11B
Fig. 11B: Effect of pravastatin on PGI
2production. Pravastatin induced
an increase in 6-keto PGF1α.95
11C
Fig. 11C: Effect of pravastatin on heme oxygenase (HO)-1 expression via PPAR-α. The increased levels of PPAR-α and HO-1 by
pravastatin was attenuated by PPAR-α siRNA. Representative results
from three independent experiments are shown.Scanning densitometry
was used for semiquantitative analysis to compare against β-actin.
11D
Fig. 11D: Effect of pravastatin on heme oxygenase (HO)-1 expression through both PGI
2and PPAR-α pathways. When the COX-2
inhibitor NS-398 was added to pravastatin-treated cells, the increase in HO-1 level was again significantly decreased, and administration of NS-398 and PPAR-α siRNA in combination showed a synergistic reduction in the level of HO-1. Representative results from three
independent experiments are shown. Scanning densitometry was used for semiquantitative analysis to compare against β-actin.
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12A
Fig. 12A: Translocation of PPAR-α in response to pravastatin
treatment. The level of cytosolic PPAR-α was not significantly
affected by pravastatin in NRK-52E cells. However, the level of nuclear PPAR-α was significantly increased in pravastatin-treated cells (Fig. 12A lane 3 vs lane 4). Representative results from twoindependent experiments are shown. Scanning densitometry was
used for semiquantitative analysis to compare against β-actin. .
12B
Fig. 12B: Subcellular localization of FLAG-PPAR-α in pravastatin treated NRK-52E cells. Immunostaining showed that FLAG-PPAR-α
translocated from the cytosol to the nucleus in NRK-52E cells treated with pravastatin (buttom left). This effect was significantly decreased in cells treated with the COX-2 inhibitor NS-398 (buttom right). Bar =200 µm.
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13A
Fig. 13A: Binding activity of PPAR-α in the PPRE binding site of the HO-1 promoter. PPAR-α DNA-binding activity was
observed in nuclear extracts of pravastatin-treated NRK-52E cells, and this was markedly suppressed in cells pretreated with
unlabeled primer P, probe alone; C, cells alone.
13B
Fig. 13B: Pravastatin enhanced expression of HO-1 by PPAR-α binding to PPRE of Ho-1 gene. ChIP experiments were carried out
with FLAG-specific antibody and primers to amplify –621 to –500 of the HO-1 locus, which contains one predicted PPAR-binding site in rats. Immunoprecipitated DNA was quantified by quantitative polymerase chain reaction with a TaqMan-labeled probe andnormalized to the quantity of DNA in 10% of the input used for ChIP for each sample. The data are representative of two to four
independent experiments.
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13C
Fig. 13C: Pravastatin induced PPRE and HO-1 promoter activity in a PPAR-α-dependent manner. PPAR-α or RXR-α alone slightly
increased luciferase activity, but combined transfection with PPAR-α and RXR-α increased luciferase activity by approximately 50-fold in cells with the PPRE reporter and approximately 6-fold in cells with the HO-1 promoter construct. In addition, treatment with pravastatin augmented luciferase activity in both the PPRE and HO-1 promoters compared to cells untreated with pravastatin or transfected with PPAR-α or RXR-α alone. Treatment with WY14643 also increased luciferase activity by approximately 50-fold in cells with the PPRE reporter and approximately 8-fold in cells with the HO-1 promoter.Pravastatin cotreatment further increased luciferase activity in both the PPRE and HO-1 promoters compared to untreated cells.
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14A
14B
Fig. 14: Effect of pravastatin on heme oxygenase (HO)-1 expression in wild-type and PPARα
–/–mice. After injection of pravastatin,
expression of PPAR-α and HO-1 was increased in renal extracts of wild-type C57/B6 (14A) and wild-type W129 mice (Fig. 14B).
However, the increase in HO-1 expression was markedly attenuated in PPAR-α–/– mice (14B). n = 7 / group; *p < 0.05 compared to sham
treatment.
Appendix
PPARα 減少TNFα產生並降低缺血及再灌注所造成之腎臟受損是同時經由內生及外源性前列 腺環素依賴路徑
Peroxisome proliferator-activated receptor alpha attenuate ischemia/reperfusion induced kidney injury via TNFαproduction by both prostacyclin-dependent endogenous and exogenous pathway
陳錫賢1.2 林繼偉1 賴怡君1 陳振文1.2 林恆3
Hsi-Hsien Chen1.2, Chi-Wei Lin1, I-Chun Lai1, Tzen-Wen Chen1, Heng Lin3
1.台北醫學大學附設醫院 腎臟科 2.台北醫學大學 臨床醫學研究所 3.慈濟大學 藥理暨毒理研究所
1.Department of Nephrology, Taipei Medical University Hospital, Taipei, Taiwan
2.Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University
3.Graduate Institute of Pharmacology and Toxicology, Tzu Chi University
台灣腎臟醫學會 九十六年度會員大會暨學術演講會 Basic 4.
