p53依赖性和非依赖性信号通路在邻苯二甲酸单（2-乙基己基）酯致肝细胞凋亡中的调控机制
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摘要
邻苯二甲酸二(2—乙基)己酯(Di-(2-ethylhexyl) phthalate, DEHP)是塑料工业中使用最广泛的一类增塑剂,它主要用来增加塑料的弹性和柔韧性。由于DEHP在塑料中以非共价键形式存在,并可随着使用时间的推移而不断释放至大气、土壤和水体中,人体可通过呼吸道、消化道和皮肤多途径持续地暴露于环境中的DEHP。为此,DEHP的潜在健康危害已倍受国内外学者的关注。
研究表明,进入机体的DEHP很快被代谢为活性中间产物—邻苯二甲酸单(2-乙基己基)酯mono-(2-ethylhexyl) phthalate, MEHP),产生肝脏毒性、肾脏毒性和生殖发育毒性等。体外研究显示,MEHP可致多种细胞的凋亡,但尚不了解其分子调控机制。
MEHP是过氧化物酶体增殖物(peroxisome proliferators, PP),它可经氧化物酶体增殖物激活受体(peroxisome proliferators activated receptors, PPAR)介导肝毒性。但是,MEHP致肝毒性的调控机制并非完全依赖PPAR途径,可能与孕烷X受体(pregnane X receptor, PXR)有一定的关联。
细胞凋亡是外环境刺激或死亡信号所触发的细胞主动死亡过程,该过程受多个基因的调控,其中抑癌基因p53发挥主要调控作用。活化p53通过转录调控下游凋亡基因,如bax-, bcl-2和puma等激活线粒体凋亡通路引起细胞凋亡。研究发现,在MEHP引起的生殖细胞凋亡过程中有p53蛋白的高表达。但是,细胞凋亡并非仅受p53介导的细胞信号通路的调控,p53非依赖信号通路,如c-Jun氨基末端激酶-促分裂素原活化蛋白激酶(c-Jun N-terminal kinases-mitogen activated protein kinases, JNK-MAPK)途径和Fas/FasL途径等也可参与其调控。不过迄今仍不清楚在MEHP致肝细胞凋亡中p53依赖性和非依赖性信号通路是否参与了调控。
基于此,本研究拟用MEHP处理人胚肝细胞L02和人肝癌细胞HepG2来研究MEHP致肝细胞毒性和氧化损伤变化,以及p53介导的线粒体通路在MEHP致肝细胞凋亡中的调控机制,并探索性研究Fas/FasL在p53非依赖性信号通路调控MEHP致肝细胞凋亡中的作用,旨在为后续深入研究MEHP致肝细胞毒性的分子机制提供科学依据。本研究包括以下三个部分：
第一部分MEHP致人肝细胞氧化性DNA损伤和细胞凋亡的研究
目的：建立MEHP染毒L02细胞和HepG2细胞模型。方法：MEHP设定浓度为6.25μM、12.50μM、25.00μM、50.00μM和100.00μM,二甲基亚砜(DMSO)为溶剂对照(终浓度≤0.1%),检测内容包括：①MTT检测细胞活力；②试剂盒检测细胞丙二醛(maleic dialdehyde, MDA)水平、超氧化物酶(superoxide dimutese, SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)的活性；③高效液相色谱仪(HPLC)检测细胞8-羟基-2’-脱氧鸟苷(8-hydroxy-2'-deoxyguanosine,8-OHdG)水平；④流式细胞仪和TUNEL检测细胞凋亡变化。研究结果如下：
(一)细胞活力
①用MEHP处理L02细胞12h,仅在50.00μM处理组检出细胞活力上升(p<0.05)；在24h时点,50.00μM和100.00μM处理组的细胞活力均显著性降低p<0.05或p<0.01)；在36h时点,较高浓度MEHP处理组(≥25.00μM)细胞的活力均显著性降低(p<0.05或p<0.01)。
②用MEHP处理HepG2细胞12h,在50.00μM和100.00μM处理组的细胞活力显著性下降(p<0.01)。在24h和36h时点,细胞活力随MEHP浓度的升高而逐渐降低,在较高浓度的MEHP处理组(≥25.00μM),细胞活力均显著性降低p<0.01)。
(二)细胞的氧化损伤
①用MEHP处理L02细胞和HepG2细胞12h,在所有处理组均未观察到MDA水平的变化p>0.05)。在24h和36h时点,较高浓度的MEHP处理L02细胞(≥25.00μM)的MDA水平均显著性升高p<0.05或p<0.01)。用MEHP处理HepG2细胞24h,所有处理组的MDA水平均显著性升高(p<0.01)；在36h时点,除6.25μM处理组外,所有MEHP处理组的MDA水平均显著性升高(p<0.01)。
②用MEHP处理L02细胞12h,所有处理组细胞的SOD活性均无显著性变化(p>0.05)；在24h时点,所有处理组细胞的SOD活性均显著性降低(p<0.05或p<0.01)；在36h时点,较高浓度MEHP处理组(≥25.00μM)细胞的SOD活性也显著性降低p<0.01)。MEHP处理HepG2细胞12h,所有MEHP处理组细胞的SOD活性均无显著性变化(p>0.05)；在24h时点,仅观察到100.00μM处理组细胞的SOD活性降低(p<0.05)；在36h时点,50.00μM和100.00μM处理组细胞的SOD活性均降低p<0.05)。
③MEHP处理L02细胞12h和24h,所有MEHP处理组均为观察到细胞GSH-Px活性的变化p>0.05)；在36h时点,除6.25μM处理组外,其他处理组细胞的GSH-Px活性均降低p<0.05)。用MEHP处理HepG2细胞12h,在所有处理组未观察到GSH-Px活性的变化(p>0.05)；在24h和36h时点,除6.25μM处理组外,其他处理组细胞的GSH-Px活性均显著性降低p<0.05)。
(三)细胞8-OHdG水平
①MEHP处理L02细胞24h和36h,仅在24h时点的50.00μM和100.00μM处理组和36h时点的100.00μM处理组8-OHdG水平有显著性升高p<0.05或p<0.01);②MEHP处理HepG2细胞24h和36h,在24h时点>25.00μM的各处理组和36h时点的100.00μM处理组细胞的8-OHdG水平有显著性升高p<0.05或p<0.01)。
(四)细胞凋亡
①流式细胞仪检测结果显示：MEHP处理L02细胞24h和36h,在50.00μM和100.00μM处理组的细胞凋亡率均显著性增加(p<0.01)。MEHP处理HepG2细胞24h和36h,观察到24h时点100.00μM处理组和36h时点>25.00μM各处理组的细胞凋亡率显著性增加(p<0.01)。
②TUNEL结果显示：MEHP处理L02细胞24h和36h,50.00μM和100.00gM处理组有绿色荧光标记的凋亡细胞数明显增多。MEHP处理HepG2细胞24h,仅发现100.00μM组绿色荧光标记的凋亡细胞略有增多；在36h时点,在25.00μM处理组已发现绿色荧光标记的凋亡细胞数有明显增多。
结论：MEHP可致L02细胞和HepG2细胞氧化应激和氧化性DNA损伤,并导致细胞凋亡。
第二部分p53介导线粒体通路在MEHP致肝细胞凋亡中的调控作用
第一部分研究结果提示：MEHP并非完全依赖于PPAR途径致肝细胞凋亡。因此,本部分将进一步研究PXR受体途径的相关基因、蛋白质表达水平和酶活性的变化,以及p53介导线粒体通路在MEHP致L02细胞和HepG2细胞凋亡中的调控作用。方法：MEHP设定浓度为6.25μM、12.50μM、25.00μM、50.00μM和100.00μM,二甲基亚砜(DMSO)为溶剂对照(终浓度≤0.1%),检测内容包括：①实时荧光定量RT-PCR检测pxr基因mRNA表达；②estern blotting检测CYP3A4蛋白表达,7-乙氧基香豆素O-去乙基酶(ECOD)检测CYP3A4酶活性；③estern blotting检测p53、p-p53、MDM2、 Bax、Bcl-2、PUMA和NOXA蛋白表达；④HPLC检测细胞ATP含量；⑤Westem blotting检测胞浆内Cytochrome c和Smac/DIABLO蛋白含量；⑥试剂盒检测caspase3,9酶活性。研究结果如下：
(一)PXR受体途径相关基因、蛋白及酶活性
①MEHP处理L02细胞24h,除6.25μM组外,其他处理组pxr基因的mRNA表达均被上调(p<0.05或p<0.01)。在36h时点,所有处理组pxr基因的mRNA表达均被上调(p<0.05或p<0.01)。MEHP处理HepG2细胞24h,在>25.00μM的各处理组pxr基因mRNA表达均被上调(p<0.05或p<0.01)。在3.6h时点,除6.25μM处理组外,其他处理组pxr基因mRNA表达均被上调(p<0.01)。
②MEHP处理L02细胞24h,50.00μM和100.00μM处理组CYP3A4蛋白表达量增加p<0.01)。在36h时点,除6.25μM处理组外,其他处理组CYP3A4蛋白表达量均增加(p<0.05或p<0.01)。MEHP处理HepG2细胞24h,除6.25μM处理组外,其他处理组CYP3A4蛋白表达量增加(p<0.05或p<0.01)。在36h时点,>25.00μM的各处理组CYP3A4蛋白表达均增加(p<0.01);③MEHP处理L02细胞24h,50.00μM和100.00gM处理组ECOD酶活性均显著性升高(p<0.05或p<0.01)；在36h时点,≥25.00μM的各处理组ECOD活性均显著性升高(p<0.01)。 MEHP处理HepG2细胞24h和36h,所有处理组ECOD活性均显著性升高(p<0.05或p<0.01)。
