Hypoxia plays an important role in the genesis and progression of renal fibrosis.The underlying mechanisms, however, have not been sufficiently elucidated. We examined the role of p53 in hypoxia-induced renal fibrosis...Hypoxia plays an important role in the genesis and progression of renal fibrosis.The underlying mechanisms, however, have not been sufficiently elucidated. We examined the role of p53 in hypoxia-induced renal fibrosis in cell culture (human and rat renal tubular epithelial cells) and a mouse unilateral ureteral obstruction (UUO) model. Cell cycle of tubular cells was determined by flow cytometry, and the expression of profibrogenic factors was determined by RT-PCR, immunohistochemistry, and western blotting. Chromatin immunoprecipitation and luciferase reporter experiments were performed to explore the effect of HIF-lα on p53 expression. We showed that, in hypoxic tubular cells, p53 upregulation suppressed the expression of CDK1 and cyclins Bl and DI, leading to cell cycle (G2/M) arrest (or delay) and higher expression of TGF-β, CTGF, collagens, and fibronectin. p53 suppression by siRNA or by a specific p53 inhibitor (PIF-α) triggered opposite effects preventing the G2/M arrest and profibrotic changes. In vivo experiments in the UUO model revealed similar antifibrotic results following intraperitoneal administration of PIF-α(2.2 mg/kg). Using gain-of-function, loss-of-function, and luciferase assays, we further identified an HRE3 region on the p53 promoter as the HIF-lα-binding site. The HIF-la-HRE3 binding resulted in a sharp transcriptional activation of p53. Collectively, we show the presence of a hypoxia-activated, p53-responsive profibrogenic pathway in the kidney. During hypoxia, p53 upregulation induced by HIF-la suppresses cell cycle progression, leading to the accumulation of G2/M cells, and activates profibrotic TGF-β and CTGF-mediated signaling pathways, causing extracellular matrix production and renal fibrosis.展开更多
目的研究低氧条件下胶质瘤细胞系U251细胞中miRNA-210的表达以及对肿瘤转移能力的影响。方法低氧条件(1%O2)培养胶质瘤细胞系U251,利用Western blot检测不同诱导时间HIF-1α水平变化,利用real time PCR检测低氧不同诱导时间miRNA-210表...目的研究低氧条件下胶质瘤细胞系U251细胞中miRNA-210的表达以及对肿瘤转移能力的影响。方法低氧条件(1%O2)培养胶质瘤细胞系U251,利用Western blot检测不同诱导时间HIF-1α水平变化,利用real time PCR检测低氧不同诱导时间miRNA-210表达变化,通过Transwell小室检测U251细胞侵袭能力,利用Western blot检测低氧条件下Vmp1表达变化。结果低氧诱导U251细胞6、12和24h,可检测到HIF-1α水平随着诱导时间而增加;在低氧诱导的不同时间点可检测到miRNA-210表达逐渐增加,给予HIF-1α抑制剂2-甲氧雌二醇(2-methoxyestradiol,2-ME2)后miRNA-210的表达受到抑制;低氧条件下U251细胞侵袭转移能力增强,在miRNA-210抑制剂antagonist miR210转染后,U251细胞转移能力受到抑制,低氧条件下U251细胞中Vmp1蛋白表达下降,antagonist miR210转染可以逆转Vmp1的表达。结论低氧可以通过HIF-1α诱导miRNA-210表达增加,miRNA-210具有促进U251细胞转移的作用,miRNA-210可能通过调节Vmp1表达发挥作用。展开更多
Advanced oxidation protein products （AOPPs）, as a novel indicator of oxidative stress, are thought to be involved in aging-related diseases. The excessive AOPPs were served as independent risk factor for coronary ar...Advanced oxidation protein products （AOPPs）, as a novel indicator of oxidative stress, are thought to be involved in aging-related diseases. The excessive AOPPs were served as independent risk factor for coronary artery disease （CAD）, atherosclerosis and carotid intima media thickness, which mainly through hypoxia inducible factor- l OL （HIF-Iot） and vascular endothelial growth factor （VEGF） pathway. Paeoniflorin, a monoterpene glycoside, ex- erts well protective effect in vascular as its good antioxidant property. However, there is no research that has reported whether Paeoniflorin has the protective effect on oxidative damage induced by AOPPs in HUVECs, and also it is little known about this underlying mechanism. The protective effect of Paeoniflorin on oxidative damage induced by AOPPs was investigated in HUVECs. The cell viability was assessed by MTT colorimetric assay. The fluorescence intensity of 2＇, 7＇-dichlorofluorescein-diacetate （DCFH-DA） staining was detected for intracellular reactive oxygen species （ROS） production. And mitochondrial membrane potential （MMP） was measured byflow cytometry and confocal mi- croscopy staining with Mito Tracker Deep Red/ MitoTracker Green. The intracellular adenosine triphosphate （ATP） was measured by ATP Determination Kit. Nox2, Nox4, HIF-lα, VEGF and NF-KB p65 protein expressions were detected by western blot. The results showed that AOPPs significantly decreased MMP and ATP in a dose-de- pendent manner. Furthermore, AOPPs increased HW-1α, VEGF protein expression, and also partly increased NF-KB p65 expression may through increase of ROS production by up-regulations of Nox2, Nox4 and RAGE expression in HUVECs. These effects were remarkably reversed by pre-treatment of Paeoniflorin, which indicated that Paeoniflorin inhibited Nox2/Nox4 expression, restored ATP depletion and mitochondria dysfunction via ROS suppression, and down-regulated HIF-lα/VEGF possibly via ROS-NF-KB axis. In conclusion, these results suggesting that Paeoniorin had a protective effect against AOPP-induced oxidative damage in HUVECs and that HIF-lα/VEGF might be inter- vention site in this process.展开更多
