本文選題：氯氰菊酯 + 凋亡　； 參考：《浙江大學》2017年博士論文

【摘要】：氯氰菊酯屬于Ⅱ型擬除蟲菊酯類殺蟲劑,被廣泛應(yīng)用于農(nóng)業(yè)生產(chǎn)及日常生活中害蟲的防治。近年來由于高毒有機磷和氨基甲酸醋農(nóng)藥的禁用,氯氰菊酯等高效、廣譜及對哺乳動物急性低毒的擬除蟲菊酯類農(nóng)藥的使用范圍不斷擴大,用量持續(xù)增加,由此造成的環(huán)境污染急劇加重。對該類農(nóng)藥的生產(chǎn)性和非生產(chǎn)性中毒病例已有報道,其相關(guān)的毒性機制研究也逐漸加深。眾所周知,免疫應(yīng)答和免疫功能失衡會造成多種疾病包括感染、腫瘤和自身免疫性疾病等的發(fā)生。先前研究已有一些關(guān)于氯氰菊酯免疫毒性的報道,如小鼠染毒氯氰菊酯后,免疫功能晝夜節(jié)律消失,表明免疫系統(tǒng)是氯氰菊酯的靶標之一。此外研究結(jié)果還顯示亞致死劑量的氯氰菊酯對動物的體液免疫和細胞免疫都能產(chǎn)生抑制作用。巨噬細胞作為機體固有免疫系統(tǒng)的重要成分之一,在炎癥、防御、修復、代謝等生理過程中發(fā)揮重要作用,同時也對機體維持自身穩(wěn)定非常關(guān)鍵。為了更加清楚的闡明氯氰菊酯的免疫毒性及其機制,本研究選用了小鼠巨噬細胞為模型,深入探討氯氰菊酯對巨噬細胞的毒性作用、極化影響及其相關(guān)分子機制。1.氯氰菊酯對巨噬細胞的毒性作用及機制研究首先我們的實驗參考氯氰菊酯防治柑橘害蟲的噴霧濃度范圍30～100mg/L,選用0-2000μM(相當83.2mg/L)的氯氰菊酯暴露巨噬細胞。研究顯示,100μM和2000μM氯氰菊酯暴露巨噬細胞48h時,MTT法檢測細胞活率相比對照組顯著下降。用流式細胞儀檢測氯氰菊酯能夠誘導巨噬細胞發(fā)生凋亡,且呈劑量效應(yīng)關(guān)系;同時我們還發(fā)現(xiàn)各氯氰菊酯暴露組細胞內(nèi)ROS高于對照組,尤其是200μM暴露組差異顯著。氯氰菊酯暴露還能使細胞周期阻滯于G1期以及DNA發(fā)生損傷。抗氧化劑N-乙酰半胱氨酸(NAC)對氯氰菊酯引起的細胞毒性具有明顯的保護作用,主要表現(xiàn)為:NAC能顯著增加細胞活率;NAC能明顯減輕細胞DNA損傷;NAC能部分抑制細胞凋亡的發(fā)生;NAC能抑制G1期細胞周期阻滯。我們還發(fā)現(xiàn)氯氰菊酯暴露使ERK1/2和JNK信號通路激活。加入NAC能抑制ERK1/2和JNK的激活。ERK1/2特異性抑制劑PD98059和JNK特異性抑制劑SP600125可有效抑制氯氰菊酯誘導的細胞凋亡。2.氯氰菊酯對巨噬細胞極化和功能的影響及相關(guān)機制巨噬細胞具有高度可塑性和異質(zhì)性。巨噬細胞在特定的微環(huán)境以及體內(nèi)外刺激作用下通常可以活化成兩種表型:被IFN-y和LPS刺激而激活分化成經(jīng)典的M1型,以及替代性活化的M2型。M1型巨噬細胞能殺滅細胞內(nèi)病原體和腫瘤細胞;M2型巨噬細胞可分泌免疫抑制因子和多種促進腫瘤生長的細胞因子,具有抑制炎癥反應(yīng)和促進腫瘤細胞生長與轉(zhuǎn)移的功能。這部分的研究是在氯氰菊酯暴露不影響巨噬細胞活率的前提下進行。氯氰菊酯處理巨噬細胞會抑制LPS誘導的M1型標志基因TNF-α,IL-6和iNOS分泌,表明氯氰菊酯會抑制M1型巨噬細胞極化;此外我們還發(fā)現(xiàn)氯氰菊酯會使巨噬細胞M2型標志分子Arg-1,Fizz1和Mg12表徶增加,并呈現(xiàn)氯氰菊酯劑量依賴效應(yīng),說明氯氰菊酯能促進M1型巨噬細胞向M2型極化。氯氰菊酯在誘導巨噬細胞M2極化過程中,主要是通過降低miRNA-155的表達。