(二)细胞凋亡相关蛋白的表达水平
①MEHP处理L02细胞36h,检出50.00μM和100.00μM处理组MDM2蛋白表达的下调(p<0.05)；除6.25μM处理组外,其他处理组p53及其磷酸化蛋白均被上调(p<0.05或p<0.01)o p53/MDM2比值从12.50μM处理组开始即被上调(p<0.01)。在≥25.00μM的各处理组均检出Bax蛋白表达上调(p<0.01),但Bcl-2蛋白在50.00μM和100.00μM处理组则下调p<0.05或p<0.01)。所有处理组的Bax/Bcl-2比值均升高p<0.05或p<0.01)o PUMA和NOXA蛋白表达在≥25.00μM的各处理组均被上调(p<0.05或p<0.01)。
②MEHP处理HepG2细胞36h,仅在100μM处理组检出MDM2蛋白表达下调(p<0.05),而在所有处理组的p53蛋白表达则检出上调(p<0.05或p<0.01),在≥25.00μM的各MEHP处理组均检出磷酸化p53蛋白表达上调(p<0.01)。所有处理组的p53/MDM2比值均被上调(p<0.01)。仅在100.00μM处理组检出Bax蛋白表达上调(p<0.05),但在100.00μM处理组则检出Bcl-2蛋白表达下调(p<0.05)。除6.25μM组外,其他处理组均检出Bax/Bcl-2比值的升高(p<0.05或p<0.01)。在≥12.50μM的各MEHP处理组均检出PUMA和NOXA蛋白表达上调(p<0.05或p<0.01)。
(三)细胞线粒体损伤指标
①MEHP处理L02细胞24h,除6.25μM和12.50μM处理组外,其他处理组的ATP含量均显著性降低(p<0.05或p<0.01)。在36h时点,50.00μM和100.00μM处理组ATP含量也显著性降低(p<0.01)。MEHP处理HepG2细胞24h和36h,除36h时点的6.25μM处理组外,其他处理组的ATP含量均显著性降低(p<0.01)。
②MEHP处理L02细胞36h,所有MEHP处理组的胞浆中细胞色素C(Cytochrome c)的含量均显著性升高(p<0.01)。除6.25μM和12.50μM处理组外,其他处理组胞浆中Smac/DIABLO的含量均显著性升高(p<0.05或p<0.01)。MEHP处理HepG2细胞36h,除6.25μM处理组外,其他处理组胞浆中Cytochrome c的含量均升高(p<0.05或p<0.01),并在所有处理组中均检出胞浆中Smac/DIABLO的含量的升高(p<0.01)。
(四)Caspase3,9酶活性
①MEHP处理L02细胞36h,在≥25.00μM各处理组的Caspase3和9酶活性均显著性升高(p<0.05或p<0.01)。
②MEHP处理HepG2细胞36h,除6.25μM处理组外,其他处理组的Caspase3和9酶活性均显著性升高(p<0.05或p<0.01)。结论:MEHP上调L02细胞和HepG2细胞pxr基因的mRNA表达水平,诱导CYP3A4蛋白表达量及其酶活性的增加；诱导p53和促凋亡蛋白(Bax、 NOXA、 PUMA)上调,以及MDM2和Bcl-2蛋白的下调,并激活了线粒体凋亡通路。
第三部分Fas/FasL在p53非依赖性信号通路调控MEHP致肝细胞凋亡中的作用
第二部分的研究结果显示：p53介导了线粒体通路调控MEHP致肝细胞的凋亡。目的：采用RNA干扰技术沉默p53,探索性研究p53非依赖性信号通路在MEHP致L02细胞和HepG2细胞凋亡中的调控作用。方法：MEHP设定浓度为50.00μM和100.00μM,二甲基亚砜(DMSO)为溶剂对照(终浓度≤0.1%),检测内容包括：检测细胞凋亡率、测定p53、 p-p53、 MDM2、 Bax. Bcl-2、 PUMA、 NOXA、 Fas和FasL蛋白的表达水平、胞浆中Cytochrome c和Smac/DIABLO蛋白含量、以及Caspase3,8,9酶活性。研究结果如下：
(一)L02细胞和HepG2细胞p53基因沉默效率
①用RNA干扰L02细胞p53基因24h后,用qRT-PCR技术检测显示：p53mRNA表达量相对于阴性对照组的表达量降低83.1%,在处理48h后,p53蛋白表达降低52.5%。
②用RNA干扰HepG2细胞p53基因24h后,P53mRNA表达量相对于阴性对照组表达量降低81.4%；在处理48h后,p53蛋白表达降低52.4%。
(二)细胞凋亡
①MEHP处理L02细胞和L02-P53细胞36h,与各自对照组相比,细胞凋亡率均显著性升高(p<0.01)。在相同MEHP处理浓度下,L02-p53细胞凋亡率与L02细胞相比均显著性升高(p<0.01)。
②MEHP处理HepG2和HepG2-p53细胞36h,与各自对照组相比,细胞凋亡率均显著性升高(p<0.01)。在相同MEHP处理浓度下,HepG2-p53细胞凋亡率与HepG2细胞相比均显著性升高p<0.01)。
(三)细胞凋亡相关蛋白
①MEHP处理L02细胞36h,各处理组与对照组相比,p53、p-p53、Bax、PUMA、 NOXA、Fas和FasL蛋白表达均上调p<0.01),但MDM2和Bcl-2蛋白表达均下调(p<0.01)。MEHP处理L02-p53细胞36h,各处理组与对照组相比,MDM2、Bax、PUMA、 NOXA、 Fas和FasL蛋白表达均上调(p<0.01), p53、p-p53和Bcl-2蛋白表达均下调。同一浓度MEHP处理的L02-p53细胞和L02细胞的结果比较显示：L02-p53细胞的MDM2、Fas和FasL蛋白表达均明显上调p<0.01),而p53和p-p53蛋白则明显下调(p<0.01),但未检出Bax、Bcl-2、PUMA和NOXA蛋白表达量的变化(p>0.05)。
②MEHP处理HepG2细胞36h,各处理组与对照组相比,p53、p-p53、Bax、PUMA、 NOXA、Fas和FasL蛋白表达均上调p<0.01),但MDM2和Bcl-2蛋白表达均下调(p<0.01)。 MEHP处理HepG2-p53细胞36h,各处理组与对照组相比,Bax、PUMA、 NOXA、Fas和FasL蛋白表达均上调(p<0.01), Bcl-2蛋白表达均下调；未检测到p53、p-p53和MDM2蛋白表达的改变(p>0.05)。同一浓度MEHP处理HepG2-p53细胞和HepG2细胞的结果比较显示：HepG2-p53细胞的MDM2、Fas和FasL蛋白表达均明显上调p<0.01),p53和p-p53蛋白表达明显下调p<0.01),但均未检出Bax、Bcl-2、PUMA和NOXA蛋白表达量的变化(p>0.05)。
(四)胞浆中Cytochrome c和Smac/DIABLO含量
①MEHP处理L02细胞和L02-p53细胞36h,各处理组与各自对照组相比,胞浆中Cytochrome c含量均显著性升高p<0.01),仅在100.00μM处理组检出Smac/DIABLO蛋白表达上调(p<0.01)。
②MEHP处理HepG2细胞和HePg2-P53细胞36h,各处理组与各种对照组相比,仅在100.00μM处理组检出胞浆Cytochrome c含量显著性升高p<0.01),在所有处理组检出Smac/DIABLO蛋白表达上调p<0.05或p<0.01)。
(五)Caspase3,8,9酶活性
MEHP处理L02细胞、HepG2细胞、L02-p53细胞和HepG2-p53细胞36h,各处理组与各自对照组相比,Caspase3,8,9酶活性均显著性升高p<0.01)。同一浓度MEHP处理L02-P53细胞和L02细胞以及HepG2-p53细胞和HepG2细胞的结果比较显示：L02-P53细胞和HepG2-p53细胞各处理组的Caspase3,8,9酶活性均诱导性升高(p<0.05或p<0.01)。结论：在MEHP致L02细胞和HepG2细胞凋亡中,p53蛋白介导的细胞信号通路并非为其唯一的调控机制,Fas/FasL通路可代偿p53介导的线粒体通路参与细胞凋亡的调控。
创新点：
1.发现p53介导的线粒体凋亡通路参与了MEHP致人肝细胞凋亡的调控。
2.发现Fas/FasL凋亡通路可部分代偿p53介导的线粒体通路参与MEHP致人肝细胞凋亡的调控。
Di-(2-ethylbexyl) phthalate (DEHP) has been widely used as a plasticizer in manufacture plastic products to increase the material elasticity and flexibility. DEHP is not covalently bound to the plastic matrix. It can leach out of products and contaminate the external environment, including air, water and soil. The three main pathways of exposure to DEHP are ingestion, inhalation and dermal. In recent years, health effects of DEHP in humans have attracted the special attention of the scientific community due to their high production volume and use in a variety of polyvinyl chloride-based consumer products.
