氯氰菊酯对巨噬细胞的毒性作用和极化的影响及其相关机制

发布时间：2018-05-26 09:23

本文选题：氯氰菊酯 + 凋亡　；参考：《浙江大学》2017年博士论文

【摘要】：氯氰菊酯属于Ⅱ型拟除虫菊酯类杀虫剂,被广泛应用于农业生产及日常生活中害虫的防治。近年来由于高毒有机磷和氨基甲酸醋农药的禁用,氯氰菊酯等高效、广谱及对哺乳动物急性低毒的拟除虫菊酯类农药的使用范围不断扩大,用量持续增加,由此造成的环境污染急剧加重。对该类农药的生产性和非生产性中毒病例已有报道,其相关的毒性机制研究也逐渐加深。众所周知,免疫应答和免疫功能失衡会造成多种疾病包括感染、肿瘤和自身免疫性疾病等的发生。先前研究已有一些关于氯氰菊酯免疫毒性的报道,如小鼠染毒氯氰菊酯后,免疫功能昼夜节律消失,表明免疫系统是氯氰菊酯的靶标之一。此外研究结果还显示亚致死剂量的氯氰菊酯对动物的体液免疫和细胞免疫都能产生抑制作用。巨噬细胞作为机体固有免疫系统的重要成分之一,在炎症、防御、修复、代谢等生理过程中发挥重要作用,同时也对机体维持自身稳定非常关键。为了更加清楚的阐明氯氰菊酯的免疫毒性及其机制,本研究选用了小鼠巨噬细胞为模型,深入探讨氯氰菊酯对巨噬细胞的毒性作用、极化影响及其相关分子机制。1.氯氰菊酯对巨噬细胞的毒性作用及机制研究首先我们的实验参考氯氰菊酯防治柑橘害虫的喷雾浓度范围30～100mg/L,选用0-2000μM(相当83.2mg/L)的氯氰菊酯暴露巨噬细胞。研究显示,100μM和2000μM氯氰菊酯暴露巨噬细胞48h时,MTT法检测细胞活率相比对照组显著下降。用流式细胞仪检测氯氰菊酯能够诱导巨噬细胞发生凋亡,且呈剂量效应关系;同时我们还发现各氯氰菊酯暴露组细胞内ROS高于对照组,尤其是200μM暴露组差异显著。氯氰菊酯暴露还能使细胞周期阻滞于G1期以及DNA发生损伤。抗氧化剂N-乙酰半胱氨酸(NAC)对氯氰菊酯引起的细胞毒性具有明显的保护作用,主要表现为:NAC能显著增加细胞活率;NAC能明显减轻细胞DNA损伤;NAC能部分抑制细胞凋亡的发生;NAC能抑制G1期细胞周期阻滞。我们还发现氯氰菊酯暴露使ERK1/2和JNK信号通路激活。加入NAC能抑制ERK1/2和JNK的激活。ERK1/2特异性抑制剂PD98059和JNK特异性抑制剂SP600125可有效抑制氯氰菊酯诱导的细胞凋亡。2.氯氰菊酯对巨噬细胞极化和功能的影响及相关机制巨噬细胞具有高度可塑性和异质性。巨噬细胞在特定的微环境以及体内外刺激作用下通常可以活化成两种表型:被IFN-y和LPS刺激而激活分化成经典的M1型,以及替代性活化的M2型。M1型巨噬细胞能杀灭细胞内病原体和肿瘤细胞;M2型巨噬细胞可分泌免疫抑制因子和多种促进肿瘤生长的细胞因子,具有抑制炎症反应和促进肿瘤细胞生长与转移的功能。这部分的研究是在氯氰菊酯暴露不影响巨噬细胞活率的前提下进行。氯氰菊酯处理巨噬细胞会抑制LPS诱导的M1型标志基因TNF-α,IL-6和iNOS分泌,表明氯氰菊酯会抑制M1型巨噬细胞极化;此外我们还发现氯氰菊酯会使巨噬细胞M2型标志分子Arg-1,Fizz1和Mg12表徶增加,并呈现氯氰菊酯剂量依赖效应,说明氯氰菊酯能促进M1型巨噬细胞向M2型极化。氯氰菊酯在诱导巨噬细胞M2极化过程中,主要是通过降低miRNA-155的表达。我们的研究也验证了,如果过表达miRNA-155,会导致氯氰菊酯处理的巨噬细胞M2型标志分子表达下调;而抑制了 miRNA-155,会引起氯氰菊酯处理的巨噬细胞M2型标志分子表达上调。同时我们还发现氯氰菊酯处理细胞使Bcl6表徶上调。通过将荧光素酶报告基因质粒,分别和miRNA-155 mimics共转染至293T细胞,结果显示野生型Bcl6 3'-UTR pMIR-Bcl6荧光素酶报告基因活性在miR-155过表达细胞中明显降低,而突变型Bcl6 3'-UTRpMIR-Bcl6-mut荧光素酶报告基因活性在miR-155过表达细胞中无明显变化,这些结果说明miR-155能够靶向结合Bcl6 3'-UTR区沉默Bcl6的表达。因此氯氰菊酯能够通过降低miR-155的表达,逆转了 miR-155对Bcl6的抑制作用,使Bcl6表达上调。当巨噬细胞沉默Bcl6的表达时,氯氰菊酯处理的巨噬细胞M2型相关基因Arg-1,Fizzl和Mgl2表徶下调,相反,当巨噬细胞过表达Bcl6时,M2型相关的基因Arg-1和Mgl2表达上调,这部分的结果说明Bcl6在氯氰菊酯介导的巨噬细胞由M1向M2极化中起着重要的作用。另外,我们还发现Bcl6可抑制MKK4的表达。当过表达Bcl6时,MKK4的表达被抑制,而MKK4是直接激活J-NK的激酶,即JNK活化也受到抑制;如果将Bcl6敲除,MKK4表达明显升高,促进其下游的JNK磷酸化而激活。在细胞转染miRNA-155前提下,同时敲除巨噬细胞MKK4的表达或者用JNK的抑制剂处理细胞时,与没有敲除MKK4或者没有加JNK抑制剂相比,氯氰菊酯诱导的M2型标志基因表达上调,使M1型基因表达下调。当细胞转染miRNA-155 inhibitor时,同时敲除Bcl6的表达,与没有敲除Bcl6相比,氯氰菊酯诱导的M2型相关基因表达下调,M1型相关基因表达上调。上述研究结果表明,氯氰菊酯可通过miRNA-155/Bcl6/MKK4/JNK通路调控巨噬细胞由M1向M2极化。体外迁移和体内成瘤实验验证了氯氰菊酯对巨噬细胞功能的影响,氯氰菊酯所诱导的M2型巨噬细胞相比LPS诱导的M1型巨噬细胞,能显著促进肺癌细胞的迁移和生长。综合以上研究结果,我们可以得出以下结论:(1)氯氰菊酯长时间(48h)暴露可以剂量依赖性的抑制巨噬细胞活率;氯氰菊酯可以诱导ROS生成,NAC预处理能降低DNA损伤、抑制G1期细胞周期阻滞以及凋亡的发生。氯氰菊酯可以激活JNK和ERK1/2通路,并通过氧化应激诱导JNK和ERK1/2通路的激活来引发凋亡。(2)氯氰菊酯在诱导巨噬细胞M2极化过程中,主要是通过降低miRNA-155的表达,从而使miRNA-155的靶基因Bcl6表达上调,进而Bcl6作为转录抑制因子抑制MKK4的表达,最终导致JNK的活化减弱,使巨噬细胞由M1向M2极化,同时可增强巨噬细胞的促肺癌细胞迁移和生长功能。本研究结果能够为系统的评估氯氰菊酯的免疫毒性提供依据。
[Abstract]:Cypermethrin is a pyrethroid insecticide type II, which is widely used in agricultural production and pest control in daily life. In recent years, the use of high toxic organophosphorus and carbamate pesticides, cypermethrin and other highly efficient, broad-spectrum and acute and low toxic pyrethroid pesticides in mammals have been widely used. The continued increase, resulting in a sharp increase in environmental pollution, has been reported in cases of productive and non productive poisoning of this kind of pesticide, and its related toxic mechanism has been gradually deepened. It is known that the immune response and immune function imbalance can cause a variety of diseases including infection, tumor and autoimmune diseases. There are some reports about the immunotoxicity of Cypermethrin, such as the cypermethrin in mice, the circadian rhythm of the immune function disappears, indicating