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SPA0355 attenuates ischemia/reperfusion-induced liver injury in mice.

Bae UJ, Yang JD, Ka SO, Koo JH, Woo SJ, Lee YR, Yu HC, Cho BH, Zhao HY, Ryu JH, Lee SM, Jeon R, Park BH - Exp. Mol. Med. (2014)

Bottom Line: Concomitantly, the expression of NF-κB target genes such as IL-1β, IL-6, TNF-α and iNOS was significantly downregulated.Lastly, the liver antioxidant enzymes superoxide dismutase, catalase and glutathione were upregulated by SPA0355 treatment, which correlated with the reduction in serum malondialdehyde.Our results suggest that SPA0355 pretreatment prior to I/R injury could be an effective method to reduce liver damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Chonbuk National University Medical School, Jeonbuk, Republic of Korea.

ABSTRACT
Hepatic ischemia/reperfusion (I/R) injury leads to oxidative stress and acute inflammatory responses that cause liver damage and have a considerable impact on the postoperative outcome. Much research has been performed to develop possible protective techniques. We aimed to investigate the efficacy of SPA0355, a synthetic thiourea analog, in an animal model of hepatic I/R injury. Male C57BL/6 mice underwent normothermic partial liver ischemia for 45 min followed by varying periods of reperfusion. The animals were divided into three groups: sham operated, I/R and SPA0355 pretreated. Pretreatment with SPA0355 protected against hepatic I/R injury, as indicated by the decreased levels of serum aminotransferase and reduced parenchymal necrosis and apoptosis. Liver synthetic function was also restored by SPA0355 as reflected by the prolonged prothrombin time. To gain insight into the mechanism involved in this protection, we measured the activity of nuclear factor-κB (NF-κB), which revealed that SPA0355 suppressed the nuclear translocation and DNA binding of NF-κB subunits. Concomitantly, the expression of NF-κB target genes such as IL-1β, IL-6, TNF-α and iNOS was significantly downregulated. Lastly, the liver antioxidant enzymes superoxide dismutase, catalase and glutathione were upregulated by SPA0355 treatment, which correlated with the reduction in serum malondialdehyde. Our results suggest that SPA0355 pretreatment prior to I/R injury could be an effective method to reduce liver damage.

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Effect of SPA0355 on I/R-induced NF-κB activation. Nuclear and cytoplasmic proteins were extracted from liver homogenates after 1 h of reperfusion and nuclear translocation of p50 and p65 subunits and cytoplasmic IκBα degradation (a), NF-κB DNA binding activity (b), and protein levels and phosphorylation of IKKα and IKKβ (c) were analyzed. After 1 h reperfusion, mRNA levels of IL-1β, IL-6, TNF-α and iNOS were analyzed by real-time RT–PCR (d). Values are the mean±s.e.m. of three independent experiments (n=9 mice per group). **P<0.01 vs sham-operated mice; #P<0.05, ##P<0.01 vs I/R mice.
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fig5: Effect of SPA0355 on I/R-induced NF-κB activation. Nuclear and cytoplasmic proteins were extracted from liver homogenates after 1 h of reperfusion and nuclear translocation of p50 and p65 subunits and cytoplasmic IκBα degradation (a), NF-κB DNA binding activity (b), and protein levels and phosphorylation of IKKα and IKKβ (c) were analyzed. After 1 h reperfusion, mRNA levels of IL-1β, IL-6, TNF-α and iNOS were analyzed by real-time RT–PCR (d). Values are the mean±s.e.m. of three independent experiments (n=9 mice per group). **P<0.01 vs sham-operated mice; #P<0.05, ##P<0.01 vs I/R mice.