Accumulated evidence shows that the DEHP administered orally is rapidly metabolized to mono (2-ethylhexyl) phthalate (MEHP). MEHP, a principle metabolite of DEHP, can cause liver toxicity, kidney toxicity, reproductive or developmental toxicity, as well as induce apoptosis in different types of cells in vitro. However, the underlying mechanism remains to be elucidated.
MEHP as a peroxisome proliferators acts ligand for the peroxisome proliferators-activated receptor (PPAR) to induce liver toxicity. Recent studies show that the active DEHP metabolite MEHP can activate the three PPAR isotypes. MEHP-induced liver damage not only related to, the active PPARa, but also did the activation of pregnane X receptor (PXR).
Apoptosis is an active form of cell death. This process happens in response to a variety of environmental factors or death signals from the extracellular environment, which regulates by multiple apoptosis-related genes. The p53tumor suppressor plays an important role in the regulation of programmed cell death. The active p53transcriptionally modulates its downstream genes, such as bax, bcl-2and puma, which induced apoptosis through the mitochondrial apoptotic pathways. The high expression of p53protein was detected in MEHP-induced germ cell apoptosis. Previous studies showed that the apoptotic process is not only modulated by p53-dependent signaling pathways, but the p53-independent signaling pathways (such as c-Jun N-terminal kinases-mitogen activated protein kinases (JNK-MAPK) pathway and Fas/FasL pathway) may participate in the process. However, it is unknown whether the p53-dependent signaling pathway and/or p53-independent signaling pathway, participates in the regulations of MEHP-induced apoptosis in human liver cell lines.
Based on the previous results, two cell lines (human embryonic liver cell (L02) and human liver cancer cell (HepG2)) were used in this study to investigate MEHP-induced oxidative stress and cytotoxicity as well as p53-mediated mitochondrial apoptosis. And the molecular mechanisms of Fas/Fas ligand pathway (one of p53-independent signaling pathways) involving in the apoptosis were further investigated. The study consists of three parts as follows:
Part One: MEHP-induced oxidative DNA damage and apoptosis in human liver cells
Objective: To build up the experimental models with MEHP-treted L02and HepG2cells. Methods:Both L02and HepG2cells were treated with serial twofold concentrations of MEHP (6.25,12.50,25.00,50.00and100.00μM) and dimethyl sulfoxide (DMSO as the solvent control, final concentration:<0.1%), respectively. The following indicators were measured:(1) cell viability was detected by MTT assay;(2) levels of maleic dialdehyde (MDA) and activities of superoxide dimutese (SOD) and glutathione peroxidase (GSH-Px) were determined by the corresponding assay kits;(3) levels of cellular8-hydroxy-2'-deoxyguanosine (8-OHdG) were analyzed by high-performance liquid chromatography (HPLC);(4) apoptosis were analyzed by flow cytometry and TUNEL staining. The results are as follows:
1. Cell viability
(1) At12h after MEHP treatment, the viability of L02cells increased only in50.00μM treatment group (p<0.05); however, at24h, MEHP significantly reduced the viability of L02cells in50.00and100.00μM treatment groups (p<0.05or p<0.01). At36h, greater reductions in the viability of L02cells were found with higher concentrations of MEHP (≥25.00μM, p<0.05or p<0.01).(2) At12h after MEHP treatment, the viability of HepG2cells decreased in50.00and100.00μM treatment groups (p<0.05or p<0.01). At24and36h, the viability of HepG2cells reduced in a concentration-dependent manner, greater reductions in the viability were found with higher concentrations of MEHP (>25.00μM, p<0.01for all).
2. Cellular oxidative damage
(1) At12h after MEHP treatment, no changes in MDA contents were found in all treatment groups of cell lines L02cells and HepG2(p>0.05for all). At24and36h after treatment, MEHP significantly increased MDA contents of L02cells with higher concentrations of MEHP (≥25.00μM, p<0.05or p<0.01). The MDA contents of HepG2cells significantly increased in all treatment groups at24h (p<0.01for all) and the other treatment groups at36h except for6.25μM treatment group (p<0.01for all).
(2) At12h after MEHP treatment, no changes in SOD activities were found in all treatment groups in both cell lines (p>0.05for all). MEHP reduced SOD activities of L02cells in all treatment groups at24h and in high concentration groups (≥25.00μM) at36h (p<0.05or p<0.01). At24h after treatment, MEHP reduced SOD activities of HepG2cells only in100.00μM treatment group (p<0.05), and in50.00and100.00μM treatment groups at36h (p<0.05for both).
(3) At12and24h after MEHP treatment, no changes in GSH-Px activities were found in all treatment groups of L02cells (p>0.05for all). At36h, MEHP significantly reduced GSH-Px activities in all treatment groups of L02cells (p<0.05for all), except for6.25μM treatment group; At12h after MEHP treatment, no changes in GSH-Px activities were found in all treatment groups of HepG2cells (p>0.05for all). At24and36h, MEHP significantly reduced GSH-Px activities of HepG2cells in all treatment groups (p<0.05for all), except for6.25μM treatment group.
3. Cellular8-OHdG levels
(1) At24and36h after MEHP treatment,8-OHdG levels of L02cells significantly increased in50.00and100.00μM treatment groups at24h but only in100.00μM treatment group at36h (p<0.05or p<0.01).
(2) At24and36h after MEHP treatment,8-OHdG levels of HepG2cells significantly increased in high concentration groups (>25μM) at24h but only in100.00μM treatment group at36h (p<0.05or p<0.01).
4. Apoptosis
(1) Flow cytometric analysis showed that in L02cells MEHP significantly increased the percentages of the apoptotic cells in50.00and100.00μM treatment groups at24and36h (p<0.01for all). In HepG2cells, MEHP significantly increased the percentages of the apoptotic cells only in100.00μM treatment group at24h (p<0.01), and in high concentration groups (≥25.00μM) at36h (p<0.0for all).
(2) The detection of apoptosis with TUNEL showed that apoptotic L02cells (labeled with green fluorescence) markedly increased in50.00and100.00μM treatment groups at24and36h. There was slight increase in apoptotic HepG2cells (labeled with green fluorescence) only in100.00μM treatment group at24h; obvious increase in apoptotic HepG2cells were found in high concentration groups (≥25.00μM) at36h (p<0.01for all). Conclusions:Under the experimental conditions, MEHP induced the oxidative stress and oxidative DNA damage and then induced apoptosis in L02and HepG2cells.
Part Two:Regulation of p53-mediated mitochondrial apoptotic signaling pathway in MEHP-induced apoptosis in human liver cells
The major results from Part One of this study suggested that MEHP induced liver toxicity and apoptosis, which did not depend only on PPAR pathway. Objective:To investigate modulations of PXR-mediated genes and the protein expressions and enzyme activities, as well as p53-mediated mitochondrial pathway in MEHP-induced apoptotic L02and HepG2cells. Methods:Cell lines L02and HepG2were treated with series twofold concentrations of MEHP (6.25,12.50,25.00,50.00and100.00μM) and DMSO (as the solvent control, final concentration:≤0.1%), respectively. The following indicators were measured:(1) Expression of pxr gene mRNA was evaluated by quantitative real-time PCR (qRT-PCR);(2) Expression of CYP3A4protein was detected by western blotting and activity of CYP3A was measured by7-ethoxycoumarin-O-deethylase (ECOD);(3) Expression of p53, p-p53, MDM2, Bax, Bcl-2, PUMA and NOXA proteins were detected by western blotting;(4) Levels of cellular ATP were analyzed by HPLC;(5) Contents of Cytochrome c and Smac/DIABLO in cytosol were detected by western blotting;(6) Caspase3,9activities were determined by the corresponding assay kits; The results are as follows:
1. Levels of pregnane X receptor mRNA, CYP3A4protein and CYP3A4activity
(1) In L02cells, MEHP up-regulated pxr mRNA levels in all treatment groups except for6.25uM treatment group at24h (p<0.05or p<0.01) and in all treatment groups at36h (p<0.05or p<0.01). In HepG2cells, MEHP up-regulated pxr gene mRNA levels in all treatment groups, except for6.25and12.50μM treatment groups at24h (p<0.05or p<0.01) and in all treatment groups, except for6.25μM treatment group at36h (p<0.01for all);
(2) In L02cells, MEHP induced expression levels of the CYP3A4protein in50.00and100.00μM treatment groups at24h (p<0.01for both) and in all treatment groups, except for6.25μM treatment group at36h (p<0.05or p<0.01). In HepG2cells, MEHP induced expression levels of the CYP3A4protein in all treatment groups, except for6.25μM treatment group at24h (p<0.05or p<0.01) and in high concentration groups (≥25.00μM) at36h (p<0.01for all).
