Interleukin-4 plays an important protective role in Alzheimer’s disease by regulating microglial phenotype,phagocytosis of amyloid-β,and secretion of anti-inflammatory and neurotrophic cytokines.Recently,increasing ...Interleukin-4 plays an important protective role in Alzheimer’s disease by regulating microglial phenotype,phagocytosis of amyloid-β,and secretion of anti-inflammatory and neurotrophic cytokines.Recently,increasing evidence has suggested that autophagy regulates innate immunity by affecting M1/M2 polarization of microglia/macrophages.However,the role of interleukin-4 in microglial autophagy is unknown.In view of this,BV2 microglia were treated with 0,10,20 or 50 ng/mL interleukin-4 for 24,48,or 72 hours.Subsequently,light chain 3-II and p62 protein expression levels were detected by western blot assay.BV2 microglia were incubated with interleukin-4(20 ng/mL,experimental group),3-methyladenine(500μM,autophagy inhibitor,negative control group),rapamycin(100 nM,autophagy inductor,positive control group),3-methyladenine+interleukin-4(rescue group),or without treatment for 24 hours,and then exposed to amyloid-β(1μM,model group)or vehicle control(control)for 24 hours.LC3-II and p62 protein expression levels were again detected by western blot assay.In addition,expression levels of multiple markers of M1 and M2 phenotype were assessed by real-time fluorescence quantitative polymerase chain reaction,while intracellular and supernatant amyloid-βprotein levels were measured by enzyme-linked immunosorbent assay.Our results showed that interleukin-4 induced microglial autophagic flux,most significantly at 20 ng/mL for 48 hours.Interleukin-4 pretreated microglia inhibited blockade of amyloid-β-induced autophagic flux,and promoted amyloid-βuptake and degradation partly through autophagic flux,but inhibited switching of amyloid-β-induced M1 phenotype independent on autophagic flux.These results indicate that interleukin-4 pretreated microglia increases uptake and degradation of amyloid-βin a process partly mediated by autophagy,which may play a protective role against Alzheimer’s disease.展开更多
Objective: Rheumatoid arthritis (RA) is an autoimmune disease in which angiogenesis represents a critical early event of synovial inflammation. The present study aimed to reveal the potential molecular mechanisms of S...Objective: Rheumatoid arthritis (RA) is an autoimmune disease in which angiogenesis represents a critical early event of synovial inflammation. The present study aimed to reveal the potential molecular mechanisms of SAA/TLR4 induction of angiogenesis through NETs in RA. Materials and methods: Firstly, immunohistochemistry and immunofluorescence were used to determinate TLR4 and NETs expression in synovial tissue, respectively. ELISA was used to detect the content of SAA, MPO and NE in serum and synovial fluid of patients. DNA quantification was done by fluorescence. DNA fluorescence staining was used to compare NETs formation in RA and HC sera, and to investigate the mechanism of NETs formation induced by SAA stimulation. PicoGreen DNA testing was used to characterize the DNA in the supernatants. Also, DNA fluorescence staining to explore whether NETs formation induced by SAA was dependent or independent on NADPH oxidase pathway. MTT assay, Wound healing assay, Tube formation assay were performed to analyze human veins umbilical cells (HUVECs) proliferation, migration, and tube vessels formation, respectively under NETs or NETs + DNase stimulants. Results: Firstly, we demonstrated that TLR4 was predominantly and widely expressed in synovial tissues with elevated serum levels of SAA, compared to osteoarthritis (OA) patients, and the similar results were observed for NETs formation. Afterwards, in a series of in vitro experiments, we reported an increased MPO and NE levels, and a relatively decreased DNA level in the sera of RA patients. Set apart, the levels of MPO and NE in RA were correlated to the disease activity. Moreover, an increased spontaneous NETs formation was observed in RA patients, enhanced under SAA stimulation and regulated by TLR4 activation. And the total DNA expressed in RA patients was partly composed of NET-DNA. Also, SAA induced NETs formation dependent on NADPH pathway. Finally, our results indicated that extracted SAA-induced NETs promoted endothelial cells (ECs) migration, proliferation, and vascular tube formation. Conclusion: Our current study highlighted the role of SAA/TLR4 interaction in the induction of angiogenesis through formed NETs. Therefore, this study offers new perspectives in the understanding of RA pathogenicity and its management.展开更多
目的探讨冠状动脉(冠脉)粥样硬化性心脏病(coronary heart disease,CHD)患者外周血细胞毒性T淋巴细胞相关抗原-4(cytotoxic T lymphocyte associated antigen-4,CTLA-4)、分化抗原簇28(cluster of differentiation 28,CD28)及淀粉样蛋白...目的探讨冠状动脉(冠脉)粥样硬化性心脏病(coronary heart disease,CHD)患者外周血细胞毒性T淋巴细胞相关抗原-4(cytotoxic T lymphocyte associated antigen-4,CTLA-4)、分化抗原簇28(cluster of differentiation 28,CD28)及淀粉样蛋白A(serum amyloid A protein,SAA)的表达。方法选择2017年7月至2018年6月南京医科大学康达学院第一附属医院CHD患者43例为研究对象,其中稳定型心绞痛患者21例,急性冠脉综合征患者22例。另外,选择同期20例经冠脉造影证实的非CHD患者为正常对照组。患者在起病入科时抽取外周血,CTLA-4、CD28及SAA的浓度采用酶联免疫吸附法(enzyme-linked immunoassay,ELISA)检测。结果与正常对照组比较,稳定型心绞痛组、急性冠脉综合征组患者外周血CTLA-4浓度降低,SAA、CD28浓度升高且急性冠脉综合征组浓度升高更高,差异有统计学意义(P<0.05);急性冠脉综合征组SAA与CD28呈正相关(r=0.871,P<0.01),与CTLA-4呈负相关(r=-0.642,P<0.01)。结论 CHD患者外周血CTLA-4浓度降低,CD28浓度升高,急性冠脉综合征患者更甚,且其与常规的SAA检测指标呈相关性,可用于评估病情的严重程度并作为CHD治疗的新靶点。展开更多