Recent evidence indicates that either prostacyclin (PGI2) or peroxisome-proliferator activated receptor (PPAR) protect against ischemia-reperfusion (I/R) injury via anti-inflammatory pathway. We utilize the over-expression and knockdown of PPARα to proved that PPARα attenuated I/R induced kidney via NF-κB induced TNFα over-expression in vitro, and I/R induced renal dysfunction and tubular cell apoptosis were significantly attenuated in PPARα agonist treated mice or more aggravated in PPARα-/- mice. We also clarify that PGI2 will induce PPARα translocation from cytosolic part into nucleic part and PGI2-PPARα associated complex will attenuate TNFα promoter activity by NF-κB binding. In addition, PGI2 also bind with PGI2 receptor (IP receptor) to inhibit TNFα production via cAMP dependent pathway. Together, our study demonstrates for the first time that associated complex of PGI2 and PPARα can attenuate NFκB–induced TNFα activation following I/R. Strategies aim at augmentation both of PGI2 and PPARα or its signal pathway may be beneficial in conditions associated with renal I/R injury.
Introduction
PPAR-α is a member of the nuclear hormone receptor superfamily and ligand-activated transcription factor which is expressed at a relatively high level in the kidney.
More recently, studies have revealed that PPAR-α ligands exert anti-inflammatory actions. Although it is documented that PPAR-mediated modulation of gene transcription by PGI2 may form the basis for their novel role as regulators of gene expression, whereas a stable analog of PGI2 has been shown to activate PPARδ, but less known about the relationship between PGI2 and PPARα in kidney.
Specific Aims The aim of this study was to characterize the in vitro effect of PPARα in a renal I/R model, with special emphasis on the influence of endogenous PGI2 on PPARα induction following I/R-induced apoptosis by TNFα. Furthermore, we aim to study the effect of PPARα on the apoptosis response in the course of renal I/R in a mouse model.
Results
Effect of PGI2 on PPARα associated with NFκB and apoptosis.
Effect of PPARα on TNFα transcription and translation level in NRK-52E cells following I/R.
Effect of I/R on renal function in WT and PPARα-/- mice.
Fig 6
Effect of PGI2 on TNFα activity via both PPARα dependent and independent pathway.
Effect of PPARα in NRK-52E
cells following I/R Effect of PGI2 on PPARα translocation.
Conclusion:
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Pravastatin 藉由調控HO-1來減緩Carboplatin之腎毒性
PPAR-α–Regulated HO-1 Mediated the Protective Effect of Pravastatin in Carboplatin Nephrotoxicity
陳錫賢1.2 林繼偉1 吳明燦1 吳岳霖1 李玠樺1 陳振文1,2 林恆3
His-Hsien Chen1,2Chi-Wei Lin1, Ming-Tsan Wu1, Yueh-Lin Wu1, Chieh-Hua Lee1, Tzen-Wen Chen1,2, Heng Lin3
1.台北醫學大學附設醫院 腎臟科 2.台北醫學大學 臨床醫學研究所 3.慈濟大學 藥理暨毒理研究所
1.Department of Nephrology, Taipei Medical University Hospital
2.Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University
3.Graduate Institute of Pharmacology and Toxicology, Tzu Chi University
The aim of this study was to explore the molecular mechanism(s) underlying the protective effect of pravastatin against carboplatin-induced nephrotoxicity. In NRK-52E cells, pravastatin increased expression of heme oxygenase-1 (HO-1), cyclooxygenase (COX)-2, and 6-keto prostaglandin (PG)F1α, and enhanced nuclear translocation of peroxisome proliferator-activated receptor-α (PPARα). In transcriptional assays, pravastatin increased HO-1 promoter activities. Interaction of PPARα with PPRE on the HO-1 promoter was demonstrated in nuclear extracts from pravastatin-treated NRK-52E cells and confirmed by chromatin immunoprecipitation. For in vivo experiments, injection of pravastatin induced overexpression of HO-1 in wild-type mice, and HO-HO-1 expression was significantly attenuated in PPARα knock-out mice. These results indicate that pravastatin upregulates HO-1 and protects against carboplatin-induced renal dysfunction via a PGI2/PPARα-dependent pathway.
Introduction Statins have demonstrated to activation of the promoter for the antioxidant defense protein heme oxygenase-1 (HO-1), which may help to explain the pleiotropic antioxidant and anti-inflammatory actions of statins.
HO-1, an inducible enzyme that catabolizes heme to biliverdin, ferrous iron, and carbon monoxide, exhibits cytoprotective effects against oxidative insults. HO-1 has been shown to be upregulated and to ameliorate the severity of renal damage in a variety of kidney injury models, including kidney transplant rejection, acute renal injury owing to I/R, and in response to nephrotoxins such as cisplatin and cyclosporine.
Specific Aims
Pravastatin induction of HO-1 in NRK-52E cells involved COX-2 and a PPARα pathway.
Pravastatin-activated HO-1 gene expression involved a PPARα-dependent pathway in NRK-52E
Pravastatin induced HO-1 expression via a PPARα-dependent pathway in mice.
Inducers of HO-1 are many, and include UV-A radiation, hydrogen peroxide, cytokines, growth factors, oxidized lipids, hyperoxia, nitric oxide (NO), prostacyclin and PPARα and γ. We hypothesized that the protective effects of statins against carboplatin-induced renal injury may be mediated by PPARα induced HO-1.
Results Pravastatin enhanced PPARα nuclear translocation in NRK-52E cells.
Pravastatin attenuated carboplatin-induced renal cell apoptosis, and caspase-3 expression.
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