【目的】探究猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)感染对先天性免疫的影响及免疫信号传导的干扰机制,重点解析信号传导转录激活因子1(signal transducer and activator of transcription 1,STAT1)与三方基序蛋白21(tripar...【目的】探究猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)感染对先天性免疫的影响及免疫信号传导的干扰机制,重点解析信号传导转录激活因子1(signal transducer and activator of transcription 1,STAT1)与三方基序蛋白21(tripartite-motif protein 21,TRIM21)在此过程中的作用。【方法】通过倒置显微镜观察感染复数(multiplicity of infection,MOI)为1的PDCoV感染IPEC-J2细胞1、12、24、48 h后细胞形态学变化,采用实时荧光定量PCR和Western blotting分别检测PDCoV-N基因及蛋白的表达水平,以此筛选PDCoV感染IPEC-J2细胞模型的最佳条件。在PDCoV感染峰值时,通过实时荧光定量PCR和Western blotting检测STAT1、TRIM21及干扰素刺激基因(interferon-stimulated genes,ISGs)的mRNA和蛋白表达水平;通过双荧光素酶报告基因系统检测过表达或抑制STAT1对PDCoV复制及干扰素激活反应元件(interferon-stimulated response element,ISRE)启动子活性的影响;将TRIM21与STAT1真核表达载体共转染至IPEC-J2细胞,结合激光共聚焦显微镜、免疫共沉淀技术(coimmunoprecipitation,Co-IP)和Western blotting验证二者的相互作用及TRIM21对STAT1的调控效应。【结果】PDCoV在感染IPEC-J2细胞24 h达复制高峰,同时试验组细胞中STAT1、TRIM21蛋白表达及ISGs转录水平均极显著高于对照组(P<0.01)。与对照组相比,过表达STAT1后ISRE启动子活性极显著增强,PDCoV-N基因及其蛋白表达量极显著降低(P<0.01),而氟达拉滨抑制STAT1后则极显著促进病毒复制(P<0.01)。激光共聚焦显微镜观察与Co-IP结果显示,STAT1与TRIM21存在共定位及相互作用,且与对照组相比,过表达TRIM21后STAT1蛋白表达量极显著降低(P<0.01)。【结论】本研究解析了PDCoV与宿主IPEC-J2细胞先天性免疫的相互作用机制,STAT1作为关键抗病毒因子通过ISRE通路增强先天性免疫来抑制病毒复制,TRIM21则靶向STAT1并下调其表达从而帮助病毒免疫逃逸。研究结果不仅为深入理解PDCoV的致病分子机制提供了新见解,也为制定PDCoV防控策略奠定了基础。展开更多
信号转导子和转录激活子1(signal transducer and activator of transcription 1,STAT1)作为JAK-STAT信号通路的核心成员,通过调控干扰素和其他细胞因子的信号转导,在肿瘤免疫微环境(tumor immune microenvironment,TIME)中起双重作用。...信号转导子和转录激活子1(signal transducer and activator of transcription 1,STAT1)作为JAK-STAT信号通路的核心成员,通过调控干扰素和其他细胞因子的信号转导,在肿瘤免疫微环境(tumor immune microenvironment,TIME)中起双重作用。STAT1激活一方面可以促进抗肿瘤免疫反应,增强T细胞、自然杀伤细胞和巨噬细胞的杀伤功能;另一方面,STAT1的持续激活可能会诱发免疫抑制性微环境的形成,促使肿瘤发生免疫逃逸。本综述着重介绍STAT1在肿瘤免疫微环境中的调控机制及其对免疫治疗的影响,并对靶向STAT1的潜在治疗策略进行了探讨。展开更多
Objective Inflammation and fibrosis are key features of diabetic nephropathy(DN).Triptolide(TP)exhibits anti-inflammatory and anti-fibrotic properties,though its mechanisms of action in DN remain unclear.CREKA(Cys-Arg...Objective Inflammation and fibrosis are key features of diabetic nephropathy(DN).Triptolide(TP)exhibits anti-inflammatory and anti-fibrotic properties,though its mechanisms of action in DN remain unclear.CREKA(Cys-Arg-Glu-Lys-Ala)is a pentapeptide that specifically binds to fibronectin(FN),and the CREKA-modified liposome(CREKA-Lip)represents a novel FN-targeted drug delivery system.This study aimed to investigate the role of TP in diabetic db/db mice and determine whether encapsulation within CREKA-Lip enhances therapeutic efficacy while reducing the multi-organ toxicity of TP.Methods Eight-week-old diabetic db/db mice received tail vein injections twice weekly with vehicle,free TP,or CREKA-Lip/TP for 10 weeks.Urine and serum parameters were measured,and kidney,heart,liver,and testis tissues were collected for pathological evaluation.Protein-protein interaction networks were constructed using Cytoscape and its plug-ins to identify core targets and elucidate the therapeutic mechanism of TP against DN.Inflammatory,fibrotic,apoptotic,and lipid metabolism markers were evaluated in the kidneys of diabetic mice with DN and in high glucose-treated mouse mesangial cells and podocytes using qPCR,Western blot,immunohistochemistry,and immunofluorescence assays.Results TP administration reduced fasting blood glucose levels and glomerular mesangial expansion in diabetic mice.TP significantly suppressed renal inflammation,fibrosis,and apoptosis while enhancing lipid metabolism.Integration of network pharmacology,molecular docking,and transcriptomics revealed that TP ameliorated DN by inhibiting the JAK2-STAT1 signaling pathway.In vitro,TP inhibited high glucose-induced phosphorylation of JAK2 and STAT1,reduced collagen production in mesangial cells,decreased apoptosis,and improved lipid metabolism in podocytes.Moreover,CREKA-Lip/TP exhibited superior efficacy compared with free TP,with a more sustained reduction in urine albumin-to-creatinine ratio and greater inhibition of mesangial expansion.Notably,CREKA-Lip/TP treatment did not induce systemic toxicity.Conclusion TP improves renal inflammation,fibrosis,apoptosis,and lipid homeostasis,thereby ameliorating DN by inhibiting JAK2-STAT1 activation.Targeted delivery of TP via FN-binding CREKA-Lip enhances therapeutic efficacy while minimizing multi-organ toxicity.展开更多
