HMGB1在猪流行性腹泻病毒感染中作用机制的研究
发布时间：2019-05-29 20:09
【摘要】：猪流行性腹泻(porcine epidemic diarrhea, PED)是由猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)引起的一种以水样腹泻、呕吐、脱水为主要特征的猪肠道传染性疾病,给世界养猪业造成了巨大的经济损失。尽管对PEDV致病机制进行了一定的研究,取得了一系列有重要意义的成果,但在病毒的侵染机制及与宿主细胞之间的相互作用机制等方面,研究的较少,是近年来被广泛研究的热点。本研究主要针对HMGB1在PEDV感染中作用机制进行了深入探讨,系统分析了 PEDV感染与宿主蛋白HMGB1相互关系的机制。主要研究内容包括:1.甘草素对PEDV的抗病毒效果甘草素是一个来源于传统中药甘草的天然产物。通过western blot、荧光定量PCR和噬斑形成试验证明了甘草素具有抑制PEDV感染Vero细胞的效果。此外,探索了甘草素抑制PEDV感染的作用过程,发现甘草素对PEDV的入胞和复制有影响,而对细胞、病毒粒子本身、病毒的组装和释放没有影响。2. HMGB1在甘草素的抗病毒过程中起着重要的作用HMGB1是一个细胞中含量非常丰富的核蛋白,能作为一个重要的炎性介质释放到胞外空间调节宿主的炎症反应。我们发现甘草素能降低由PEDV感染引起的促炎性因子(IL-1β,IL-6,IL-8,TNF-α)的升高,且Poly(I:C)引起促炎性因子的升高且促进了PEDV的感染。甘草素是HMGB1的竞争性抑制剂。通过HMGB1抗体中和试验和siRNA干扰HMGB1表达,发现HMGB1在感染条件下对促炎性因子的升高起着重要的作用。HMGB1可与TLR4和RAGE等受体相结合,因此利用TLR4的抑制剂和RAGE的siRNA处理细胞,发现TLR4抑制剂和siRAGE能降低促炎性因子的升高和病毒的感染,且过表达HMGB1的突变体(PCA-C45S、PCA-C106S、PCA-C45106S)可降低促炎性因子的升高和PEDV的感染。此外,我们还证明了 HMGB1与TLR4和RAGE结合后可激活P38MAPK来诱导促炎性因子的升高进而有利于PEDV的感染。总之,甘草素与胞外HMGB1结合后,降低了其与TLR4和RAGE受体的结合,从而降低了P38MAPK的激活,使促炎性因子降低来抑制PEDV的增殖。3.硫酸类肝素是猪流行性腹泻病毒感染Vero细胞的吸附因子许多病毒利用硫酸类肝素作为它们的吸附因子。在该研究中,证明了 PEDV利用硫酸类肝素吸附Vero细胞。Western blot分析、荧光定量PCR和噬斑形成试验揭示被肝素预处理的病毒,共感染细胞的能力受到抑制;而用肝素预处理细胞,对PEDV的感染没有抑制效应。其次,我们发现肝素酶Ⅰ处理细胞去除硫酸类肝素后,细胞对PEDV的易感性下降。另外,我们证明抑制硫酸类肝素生物合成的氯酸钠处理细胞,能够有效抑制PEDV的感染。我们还检测了不同程度硫酸化的两个肝素类似物对PEDV感染的影响,数据表明N-去硫酸化肝素(不是N-乙酰化O-去硫酸化肝素)预处理病毒能够降低病毒感染细胞的能力。此外,肝素可抑制PEDV的复制和HMGB1 mRNA水平。总之,研究表明硫酸类肝素是PEDV感染Vero细胞的吸附因子。4. PEDV N蛋白通过与C/EBP-β相互作用来正调HMGB1的转录及乙酰化和释放HMGB1是一个重要的促炎性介质并且促进多种炎性疾病的发生。发现了 PEDV感染导致HMGB1的乙酰化和释放,PEDV的感染和PEDV衣壳蛋白通过SIRT1、组蛋白乙酰转移酶和NF-κB调节HMGB1的乙酰化。过表达PEDVN蛋白能促进HMGB1的乙酰化和释放。CHIP试验表明PEDVN能与HMGB1启动子区DNA相互作用。双荧光素酶报告基因试验证明了 PEDVN蛋白介导的HMGB1的转录与转录因子C/EBP结合基序有关。用Co-IP试验进一步表明病毒的衣壳蛋白与C/EBP-β相互作用来正调HMGB1基因的转录。总之,PEDV感染能引起HMGB1的乙酰化和释放,且主要是PEDV N蛋白起作用。我们的结果对PEDV的发病提供了新的视角并且为发展以HMGB1为靶点的治疗方法和药物提供了理论基础。5.猪流行性腹泻病毒通过DNA损伤引起HMGB1的释放由DNA病毒激活的DNA损伤反应被广泛的研究。然而,RNA病毒引起DNA损伤反应的研究很少。因此通过western blot和间接免疫荧光揭示了 PEDV快速激活H2AX的磷酸化,且HY增加了 PEDVN蛋白的表达。此外,通过western blot、qRT-PCR和噬斑形成试验证明DNA-PK抑制剂(NU7441)和ATM抑制剂(KU-55933 )抑制了 HMGB1的释放和PEDV的感染,且DNA-PK的抑制剂效果更好。但是ATR的抑制剂(VE-821)对PEDV感染没有影响。此外,PEDV引起PARP1的激活,PARP1的抑制剂3-AB抑制了 HMGB1的释放及PEDV的感染。DNA-PK抑制剂(NU7441 )和ATM抑制剂(KU-55933)抑制了 PARP1的活性,表明PEDV主要通过诱导DNA-PK依赖的DNA损伤和部分ATM依赖的DNA损伤激活PARP1和PEDV的增殖。通过流式细胞术证明了 PEDV引起ROS的升高,且抑制DNA损伤和PARP1的激活能降低ROS。总之,PEDV通过DNA损伤/PARP1/ROS引起HMGB1的释放从而有利于自身的复制。
[Abstract]:Porcine epidemic diarrhea (PED), which is caused by the porcine epidemic diarrhea virus (PEDV), is an infectious disease of the intestinal tract, which is characterized by watery diarrhea, vomiting and dehydration, and has caused great economic losses to the pig industry in the world. Although some research has been carried out on the pathogenesis of the PEDV, a series of important results have been obtained. However, the research on the mechanism of the infection of the virus and the mechanism of the interaction with the host cell is a hotspot in recent years. The mechanism of the relationship between the PEDV infection and the host protein HMGB1 was analyzed. The main research contents include:1. The antiviral effect of the glycyrrhizin on the PEDV is a natural product derived from the traditional Chinese medicine licorice. The results of western blot, fluorescence quantitative PCR and plaque formation demonstrated that the glycyrrhizin had the effect of inhibiting the infection of Vero cells with PEDV. In addition, the effect of glycyrrhizin on the inhibition of PEDV infection was explored, and it was found that the glycyrrhizin had an effect on the cell and replication of the PEDV, and no effect on the cell, viral particles and the assembly and release of the virus. HMGB1 plays an important role in the antiviral process of the glycyrrhizin, and the HMGB1 is a very abundant nucleoprotein in a cell and can be released into the extracellular space as an important inflammatory medium to regulate the inflammatory reaction of the host. We found that the increase of the pro-inflammatory factor (IL-1, IL-6, IL-8, TNF-1) caused by PEDV infection and the increase in the pro-inflammatory factor by Poly (I: C) and the promotion of the infection of