JNK抑制剂对PDGF诱导的肝星状细胞自噬的影响

发布时间：2018-05-16 10:45

  本文选题：肝星状细胞 + 自噬　； 参考：《河北医科大学》2014年硕士论文

【摘要】：肝纤维化(hepatic fibrosis, HF)是嗜肝病毒、酒精、药物和毒物等致病因素引起各种慢性肝病进而逐渐发展为肝硬化的必经病理过程，是机体对各种持续的急慢性肝损伤产生的一种损伤修复反应。肝星状细胞(hepaticstellate cells, HSCs)在肝纤维化的形成过程中发挥着关键作用。HSCs在正常情况时处于静止状态具有大脂滴结构，合成少量胶原，活化后的HSCs转化为肌成纤维样细胞，大脂滴结构消失同时自身增殖和胶原合成增加，这个活化过程与自噬的发生有关。自噬(autophagy)是细胞将自身损伤或过剩的结构通过溶酶体机制而被分解使之可再利用的过程。自噬的发生包括粗面内质网的无核糖体附着区、高尔基体等来源的双层磷脂结构的膜包裹部分胞质、细胞内需降解的细胞器和衰老或错误折叠的蛋白质等成分形成自噬体(autophagosome)，再通过与溶酶体融合利用其内的水解酶降解内容物，这个融合后的状态叫做自噬溶酶体(autolysosome)。在组织细胞受到各种理化因素损伤时，自噬性溶酶体大量增加，使细胞能够适应环境变化同时修复损伤。 C-Jun氨基末端激酶(c-Jun N-terminal kinase, JNK)是丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)家族中的一员。JNK的激活通过三级激酶模式，包括MAP kinase kinase kinases (MAP3Ks orMKKKs)，MAP kinase kinases (MAP2Ks or MKKs)和MAP kinases (JNKs)。JNK可以被许多刺激激活，包括细胞因子、活性氧(ROS)、病原体、毒素、药物、内质网应激、游离脂肪酸和代谢环境改变。血小板衍生生长因子(platelet-derived growth factor, PDGF)是HSCs最强有力的促有丝分裂因子。肝纤维化时PDGF及其受体表达量均有增加，PDGF可以与其受体结合后激活受体，活化的PDGF受体与许多能够激活RaS的信号分子有很强的亲和力，通过间接激活RaS从而可以进一步激活下游的Raf，进而激活包括MAPKs在内的多条信号通路。目前已知的JNK下游蛋白至少有40种。其中，c-Jun（一种转录调节因子，属亮氨酸拉链家族成员）是特征性的下游蛋白，可以与JunB、JunD或者Fos结合参与转录因子AP-1的形成，实现包括细胞代谢、增殖、分化等在内的多种生物学效应。 迄今，关于JNK与HSCs的自噬之间的关系的研究甚少，对于JNK的干预能否成为抗纤维化治疗的一个靶点？为此，我们运用JNK抑制剂SP600125特异性阻断JNK活性，同时在PDGF诱导下，对JNK在HSCs自噬中的作用做了初步研究。 目的：通过应用JNK抑制剂SP600125探讨JNK在PDGF诱导肝星状细胞自噬及活化中的作用。 方法：应用体外HSCs培养技术，利用PDGF-BB刺激人肝星状细胞系HSC-LX2后，以一定浓度的SP600125阻断JNK通路。分组如下①对照组；②P DGF组；③S P600125组；④SP600125+PDGF组。采用免疫细胞化学方法检测HSCs中α-SMA的表达，Westem bloting方法检测JNK、p-JNK、c-Jun、p-c-Jun、α-SMA、LC3BⅡ（检测自噬发生的标志性蛋白）、LC3BⅠ、Agt5和Beclin1（自噬起始阶段的两个关键蛋白）蛋白表达，，RT-PCR法检测Agt5和Beclin1的基因水平的表达；采用吖啶橙染色荧光显微镜观察HSCs自噬情况。 结果： 1SP600125对PDGF诱导的HSCs的JNK和c-Jun磷酸化具有抑制作用。应用Western blot技术检测各组JNK和p-JNK、c-Jun和p-c-Jun蛋白的表达情况。统计p-JNK与JNK、p-c-Jun与c-Jun蛋白灰度的比值，PDGF组较对照组JNK、c-Jun磷酸化水平明显升高(0.77±0.05vs0.34±0.02,0.92±0.52vs0.51±0.05)(p0.01)，SP600125+PDGF组和SP600125组分别较PDGF组和对照组JNK (0.39±0.04vs0.77±0.05,0.24±0.02vs0.34±0.02)、c-Jun (0.21±0.04vs0.92±0.52,0.14±0.01vs0.51±0.05)磷酸化水平显著降低(p0.05)。 2SP600125对PDGF诱导的HSCs的自噬具有抑制作用。应用吖啶橙染色方法结果显示，PDGF组较对照组红色荧光面积明显升高(130.0±24.1vs54.6±8.3)(p0.05)，SP600125+PDGF组较PDGF组红色荧光的面积显著降低(50.2±6.6vs130.0±24.1)(p0.05)；应用Western blot技术检测各组LC3BⅡ、LC3BⅠ、Beclin-1和Atg-5蛋白的表达情况，统计LC3BⅡ与LC3BⅠ的比值和Beclin-1和Atg-5蛋白表达量。PDGF组较对照组LC3BⅡ与LC3BⅠ的比值、Beclin-1和Atg-5蛋白的表达均明显升高(1.29±0.21vs0.66±0.05,746.10±46.33vs310.20±31.80,1778.00±130.00vs562.90±60.90)(p0.05)。SP600125+PDGF组和SP600125组分别较PDGF组和对照组LC3BⅡ与LC3BⅠ的比值(0.35±0.03vs1.29±0.21,0.33±0.05vs0.66±0.05)、Beclin-1蛋白的表达(197.50±26.99vs746.10±46.33,133.90±17.96vs310.20±31.80)均显著降低(p0.05)。SP600125+PDGF组较PDGF组Atg-5蛋白的表达显著降低(714.20±47.75vs1778.00±130.00)(p0.01)，而SP600125组较对照组Atg-5蛋白的表达量无显著性差异(p0.05)；应用Real-time PCR方法检测Beclin-1mRNA和Atg-5mRNA的表达，其结果与Western blot结构一致。 3SP600125能够抑制HSCs α-SMA的表达。应用免疫细胞化学和Western blot检测α-SMA蛋白的表达情况。PDGF组较对照组α-SMA蛋白的表达水平明显升高(1566.00±116.50vs757.70±72.87)(p0.01)，SP600125+PDGF组较PDGF组α-SMA蛋白的表达显著降低(582.10±60.48vs1566.00±116.50)(p0.01)。 结论：特异性地阻断JNK通路能够抑制PDGF诱导的肝星状细胞的自噬，抑制肝星状细胞活化。
[Abstract]:Hepatic fibrosis (HF) is an essential pathological process that causes various chronic liver diseases, such as hepatoviruses, alcohol, drugs and toxicants, and then gradually develops into liver cirrhosis. It is a kind of injury repair response to various persistent acute and chronic liver damage. The hepatic stellate cells (hepaticstellate cells, HSCs) are in the liver fiber. In the process of formation,.HSCs plays a key role in the normal condition with a large lipid droplet structure, a small amount of collagen is synthesized, and the activated HSCs is converted into myofibroblast like cells. The large fat