目的:探究核受体亚家族4A组成员1(nuclear receptor subfamily 4 group A member 1,Nr4a1)Nr4a1激动剂胞孢子酮B(cytosporone B,Csn-B)对小鼠噪声暴露后听力损失的治疗作用。方法:采用双氧水刺激HEI-OC1毛细胞系的方法构建氧化应激细胞...目的:探究核受体亚家族4A组成员1(nuclear receptor subfamily 4 group A member 1,Nr4a1)Nr4a1激动剂胞孢子酮B(cytosporone B,Csn-B)对小鼠噪声暴露后听力损失的治疗作用。方法:采用双氧水刺激HEI-OC1毛细胞系的方法构建氧化应激细胞模型;通过实时荧光定量PCR(quantitative real-time PCR,q PCR)检测细胞中Nr4a1的mRNA表达水平;分别通过细胞计数试剂盒(cell counting kit-8,CCK8)及流式细胞术的方法检测细胞活力和细胞凋亡水平以评估Csn-B预处理后经双氧水刺激的细胞状态。构建小鼠噪声性听力损失模型,运用qPCR和免疫荧光技术检测噪声暴露后Nr4a1在小鼠耳蜗中的表达;通过检测听性脑干反应(auditory brainstem response,ABR)评估噪声暴露后以及Csn-B连续治疗13 d后小鼠听力情况。结果:双氧水刺激后HEI-OC1毛细胞中Nr4a1表达上升,细胞活力显著下降,凋亡水平显著升高;Csn-B预处理HEI-OC1毛细胞经双氧水刺激,细胞活力显著高于对照组而凋亡水平则显著低于对照组。在体研究结果显示,噪声暴露后小鼠听力显著降低,Nr4a1在小鼠耳蜗中的表达水平显著升高。噪声暴露后经Csn-B治疗小鼠听力得到改善,主要表现为Click-ABR以及Tone Burst-ABR(4000、8000Hz处)阈值下降。结论:Nr4a1激动剂Csn-B增强内耳毛细胞对氧化应激损伤的抵御能力,部分改善噪声暴露后的小鼠听力。展开更多
AIM:To explore the DNA methylation of COL4A1 in ultraviolet-B(UVB)-induced age-related cataract(ARC)models in vitro and in vivo.METHODS:Human lens epithelium B3(HLEB3)cells and Sprague Dawley rats were exposure to UVB...AIM:To explore the DNA methylation of COL4A1 in ultraviolet-B(UVB)-induced age-related cataract(ARC)models in vitro and in vivo.METHODS:Human lens epithelium B3(HLEB3)cells and Sprague Dawley rats were exposure to UVB respectively.The MTT assay was utilized to evaluate cell proliferation.Flow cytometry was employed for analysis of cell apoptosis and cell cycle.COL4A1 expression in HLEB3 cells and anterior lens capsules were assessed using Western blot and reverse transcription-polymerase chain reaction(RTPCR).The localization of COL4A1 in HLEB3 cells was determined by immunofluorescence.The methylation status of CpG islands located in COL4A1 promoter was verified using bisulfite-sequencing PCR(BSP).DNMTs and TETs mRNA levels was examined by RT-PCR.RESULTS:UVB exposure decreased HLEB3 cells proliferation,while increased the apoptosis rate and cells were arrested in G0/G1 phase.COL4A1 expression was markedly inhibited in UVB treated cells compared to the controls.Hypermethylation status was detected in the CpG islands within COL4A1 promoter in HLEB3 cells subjected to UVB exposure.Expressions of DNMTs including DNMT1/2/3 were elevated in UVB treated HLEB3 cells compared to that in the controls,while expressions of TETs including TET1/2/3 showed the opposite trend.Results from the UVB treated rat model further confirmed the decreased expression of COL4A1,hypermethylation status of the CpG islands at promoter of COL4A1 and abnormal expression of DNMT1/2/3 and TET1/2/in UVB exposure group.CONCLUSION:DNA hypermethylation of COL4A1 promoter CpG islands is correlated with decreased COL4A1 expression in UVB induced HLEB3 cells and anterior lens capsules of rats.展开更多
AIM:To evaluate the regulation of the aberrant expression of collagen typeⅣalpha 1 chain(COL4A1)in the development of age-related cataract(ARC).METHODS:Quantitative reverse transcription-polymerase chain reaction(qRT...AIM:To evaluate the regulation of the aberrant expression of collagen typeⅣalpha 1 chain(COL4A1)in the development of age-related cataract(ARC).METHODS:Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blot analysis were employed to evaluate the expression of COL4A1 in ARC patients and healthy controls.The proliferation,apoptosis,cell cycle and epithelial-mesenchymal transition(EMT)of human lens epithelial cell(HLE-B3)were further analyzed under the condition of COL4A1 gene silence.Alteration of gene expression at mRNA level after knockdown COL4A1 were also evaluated by qRT-PCR on HLE-B3 cells.RESULTS:The aberrant expression of COL4A1 was identified a clinically associated with the ARC.Silencing of COL4A1 promoted the apoptosis and inhibited the proliferation of HLE-B3 by blocking the cell cycle.Moreover,COL4A1 gene silence didn’t affect the cytoskeleton of HLE-B3 but down-regulated the Collagen typeⅣAlpha 2 Chain(COL4A2),paired box 6(PAX6),procollagen-lysine 2-oxoglutarate 5-dioxygenases 1(PLOD1)and procollagenlysine 2-oxoglutarate 5-dioxygenases 2(PLOD2)expression levels in HLE-B3 cells.Silencing the COL4A1 gene induced EMT of the HLE-B3 cells by promoting the transforming growth factor beta(TGF-β)expression.CONCLUSION:Silencing of COL4A1 induces S-phase arrest,also inhibits the proliferation and enhance HLE-B3 apoptosis and EMT,and down-regulates the expression of COL4A2,PAX6,PLOD1 and PLOD2.Thus,the expression alteration of COL4A1 may play a critical role in the pathogenesis of ARC.展开更多