PEDV. Licorice is a competitive inhibitor of HMGB1. HMGB1 was found to play an important role in the increase of the pro-inflammatory factor under the condition of infection by the HMGB1 antibody and the expression of HMGB1 in the test and siRNA. The HMGB1 can be combined with a receptor such as TLR4 and RAGE, so that the cell is treated with a TLR4 inhibitor and a siRNA of RAGE, and it is found that the TLR4 inhibitor and siRAGE can reduce the increase in the proinflammatory factor and the infection of the virus, and that the mutant (PCA-C45S, PCA-C106S, PCA-C45106S) overexpressing the HMGB1 may reduce the increase in the proinflammatory factor and the infection of the PEDV. In addition, we have shown that the combination of HMGB1 with TLR4 and RAGE can activate the P38MAPK to induce the increase of the pro-inflammatory factor, which is beneficial to the infection of the PEDV. In conclusion, after the combination of the licorice and the extracellular HMGB1, the combination with the TLR4 and the RAGE receptor is reduced, so that the activation of the P38MAPK is reduced, and the proinflammatory factor is reduced to inhibit the proliferation of the PEDV. The sulfate-like heparin is the adsorption factor of the Vero cells infected with the porcine epidemic diarrhea virus, and the sulfate-like heparin is used as the adsorption factor of the virus. In this study, it was demonstrated that PEDV was used to adsorb Vero cells using heparan sulfate. Western blot analysis, fluorescence quantitative PCR and plaque formation assay revealed that the ability of the co-infected cells to be pre-treated by heparin was inhibited; while the cells were pretreated with heparin, the infection of the PEDV was not inhibited. Second, we found that the susceptibility of the cells to the PEDV decreased after the heparin enzyme I was treated to remove the sulfate-like heparin. In addition, we have shown that the inhibition of the sodium chlorate-treated cells of the sulfate-like heparin biosynthesis can effectively inhibit the infection of the PEDV. We also examined the effect of two heparin analogs of varying degrees of sulfation on the PEDV infection, which indicated that the N-desulphated heparin (not N-ethionized O-desulphonated heparin) pretreated the virus to reduce the ability of the virus to infect cells. In addition, heparin can inhibit the replication of PEDV and the level of HMGB1 mRNA. In conclusion, the study shows that the sulfate-like heparin is an adsorption factor for the Vero cells infected with PEDV. The PEDV N protein is an important pro-inflammatory mediator by interacting with C/ EBP-1 to regulate the transcription of HMGB1 and to release HMGB1, and to promote the occurrence of a variety of inflammatory diseases. The infection of the PEDV and the envelope protein of the PEDV were detected by SIRT1, histone B-transferase and NF-B-B to regulate the ethylation of HMGB1. Overexpression of the PEDVN protein can promote the ethylation and release of HMGB1. The CHIP test shows that the PEDVN can interact with the DNA of the HMGB1 promoter region. The double luciferase reporter gene test demonstrated that the transcription of the PEDVN protein-mediated HMGB1 was related to the transcription factor C/ EBP binding motif. The interaction of the capsid protein of the virus with the C/ EBP-1 was further shown by the Co-IP test to regulate the transcription of the HMGB1 gene. In conclusion, PEDV infection can cause the ethylation and release of HMGB1, and is mainly the function of the PEDV N protein. Our results provide a new perspective for the development of PEDV and provide a theoretical basis for the development of therapeutic methods and drugs targeting HMGB1 as a target. The release of HMGB1 caused by the DNA damage of the porcine epidemic diarrhea virus is widely studied by the DNA damage reaction activated by the DNA virus. However, there are few studies on the reaction of DNA damage caused by the RNA virus. Therefore, by western blot and indirect immunofluorescence, the phosphorylation of H2AX was rapidly activated by PEDV, and the expression