1 材料与方法
1.1 材料
DMEM培养基为Gibco产品;胎牛血清购自四季青公司;RIPA裂解液、蛋白酶抑制剂和BCA蛋白质定量试剂盒为康为世纪产品;化学发光辣根过氧化物酶底物和PVDF膜为Millipore产品;LC3、p62、Wnt5a 抗体均购自Abcam公司;兔抗PKCδ抗体、鼠抗STAT3抗体、兔抗mTOR抗体均购自Cell Signaling Technology;山羊抗兔IgG为Proteintech产品;磷酸酶抑制剂购自Thermo Fisher Scientific.饱和油红O染液购自北京索莱宝科技有限公司;总胆固醇酶法测定试剂盒购自北京普利莱基因技术有限公司.
1.2 细胞培养与处理
鼠源RAW264.7巨噬细胞用含10%胎牛血清的DMEM培养基在37℃、5% C
O2 的培养箱中培养. 待培养瓶中细胞覆盖瓶底约80%时,用0.25%胰酶将细胞消化,接种于6孔板中,细胞每24 h换液,以6孔板每孔2 ml培养基计. 细胞处理前需换无血清DMEM培养基同步化12 h.1.3 Ox-LDL的制备
采用梯度超速离心法分离血清中低密度脂蛋白(low density lipoprotein,LDL),PBS透析48 h. 随后加入50 μl浓度为10 μmol/L的CuS
O4 溶液氧化LDL,37℃温育24 h. 在含200 μmol/L EDTA的PBS中透析24 h以去除Cu2+ ,用PBS透析24 h除去EDTA,0.22 μm微孔滤膜过滤除菌. 最后用BCA法测定LDL蛋白浓度,调整蛋白质终浓度至1 g/L,4℃储存.1.4 细胞转染
从吉玛基因公司购得Wnt5a干扰链和对照干扰链(表1).
Table 1 Interference chain sequences
Gene name Forward primer (5'-3') Reverse primer (5'-3') Wnt5a-mus-748 GGUUGUUAUAGAAGCUAAUTT AUUAGCUUCUAUAACAACCTT Wnt5a-mus-1227 GGACGCUAGAGAAAGGGAATT UUCCCUUUCUCUAGCGUCCTT Wnt5a-mus-1659 GCGUGGCUAUGACCAGUUUTT AAACUGGUCAUAGCCACGCTT 将细胞种到6孔板上,待细胞融合至40%时,各孔加入2 ml培养基. 首先在50 μl培养基中加入 5 μl Wnt5a干扰试剂,孵育5 min. 然后加入5 μl转染试剂Lipofectamine 2000,孵育15 min. 将上述液体混匀后加入至培养板各孔. 转染效果采用蛋白质印迹(Western blot)鉴定.
1.5 蛋白质印迹
蛋白质印迹(Western blot)检测细胞中 LC3Ⅰ、LC3Ⅱ、Wnt5a、PKCδ、mTOR、STAT3蛋白的表达. 细胞进行相应处理后,收集细胞,PBS洗涤3次,使用RIPA蛋白裂解液抽提总蛋白质,采用BCA蛋白质定量法测定蛋白质浓度. 进行6%~15% SDS聚丙烯酰胺凝胶电泳,蛋白质分离后恒定电流300 mA转膜至PVDF膜上;室温下5%脱脂奶粉封闭膜2 h,用相应的一抗4℃孵育膜过夜;洗涤后换二抗室温孵育2 h. 洗涤后采用化学发光辣根过氧化物酶底物显影;使用Image-Pro Plus软件分析蛋白质条带积分吸光度(integrated absorbance,IA)值,以靶蛋白IA值与β-actin IA值的比值反映靶蛋白相对表达水平.
1.6 油红O染色
将细胞培养于放有无菌盖玻片的6孔培养板内,细胞经处理后,PBS润洗,4%多聚甲醛溶液固定,油红O染色液染色20 min,苏木素复染. Olympus显微镜观察,细胞内脂质呈红色,细胞核呈蓝色. 每张爬片随机选取5个区域,采用Image-Pro Plus 6.0(Media Cybernetics)图像分析软件分析实验结果.
1.7 胆固醇酶法
细胞进行相应处理后,收集细胞,PBS洗涤3次,裂解后离心,进行胆固醇含量测定. 根据标准曲线(采用已知浓度的胆固醇)计算得出巨噬细胞内总胆固醇(total cholesterol,TC)的含量. 同时检测细胞蛋白质含量(BCA法),用以标准化各样本的总胆固醇值.