基金supported by the Natural Science Foundation of Liaoning Province of China,No.20170541036(to HYL)
文摘Interleukin-4 plays an important protective role in Alzheimer’s disease by regulating microglial phenotype,phagocytosis of amyloid-β,and secretion of anti-inflammatory and neurotrophic cytokines.Recently,increasing evidence has suggested that autophagy regulates innate immunity by affecting M1/M2 polarization of microglia/macrophages.However,the role of interleukin-4 in microglial autophagy is unknown.In view of this,BV2 microglia were treated with 0,10,20 or 50 ng/mL interleukin-4 for 24,48,or 72 hours.Subsequently,light chain 3-II and p62 protein expression levels were detected by western blot assay.BV2 microglia were incubated with interleukin-4(20 ng/mL,experimental group),3-methyladenine(500μM,autophagy inhibitor,negative control group),rapamycin(100 nM,autophagy inductor,positive control group),3-methyladenine+interleukin-4(rescue group),or without treatment for 24 hours,and then exposed to amyloid-β(1μM,model group)or vehicle control(control)for 24 hours.LC3-II and p62 protein expression levels were again detected by western blot assay.In addition,expression levels of multiple markers of M1 and M2 phenotype were assessed by real-time fluorescence quantitative polymerase chain reaction,while intracellular and supernatant amyloid-βprotein levels were measured by enzyme-linked immunosorbent assay.Our results showed that interleukin-4 induced microglial autophagic flux,most significantly at 20 ng/mL for 48 hours.Interleukin-4 pretreated microglia inhibited blockade of amyloid-β-induced autophagic flux,and promoted amyloid-βuptake and degradation partly through autophagic flux,but inhibited switching of amyloid-β-induced M1 phenotype independent on autophagic flux.These results indicate that interleukin-4 pretreated microglia increases uptake and degradation of amyloid-βin a process partly mediated by autophagy,which may play a protective role against Alzheimer’s disease.
文摘Objective: Rheumatoid arthritis (RA) is an autoimmune disease in which angiogenesis represents a critical early event of synovial inflammation. The present study aimed to reveal the potential molecular mechanisms of SAA/TLR4 induction of angiogenesis through NETs in RA. Materials and methods: Firstly, immunohistochemistry and immunofluorescence were used to determinate TLR4 and NETs expression in synovial tissue, respectively. ELISA was used to detect the content of SAA, MPO and NE in serum and synovial fluid of patients. DNA quantification was done by fluorescence. DNA fluorescence staining was used to compare NETs formation in RA and HC sera, and to investigate the mechanism of NETs formation induced by SAA stimulation. PicoGreen DNA testing was used to characterize the DNA in the supernatants. Also, DNA fluorescence staining to explore whether NETs formation induced by SAA was dependent or independent on NADPH oxidase pathway. MTT assay, Wound healing assay, Tube formation assay were performed to analyze human veins umbilical cells (HUVECs) proliferation, migration, and tube vessels formation, respectively under NETs or NETs + DNase stimulants. Results: Firstly, we demonstrated that TLR4 was predominantly and widely expressed in synovial tissues with elevated serum levels of SAA, compared to osteoarthritis (OA) patients, and the similar results were observed for NETs formation. Afterwards, in a series of in vitro experiments, we reported an increased MPO and NE levels, and a relatively decreased DNA level in the sera of RA patients. Set apart, the levels of MPO and NE in RA were correlated to the disease activity. Moreover, an increased spontaneous NETs formation was observed in RA patients, enhanced under SAA stimulation and regulated by TLR4 activation. And the total DNA expressed in RA patients was partly composed of NET-DNA. Also, SAA induced NETs formation dependent on NADPH pathway. Finally, our results indicated that extracted SAA-induced NETs promoted endothelial cells (ECs) migration, proliferation, and vascular tube formation. Conclusion: Our current study highlighted the role of SAA/TLR4 interaction in the induction of angiogenesis through formed NETs. Therefore, this study offers new perspectives in the understanding of RA pathogenicity and its management.
文摘目的探讨冠状动脉(冠脉)粥样硬化性心脏病(coronary heart disease,CHD)患者外周血细胞毒性T淋巴细胞相关抗原-4(cytotoxic T lymphocyte associated antigen-4,CTLA-4)、分化抗原簇28(cluster of differentiation 28,CD28)及淀粉样蛋白A(serum amyloid A protein,SAA)的表达。方法选择2017年7月至2018年6月南京医科大学康达学院第一附属医院CHD患者43例为研究对象,其中稳定型心绞痛患者21例,急性冠脉综合征患者22例。另外,选择同期20例经冠脉造影证实的非CHD患者为正常对照组。患者在起病入科时抽取外周血,CTLA-4、CD28及SAA的浓度采用酶联免疫吸附法(enzyme-linked immunoassay,ELISA)检测。结果与正常对照组比较,稳定型心绞痛组、急性冠脉综合征组患者外周血CTLA-4浓度降低,SAA、CD28浓度升高且急性冠脉综合征组浓度升高更高,差异有统计学意义(P<0.05);急性冠脉综合征组SAA与CD28呈正相关(r=0.871,P<0.01),与CTLA-4呈负相关(r=-0.642,P<0.01)。结论 CHD患者外周血CTLA-4浓度降低,CD28浓度升高,急性冠脉综合征患者更甚,且其与常规的SAA检测指标呈相关性,可用于评估病情的严重程度并作为CHD治疗的新靶点。
