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Anticancer drug 2-methoxyestradiol protects against renal ischemia/reperfusion injury by reducing inflammatory cytokines expression.

Chen YY, Yeh CH, So EC, Sun DP, Wang LY, Hsing CH - Biomed Res Int (2014)

Bottom Line: In vitro, we determined the effect of 2ME2 on reactive oxygen species (ROS) production and cell viability in antimycin-A-treated renal mesangial (RMC) and tubular (NRK52E) cells.Western blotting showed that 2ME2 treatment substantially decreased the expression of activated caspase-9, NF-κB, and HIF-1α but increased the antiapoptotic proteins BCL-2 and BCL-xL in kidney of I/R injury.In vitro, 2MR2 decreased ROS production and increased cell viability in antimycin-A-treated RMC and NRK52E cells. 2ME2 reduces renal I/R injury in mice because it inhibits the expression of ROS and proinflammatory cytokines and induces antiapoptotic proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Research, Chi-Mei Medical Center, Tainan 710, Taiwan ; Department of Biotechnology, National Formosa University, Yunlin County 632, Taiwan.

ABSTRACT

Background: Ischemia/reperfusion (I/R) injury is a major cause of acute renal failure and allograft dysfunction in kidney transplantation. ROS/inflammatory cytokines are involved in I/R injury. 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol, inhibits inflammatory cytokine expression and is an antiangiogenic and antitumor agent. We investigated the inhibitory effect of 2ME2 on renal I/R injury and possible molecular actions.

Methods: BALB/c mice were intraperitoneally injected with 2ME2 (10 or 20 mg/kg) or vehicle 12 h before and immediately after renal I/R experiments. The kidney weight, renal function, tubular damages, and apoptotic response were examined 24 h after I/R injury. The expression of mRNA of interleukin-1β, tumor necrosis factor- (TNF) α, caspase-3, hypoxia inducible factor- (HIF) 1α, and proapoptotic Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) in kidney tissue was determined using RT-PCR, while the expression of nuclear factor κB (NF-κB), BCL-2, and BCL-xL, activated caspase-9, and HIF-1α was determined using immunoblotting. In vitro, we determined the effect of 2ME2 on reactive oxygen species (ROS) production and cell viability in antimycin-A-treated renal mesangial (RMC) and tubular (NRK52E) cells.

Results: Serum creatinine and blood urea nitrogen were significantly higher in mice with renal I/R injury than in sham control and in I/R+2ME2-treated mice. Survival in I/R+2ME2-treated mice was higher than in I/R mice. Histological examination showed that 2ME2 attenuated tubular damage in I/R mice, which was associated with lower expression TNF-α, IL-1β, caspase-9, HIF-1α, and BNIP3 mRNA in kidney tissue. Western blotting showed that 2ME2 treatment substantially decreased the expression of activated caspase-9, NF-κB, and HIF-1α but increased the antiapoptotic proteins BCL-2 and BCL-xL in kidney of I/R injury. In vitro, 2MR2 decreased ROS production and increased cell viability in antimycin-A-treated RMC and NRK52E cells.

Conclusions: 2ME2 reduces renal I/R injury in mice because it inhibits the expression of ROS and proinflammatory cytokines and induces antiapoptotic proteins.