1.8 统计学处理
数据用均数±标准差(mean±SD)表示,应用SPSS 13.0软件处理,组间比较用t检验,P<0.05为差异有统计学意义,P<0.01时为组间具有非常显著差异,结果具有统计学意义.
2 结果
2.1 Ox-LDL诱导Raw264.7细胞自噬的量效与时效关系
以浓度为0、30、60、90 mg/L的ox-LDL处理Raw264.7细胞6 h. 随着ox-LDL浓度增大, LC3Ⅱ/Ⅰ表达显著增多,而当浓度达到90 mg/L时LC3Ⅱ/Ⅰ表达降低(图1a). 因此选择60 mg/L ox-LDL作为后续处理浓度. 以60 mg/L ox-LDL处理细胞0、3、6、12、24 h. 随着ox-LDL处理时间增长,LC3Ⅱ/Ⅰ增加,p62表达减少(图1b),表明ox-LDL可诱导细胞发生自噬反应.
2.2 Ox-LDL诱导巨噬细胞中Wnt5a、PKCδ、STAT3蛋白质表达
分别采用30、60、90 mg/L ox-LDL处理Raw264.7细胞6 h后进行Western blot检测. 结果显示,与对照组比较,60 mg/L ox-LDL诱导的Raw264.7细胞中Wnt5a、PKCδ和STAT3的表达显著上调(P<0.05)(图2). 提示60 mg/L ox-LDL刺激Raw264.7细胞发生的自噬可能依赖Wnt5a、PKCδ和STAT3.
2.3 敲低Wnt5a抑制ox-LDL诱导的Raw264.7细胞自噬
为了验证Wnt5a在ox-LDL诱导的Raw264.7细胞自噬中的作用,我们首先筛选了有效的Wnt5a干扰链. 结果显示,Wnt5a-mus-748干扰效果最佳 (P<0.05)(图3a),因此选用此干扰链进行后续实验. Wnt5a敲低后,LC3Ⅱ/Ⅰ、PKCδ和STAT3蛋白表达均显著降低(P<0.05),p62表达显著增加(P<0.01)(图3b). 油红O染色检测结果显示, ox-LDL处理后,Raw264.7细胞中脂滴明显增多(图3c). 随后,在敲低Wnt5a的细胞中观察荷脂巨噬细胞的脂质蓄积情况. 结果显示,与si-NC细胞相比,敲低Wnt5a细胞内脂滴显著减少(图3c),细胞内总胆固醇(total cholesterol,TC)水平明显下降(图3d). 因此,敲低Wnt5a抑制自噬发生可能与PKCδ、STAT3相关.
Fig. 3 Knockdown of Wnt5a suppresses autophagy induced by ox-LDL in Raw264.7 cells
NOTE: (a)The protein expression of Wnt5a was measured by Western blot assay in knock-down of Wnt5a Raw264.7 cells
;* P<0.05 compared to negative group (NEG).(b)The protein expressions of LC3Ⅱ/Ⅰ,p62,PKCδ and STAT3 were detected by Western blot assay.(c)The intracellular lipid droplet was detected by Oil red O staining (×40).(d)TC content in macrophages was detected by cholesterol enzyme link assay. Mean±SD,n=3,# P<0.05,## P<0.01,### P<0.01 compared to control group,* P<0.05 compared to si-NC group.2.4 PKCδ介导ox-LDL诱导的Raw264.7细胞自噬
为进一步证实PKCδ是否参与ox-LDL诱导Raw264.7细胞自噬,采用其抑制剂Rottlerin阻断PKCδ磷酸化. 结果显示,与加入ox-LDL与DMSO组相比,Rottlerin抑制PKCδ后,LC3Ⅱ/Ⅰ和STAT3的表达均显著降低,p62的表达显著增加(图4a). 进一步采用油红O染色和胆固醇酶法检测脂质蓄积情况. 结果显示,与ox-LDL+DMSO组相比,Rottlerin处理后细胞内脂质蓄积显著降低(图4b),且细胞内TC的含量显著减少(图4c). 提示PKCδ可介导ox-LDL诱导的Raw264.7细胞自噬,促进脂质降解.
