX-2蛋白表达。

Figure 5 The effects of baicalein on the expression levels of inducible nitric oxide synthase (iNOS), cyclo-oxygenase-2 (COX-2) in the PC12 cells induced by Aβ25-35. n = 3, x ± s. *P < 0.05, **P < 0.01 vs control group; #P < 0.05, ##P < 0.01 vs Aβ25-35 group
讨论

细胞凋亡[29]是由基因控制的程序性细胞死亡, 受Bcl-2家族、caspase家族、癌基因和抑癌基因等基因调控。其中, caspases介导的细胞凋亡是主要的凋亡途径[30]。研究表明[31], AD与神经元细胞凋亡密切相关, 而阻断细胞凋亡的药物可在早期预防神经元细胞死亡[32]。本研究发现, Aβ25-35处理组caspase-3和cleaved caspase-3的水平升高, 而给予黄芩素(80 μmol·L-1) 后可抑制caspase-3和cleaved caspase-3的水平, 结合Hoechst染色结果, 表明黄芩素可抑制caspase-3介导的细胞凋亡。

AD与炎症反应密切相关[33]。Aβ沉积可诱发炎症反应; 而非甾体类抗炎药可以降低AD风险[34-36]。通过对AD患者尸检的研究结果发现, 炎症与AD病理相关[37, 38], 而且在AD小鼠大脑皮层中发现处于活化状态的炎症因子[39]。促炎细胞因子[40] (如IL-8) 在神经系统的生长发育、分化调节中具有重要作用, 参与一系列生理功能的调节。因此, 抑制炎症可能是治疗许多慢性神经退行性疾病的关键[41]。本研究发现, 黄芩素给药后可显著降低炎症因子IL-8和TNF-α释放以及炎症蛋白iNOS和COX-2的表达, 表明黄芩素抑制Aβ25-35诱导的PC12细胞炎症。

JAK/STATs信号通路由酪氨酸激酶JAK和转录因子STAT级联组成, 该通路不仅参与炎症反应, 同时也与细胞损伤、凋亡和氧化应激有关[42, 43]。JAK/STATs信号转导失调是各种实体癌和造血系统恶性肿瘤发病的驱动力[44], 也与自身免疫性疾病(类风湿性关节炎、牛皮癣和炎症性肠病)[45]和过敏性疾病(哮喘和过敏性鼻炎)[46]的发生有关。Nevado-Holgado等[47]使用啮齿动物模型和体外模型证明了JAK/STATs信号通路异常是AD的病因之一。研究表明[48], JAK/STATs信号通路的激活会引起IL-1β、TNF-α和IL-6等炎症因子释放, 并激活下游靶蛋白iNOS和COX-2, 使促炎因子释放增多, 导致炎症反应发生[49]。本研究发现, 黄芩素给药后可显著抑制JAK2和STAT1的磷酸化, 表明黄芩素可通过JAK2/STAT1通路抑制Aβ25-35诱导的PC12细胞炎症。

综上所述, 本研究结果表明, 黄芩素可以有效地抑制Aβ25-35诱导PC12细胞中JAK2/STAT1信号活化, 从而抑制炎症因子IL-8释放和炎性靶蛋白iNOS和COX-2的表达, 最终阻断炎症的级联反应。

作者贡献: 郑文鸽和周峰负责进行实验和数据分析, 完成文章撰写; 高丽和秦雪梅负责指导实验思路; 所有作者均对本文有所贡献。

利益冲突: 无利益冲突。

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