(3) In L02cells, the ECOD enzyme activities were significantly increased in50.00and100.00μM treatment groups at24h (p<0.05or p<0.01), and in high concentration groups (>25.00μM) at36h (p<0.01for all). In HepG2cells, at24and36h after treatment, MEHP induced increases in the ECOD activities in all treatment groups (p<0.05or p<0.01).
2. Expression of apoptosis-related proteins
(1) In L02cells, at36h after treatment, MEHP decreased levels of MDM2protein in50.00and100.00μM treatment groups (p<0.05for both), but increased levels of p53and phosphorylated p53proteins in all treatment groups, except for6.25μM treatment group (p<0.05or p<0.01). MEHP increased the p53/MDM2protein ratios in all treatment groups, except for6.25μM treatment group (p<0.01for all), and Bax protein levels in high concentration groups (≥25.00μM,p<0.01), but decreased Bcl-2protein levels in50.00and100.00μM treatment groups (p<0.05or p<0.01). In addition, MEHP increased the Bax/Bcl-2protein ratios in all treatment groups (p<0.05or p<0.01) as well as Levels of PUMA and NOXA proteins in high concentration groups (≥25.00μM,p<0.05or p<0.01).
(2) In HepG2cells, at36h after treatment, MEHP induced the decrease in MDM2protein level only in100.00μM treatment group (p<0.05), but increased p53protein levels in all treatment groups (p<0.05or p<0.01) and phosphorylaed p53protein levels in high concentration groups (>25.00μM,p<0.01for all). MEHP increased the p53/MDM2protein ratios in all treatment groups (p<0.01for all), and Bax protein level only in100.00μM treatment group but decreased Bcl-2protein level (p<0.05for both). In addition, MEHP increased the Bax/Bcl-2protein ratios in all treatment groups except for6.25μM treatment group (p<0.05or p<0.01) and levels of PUMA and NOXA proteins in high concentration groups (≥12.50μM,p<0.05or<0.01).
3. Indicators of mitochondrial damage indicators MEHP increased levels of cytosolic Cytochrome c and Smac/DIABOL proteins, but decreased the intracellular ATP contents:
(1) MEHP decreased intracellular ATP contents of L02cells in high concentration groups (≥25.00μM) at24h (p<0.05or p<0.01) and in50.00and100.00μM treatment groups at36h (p<0.01for all), but decreased intracellular ATP contents of HepG2cells in all treatment groups (p <0.01for all) at24h and in all treatment groups except for6.25μM treatment group at36h (p<0.01for all).
(2) In L02cells at36h after treatment, MEHP increased the levels of cytosolic Cytochrome c in all treatment groups (p<0.01for all) and Smac/DIABLO proteins in high concentrations (>25.00μM, p<0.05or p<0.01). In HepG2cells, at36h after treatment, MEHP increased the levels of cytosolic Cytochrome c in all treatment except for6.25μM treatment group (p<0.05or p<0.01) and the Smac/DIABOL protein at all treatment groups (p<0.01).
4. Caspase3,9activities
(1) At36h after treatment, MEHP significantly increased the activities of Caspase3,9in high concentration groups (≥25.00μM) in L02cells (p<0.05or p<0.01) and in all treatment groups except for6.25μM treatment group in HepG2cells (p<0.05or p<0.01).
Conclusions:In cell lines L02and HepG2, MEHP up-regulated the pxr at mRNA level and induced enzyme activity and CYP3A4protein expression, and increases the levels of p53, p-p53, Bax, PUMA and NOXA proteins, but decreased the levels of MDM2and Bcl-2proteins. Additionally, the mitochondrial apoptosis signaling pathway was activated in the MEHP-treated cell lines.
Part three:Regulation of Fas/FasL mediated p53-independent apoptotic pathway in MEHP-induced apoptosis in human liver cells
The major results from Part Two of this study suggested that p53mediated mitochondrial apoptotic pathway in MEHP-treated cell lines L02and HepG2. Objective:In this part, the technique of RNA interference (RNAi) was used to investigate the regulation of p53-independent pathway in HepG2and L02cell lines. Methods:L02, L02-p53, HepG2and HepG2-p53cells were treated with different concentrations of MEHP (50.00and100.00μM) and DMSO (as the solvent control, final concentration:≤0.1%), respectively. The following indicators were measured at36h: the percentage of apoptotic cells, expression of p53, p-p53, MDM2, Bax, Bcl-2, PUMA, NOXA, Fas/FasL and Smac/DIABOL proteins, cytosolic Cytochrome c concentration, the activities of Caspase3,8,9. The results are as follows:
1. The p53silence efficiency in L02-p53and HepG2-p53cells by RNA interference
(1) To evaluate the p53silence efficiency, qRT-PCR method was used to measure the levels of the decreasing p53in in L02-p53and HepG2-p53cells. The results showed that in L02-p53cells the p53mRNA and protein levels was decreased by83.1%at24h and52.5%at48h, compared with the corresponding negative controls, respectively;(2) in HepG2-p53cells, the p53mRNA and protein levels were decreased by81.4%at24h and52.4%at48h, compared with the corresponding negative controls, respectively.
2. The cell Apoptosis
(1) At36h after treatment, MEHP increased the percentages of the apoptotic cells of both L02and L02-p53cells in all treatment groups (p<0.01for all). At36h after L02and L02-p53cells were treated with the same concentration of MEHP, the higher percentages of the L02-p53apoptotic cells were found in all treatment groups compared with the L02cells (p<0.01for all).(2) At36h after treatment, MEHP increased the percentages of both HepG2and HepG2-p53cells in all treatment groups (p<0.01for all). At36h after HepG2and HepG2-p53cells were treated with the same concentration of MEHP, the higher percentages of the HepG2-p53apoptotic cells were found in all treatment groups compared with the HepG2cells (p<0.01for all).
3. The expression of apoptosis-related proteins
(1) At36h after treatment of L02cells, MEHP up-regulated the expression of p53, p-p53, Bax, PUMA, NOXA, Fas and FasL proteins, but down-regulated the expression of MDM2and Bcl-2proteins in all treatment groups (p<0.01for all); at36h after the treatment of L02-p53cells, MEHP up-regulated the expression of MDM2, Bax, PUMA, NOXA, Fas and FasL proteins (p<0.01for all), but decreased the expression of p53, p-p53and Bcl-2proteins in all treatment groups, compared with the corresponding controls (p<0.01for all). At36h after treatments of L02and L02-p53cells at the same concentrations of MEHP, the results suggested that the expression of MDM2, Fas and FasL proteins were up-regulated in all treatment groups (p<0.01for all), but the expression of p53and p-p53proteins were down-regulated in all treatment groups (p<0.01for all). No changes in the levels of Bax, Bcl-2, PUMA and NOXA proteins were found in all treatment groups of either L02-p53or L02cells (p>0.05for all).
(2) At36h after treatment of HepG2cells, MEHP up-regulated the expression of p53, p-p53, Bax, PUMA, NOXA, Fas and FasL proteins, but down-regulated the expression of MDM2and Bcl-2protein in all treatment groups (p<0.01for all); at36h after treatment of HepG2-p53cells, MEHP up-regulated the expression of Bax, PUMA, NOXA, Fas and FasL proteins (p<0.01for all), but down-regulated the expression of Bcl-2protein compared with the corresponding negative controls (p<0.01for all). No changes in the levels of the p53, p-p53and MDM2proteins were found in all treatment groups of HepG2-p53cells (p>0.05for all). At36h after treatments of HepG2and HepG2-p53cells at the same concentrations of MEHP, the results suggested that the levels of MDM2, Fas and FasL proteins were up-regulated (p<0.01for all), but the levels of p53and p-p53protiens were down-regulated in all treatment groups (p<0.01for all). There was no changes in the levels of Bax, Bcl-2, PUMA and NOXA proteins in all treatment groups of either HepG2"p53or HepG2cells (p>0.05for all).
4. The levels of cytosolic Cytochrome c and Smac/DIABOL protein
(1) At36h after treatments of L02and L02-p53cells, MEHP increased the levels of cytosolic Cytochrome c in all treatment groups and the Smac/DIABOL protein in100.00μM treatment group compared with the corresponding controls (p<0.01for all).(2) At36h after treatment of HepG2and HepG2"p53cells, MEHP increased the levels of cytosolic Cytochrome c in100.00μM treatment group (p<0.01for all) and the Smac/DIABLO protein in all treatment groups (p<0.05or p<0.01), compared with the corresponding controls.
5. The activities of Caspase3,8, and9At36h after treatment, MEHP increased Caspase3,8,9activities in all treatment groups of L02, HepG2, L02-p53and HepG2-p53cells (p<0.01for all). At36h after treatments either between L02and L02-p53cells or between HepG2and HepG2"p53cells at the same concentrations of MEHP, the results suggested that MEHP induced the activities of Caspase3,8, and9in all treatment groups (p<0.05or p<0.01). Conclusions:The modulation of p53-mediated pathway is not only in MEHP-induced apoptosis in cell lines L02and HepG2. Fas/FasL pathway may partially replace the regulation of the p53-mediated mitochondrial apoptotic pathway in MEHP-induced apoptosis of the cell lines L02and HepG2.