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Related in: MedlinePlus

2ME2 reduced ROS production and increased cell survival in antimycin-A (Aa)-treated renal cells. (a) Direct free radical scavenging activity of 2ME2 determined by the DPPH radical scavenging assay. Values represent the data of three independent experiments. Vitamin C was used as a positive control compound. (b) Cells were treated with antimycin-A 10 μM with saline or 2ME2 (2.5 or 5 μM) for 1 h. Quantitation of ROS generation in RMCs and NRK52E cells using CM-H2DCFDA assay. (c) RMCs and NRK52E cells were treated with antimycin-A 10 μM with saline or 2ME2 2.5 μM for 16 h. Cell survival was determined using MTT assay. All groups: n = 3. #P < 0.05 compared to untreated group and *P < 0.05 compared to antimycin-A group.
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fig6: 2ME2 reduced ROS production and increased cell survival in antimycin-A (Aa)-treated renal cells. (a) Direct free radical scavenging activity of 2ME2 determined by the DPPH radical scavenging assay. Values represent the data of three independent experiments. Vitamin C was used as a positive control compound. (b) Cells were treated with antimycin-A 10 μM with saline or 2ME2 (2.5 or 5 μM) for 1 h. Quantitation of ROS generation in RMCs and NRK52E cells using CM-H2DCFDA assay. (c) RMCs and NRK52E cells were treated with antimycin-A 10 μM with saline or 2ME2 2.5 μM for 16 h. Cell survival was determined using MTT assay. All groups: n = 3. #P < 0.05 compared to untreated group and *P < 0.05 compared to antimycin-A group.

Mentions: ROS play a critical role in I/R-induced renal injury; however, the effect of 2ME2 on ROS production has not been reported. To determine whether oxidative stress is involved in 2ME2's renal protective mechanism under I/R condition, we evaluated the direct ROS scavenging effect of 2ME2 in antimycin-A-treated RMCs and NRK52E. Antimycin-A is an inhibitor of oxidative phosphorylation used for chemical hypoxia. As shown in the DPPH radical scavenging assay (Figure 6(a)), 2ME2 scavenged the free radicals in a concentration-dependent manner similar to the antioxidant vitamin C. The antioxidative ability of 2EM2 was further measured by CM-H2DCFDA staining. ROS was noted to increase in RMCs and NRK52E under antimycin-A exposure compared with untreated control, but its levels were markedly inhibited by 2ME2 (Figure 6(b)). Moreover, we treated RMCs and NRK52E cells with antimycin-A 10 μM combined with saline or 2ME2 2.5 μM for 16 h and determined the cell viability using MTT assay. 2ME2 significantly reduced cell death in antimycin-A-treated cells (Figure 6(c)).


Anticancer drug 2-methoxyestradiol protects against renal ischemia/reperfusion injury by reducing inflammatory cytokines expression.

Chen YY, Yeh CH, So EC, Sun DP, Wang LY, Hsing CH - Biomed Res Int (2014)

2ME2 reduced ROS production and increased cell survival in antimycin-A (Aa)-treated renal cells. (a) Direct free radical scavenging activity of 2ME2 determined by the DPPH radical scavenging assay. Values represent the data of three independent experiments. Vitamin C was used as a positive control compound. (b) Cells were treated with antimycin-A 10 μM with saline or 2ME2 (2.5 or 5 μM) for 1 h. Quantitation of ROS generation in RMCs and NRK52E cells using CM-H2DCFDA assay. (c) RMCs and NRK52E cells were treated with antimycin-A 10 μM with saline or 2ME2 2.5 μM for 16 h. Cell survival was determined using MTT assay. All groups: n = 3. #P < 0.05 compared to untreated group and *P < 0.05 compared to antimycin-A group.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4151070&req=5

fig6: 2ME2 reduced ROS production and increased cell survival in antimycin-A (Aa)-treated renal cells. (a) Direct free radical scavenging activity of 2ME2 determined by the DPPH radical scavenging assay. Values represent the data of three independent experiments. Vitamin C was used as a positive control compound. (b) Cells were treated with antimycin-A 10 μM with saline or 2ME2 (2.5 or 5 μM) for 1 h. Quantitation of ROS generation in RMCs and NRK52E cells using CM-H2DCFDA assay. (c) RMCs and NRK52E cells were treated with antimycin-A 10 μM with saline or 2ME2 2.5 μM for 16 h. Cell survival was determined using MTT assay. All groups: n = 3. #P < 0.05 compared to untreated group and *P < 0.05 compared to antimycin-A group.
Mentions: ROS play a critical role in I/R-induced renal injury; however, the effect of 2ME2 on ROS production has not been reported. To determine whether oxidative stress is involved in 2ME2's renal protective mechanism under I/R condition, we evaluated the direct ROS scavenging effect of 2ME2 in antimycin-A-treated RMCs and NRK52E. Antimycin-A is an inhibitor of oxidative phosphorylation used for chemical hypoxia. As shown in the DPPH radical scavenging assay (Figure 6(a)), 2ME2 scavenged the free radicals in a concentration-dependent manner similar to the antioxidant vitamin C. The antioxidative ability of 2EM2 was further measured by CM-H2DCFDA staining. ROS was noted to increase in RMCs and NRK52E under antimycin-A exposure compared with untreated control, but its levels were markedly inhibited by 2ME2 (Figure 6(b)). Moreover, we treated RMCs and NRK52E cells with antimycin-A 10 μM combined with saline or 2ME2 2.5 μM for 16 h and determined the cell viability using MTT assay. 2ME2 significantly reduced cell death in antimycin-A-treated cells (Figure 6(c)).