Fig. 4 Ox-LDL-induced autophagy is suppressed by PKCδ inhibitor (Rottlerin) in Raw264.7 cells
NOTE: (a)The levels of LC3Ⅱ/Ⅰ and STAT3 in Raw264.7 cells were examined by Western blot assay.(b)The intracellular lipid droplet was measured by Oil red O staining(×40).(c)Cellular TC content was detected by cholesterol enzyme link assay.
* P<0.05,** P<0.01,*** P<0.001 compared to control group,### P<0.001 compared to ox-LDL+DMSO group.3 讨论
晚期动脉粥样硬化病变的特点之一表现为纤维帽薄、脂质核大、巨噬细胞积聚丰富,因此被称为“易损斑块
”[11] . 当巨噬细胞荷脂超过其代谢能力时,细胞内聚集大量脂质,促进血管内膜生长与坏死核的形成,增加斑块破裂的风险. 巨噬细胞凋亡及其缺陷功能促进斑块坏死,导致斑块不稳定和血栓形成,并最终引发心肌梗死和脑梗塞[12] . 以往有研究表明巨噬细胞自噬通过促进胆固醇流出发挥斑块稳定性作用[13] . 巨噬细胞自噬是由溶酶体系统降解胞内受损、变性及衰老的蛋白质与细胞器的过程,基础水平的自噬可保护细胞免受环境刺激影响. 自噬在巨噬细胞胆固醇流出所发挥的积极作用,这种特殊的选择性自噬称为脂噬 (lipophagy)[14] . 此外,脂质负荷巨噬细胞中,自噬标记物明显增加,反之过度自噬则导致细胞死亡、斑块失稳[15] . 因此巨噬细胞自噬作为一种代偿机制,对于控制动脉粥样硬化进程具有重要意义.LC3前体合成以后,立即被Atg4水解为活化的胞质可溶性的LC3Ⅰ,随后Atg7催化E2泛素结合酶Atg3,促进LC3Ⅰ与磷脂酰乙醇胺结合形成 LC3Ⅱ. 细胞内LC3的含量及LC3Ⅰ向LC3Ⅱ转化量被认为是自噬发生的指
标[16] . 低浓度ox-LDL诱导总LC3B增加而LC3Ⅱ/Ⅰ几乎不变. 随着ox-LDL浓度增加,LC3Ⅰ的生成增多,同时LC3Ⅱ含量也增多. 由于此时溶酶体较多,所以LC3Ⅰ向LC3Ⅱ的转变成为限速步骤,LC3Ⅱ/Ⅰ也保持恒定. 而当溶酶体缺乏时,LC3Ⅱ开始累积,此时LC3Ⅱ/Ⅰ与总LC3B的含量都增加. 然而当ox-LDL浓度达到90 mg/L时则抑制自噬. 在自噬流出现抑制时,总LC3B蛋白水平改变较为迅速,而作为自噬底物,p62的变化具有一定的滞后性,积累所需时间则较长.研究显示,AS斑块处炎症反应与自噬成负相关,自噬的发生有利于减轻炎症,延缓AS,另一方面,也有研究认为抑制自噬可以避免凋亡,减轻炎
症[17] . 同时自噬本身对AS的效应也是双面的,病变早期,自噬作为潜在的补偿机制,维持体内平衡,病变后期,自噬作为细胞持续应激或无法防御的致病过程的适应性反应,失调或过度激活直接影响细胞的存活[18] . 在自噬活动可以有效清除脂质时,自噬对AS进程有益;在自噬失调后,无法形成有效的自噬流,凋亡途径被激发,进而导致细胞死亡. 自噬可降低巨噬细胞内胆固醇和胆固醇酯水平,敲除自噬基因后胆固醇酯明显增多,同时泡沫细胞形成增加. 在AS斑块处,炎性因子表达较高,其中Wnt5a是一种与炎症因子和炎症信号通路密切相关的蛋白质,且在斑块处表达较高. Wnt5a可专一性激活PKCδ,PKCδ则是一种可以直接激活STAT3从而启动自噬的关键分子[10,19] . STAT3参与自噬过程的多个方面,其不同亚细胞定位模式激活自噬途径不同[20] . STAT3直接与beclin-1启动子区域结合,最终抑制其基因及蛋白质表达[21] . STAT3可上调缺氧环境下缺氧诱导因子1α(hypoxia inducible factor-1α,HIF-1α)表达,激活beclin-1,从而诱导自噬[22] . 我们的结果表明, 60 mg/L ox-LDL诱导的Raw264.7细胞中Wnt5a、PKCδ和STAT3表达增加,敲低Wnt5a则使ox-LDL诱导的Raw264.7细胞的自噬减少,也降低了PKCδ表达,而PKCδ可能激活自噬. 进一步使用Rottlerin专一性抑制PKCδ后,LC3Ⅱ/Ⅰ显著降低,STAT3的表达也降低,表明抑制了Raw264.7细胞自噬.综上,ox-LDL可通过诱导Wnt5a/PKCδ信号通路激活自噬. 随着ox-LDL的浓度逐渐升高,自噬被过度激活,导致斑块稳定性降低. 因此,深入研究Wnt5a/PKCδ信号通路在巨噬细胞自噬及AS发生发展中的作用机制,可为自噬机制研究提供新的着力点,并为药物干预提供新的靶点.