The innovation points of this study:
(1) This is the first report on the modulation of p53-mediated mitochondrial pathway in MEHP-induced apoptosis in cell lines L02and HepG2.
(2) This is the first report on the Fas/FasL pathway may partially replace the regulation of the p53-mediated mitochondrial pathway in MEHP-induced apoptosis in cell lines L02and HepG2.
研究表明,进入机体的DEHP很快被代谢为活性中间产物—邻苯二甲酸单(2-乙基己基)酯mono-(2-ethylhexyl) phthalate, MEHP),产生肝脏毒性、肾脏毒性和生殖发育毒性等。体外研究显示,MEHP可致多种细胞的凋亡,但尚不了解其分子调控机制。
MEHP是过氧化物酶体增殖物(peroxisome proliferators, PP),它可经氧化物酶体增殖物激活受体(peroxisome proliferators activated receptors, PPAR)介导肝毒性。但是,MEHP致肝毒性的调控机制并非完全依赖PPAR途径,可能与孕烷X受体(pregnane X receptor, PXR)有一定的关联。
细胞凋亡是外环境刺激或死亡信号所触发的细胞主动死亡过程,该过程受多个基因的调控,其中抑癌基因p53发挥主要调控作用。活化p53通过转录调控下游凋亡基因,如bax-, bcl-2和puma等激活线粒体凋亡通路引起细胞凋亡。研究发现,在MEHP引起的生殖细胞凋亡过程中有p53蛋白的高表达。但是,细胞凋亡并非仅受p53介导的细胞信号通路的调控,p53非依赖信号通路,如c-Jun氨基末端激酶-促分裂素原活化蛋白激酶(c-Jun N-terminal kinases-mitogen activated protein kinases, JNK-MAPK)途径和Fas/FasL途径等也可参与其调控。不过迄今仍不清楚在MEHP致肝细胞凋亡中p53依赖性和非依赖性信号通路是否参与了调控。
基于此,本研究拟用MEHP处理人胚肝细胞L02和人肝癌细胞HepG2来研究MEHP致肝细胞毒性和氧化损伤变化,以及p53介导的线粒体通路在MEHP致肝细胞凋亡中的调控机制,并探索性研究Fas/FasL在p53非依赖性信号通路调控MEHP致肝细胞凋亡中的作用,旨在为后续深入研究MEHP致肝细胞毒性的分子机制提供科学依据。本研究包括以下三个部分：
第一部分MEHP致人肝细胞氧化性DNA损伤和细胞凋亡的研究
目的：建立MEHP染毒L02细胞和HepG2细胞模型。方法：MEHP设定浓度为6.25μM、12.50μM、25.00μM、50.00μM和100.00μM,二甲基亚砜(DMSO)为溶剂对照(终浓度≤0.1%),检测内容包括：①MTT检测细胞活力；②试剂盒检测细胞丙二醛(maleic dialdehyde, MDA)水平、超氧化物酶(superoxide dimutese, SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)的活性；③高效液相色谱仪(HPLC)检测细胞8-羟基-2’-脱氧鸟苷(8-hydroxy-2'-deoxyguanosine,8-OHdG)水平；④流式细胞仪和TUNEL检测细胞凋亡变化。研究结果如下：
(一)细胞活力
①用MEHP处理L02细胞12h,仅在50.00μM处理组检出细胞活力上升(p<0.05)；在24h时点,50.00μM和100.00μM处理组的细胞活力均显著性降低p<0.05或p<0.01)；在36h时点,较高浓度MEHP处理组(≥25.00μM)细胞的活力均显著性降低(p<0.05或p<0.01)。
②用MEHP处理HepG2细胞12h,在50.00μM和100.00μM处理组的细胞活力显著性下降(p<0.01)。在24h和36h时点,细胞活力随MEHP浓度的升高而逐渐降低,在较高浓度的MEHP处理组(≥25.00μM),细胞活力均显著性降低p<0.01)。
(二)细胞的氧化损伤
①用MEHP处理L02细胞和HepG2细胞12h,在所有处理组均未观察到MDA水平的变化p>0.05)。在24h和36h时点,较高浓度的MEHP处理L02细胞(≥25.00μM)的MDA水平均显著性升高p<0.05或p<0.01)。用MEHP处理HepG2细胞24h,所有处理组的MDA水平均显著性升高(p<0.01)；在36h时点,除6.25μM处理组外,所有MEHP处理组的MDA水平均显著性升高(p<0.01)。
②用MEHP处理L02细胞12h,所有处理组细胞的SOD活性均无显著性变化(p>0.05)；在24h时点,所有处理组细胞的SOD活性均显著性降低(p<0.05或p<0.01)；在36h时点,较高浓度MEHP处理组(≥25.00μM)细胞的SOD活性也显著性降低p<0.01)。MEHP处理HepG2细胞12h,所有MEHP处理组细胞的SOD活性均无显著性变化(p>0.05)；在24h时点,仅观察到100.00μM处理组细胞的SOD活性降低(p<0.05)；在36h时点,50.00μM和100.00μM处理组细胞的SOD活性均降低p<0.05)。
③MEHP处理L02细胞12h和24h,所有MEHP处理组均为观察到细胞GSH-Px活性的变化p>0.05)；在36h时点,除6.25μM处理组外,其他处理组细胞的GSH-Px活性均降低p<0.05)。用MEHP处理HepG2细胞12h,在所有处理组未观察到GSH-Px活性的变化(p>0.05)；在24h和36h时点,除6.25μM处理组外,其他处理组细胞的GSH-Px活性均显著性降低p<0.05)。
(三)细胞8-OHdG水平
①MEHP处理L02细胞24h和36h,仅在24h时点的50.00μM和100.00μM处理组和36h时点的100.00μM处理组8-OHdG水平有显著性升高p<0.05或p<0.01);②MEHP处理HepG2细胞24h和36h,在24h时点>25.00μM的各处理组和36h时点的100.00μM处理组细胞的8-OHdG水平有显著性升高p<0.05或p<0.01)。
(四)细胞凋亡
①流式细胞仪检测结果显示：MEHP处理L02细胞24h和36h,在50.00μM和100.00μM处理组的细胞凋亡率均显著性增加(p<0.01)。MEHP处理HepG2细胞24h和36h,观察到24h时点100.00μM处理组和36h时点>25.00μM各处理组的细胞凋亡率显著性增加(p<0.01)。
②TUNEL结果显示：MEHP处理L02细胞24h和36h,50.00μM和100.00gM处理组有绿色荧光标记的凋亡细胞数明显增多。MEHP处理HepG2细胞24h,仅发现100.00μM组绿色荧光标记的凋亡细胞略有增多；在36h时点,在25.00μM处理组已发现绿色荧光标记的凋亡细胞数有明显增多。
结论：MEHP可致L02细胞和HepG2细胞氧化应激和氧化性DNA损伤,并导致细胞凋亡。
第二部分p53介导线粒体通路在MEHP致肝细胞凋亡中的调控作用
第一部分研究结果提示：MEHP并非完全依赖于PPAR途径致肝细胞凋亡。因此,本部分将进一步研究PXR受体途径的相关基因、蛋白质表达水平和酶活性的变化,以及p53介导线粒体通路在MEHP致L02细胞和HepG2细胞凋亡中的调控作用。方法：MEHP设定浓度为6.25μM、12.50μM、25.00μM、50.00μM和100.00μM,二甲基亚砜(DMSO)为溶剂对照(终浓度≤0.1%),检测内容包括：①实时荧光定量RT-PCR检测pxr基因mRNA表达；②estern blotting检测CYP3A4蛋白表达,7-乙氧基香豆素O-去乙基酶(ECOD)检测CYP3A4酶活性；③estern blotting检测p53、p-p53、MDM2、 Bax、Bcl-2、PUMA和NOXA蛋白表达；④HPLC检测细胞ATP含量；⑤Westem blotting检测胞浆内Cytochrome c和Smac/DIABLO蛋白含量；⑥试剂盒检测caspase3,9酶活性。研究结果如下：
(一)PXR受体途径相关基因、蛋白及酶活性
①MEHP处理L02细胞24h,除6.25μM组外,其他处理组pxr基因的mRNA表达均被上调(p<0.05或p<0.01)。在36h时点,所有处理组pxr基因的mRNA表达均被上调(p<0.05或p<0.01)。MEHP处理HepG2细胞24h,在>25.00μM的各处理组pxr基因mRNA表达均被上调(p<0.05或p<0.01)。在3.6h时点,除6.25μM处理组外,其他处理组pxr基因mRNA表达均被上调(p<0.01)。
②MEHP处理L02细胞24h,50.00μM和100.00μM处理组CYP3A4蛋白表达量增加p<0.01)。在36h时点,除6.25μM处理组外,其他处理组CYP3A4蛋白表达量均增加(p<0.05或p<0.01)。MEHP处理HepG2细胞24h,除6.25μM处理组外,其他处理组CYP3A4蛋白表达量增加(p<0.05或p<0.01)。在36h时点,>25.00μM的各处理组CYP3A4蛋白表达均增加(p<0.01);③MEHP处理L02细胞24h,50.00μM和100.00gM处理组ECOD酶活性均显著性升高(p<0.05或p<0.01)；在36h时点,≥25.00μM的各处理组ECOD活性均显著性升高(p<0.01)。 MEHP处理HepG2细胞24h和36h,所有处理组ECOD活性均显著性升高(p<0.05或p<0.01)。