that the immune system is one of the targets of cypermethrin. In addition, the sublethal dose of Cypermethrin can inhibit the humoral and cellular immunity of animals. As one of the important components of the body's inherent immune system, it plays an important role in the physiological processes such as inflammation, defense, repair and metabolism, and it is also crucial for the body to maintain its own stability. In order to clarify the immune toxicity and mechanism of Cypermethrin, the mouse macrophage is selected as a model to explore cyanogen in depth. Toxic effects of permethrin on macrophages, polarization effect and related molecular mechanism.1. cypermethrin's toxicity and mechanism to macrophages. First of all, our experimental reference to the spray concentration of Cypermethrin against citrus pests ranged from 30 to 100mg/L, and 0-2000 micron (quite 83.2mg/L) of Cypermethrin exposed macrophages. When 100 M and 2000 M cypermethrin exposed macrophage 48h, the cell viability was significantly lower than that in the control group. Cypermethrin detected by flow cytometry could induce macrophage apoptosis and showed a dose effect relationship. At the same time, we found that the intracellular ROS of permethrin exposed group was higher than that of the control group, especially the 200 micron M storm. The exposure group was significantly different. Cypermethrin exposure could block the cell cycle in G1 and DNA. The antioxidant N- acetylcysteine (NAC) has a significant protective effect on Cypermethrin induced cytotoxicity. The main manifestation is that NAC can significantly increase the cell viability; NAC can significantly reduce the cell DNA damage; NAC can partially inhibit the refinement of the cell. NAC can inhibit cell cycle arrest in G1 phase. We also found that cypermethrin exposes ERK1/2 and JNK signaling pathways. Addition of NAC can inhibit ERK1/2 and JNK activation of.ERK1/2 specific inhibitors PD98059 and JNK specific inhibitors, SP600125 can effectively inhibit cypermethrin induced apoptosis of Cypermethrin to macrophages The effects of cell polarization and function and related mechanisms of macrophages have high plasticity and heterogeneity. Macrophages can normally be activated into two types of phenotypes under specific microenvironment and in vivo and in vitro: stimulated by IFN-y and LPS and differentiated into classical M1 type, and the replacement of M2 type.M1 macrophages can be killed. Intracellular pathogens and tumor cells; M2 macrophages secrete immunosuppressive factors and a variety of cytokines that promote tumor growth. It has the function of inhibiting inflammatory response and promoting tumor cell growth and metastasis. This part of this study is conducted on the premise of Cypermethrin exposure without affecting the macrophage survival rate. Cypermethrin treatment Macrophages inhibit the LPS induced M1 marker gene TNF- alpha, IL-6 and iNOS secretion, indicating that cypermethrin inhibits the polarization of M1 type macrophages. In addition, cypermethrin may increase the M2 type molecule Arg-1, Fizz1 and Mg12, and present a dose-dependent effect of Cypermethrin, indicating that cypermethrin can promote M1 type. Macrophages are polarized to M2 type. Cypermethrin is mainly induced by reducing the expression of miRNA-155 in the process of inducing M2 polarization in macrophages. Our study also demonstrated that if miRNA-155 was expressed, the expression of M2 marker molecules in cypermethrin treated macrophages was downregulated, and miRNA-155 was inhibited, which