Tel: 86-731-88458238, E-mail: Lqin@hnucm.edu.cn
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摘要
研究发现动脉粥样硬化(atherosclerosis,AS)斑块中巨噬细胞摄取氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)和巨噬细胞极化等关键变化与失调性自噬关系密切. Wnt5a(wingless-type MMTV integration site family member 5a)在AS病变的富含巨噬细胞区域中高表达,然而Wnt5a是否参与巨噬细胞自噬尚未明确. 本研究发现, 60 mg/L ox-LDL处理Raw264.7细胞6 h时,自噬标志物LC3Ⅱ/Ⅰ显著增加,p62显著减少,且Wnt5a、PKCδ及STAT3的表达均增加. 小分子干扰RNA(small interference RNA,siRNA)敲低Wnt5a后,逆转ox-LDL诱导的LC3Ⅱ/Ⅰ和PKCδ表达,上调p62表达,减少细胞内脂质蓄积. PKCδ抑制剂Rottlerin干预后,LC3Ⅱ/Ⅰ和STAT3减少,p62增加,降低细胞内脂质含量. 综上,ox-LDL可能通过Wnt5a/PKCδ信号通路诱导巨噬细胞自噬. 因此,深入研究Wnt5a/PKCδ通路在巨噬细胞及AS发生发展中的作用,是研究自噬机制新的着力点,并为药物干预提供新的靶点.
Abstract
Evidence indicated that key changes in macrophage uptake of oxidized low density lipoprotein (ox-LDL) and macrophage polarization in atherosclerotic plaques are closely related to dysfunctional autophagy. Wnt5a (wingless-type MMTV integration site family member 5a) is highly expressed in the macrophage-rich region of atherosclerosis (AS) lesions. However, whether Wnt5a is involved in macrophages autophagy is not clear. In this study, we established macrophages-derived foam cell induced by ox-LDL to explore the effects of Wnt5a/PKCδ pathway on autophagy. RAW 264.7 macrophages were incubated with 60 mg/L ox-LDL for 6h. The expression of autophagy marker, LC3Ⅱ/Ⅰ was significantly increased and p62 was decreased obviously. Moreover, the expressions of Wnt5a, PCKδ and STAT3 were also elevated. Knockdown of Wnt5a reduced the expressions of LC3Ⅱ/Ⅰ and PKCδ, induced the expression of p62, inhibited cellular lipid accumulation. Furthermore, PKCδ inhibitor (Rottlerin) was downregulated the levels of LC3Ⅱ/Ⅰ and STAT3, upregulated p62 level, inhibited cellular lipid accumulation. Therefore, ox-LDL induces autophagy in macrophages may be associated with Wnt5a/PKCδ signaling pathway. The present study indicates that Wnt5a/PKCδ signaling pathway may be underlying target for autophagy and drug intervention.
动脉粥样硬化(atherosclerosis,AS)是一种以动脉血管中过量脂质蓄积为主要特征的疾病,已成为全球首位死亡原因之
自噬是真核生物细胞普遍存在的一种保守的自我消化过程,是损坏和衰老的细胞器、蛋白质等降解的途径. 当细胞受到刺激之后,在胞质内形成半月形双层膜结构自噬体,自噬体逐渐延伸包裹住自噬刺激物与细胞器,并转运至溶酶体降解,实现物质代谢和细胞器更新,有利于维持细胞内环境稳
Wnt5a (wingless-type MMTV integration site family member 5a)是一种与炎症密切相关的蛋白质,在炎性组织中广泛高表