Background:Pulmonary arterial hypertension(PAH)is a chronic and progressive disease that is strongly associated with dysregulation of glucose metabolism.Alterations in nuclear receptor subfamily 4 group A member 1(NR4...Background:Pulmonary arterial hypertension(PAH)is a chronic and progressive disease that is strongly associated with dysregulation of glucose metabolism.Alterations in nuclear receptor subfamily 4 group A member 1(NR4A1)activity alter the outcome of PAH.This study aimed to investigate the effects of NR4A1 on glycolysis in PAH and its underlying mechanisms.Methods:This study included twenty healthy volunteers and twenty-three PAH patients,and plasma samples were collected from the participants.To mimic the conditions of PAH in vitro,a hypoxia-induced model of pulmonary artery smooth muscle cell(PASMC)model was established.The proliferation of PASMCs was assessed using CCK8 assays.Results:Levels of NR4A1,hypoxia-inducible factor-1α(HIF-1α),and various glycolysis-related enzymes were measured.In addition,extracellular glucose and lactate production were assessed.The interaction between NR4A1 and HIF-1αwas evaluated by co-immunoprecipitation assays.Levels of NR4A1 and HIF-1αwas increased in PAH patients,and exposure to hypoxia resulted in increased levels of NR4A1 and HIF-1αin PASMCs.NR4A1 interacted with HIF-1α.NR4A1 overexpression enhanced hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,decreased glucose levels,increased lactate levels and promoted hypoxic PASMC viability.Conversely,silencing NR4A1 decreased hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,promoted glucose production,reduced lactate levels and inhibited hypoxic PASMC viability.Furthermore,overexpression of HIF-1αreversed the regulation of glycolysis caused by NR4A1 knockdown.Conclusion:NR4A1 enhances glycolysis in hypoxia-induced PASMCs by upregulating HIF-1α.Our findings indicate that the management of NR4A1 activity may be a promising strategy for PAH therapy.展开更多
基金Supported by the Shaanxi Provincial Department of Science and Technology Agency Project(No.2022SF-502)Xi’an Medical University Scientific Research Capacity Improvement Project(No.2022NLTS104)+2 种基金The Fifth Batch of Key Disciplines of Xi’an Medical University(No.medical technology12202306)Yunzhen Optometry Special Fund(No.2021HXZR10)Innovation and Entrepreneurship Training Program for College Students(No.S202211840043).
文摘AIM:To explore the DNA methylation of COL4A1 in ultraviolet-B(UVB)-induced age-related cataract(ARC)models in vitro and in vivo.METHODS:Human lens epithelium B3(HLEB3)cells and Sprague Dawley rats were exposure to UVB respectively.The MTT assay was utilized to evaluate cell proliferation.Flow cytometry was employed for analysis of cell apoptosis and cell cycle.COL4A1 expression in HLEB3 cells and anterior lens capsules were assessed using Western blot and reverse transcription-polymerase chain reaction(RTPCR).The localization of COL4A1 in HLEB3 cells was determined by immunofluorescence.The methylation status of CpG islands located in COL4A1 promoter was verified using bisulfite-sequencing PCR(BSP).DNMTs and TETs mRNA levels was examined by RT-PCR.RESULTS:UVB exposure decreased HLEB3 cells proliferation,while increased the apoptosis rate and cells were arrested in G0/G1 phase.COL4A1 expression was markedly inhibited in UVB treated cells compared to the controls.Hypermethylation status was detected in the CpG islands within COL4A1 promoter in HLEB3 cells subjected to UVB exposure.Expressions of DNMTs including DNMT1/2/3 were elevated in UVB treated HLEB3 cells compared to that in the controls,while expressions of TETs including TET1/2/3 showed the opposite trend.Results from the UVB treated rat model further confirmed the decreased expression of COL4A1,hypermethylation status of the CpG islands at promoter of COL4A1 and abnormal expression of DNMT1/2/3 and TET1/2/in UVB exposure group.CONCLUSION:DNA hypermethylation of COL4A1 promoter CpG islands is correlated with decreased COL4A1 expression in UVB induced HLEB3 cells and anterior lens capsules of rats.