of PEDVN protein was increased by HY. In addition, the DNA-PK inhibitor (NU7441) and the ATM inhibitor (KU-55933) have been shown to inhibit the release of HMGB1 and the infection of the PEDV by western blot, qRT-PCR and plaque formation test, and the effect of the inhibitor of DNA-PK is better. However, the ATR inhibitor (VE-821) has no effect on the PEDV infection. In addition, PEDV is responsible for the activation of PARP1, and the inhibitor 3-AB of PARP1 inhibits the release of HMGB1 and the infection of the PEDV. The activity of PARP1 was inhibited by the DNA-PK inhibitor (NU7441) and the ATM inhibitor (KU-55933), indicating that the PEDV primarily activates the proliferation of PARP1 and PEDV by inducing DNA-PK-dependent DNA damage and partial ATM-dependent DNA damage. The increase of ROS caused by PEDV was demonstrated by flow cytometry, and the inhibition of DNA damage and the activation of PARP1 could reduce ROS. In conclusion, PEDV is responsible for the release of HMGB1 by DNA damage/ PARP1/ ROS to facilitate its own replication.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.28
，
本文编号：2488193
[Abstract]:Porcine epidemic diarrhea (PED), which is caused by the porcine epidemic diarrhea virus (PEDV), is an infectious disease of the intestinal tract, which is characterized by watery diarrhea, vomiting and dehydration, and has caused great economic losses to the pig industry in the world. Although some research has been carried out on the pathogenesis of the PEDV, a series of important results have been obtained. However, the research on the mechanism of the infection of the virus and the mechanism of the interaction with the host cell is a hotspot in recent years. The mechanism of the relationship between the PEDV infection and the host protein HMGB1 was analyzed. The main research contents include:1. The antiviral effect of the glycyrrhizin on the PEDV is a natural product derived from the traditional Chinese medicine licorice. The results of western blot, fluorescence quantitative PCR and plaque formation demonstrated that the glycyrrhizin had the effect of inhibiting the infection of Vero cells with PEDV. In addition, the effect of glycyrrhizin on the inhibition of PEDV infection was explored, and it was found that the glycyrrhizin had an effect on the cell and replication of the PEDV, and no effect on the cell, viral particles and the assembly and release of the virus. HMGB1 plays an important role in the antiviral process of the glycyrrhizin, and the HMGB1 is a very abundant nucleoprotein in a cell and can be released into the extracellular space as an important inflammatory medium to regulate the inflammatory reaction of the host. We found that the increase of the pro-inflammatory factor (IL-1, IL-6, IL-8, TNF-1) caused by PEDV infection and the increase in the pro-inflammatory factor by Poly (I: C) and the promotion of the infection of PEDV. Licorice is a competitive inhibitor of HMGB1. HMGB1 was found to play an important role in the increase of the pro-inflammatory factor under the condition of infection by the HMGB1 antibody and the expression of HMGB1 in the test and siRNA. The HMGB1 can be combined with a receptor such as TLR4 and RAGE, so that the cell is treated with a TLR4 inhibitor and a siRNA of RAGE, and it is found that the TLR4 inhibitor and siRAGE can reduce the increase in the proinflammatory factor and the infection of the virus, and that the mutant (PCA-C45S, PCA-C106S, PCA-C45106S) overexpressing the HMGB1 may reduce the increase in the proinflammatory factor and the infection of the PEDV. In addition, we have shown that the combination of HMGB1 with TLR4 and RAGE can activate the P38MAPK to induce the increase of the pro-inflammatory factor, which is beneficial to the infection of the PEDV. In conclusion, after the combination of the licorice and the extracellular HMGB1, the combination with the TLR4 and the RAGE receptor is reduced, so that the activation of the P38MAPK is reduced, and the proinflammatory factor is reduced to inhibit the proliferation of the PEDV. The sulfate-like