Bottom Line: In vitro, we determined the effect of 2ME2 on reactive oxygen species (ROS) production and cell viability in antimycin-A-treated renal mesangial (RMC) and tubular (NRK52E) cells.Western blotting showed that 2ME2 treatment substantially decreased the expression of activated caspase-9, NF-κB, and HIF-1α but increased the antiapoptotic proteins BCL-2 and BCL-xL in kidney of I/R injury.In vitro, 2MR2 decreased ROS production and increased cell viability in antimycin-A-treated RMC and NRK52E cells. 2ME2 reduces renal I/R injury in mice because it inhibits the expression of ROS and proinflammatory cytokines and induces antiapoptotic proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Research, Chi-Mei Medical Center, Tainan 710, Taiwan ; Department of Biotechnology, National Formosa University, Yunlin County 632, Taiwan.

ABSTRACT

Background: Ischemia/reperfusion (I/R) injury is a major cause of acute renal failure and allograft dysfunction in kidney transplantation. ROS/inflammatory cytokines are involved in I/R injury. 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol, inhibits inflammatory cytokine expression and is an antiangiogenic and antitumor agent. We investigated the inhibitory effect of 2ME2 on renal I/R injury and possible molecular actions.

Methods: BALB/c mice were intraperitoneally injected with 2ME2 (10 or 20 mg/kg) or vehicle 12 h before and immediately after renal I/R experiments. The kidney weight, renal function, tubular damages, and apoptotic response were examined 24 h after I/R injury. The expression of mRNA of interleukin-1β, tumor necrosis factor- (TNF) α, caspase-3, hypoxia inducible factor- (HIF) 1α, and proapoptotic Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) in kidney tissue was determined using RT-PCR, while the expression of nuclear factor κB (NF-κB), BCL-2, and BCL-xL, activated caspase-9, and HIF-1α was determined using immunoblotting. In vitro, we determined the effect of 2ME2 on reactive oxygen species (ROS) production and cell viability in antimycin-A-treated renal mesangial (RMC) and tubular (NRK52E) cells.

Results: Serum creatinine and blood urea nitrogen were significantly higher in mice with renal I/R injury than in sham control and in I/R+2ME2-treated mice. Survival in I/R+2ME2-treated mice was higher than in I/R mice. Histological examination showed that 2ME2 attenuated tubular damage in I/R mice, which was associated with lower expression TNF-α, IL-1β, caspase-9, HIF-1α, and BNIP3 mRNA in kidney tissue. Western blotting showed that 2ME2 treatment substantially decreased the expression of activated caspase-9, NF-κB, and HIF-1α but increased the antiapoptotic proteins BCL-2 and BCL-xL in kidney of I/R injury. In vitro, 2MR2 decreased ROS production and increased cell viability in antimycin-A-treated RMC and NRK52E cells.

Conclusions: 2ME2 reduces renal I/R injury in mice because it inhibits the expression of ROS and proinflammatory cytokines and induces antiapoptotic proteins.

Show MeSH
Related in: MedlinePlus