(二)细胞凋亡相关蛋白的表达水平
①MEHP处理L02细胞36h,检出50.00μM和100.00μM处理组MDM2蛋白表达的下调(p<0.05)；除6.25μM处理组外,其他处理组p53及其磷酸化蛋白均被上调(p<0.05或p<0.01)o p53/MDM2比值从12.50μM处理组开始即被上调(p<0.01)。在≥25.00μM的各处理组均检出Bax蛋白表达上调(p<0.01),但Bcl-2蛋白在50.00μM和100.00μM处理组则下调p<0.05或p<0.01)。所有处理组的Bax/Bcl-2比值均升高p<0.05或p<0.01)o PUMA和NOXA蛋白表达在≥25.00μM的各处理组均被上调(p<0.05或p<0.01)。
②MEHP处理HepG2细胞36h,仅在100μM处理组检出MDM2蛋白表达下调(p<0.05),而在所有处理组的p53蛋白表达则检出上调(p<0.05或p<0.01),在≥25.00μM的各MEHP处理组均检出磷酸化p53蛋白表达上调(p<0.01)。所有处理组的p53/MDM2比值均被上调(p<0.01)。仅在100.00μM处理组检出Bax蛋白表达上调(p<0.05),但在100.00μM处理组则检出Bcl-2蛋白表达下调(p<0.05)。除6.25μM组外,其他处理组均检出Bax/Bcl-2比值的升高(p<0.05或p<0.01)。在≥12.50μM的各MEHP处理组均检出PUMA和NOXA蛋白表达上调(p<0.05或p<0.01)。
(三)细胞线粒体损伤指标
①MEHP处理L02细胞24h,除6.25μM和12.50μM处理组外,其他处理组的ATP含量均显著性降低(p<0.05或p<0.01)。在36h时点,50.00μM和100.00μM处理组ATP含量也显著性降低(p<0.01)。MEHP处理HepG2细胞24h和36h,除36h时点的6.25μM处理组外,其他处理组的ATP含量均显著性降低(p<0.01)。
②MEHP处理L02细胞36h,所有MEHP处理组的胞浆中细胞色素C(Cytochrome c)的含量均显著性升高(p<0.01)。除6.25μM和12.50μM处理组外,其他处理组胞浆中Smac/DIABLO的含量均显著性升高(p<0.05或p<0.01)。MEHP处理HepG2细胞36h,除6.25μM处理组外,其他处理组胞浆中Cytochrome c的含量均升高(p<0.05或p<0.01),并在所有处理组中均检出胞浆中Smac/DIABLO的含量的升高(p<0.01)。
(四)Caspase3,9酶活性
①MEHP处理L02细胞36h,在≥25.00μM各处理组的Caspase3和9酶活性均显著性升高(p<0.05或p<0.01)。
②MEHP处理HepG2细胞36h,除6.25μM处理组外,其他处理组的Caspase3和9酶活性均显著性升高(p<0.05或p<0.01)。结论:MEHP上调L02细胞和HepG2细胞pxr基因的mRNA表达水平,诱导CYP3A4蛋白表达量及其酶活性的增加；诱导p53和促凋亡蛋白(Bax、 NOXA、 PUMA)上调,以及MDM2和Bcl-2蛋白的下调,并激活了线粒体凋亡通路。
第三部分Fas/FasL在p53非依赖性信号通路调控MEHP致肝细胞凋亡中的作用
第二部分的研究结果显示：p53介导了线粒体通路调控MEHP致肝细胞的凋亡。目的：采用RNA干扰技术沉默p53,探索性研究p53非依赖性信号通路在MEHP致L02细胞和HepG2细胞凋亡中的调控作用。方法：MEHP设定浓度为50.00μM和100.00μM,二甲基亚砜(DMSO)为溶剂对照(终浓度≤0.1%),检测内容包括：检测细胞凋亡率、测定p53、 p-p53、 MDM2、 Bax. Bcl-2、 PUMA、 NOXA、 Fas和FasL蛋白的表达水平、胞浆中Cytochrome c和Smac/DIABLO蛋白含量、以及Caspase3,8,9酶活性。研究结果如下：
(一)L02细胞和HepG2细胞p53基因沉默效率
①用RNA干扰L02细胞p53基因24h后,用qRT-PCR技术检测显示：p53mRNA表达量相对于阴性对照组的表达量降低83.1%,在处理48h后,p53蛋白表达降低52.5%。
②用RNA干扰HepG2细胞p53基因24h后,P53mRNA表达量相对于阴性对照组表达量降低81.4%；在处理48h后,p53蛋白表达降低52.4%。
(二)细胞凋亡
①MEHP处理L02细胞和L02-P53细胞36h,与各自对照组相比,细胞凋亡率均显著性升高(p<0.01)。在相同MEHP处理浓度下,L02-p53细胞凋亡率与L02细胞相比均显著性升高(p<0.01)。
②MEHP处理HepG2和HepG2-p53细胞36h,与各自对照组相比,细胞凋亡率均显著性升高(p<0.01)。在相同MEHP处理浓度下,HepG2-p53细胞凋亡率与HepG2细胞相比均显著性升高p<0.01)。
(三)细胞凋亡相关蛋白
①MEHP处理L02细胞36h,各处理组与对照组相比,p53、p-p53、Bax、PUMA、 NOXA、Fas和FasL蛋白表达均上调p<0.01),但MDM2和Bcl-2蛋白表达均下调(p<0.01)。MEHP处理L02-p53细胞36h,各处理组与对照组相比,MDM2、Bax、PUMA、 NOXA、 Fas和FasL蛋白表达均上调(p<0.01), p53、p-p53和Bcl-2蛋白表达均下调。同一浓度MEHP处理的L02-p53细胞和L02细胞的结果比较显示：L02-p53细胞的MDM2、Fas和FasL蛋白表达均明显上调p<0.01),而p53和p-p53蛋白则明显下调(p<0.01),但未检出Bax、Bcl-2、PUMA和NOXA蛋白表达量的变化(p>0.05)。
②MEHP处理HepG2细胞36h,各处理组与对照组相比,p53、p-p53、Bax、PUMA、 NOXA、Fas和FasL蛋白表达均上调p<0.01),但MDM2和Bcl-2蛋白表达均下调(p<0.01)。 MEHP处理HepG2-p53细胞36h,各处理组与对照组相比,Bax、PUMA、 NOXA、Fas和FasL蛋白表达均上调(p<0.01), Bcl-2蛋白表达均下调；未检测到p53、p-p53和MDM2蛋白表达的改变(p>0.05)。同一浓度MEHP处理HepG2-p53细胞和HepG2细胞的结果比较显示：HepG2-p53细胞的MDM2、Fas和FasL蛋白表达均明显上调p<0.01),p53和p-p53蛋白表达明显下调p<0.01),但均未检出Bax、Bcl-2、PUMA和NOXA蛋白表达量的变化(p>0.05)。
(四)胞浆中Cytochrome c和Smac/DIABLO含量
①MEHP处理L02细胞和L02-p53细胞36h,各处理组与各自对照组相比,胞浆中Cytochrome c含量均显著性升高p<0.01),仅在100.00μM处理组检出Smac/DIABLO蛋白表达上调(p<0.01)。
②MEHP处理HepG2细胞和HePg2-P53细胞36h,各处理组与各种对照组相比,仅在100.00μM处理组检出胞浆Cytochrome c含量显著性升高p<0.01),在所有处理组检出Smac/DIABLO蛋白表达上调p<0.05或p<0.01)。
(五)Caspase3,8,9酶活性
MEHP处理L02细胞、HepG2细胞、L02-p53细胞和HepG2-p53细胞36h,各处理组与各自对照组相比,Caspase3,8,9酶活性均显著性升高p<0.01)。同一浓度MEHP处理L02-P53细胞和L02细胞以及HepG2-p53细胞和HepG2细胞的结果比较显示：L02-P53细胞和HepG2-p53细胞各处理组的Caspase3,8,9酶活性均诱导性升高(p<0.05或p<0.01)。结论：在MEHP致L02细胞和HepG2细胞凋亡中,p53蛋白介导的细胞信号通路并非为其唯一的调控机制,Fas/FasL通路可代偿p53介导的线粒体通路参与细胞凋亡的调控。
创新点：
1.发现p53介导的线粒体凋亡通路参与了MEHP致人肝细胞凋亡的调控。
2.发现Fas/FasL凋亡通路可部分代偿p53介导的线粒体通路参与MEHP致人肝细胞凋亡的调控。
Di-(2-ethylbexyl) phthalate (DEHP) has been widely used as a plasticizer in manufacture plastic products to increase the material elasticity and flexibility. DEHP is not covalently bound to the plastic matrix. It can leach out of products and contaminate the external environment, including air, water and soil. The three main pathways of exposure to DEHP are ingestion, inhalation and dermal. In recent years, health effects of DEHP in humans have attracted the special attention of the scientific community due to their high production volume and use in a variety of polyvinyl chloride-based consumer products.