droplet structure disappears and increases in self proliferation and collagen synthesis. This activation process is related to the occurrence of autophagy. Autophagy (autophagy) is thin. The process of decomposing the structure of its own damage or excess through the lysosome mechanism. The occurrence of autophagy includes the unribosomal attachment area of the rough endoplasmic reticulum, the membrane of the bilayer phospholipid structure, such as the Golgi body, the part of the cytoplasm, the cellular organelles that degrade in the cells, and the aging or erroneous folding proteins. The formation of autophagosome (autophagosome) is formed by fusion of lysosomes with lysosomes to degrade content. The fusion state is called autophagic lysosome (autolysosome). When tissue cells are damaged by various physical and chemical factors, autophagic lysosomes are greatly increased to enable cells to adapt to environmental changes and repair damage.
The C-Jun amino terminal kinase (c-Jun N-terminal kinase, JNK) is a member of the mitogen activated protein kinase (mitogen-activated protein kinase, MAPK) family, which is activated by the three stage kinase mode, including MAP kinase. Many stimuli are activated, including cytokines, reactive oxygen species (ROS), pathogens, toxins, drugs, endoplasmic reticulum stress, free fatty acids and metabolic environment changes. Platelet-derived growth factor (PDGF) is the most powerful fibroblast stimulating factor of HSCs. The expression of PDGF and its receptor in liver fibrosis is increased, PDGF can be increased. Activating the receptor after binding to its receptor, the activated PDGF receptor has a strong affinity with many signal molecules that can activate RaS. By activating the RaS indirectly, the downstream Raf can be activated further, and then multiple signal pathways, including MAPKs, are activated. At least 40 known downstream proteins of the JNK are known, of which, c-Jun (a transcription) Regulatory factor, a member of the leucine zipper family) is a characteristic downstream protein that can be combined with JunB, JunD or Fos to participate in the formation of transcription factor AP-1, and to achieve a variety of biological effects, including cell metabolism, proliferation, and differentiation.
So far, there is little research on the relationship between autophagy between JNK and HSCs. Can the intervention of JNK be a target for anti fibrosis therapy? To this end, we use JNK inhibitor SP600125 to specifically block the activity of JNK. At the same time, we have made a preliminary study on the use of JNK in HSCs autophagy under the induction of PDGF.
Objective: To explore the role of JNK in PDGF induced autophagy and activation of hepatic stellate cells by using JNK inhibitor SP600125.
Methods: using the HSCs culture technique in vitro and using PDGF-BB to stimulate the human hepatic stellate cell line HSC-LX2, the JNK pathway was blocked with a certain concentration of SP600125. The groups were grouped as follows: the control group, (2) P DGF group; (3) S P600125 group; (4) SP600125+PDGF group. The expression of alpha -SMA in HSCs was detected by immunocytochemical method. P-JNK, c-Jun, p-c-Jun, alpha -SMA, LC3B II (detection of autophagy), LC3B I, Agt5 and Beclin1 (two key proteins at the beginning of autophagy) protein expression, RT-PCR method for detecting the expression of Agt5 and Beclin1 gene levels, and using acridine orange staining fluorescence microscopy to observe the status of HSCs autophagy.
Result锛

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