Background:Intrahepatic cholangiocarcinoma(ICC)is the second most frequent primary liver cancer.The involvement of Y-box binding protein 1(YBX1)in tumor advancement is well-documented.However,its function in ICC is no...Background:Intrahepatic cholangiocarcinoma(ICC)is the second most frequent primary liver cancer.The involvement of Y-box binding protein 1(YBX1)in tumor advancement is well-documented.However,its function in ICC is not fully understood.This study aimed to explore the function and regulatory mechanism of YBX1 in ICC and provide evidence for YBX1 as a potential new approach for immunotherapy in ICC.Methods:Tissue immunohistochemistry,TCGA,and GEO databases were used to analyze the expression of YBX1 in ICC.The expression of YBX1 was silenced and overexpressed in cell lines.Both in vitro and in vivo assays were conducted to examine the antitumor T-cell responses.Actinomycin D,RNA immunoprecipitation,and methylated RNA immunoprecipitation assays were used to identify mechanism of YBX1 on downstream genes.Immunofluorescence assay was used to validate the association between YBX1 and relevant genes in clinical specimens of ICC.Results:The research findings indicated that ICC exhibited high levels of YBX1 expression,which was strongly associated with unfavorable outcomes.YBX1 promoted tumor progression by suppressing antitumor T-cell responses.YBX1 enhanced signal transducer and activator of transcription 1(STAT1)translation by serving as a 5-methylated cytosine(m5C)reader and activating the STAT1/PD-L1 pathway.Mouse experiments and clinical samples of ICC confirmed the strong correlation between the levels of YBX1,STAT1,and PD-L1 expression.Conclusions:YBX1 regulates STAT1 stability in an m5C dependent manner and maintains PD-L1 expression in ICC.展开更多
文摘【目的】探究猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)感染对先天性免疫的影响及免疫信号传导的干扰机制,重点解析信号传导转录激活因子1(signal transducer and activator of transcription 1,STAT1)与三方基序蛋白21(tripartite-motif protein 21,TRIM21)在此过程中的作用。【方法】通过倒置显微镜观察感染复数(multiplicity of infection,MOI)为1的PDCoV感染IPEC-J2细胞1、12、24、48 h后细胞形态学变化,采用实时荧光定量PCR和Western blotting分别检测PDCoV-N基因及蛋白的表达水平,以此筛选PDCoV感染IPEC-J2细胞模型的最佳条件。在PDCoV感染峰值时,通过实时荧光定量PCR和Western blotting检测STAT1、TRIM21及干扰素刺激基因(interferon-stimulated genes,ISGs)的mRNA和蛋白表达水平;通过双荧光素酶报告基因系统检测过表达或抑制STAT1对PDCoV复制及干扰素激活反应元件(interferon-stimulated response element,ISRE)启动子活性的影响;将TRIM21与STAT1真核表达载体共转染至IPEC-J2细胞,结合激光共聚焦显微镜、免疫共沉淀技术(coimmunoprecipitation,Co-IP)和Western blotting验证二者的相互作用及TRIM21对STAT1的调控效应。【结果】PDCoV在感染IPEC-J2细胞24 h达复制高峰,同时试验组细胞中STAT1、TRIM21蛋白表达及ISGs转录水平均极显著高于对照组(P<0.01)。与对照组相比,过表达STAT1后ISRE启动子活性极显著增强,PDCoV-N基因及其蛋白表达量极显著降低(P<0.01),而氟达拉滨抑制STAT1后则极显著促进病毒复制(P<0.01)。激光共聚焦显微镜观察与Co-IP结果显示,STAT1与TRIM21存在共定位及相互作用,且与对照组相比,过表达TRIM21后STAT1蛋白表达量极显著降低(P<0.01)。【结论】本研究解析了PDCoV与宿主IPEC-J2细胞先天性免疫的相互作用机制,STAT1作为关键抗病毒因子通过ISRE通路增强先天性免疫来抑制病毒复制,TRIM21则靶向STAT1并下调其表达从而帮助病毒免疫逃逸。研究结果不仅为深入理解PDCoV的致病分子机制提供了新见解,也为制定PDCoV防控策略奠定了基础。
文摘信号转导子和转录激活子1(signal transducer and activator of transcription 1,STAT1)作为JAK-STAT信号通路的核心成员,通过调控干扰素和其他细胞因子的信号转导,在肿瘤免疫微环境(tumor immune microenvironment,TIME)中起双重作用。STAT1激活一方面可以促进抗肿瘤免疫反应,增强T细胞、自然杀伤细胞和巨噬细胞的杀伤功能;另一方面,STAT1的持续激活可能会诱发免疫抑制性微环境的形成,促使肿瘤发生免疫逃逸。本综述着重介绍STAT1在肿瘤免疫微环境中的调控机制及其对免疫治疗的影响,并对靶向STAT1的潜在治疗策略进行了探讨。