Accumulated evidence shows that the DEHP administered orally is rapidly metabolized to mono (2-ethylhexyl) phthalate (MEHP). MEHP, a principle metabolite of DEHP, can cause liver toxicity, kidney toxicity, reproductive or developmental toxicity, as well as induce apoptosis in different types of cells in vitro. However, the underlying mechanism remains to be elucidated.
MEHP as a peroxisome proliferators acts ligand for the peroxisome proliferators-activated receptor (PPAR) to induce liver toxicity. Recent studies show that the active DEHP metabolite MEHP can activate the three PPAR isotypes. MEHP-induced liver damage not only related to, the active PPARa, but also did the activation of pregnane X receptor (PXR).
Apoptosis is an active form of cell death. This process happens in response to a variety of environmental factors or death signals from the extracellular environment, which regulates by multiple apoptosis-related genes. The p53tumor suppressor plays an important role in the regulation of programmed cell death. The active p53transcriptionally modulates its downstream genes, such as bax, bcl-2and puma, which induced apoptosis through the mitochondrial apoptotic pathways. The high expression of p53protein was detected in MEHP-induced germ cell apoptosis. Previous studies showed that the apoptotic process is not only modulated by p53-dependent signaling pathways, but the p53-independent signaling pathways (such as c-Jun N-terminal kinases-mitogen activated protein kinases (JNK-MAPK) pathway and Fas/FasL pathway) may participate in the process. However, it is unknown whether the p53-dependent signaling pathway and/or p53-independent signaling pathway, participates in the regulations of MEHP-induced apoptosis in human liver cell lines.
Based on the previous results, two cell lines (human embryonic liver cell (L02) and human liver cancer cell (HepG2)) were used in this study to investigate MEHP-induced oxidative stress and cytotoxicity as well as p53-mediated mitochondrial apoptosis. And the molecular mechanisms of Fas/Fas ligand pathway (one of p53-independent signaling pathways) involving in the apoptosis were further investigated. The study consists of three parts as follows:
Part One: MEHP-induced oxidative DNA damage and apoptosis in human liver cells
Objective: To build up the experimental models with MEHP-treted L02and HepG2cells. Methods:Both L02and HepG2cells were treated with serial twofold concentrations of MEHP (6.25,12.50,25.00,50.00and100.00μM) and dimethyl sulfoxide (DMSO as the solvent control, final concentration:<0.1%), respectively. The following indicators were measured:(1) cell viability was detected by MTT assay;(2) levels of maleic dialdehyde (MDA) and activities of superoxide dimutese (SOD) and glutathione peroxidase (GSH-Px) were determined by the corresponding assay kits;(3) levels of cellular8-hydroxy-2'-deoxyguanosine (8-OHdG) were analyzed by high-performance liquid chromatography (HPLC);(4) apoptosis were analyzed by flow cytometry and TUNEL staining. The results are as follows:
1. Cell viability
(1) At12h after MEHP treatment, the viability of L02cells increased only in50.00μM treatment group (p<0.05); however, at24h, MEHP significantly reduced the viability of L02cells in50.00and100.00μM treatment groups (p<0.05or p<0.01). At36h, greater reductions in the viability of L02cells were found with higher concentrations of MEHP (≥25.00μM, p<0.05or p<0.01).(2) At12h after MEHP treatment, the viability of HepG2cells decreased in50.00and100.00μM treatment groups (p<0.05or p<0.01). At24and36h, the viability of HepG2cells reduced in a concentration-dependent manner, greater reductions in the viability were found with higher concentrations of MEHP (>25.00μM, p<0.01for all).
2. Cellular oxidative damage
(1) At12h after MEHP treatment, no changes in MDA contents were found in all treatment groups of cell lines L02cells and HepG2(p>0.05for all). At24and36h after treatment, MEHP significantly increased MDA contents of L02cells with higher concentrations of MEHP (≥25.00μM, p<0.05or p<0.01). The MDA contents of HepG2cells significantly increased in all treatment groups at24h (p<0.01for all) and the other treatment groups at36h except for6.25μM treatment group (p<0.01for all).
(2) At12h after MEHP treatment, no changes in SOD activities were found in all treatment groups in both cell lines (p>0.05for all). MEHP reduced SOD activities of L02cells in all treatment groups at24h and in high concentration groups (≥25.00μM) at36h (p<0.05or p<0.01). At24h after treatment, MEHP reduced SOD activities of HepG2cells only in100.00μM treatment group (p<0.05), and in50.00and100.00μM treatment groups at36h (p<0.05for both).
(3) At12and24h after MEHP treatment, no changes in GSH-Px activities were found in all treatment groups of L02cells (p>0.05for all). At36h, MEHP significantly reduced GSH-Px activities in all treatment groups of L02cells (p<0.05for all), except for6.25μM treatment group; At12h after MEHP treatment, no changes in GSH-Px activities were found in all treatment groups of HepG2cells (p>0.05for all). At24and36h, MEHP significantly reduced GSH-Px activities of HepG2cells in all treatment groups (p<0.05for all), except for6.25μM treatment group.
3. Cellular8-OHdG levels
(1) At24and36h after MEHP treatment,8-OHdG levels of L02cells significantly increased in50.00and100.00μM treatment groups at24h but only in100.00μM treatment group at36h (p<0.05or p<0.01).
(2) At24and36h after MEHP treatment,8-OHdG levels of HepG2cells significantly increased in high concentration groups (>25μM) at24h but only in100.00μM treatment group at36h (p<0.05or p<0.01).
4. Apoptosis
(1) Flow cytometric analysis showed that in L02cells MEHP significantly increased the percentages of the apoptotic cells in50.00and100.00μM treatment groups at24and36h (p<0.01for all). In HepG2cells, MEHP significantly increased the percentages of the apoptotic cells only in100.00μM treatment group at24h (p<0.01), and in high concentration groups (≥25.00μM) at36h (p<0.0for all).
(2) The detection of apoptosis with TUNEL showed that apoptotic L02cells (labeled with green fluorescence) markedly increased in50.00and100.00μM treatment groups at24and36h. There was slight increase in apoptotic HepG2cells (labeled with green fluorescence) only in100.00μM treatment group at24h; obvious increase in apoptotic HepG2cells were found in high concentration groups (≥25.00μM) at36h (p<0.01for all). Conclusions:Under the experimental conditions, MEHP induced the oxidative stress and oxidative DNA damage and then induced apoptosis in L02and HepG2cells.
Part Two:Regulation of p53-mediated mitochondrial apoptotic signaling pathway in MEHP-induced apoptosis in human liver cells
The major results from Part One of this study suggested that MEHP induced liver toxicity and apoptosis, which did not depend only on PPAR pathway. Objective:To investigate modulations of PXR-mediated genes and the protein expressions and enzyme activities, as well as p53-mediated mitochondrial pathway in MEHP-induced apoptotic L02and HepG2cells. Methods:Cell lines L02and HepG2were treated with series twofold concentrations of MEHP (6.25,12.50,25.00,50.00and100.00μM) and DMSO (as the solvent control, final concentration:≤0.1%), respectively. The following indicators were measured:(1) Expression of pxr gene mRNA was evaluated by quantitative real-time PCR (qRT-PCR);(2) Expression of CYP3A4protein was detected by western blotting and activity of CYP3A was measured by7-ethoxycoumarin-O-deethylase (ECOD);(3) Expression of p53, p-p53, MDM2, Bax, Bcl-2, PUMA and NOXA proteins were detected by western blotting;(4) Levels of cellular ATP were analyzed by HPLC;(5) Contents of Cytochrome c and Smac/DIABLO in cytosol were detected by western blotting;(6) Caspase3,9activities were determined by the corresponding assay kits; The results are as follows:
1. Levels of pregnane X receptor mRNA, CYP3A4protein and CYP3A4activity
(1) In L02cells, MEHP up-regulated pxr mRNA levels in all treatment groups except for6.25uM treatment group at24h (p<0.05or p<0.01) and in all treatment groups at36h (p<0.05or p<0.01). In HepG2cells, MEHP up-regulated pxr gene mRNA levels in all treatment groups, except for6.25and12.50μM treatment groups at24h (p<0.05or p<0.01) and in all treatment groups, except for6.25μM treatment group at36h (p<0.01for all);
(2) In L02cells, MEHP induced expression levels of the CYP3A4protein in50.00and100.00μM treatment groups at24h (p<0.01for both) and in all treatment groups, except for6.25μM treatment group at36h (p<0.05or p<0.01). In HepG2cells, MEHP induced expression levels of the CYP3A4protein in all treatment groups, except for6.25μM treatment group at24h (p<0.05or p<0.01) and in high concentration groups (≥25.00μM) at36h (p<0.01for all).
(3) In L02cells, the ECOD enzyme activities were significantly increased in50.00and100.00μM treatment groups at24h (p<0.05or p<0.01), and in high concentration groups (>25.00μM) at36h (p<0.01for all). In HepG2cells, at24and36h after treatment, MEHP induced increases in the ECOD activities in all treatment groups (p<0.05or p<0.01).