我們的研究也驗證了,如果過表達miRNA-155,會導致氯氰菊酯處理的巨噬細胞M2型標志分子表達下調(diào);而抑制了 miRNA-155,會引起氯氰菊酯處理的巨噬細胞M2型標志分子表達上調(diào)。同時我們還發(fā)現(xiàn)氯氰菊酯處理細胞使Bcl6表徶上調(diào)。通過將熒光素酶報告基因質(zhì)粒,分別和miRNA-155 mimics共轉(zhuǎn)染至293T細胞,結(jié)果顯示野生型Bcl6 3'-UTR pMIR-Bcl6熒光素酶報告基因活性在miR-155過表達細胞中明顯降低,而突變型Bcl6 3'-UTRpMIR-Bcl6-mut熒光素酶報告基因活性在miR-155過表達細胞中無明顯變化,這些結(jié)果說明miR-155能夠靶向結(jié)合Bcl6 3'-UTR區(qū)沉默Bcl6的表達。因此氯氰菊酯能夠通過降低miR-155的表達,逆轉(zhuǎn)了 miR-155對Bcl6的抑制作用,使Bcl6表達上調(diào)。當巨噬細胞沉默Bcl6的表達時,氯氰菊酯處理的巨噬細胞M2型相關(guān)基因Arg-1,Fizzl和Mgl2表徶下調(diào),相反,當巨噬細胞過表達Bcl6時,M2型相關(guān)的基因Arg-1和Mgl2表達上調(diào),這部分的結(jié)果說明Bcl6在氯氰菊酯介導的巨噬細胞由M1向M2極化中起著重要的作用。另外,我們還發(fā)現(xiàn)Bcl6可抑制MKK4的表達。當過表達Bcl6時,MKK4的表達被抑制,而MKK4是直接激活J-NK的激酶,即JNK活化也受到抑制;如果將Bcl6敲除,MKK4表達明顯升高,促進其下游的JNK磷酸化而激活。在細胞轉(zhuǎn)染miRNA-155前提下,同時敲除巨噬細胞MKK4的表達或者用JNK的抑制劑處理細胞時,與沒有敲除MKK4或者沒有加JNK抑制劑相比,氯氰菊酯誘導的M2型標志基因表達上調(diào),使M1型基因表達下調(diào)。當細胞轉(zhuǎn)染miRNA-155 inhibitor時,同時敲除Bcl6的表達,與沒有敲除Bcl6相比,氯氰菊酯誘導的M2型相關(guān)基因表達下調(diào),M1型相關(guān)基因表達上調(diào)。上述研究結(jié)果表明,氯氰菊酯可通過miRNA-155/Bcl6/MKK4/JNK通路調(diào)控巨噬細胞由M1向M2極化。體外遷移和體內(nèi)成瘤實驗驗證了氯氰菊酯對巨噬細胞功能的影響,氯氰菊酯所誘導的M2型巨噬細胞相比LPS誘導的M1型巨噬細胞,能顯著促進肺癌細胞的遷移和生長。綜合以上研究結(jié)果,我們可以得出以下結(jié)論:(1)氯氰菊酯長時間(48h)暴露可以劑量依賴性的抑制巨噬細胞活率;氯氰菊酯可以誘導ROS生成,NAC預處理能降低DNA損傷、抑制G1期細胞周期阻滯以及凋亡的發(fā)生。氯氰菊酯可以激活JNK和ERK1/2通路,并通過氧化應(yīng)激誘導JNK和ERK1/2通路的激活來引發(fā)凋亡。(2)氯氰菊酯在誘導巨噬細胞M2極化過程中,主要是通過降低miRNA-155的表達,從而使miRNA-155的靶基因Bcl6表達上調(diào),進而Bcl6作為轉(zhuǎn)錄抑制因子抑制MKK4的表達,最終導致JNK的活化減弱,使巨噬細胞由M1向M2極化,同時可增強巨噬細胞的促肺癌細胞遷移和生長功能。本研究結(jié)果能夠為系統(tǒng)的評估氯氰菊酯的免疫毒性提供依據(jù)。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R114

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