heparin is the adsorption factor of the Vero cells infected with the porcine epidemic diarrhea virus, and the sulfate-like heparin is used as the adsorption factor of the virus. In this study, it was demonstrated that PEDV was used to adsorb Vero cells using heparan sulfate. Western blot analysis, fluorescence quantitative PCR and plaque formation assay revealed that the ability of the co-infected cells to be pre-treated by heparin was inhibited; while the cells were pretreated with heparin, the infection of the PEDV was not inhibited. Second, we found that the susceptibility of the cells to the PEDV decreased after the heparin enzyme I was treated to remove the sulfate-like heparin. In addition, we have shown that the inhibition of the sodium chlorate-treated cells of the sulfate-like heparin biosynthesis can effectively inhibit the infection of the PEDV. We also examined the effect of two heparin analogs of varying degrees of sulfation on the PEDV infection, which indicated that the N-desulphated heparin (not N-ethionized O-desulphonated heparin) pretreated the virus to reduce the ability of the virus to infect cells. In addition, heparin can inhibit the replication of PEDV and the level of HMGB1 mRNA. In conclusion, the study shows that the sulfate-like heparin is an adsorption factor for the Vero cells infected with PEDV. The PEDV N protein is an important pro-inflammatory mediator by interacting with C/ EBP-1 to regulate the transcription of HMGB1 and to release HMGB1, and to promote the occurrence of a variety of inflammatory diseases. The infection of the PEDV and the envelope protein of the PEDV were detected by SIRT1, histone B-transferase and NF-B-B to regulate the ethylation of HMGB1. Overexpression of the PEDVN protein can promote the ethylation and release of HMGB1. The CHIP test shows that the PEDVN can interact with the DNA of the HMGB1 promoter region. The double luciferase reporter gene test demonstrated that the transcription of the PEDVN protein-mediated HMGB1 was related to the transcription factor C/ EBP binding motif. The interaction of the capsid protein of the virus with the C/ EBP-1 was further shown by the Co-IP test to regulate the transcription of the HMGB1 gene. In conclusion, PEDV infection can cause the ethylation and release of HMGB1, and is mainly the function of the PEDV N protein. Our results provide a new perspective for the development of PEDV and provide a theoretical basis for the development of therapeutic methods and drugs targeting HMGB1 as a target. The release of HMGB1 caused by the DNA damage of the porcine epidemic diarrhea virus is widely studied by the DNA damage reaction activated by the DNA virus. However, there are few studies on the reaction of DNA damage caused by the RNA virus. Therefore, by western blot and indirect immunofluorescence, the phosphorylation of H2AX was rapidly activated by PEDV, and the expression of PEDVN protein was increased by HY. In addition, the DNA-PK inhibitor (NU7441) and the ATM inhibitor (KU-55933) have been shown to inhibit the release of HMGB1 and the infection of the PEDV by western blot, qRT-PCR and plaque formation test, and the effect of the inhibitor of DNA-PK is better. However, the ATR inhibitor (VE-821) has no effect on the PEDV infection. In addition, PEDV is responsible for the activation of PARP1, and the inhibitor 3-AB of PARP1 inhibits the release of HMGB1 and the infection of the PEDV. The activity of PARP1 was inhibited by the DNA-PK inhibitor (NU7441) and the ATM inhibitor (KU-55933), indicating that the PEDV primarily activates the proliferation of PARP1 and PEDV by inducing DNA-PK-dependent DNA damage and partial ATM-dependent DNA damage. The increase of ROS caused by PEDV was demonstrated by flow cytometry, and the inhibition of DNA damage and the activation of PARP1 could reduce ROS. In conclusion, PEDV is responsible for the release of HMGB1 by DNA damage/ PARP1/ ROS to facilitate its own replication.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.28
，
本文编号：2488193
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