基金Supported by Supporting Fund Project of Shaanxi Provincial Department of Science and Technology Agency Project(No.2022SF-502)Special Scientific Research Program of Education Department of Shaanxi Provincial Government(No.21JK0891)+1 种基金Young Talent Lifting Project of Xi’an Science and Technology Association(No.095920221365)Innovation and Entrepreneurship Training Program for College students of Xi’an Medical University(No.121521113)。
文摘AIM:To evaluate the regulation of the aberrant expression of collagen typeⅣalpha 1 chain(COL4A1)in the development of age-related cataract(ARC).METHODS:Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blot analysis were employed to evaluate the expression of COL4A1 in ARC patients and healthy controls.The proliferation,apoptosis,cell cycle and epithelial-mesenchymal transition(EMT)of human lens epithelial cell(HLE-B3)were further analyzed under the condition of COL4A1 gene silence.Alteration of gene expression at mRNA level after knockdown COL4A1 were also evaluated by qRT-PCR on HLE-B3 cells.RESULTS:The aberrant expression of COL4A1 was identified a clinically associated with the ARC.Silencing of COL4A1 promoted the apoptosis and inhibited the proliferation of HLE-B3 by blocking the cell cycle.Moreover,COL4A1 gene silence didn’t affect the cytoskeleton of HLE-B3 but down-regulated the Collagen typeⅣAlpha 2 Chain(COL4A2),paired box 6(PAX6),procollagen-lysine 2-oxoglutarate 5-dioxygenases 1(PLOD1)and procollagenlysine 2-oxoglutarate 5-dioxygenases 2(PLOD2)expression levels in HLE-B3 cells.Silencing the COL4A1 gene induced EMT of the HLE-B3 cells by promoting the transforming growth factor beta(TGF-β)expression.CONCLUSION:Silencing of COL4A1 induces S-phase arrest,also inhibits the proliferation and enhance HLE-B3 apoptosis and EMT,and down-regulates the expression of COL4A2,PAX6,PLOD1 and PLOD2.Thus,the expression alteration of COL4A1 may play a critical role in the pathogenesis of ARC.
基金supported by the National Natural Science Foundation of China(No.82000300).
文摘Background:Pulmonary arterial hypertension(PAH)is a chronic and progressive disease that is strongly associated with dysregulation of glucose metabolism.Alterations in nuclear receptor subfamily 4 group A member 1(NR4A1)activity alter the outcome of PAH.This study aimed to investigate the effects of NR4A1 on glycolysis in PAH and its underlying mechanisms.Methods:This study included twenty healthy volunteers and twenty-three PAH patients,and plasma samples were collected from the participants.To mimic the conditions of PAH in vitro,a hypoxia-induced model of pulmonary artery smooth muscle cell(PASMC)model was established.The proliferation of PASMCs was assessed using CCK8 assays.Results:Levels of NR4A1,hypoxia-inducible factor-1α(HIF-1α),and various glycolysis-related enzymes were measured.In addition,extracellular glucose and lactate production were assessed.The interaction between NR4A1 and HIF-1αwas evaluated by co-immunoprecipitation assays.Levels of NR4A1 and HIF-1αwas increased in PAH patients,and exposure to hypoxia resulted in increased levels of NR4A1 and HIF-1αin PASMCs.NR4A1 interacted with HIF-1α.NR4A1 overexpression enhanced hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,decreased glucose levels,increased lactate levels and promoted hypoxic PASMC viability.Conversely,silencing NR4A1 decreased hypoxia-induced expression of HIF-1α,GLUT1,PKM2,HK2,and CD36,promoted glucose production,reduced lactate levels and inhibited hypoxic PASMC viability.Furthermore,overexpression of HIF-1αreversed the regulation of glycolysis caused by NR4A1 knockdown.Conclusion:NR4A1 enhances glycolysis in hypoxia-induced PASMCs by upregulating HIF-1α.Our findings indicate that the management of NR4A1 activity may be a promising strategy for PAH therapy.