文摘Hypoxia plays an important role in the genesis and progression of renal fibrosis.The underlying mechanisms, however, have not been sufficiently elucidated. We examined the role of p53 in hypoxia-induced renal fibrosis in cell culture (human and rat renal tubular epithelial cells) and a mouse unilateral ureteral obstruction (UUO) model. Cell cycle of tubular cells was determined by flow cytometry, and the expression of profibrogenic factors was determined by RT-PCR, immunohistochemistry, and western blotting. Chromatin immunoprecipitation and luciferase reporter experiments were performed to explore the effect of HIF-lα on p53 expression. We showed that, in hypoxic tubular cells, p53 upregulation suppressed the expression of CDK1 and cyclins Bl and DI, leading to cell cycle (G2/M) arrest (or delay) and higher expression of TGF-β, CTGF, collagens, and fibronectin. p53 suppression by siRNA or by a specific p53 inhibitor (PIF-α) triggered opposite effects preventing the G2/M arrest and profibrotic changes. In vivo experiments in the UUO model revealed similar antifibrotic results following intraperitoneal administration of PIF-α(2.2 mg/kg). Using gain-of-function, loss-of-function, and luciferase assays, we further identified an HRE3 region on the p53 promoter as the HIF-lα-binding site. The HIF-la-HRE3 binding resulted in a sharp transcriptional activation of p53. Collectively, we show the presence of a hypoxia-activated, p53-responsive profibrogenic pathway in the kidney. During hypoxia, p53 upregulation induced by HIF-la suppresses cell cycle progression, leading to the accumulation of G2/M cells, and activates profibrotic TGF-β and CTGF-mediated signaling pathways, causing extracellular matrix production and renal fibrosis.
文摘Advanced oxidation protein products （AOPPs）, as a novel indicator of oxidative stress, are thought to be involved in aging-related diseases. The excessive AOPPs were served as independent risk factor for coronary artery disease （CAD）, atherosclerosis and carotid intima media thickness, which mainly through hypoxia inducible factor- l OL （HIF-Iot） and vascular endothelial growth factor （VEGF） pathway. Paeoniflorin, a monoterpene glycoside, ex- erts well protective effect in vascular as its good antioxidant property. However, there is no research that has reported whether Paeoniflorin has the protective effect on oxidative damage induced by AOPPs in HUVECs, and also it is little known about this underlying mechanism. The protective effect of Paeoniflorin on oxidative damage induced by AOPPs was investigated in HUVECs. The cell viability was assessed by MTT colorimetric assay. The fluorescence intensity of 2＇, 7＇-dichlorofluorescein-diacetate （DCFH-DA） staining was detected for intracellular reactive oxygen species （ROS） production. And mitochondrial membrane potential （MMP） was measured byflow cytometry and confocal mi- croscopy staining with Mito Tracker Deep Red/ MitoTracker Green. The intracellular adenosine triphosphate （ATP） was measured by ATP Determination Kit. Nox2, Nox4, HIF-lα, VEGF and NF-KB p65 protein expressions were detected by western blot. The results showed that AOPPs significantly decreased MMP and ATP in a dose-de- pendent manner. Furthermore, AOPPs increased HW-1α, VEGF protein expression, and also partly increased NF-KB p65 expression may through increase of ROS production by up-regulations of Nox2, Nox4 and RAGE expression in HUVECs. These effects were remarkably reversed by pre-treatment of Paeoniflorin, which indicated that Paeoniflorin inhibited Nox2/Nox4 expression, restored ATP depletion and mitochondria dysfunction via ROS suppression, and down-regulated HIF-lα/VEGF possibly via ROS-NF-KB axis. In conclusion, these results suggesting that Paeoniorin had a protective effect against AOPP-induced oxidative damage in HUVECs and that HIF-lα/VEGF might be inter- vention site in this process.