文摘Objective Inflammation and fibrosis are key features of diabetic nephropathy(DN).Triptolide(TP)exhibits anti-inflammatory and anti-fibrotic properties,though its mechanisms of action in DN remain unclear.CREKA(Cys-Arg-Glu-Lys-Ala)is a pentapeptide that specifically binds to fibronectin(FN),and the CREKA-modified liposome(CREKA-Lip)represents a novel FN-targeted drug delivery system.This study aimed to investigate the role of TP in diabetic db/db mice and determine whether encapsulation within CREKA-Lip enhances therapeutic efficacy while reducing the multi-organ toxicity of TP.Methods Eight-week-old diabetic db/db mice received tail vein injections twice weekly with vehicle,free TP,or CREKA-Lip/TP for 10 weeks.Urine and serum parameters were measured,and kidney,heart,liver,and testis tissues were collected for pathological evaluation.Protein-protein interaction networks were constructed using Cytoscape and its plug-ins to identify core targets and elucidate the therapeutic mechanism of TP against DN.Inflammatory,fibrotic,apoptotic,and lipid metabolism markers were evaluated in the kidneys of diabetic mice with DN and in high glucose-treated mouse mesangial cells and podocytes using qPCR,Western blot,immunohistochemistry,and immunofluorescence assays.Results TP administration reduced fasting blood glucose levels and glomerular mesangial expansion in diabetic mice.TP significantly suppressed renal inflammation,fibrosis,and apoptosis while enhancing lipid metabolism.Integration of network pharmacology,molecular docking,and transcriptomics revealed that TP ameliorated DN by inhibiting the JAK2-STAT1 signaling pathway.In vitro,TP inhibited high glucose-induced phosphorylation of JAK2 and STAT1,reduced collagen production in mesangial cells,decreased apoptosis,and improved lipid metabolism in podocytes.Moreover,CREKA-Lip/TP exhibited superior efficacy compared with free TP,with a more sustained reduction in urine albumin-to-creatinine ratio and greater inhibition of mesangial expansion.Notably,CREKA-Lip/TP treatment did not induce systemic toxicity.Conclusion TP improves renal inflammation,fibrosis,apoptosis,and lipid homeostasis,thereby ameliorating DN by inhibiting JAK2-STAT1 activation.Targeted delivery of TP via FN-binding CREKA-Lip enhances therapeutic efficacy while minimizing multi-organ toxicity.
基金supported by a grant from the National Natural Science Foundation of China(No.82070643)。
文摘Background:Intrahepatic cholangiocarcinoma(ICC)is the second most frequent primary liver cancer.The involvement of Y-box binding protein 1(YBX1)in tumor advancement is well-documented.However,its function in ICC is not fully understood.This study aimed to explore the function and regulatory mechanism of YBX1 in ICC and provide evidence for YBX1 as a potential new approach for immunotherapy in ICC.Methods:Tissue immunohistochemistry,TCGA,and GEO databases were used to analyze the expression of YBX1 in ICC.The expression of YBX1 was silenced and overexpressed in cell lines.Both in vitro and in vivo assays were conducted to examine the antitumor T-cell responses.Actinomycin D,RNA immunoprecipitation,and methylated RNA immunoprecipitation assays were used to identify mechanism of YBX1 on downstream genes.Immunofluorescence assay was used to validate the association between YBX1 and relevant genes in clinical specimens of ICC.Results:The research findings indicated that ICC exhibited high levels of YBX1 expression,which was strongly associated with unfavorable outcomes.YBX1 promoted tumor progression by suppressing antitumor T-cell responses.YBX1 enhanced signal transducer and activator of transcription 1(STAT1)translation by serving as a 5-methylated cytosine(m5C)reader and activating the STAT1/PD-L1 pathway.Mouse experiments and clinical samples of ICC confirmed the strong correlation between the levels of YBX1,STAT1,and PD-L1 expression.Conclusions:YBX1 regulates STAT1 stability in an m5C dependent manner and maintains PD-L1 expression in ICC.