2. Expression of apoptosis-related proteins
(1) In L02cells, at36h after treatment, MEHP decreased levels of MDM2protein in50.00and100.00μM treatment groups (p<0.05for both), but increased levels of p53and phosphorylated p53proteins in all treatment groups, except for6.25μM treatment group (p<0.05or p<0.01). MEHP increased the p53/MDM2protein ratios in all treatment groups, except for6.25μM treatment group (p<0.01for all), and Bax protein levels in high concentration groups (≥25.00μM,p<0.01), but decreased Bcl-2protein levels in50.00and100.00μM treatment groups (p<0.05or p<0.01). In addition, MEHP increased the Bax/Bcl-2protein ratios in all treatment groups (p<0.05or p<0.01) as well as Levels of PUMA and NOXA proteins in high concentration groups (≥25.00μM,p<0.05or p<0.01).
(2) In HepG2cells, at36h after treatment, MEHP induced the decrease in MDM2protein level only in100.00μM treatment group (p<0.05), but increased p53protein levels in all treatment groups (p<0.05or p<0.01) and phosphorylaed p53protein levels in high concentration groups (>25.00μM,p<0.01for all). MEHP increased the p53/MDM2protein ratios in all treatment groups (p<0.01for all), and Bax protein level only in100.00μM treatment group but decreased Bcl-2protein level (p<0.05for both). In addition, MEHP increased the Bax/Bcl-2protein ratios in all treatment groups except for6.25μM treatment group (p<0.05or p<0.01) and levels of PUMA and NOXA proteins in high concentration groups (≥12.50μM,p<0.05or<0.01).
3. Indicators of mitochondrial damage indicators MEHP increased levels of cytosolic Cytochrome c and Smac/DIABOL proteins, but decreased the intracellular ATP contents:
(1) MEHP decreased intracellular ATP contents of L02cells in high concentration groups (≥25.00μM) at24h (p<0.05or p<0.01) and in50.00and100.00μM treatment groups at36h (p<0.01for all), but decreased intracellular ATP contents of HepG2cells in all treatment groups (p <0.01for all) at24h and in all treatment groups except for6.25μM treatment group at36h (p<0.01for all).
(2) In L02cells at36h after treatment, MEHP increased the levels of cytosolic Cytochrome c in all treatment groups (p<0.01for all) and Smac/DIABLO proteins in high concentrations (>25.00μM, p<0.05or p<0.01). In HepG2cells, at36h after treatment, MEHP increased the levels of cytosolic Cytochrome c in all treatment except for6.25μM treatment group (p<0.05or p<0.01) and the Smac/DIABOL protein at all treatment groups (p<0.01).
4. Caspase3,9activities
(1) At36h after treatment, MEHP significantly increased the activities of Caspase3,9in high concentration groups (≥25.00μM) in L02cells (p<0.05or p<0.01) and in all treatment groups except for6.25μM treatment group in HepG2cells (p<0.05or p<0.01).
Conclusions:In cell lines L02and HepG2, MEHP up-regulated the pxr at mRNA level and induced enzyme activity and CYP3A4protein expression, and increases the levels of p53, p-p53, Bax, PUMA and NOXA proteins, but decreased the levels of MDM2and Bcl-2proteins. Additionally, the mitochondrial apoptosis signaling pathway was activated in the MEHP-treated cell lines.
Part three:Regulation of Fas/FasL mediated p53-independent apoptotic pathway in MEHP-induced apoptosis in human liver cells
The major results from Part Two of this study suggested that p53mediated mitochondrial apoptotic pathway in MEHP-treated cell lines L02and HepG2. Objective:In this part, the technique of RNA interference (RNAi) was used to investigate the regulation of p53-independent pathway in HepG2and L02cell lines. Methods:L02, L02-p53, HepG2and HepG2-p53cells were treated with different concentrations of MEHP (50.00and100.00μM) and DMSO (as the solvent control, final concentration:≤0.1%), respectively. The following indicators were measured at36h: the percentage of apoptotic cells, expression of p53, p-p53, MDM2, Bax, Bcl-2, PUMA, NOXA, Fas/FasL and Smac/DIABOL proteins, cytosolic Cytochrome c concentration, the activities of Caspase3,8,9. The results are as follows:
1. The p53silence efficiency in L02-p53and HepG2-p53cells by RNA interference
(1) To evaluate the p53silence efficiency, qRT-PCR method was used to measure the levels of the decreasing p53in in L02-p53and HepG2-p53cells. The results showed that in L02-p53cells the p53mRNA and protein levels was decreased by83.1%at24h and52.5%at48h, compared with the corresponding negative controls, respectively;(2) in HepG2-p53cells, the p53mRNA and protein levels were decreased by81.4%at24h and52.4%at48h, compared with the corresponding negative controls, respectively.
2. The cell Apoptosis
(1) At36h after treatment, MEHP increased the percentages of the apoptotic cells of both L02and L02-p53cells in all treatment groups (p<0.01for all). At36h after L02and L02-p53cells were treated with the same concentration of MEHP, the higher percentages of the L02-p53apoptotic cells were found in all treatment groups compared with the L02cells (p<0.01for all).(2) At36h after treatment, MEHP increased the percentages of both HepG2and HepG2-p53cells in all treatment groups (p<0.01for all). At36h after HepG2and HepG2-p53cells were treated with the same concentration of MEHP, the higher percentages of the HepG2-p53apoptotic cells were found in all treatment groups compared with the HepG2cells (p<0.01for all).
3. The expression of apoptosis-related proteins
(1) At36h after treatment of L02cells, MEHP up-regulated the expression of p53, p-p53, Bax, PUMA, NOXA, Fas and FasL proteins, but down-regulated the expression of MDM2and Bcl-2proteins in all treatment groups (p<0.01for all); at36h after the treatment of L02-p53cells, MEHP up-regulated the expression of MDM2, Bax, PUMA, NOXA, Fas and FasL proteins (p<0.01for all), but decreased the expression of p53, p-p53and Bcl-2proteins in all treatment groups, compared with the corresponding controls (p<0.01for all). At36h after treatments of L02and L02-p53cells at the same concentrations of MEHP, the results suggested that the expression of MDM2, Fas and FasL proteins were up-regulated in all treatment groups (p<0.01for all), but the expression of p53and p-p53proteins were down-regulated in all treatment groups (p<0.01for all). No changes in the levels of Bax, Bcl-2, PUMA and NOXA proteins were found in all treatment groups of either L02-p53or L02cells (p>0.05for all).
(2) At36h after treatment of HepG2cells, MEHP up-regulated the expression of p53, p-p53, Bax, PUMA, NOXA, Fas and FasL proteins, but down-regulated the expression of MDM2and Bcl-2protein in all treatment groups (p<0.01for all); at36h after treatment of HepG2-p53cells, MEHP up-regulated the expression of Bax, PUMA, NOXA, Fas and FasL proteins (p<0.01for all), but down-regulated the expression of Bcl-2protein compared with the corresponding negative controls (p<0.01for all). No changes in the levels of the p53, p-p53and MDM2proteins were found in all treatment groups of HepG2-p53cells (p>0.05for all). At36h after treatments of HepG2and HepG2-p53cells at the same concentrations of MEHP, the results suggested that the levels of MDM2, Fas and FasL proteins were up-regulated (p<0.01for all), but the levels of p53and p-p53protiens were down-regulated in all treatment groups (p<0.01for all). There was no changes in the levels of Bax, Bcl-2, PUMA and NOXA proteins in all treatment groups of either HepG2"p53or HepG2cells (p>0.05for all).
4. The levels of cytosolic Cytochrome c and Smac/DIABOL protein
(1) At36h after treatments of L02and L02-p53cells, MEHP increased the levels of cytosolic Cytochrome c in all treatment groups and the Smac/DIABOL protein in100.00μM treatment group compared with the corresponding controls (p<0.01for all).(2) At36h after treatment of HepG2and HepG2"p53cells, MEHP increased the levels of cytosolic Cytochrome c in100.00μM treatment group (p<0.01for all) and the Smac/DIABLO protein in all treatment groups (p<0.05or p<0.01), compared with the corresponding controls.
5. The activities of Caspase3,8, and9At36h after treatment, MEHP increased Caspase3,8,9activities in all treatment groups of L02, HepG2, L02-p53and HepG2-p53cells (p<0.01for all). At36h after treatments either between L02and L02-p53cells or between HepG2and HepG2"p53cells at the same concentrations of MEHP, the results suggested that MEHP induced the activities of Caspase3,8, and9in all treatment groups (p<0.05or p<0.01). Conclusions:The modulation of p53-mediated pathway is not only in MEHP-induced apoptosis in cell lines L02and HepG2. Fas/FasL pathway may partially replace the regulation of the p53-mediated mitochondrial apoptotic pathway in MEHP-induced apoptosis of the cell lines L02and HepG2.
The innovation points of this study:
(1) This is the first report on the modulation of p53-mediated mitochondrial pathway in MEHP-induced apoptosis in cell lines L02and HepG2.
(2) This is the first report on the Fas/FasL pathway may partially replace the regulation of the p53-mediated mitochondrial pathway in MEHP-induced apoptosis in cell lines L02and HepG2.
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