could cause the giant cypermethrin treatment. The expression of phagocytic M2 type marker was up-regulated. We also found that cypermethrin treated cells up-regulated the Bcl6. By CO transfection of luciferase reporter gene plasmid and miRNA-155 mimics to 293T cells, the results showed that the activity of the wild type Bcl6 3'-UTR pMIR-Bcl6 luciferase reporter gene was obvious in the miR-155 overexpressed cells. The activity of the mutant Bcl6 3'-UTRpMIR-Bcl6-mut luciferase reporter gene has no obvious changes in miR-155 overexpressed cells. These results indicate that miR-155 can target the expression of Bcl6 in the Bcl6 3'-UTR region. Cypermethrin can reverse the inhibition of miR-155 to Bcl6 by reducing the expression of miR-155, so that the Bcl6 table is made. When macrophages silenced the expression of Bcl6, the M2 related gene Arg-1, Fizzl and Mgl2 down regulation of Cypermethrin treated macrophages, on the contrary, when macrophages overexpressed Bcl6, the M2 related gene Arg-1 and Mgl2 were up-regulated. This part showed that Bcl6 in cypermethrin mediated macrophages from M1 to M2 polarization In addition, we also found that Bcl6 inhibits the expression of MKK4. When Bcl6 is expressed, the expression of MKK4 is inhibited, and MKK4 is a kinase that activates J-NK directly, that is, the activation of JNK is also inhibited. If Bcl6 knocks, MKK4 expression increases obviously and activates the downstream JNK phosphorylation. When the expression of macrophage MKK4 was knocked out or treated with a JNK inhibitor, the expression of the M2 type marker gene induced by cypermethrin was up-regulated and the expression of the M1 gene was down regulated compared with no knockout of MKK4 or without a JNK inhibitor. When cells transfected to miRNA-155 inhibitor, the expression of Bcl6 was knocked out at the same time, and cyanogen was compared with no knockout Bcl6. The expression of M2 type related genes induced by permethrin was down, and the expression of M1 type related genes was up-regulated. The results showed that cypermethrin could regulate the polarization of macrophages from M1 to M2 through the miRNA-155/Bcl6/MKK4/JNK pathway. In vitro migration and in vivo tumorigenesis test the effect of Cypermethrin on macrophage cell function, the M2 type induced by cypermethrin Macrophages compared with LPS induced M1 macrophages can significantly promote the migration and growth of lung cancer cells. Combined with the above results, we can conclude that (1) cypermethrin exposure (48h) exposure can inhibit macrophage viability in a dose-dependent manner; cypermethrin can induce ROS generation, and NAC pretreatment can reduce DNA damage. Inhibition of cell cycle arrest and apoptosis in G1 phase. Cypermethrin activates JNK and ERK1/2 pathways and induces apoptosis through oxidative stress induced activation of JNK and ERK1/2 pathways. (2) cypermethrin mainly induces the Bcl6 expression of the target gene of miRNA-155 in the process of inducing M2 polarization in macrophages, mainly by reducing the expression of miRNA-155. Up - regulation, and then Bcl6 as a transcriptional inhibiting factor inhibits the expression of MKK4 and eventually leads to the activation of JNK, which makes macrophages from M1 to M2, and enhances the migration and growth function of lung cancer cells in macrophages. The results of this study can provide a basis for the systematic assessment of the immunotoxicity of cypermethrin.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R114

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