Mentions: To determine whether SPA0355 exerted a liver protective effect through suppression of NF-κB activation, we measured protein levels of p65 and p50 subunits in nuclear extracts and DNA binding of NF-κB subunits prepared 1 h after reperfusion. I/R mice showed increases in nuclear p65 and p50 protein levels (Figure 5a) and binding activity of the p65 subunit to an NF-κB consensus sequence (Figure 5b) compared with sham-operated mice. The DNA-bound p65 subunit of NF-κB was resolved by supershift (Figure 5b, lane 5). In contrast, nuclear extracts prepared from mice that were pretreated with SPA0355 revealed markedly suppressed nuclear translocation and DNA binding of NF-κB. Consistently, I/R mice showed decreased levels of IκBα protein in the cytoplasm because of IκBα degradation, as compared with a similar fraction from sham mice, but IκBα degradation was markedly suppressed by SPA0355 pretreatment (Figure 5a). Next, we tested the effect of SPA0355 on IKK activation, which is required for IκB degradation. SPA0355 had no effect on IKKα and IKKβ protein levels, but suppressed I/R-induced IKK activity, as shown by decreased levels of phosphorylated forms of IKKα and IKKβ (Figure 5c). Together, these results suggested that SPA0355 inhibited the IκBα degradation by reducing IKK activities, thereby preventing subsequent NF-κB activation.


SPA0355 attenuates ischemia/reperfusion-induced liver injury in mice.

Bae UJ, Yang JD, Ka SO, Koo JH, Woo SJ, Lee YR, Yu HC, Cho BH, Zhao HY, Ryu JH, Lee SM, Jeon R, Park BH - Exp. Mol. Med. (2014)

Effect of SPA0355 on I/R-induced NF-κB activation. Nuclear and cytoplasmic proteins were extracted from liver homogenates after 1 h of reperfusion and nuclear translocation of p50 and p65 subunits and cytoplasmic IκBα degradation (a), NF-κB DNA binding activity (b), and protein levels and phosphorylation of IKKα and IKKβ (c) were analyzed. After 1 h reperfusion, mRNA levels of IL-1β, IL-6, TNF-α and iNOS were analyzed by real-time RT–PCR (d). Values are the mean±s.e.m. of three independent experiments (n=9 mice per group). **P<0.01 vs sham-operated mice; #P<0.05, ##P<0.01 vs I/R mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4150932&req=5

fig5: Effect of SPA0355 on I/R-induced NF-κB activation. Nuclear and cytoplasmic proteins were extracted from liver homogenates after 1 h of reperfusion and nuclear translocation of p50 and p65 subunits and cytoplasmic IκBα degradation (a), NF-κB DNA binding activity (b), and protein levels and phosphorylation of IKKα and IKKβ (c) were analyzed. After 1 h reperfusion, mRNA levels of IL-1β, IL-6, TNF-α and iNOS were analyzed by real-time RT–PCR (d). Values are the mean±s.e.m. of three independent experiments (n=9 mice per group). **P<0.01 vs sham-operated mice; #P<0.05, ##P<0.01 vs I/R mice.
Mentions: To determine whether SPA0355 exerted a liver protective effect through suppression of NF-κB activation, we measured protein levels of p65 and p50 subunits in nuclear extracts and DNA binding of NF-κB subunits prepared 1 h after reperfusion. I/R mice showed increases in nuclear p65 and p50 protein levels (Figure 5a) and binding activity of the p65 subunit to an NF-κB consensus sequence (Figure 5b) compared with sham-operated mice. The DNA-bound p65 subunit of NF-κB was resolved by supershift (Figure 5b, lane 5). In contrast, nuclear extracts prepared from mice that were pretreated with SPA0355 revealed markedly suppressed nuclear translocation and DNA binding of NF-κB. Consistently, I/R mice showed decreased levels of IκBα protein in the cytoplasm because of IκBα degradation, as compared with a similar fraction from sham mice, but IκBα degradation was markedly suppressed by SPA0355 pretreatment (Figure 5a). Next, we tested the effect of SPA0355 on IKK activation, which is required for IκB degradation. SPA0355 had no effect on IKKα and IKKβ protein levels, but suppressed I/R-induced IKK activity, as shown by decreased levels of phosphorylated forms of IKKα and IKKβ (Figure 5c). Together, these results suggested that SPA0355 inhibited the IκBα degradation by reducing IKK activities, thereby preventing subsequent NF-κB activation.

Bottom Line: Concomitantly, the expression of NF-κB target genes such as IL-1β, IL-6, TNF-α and iNOS was significantly downregulated.Lastly, the liver antioxidant enzymes superoxide dismutase, catalase and glutathione were upregulated by SPA0355 treatment, which correlated with the reduction in serum malondialdehyde.Our results suggest that SPA0355 pretreatment prior to I/R injury could be an effective method to reduce liver damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Chonbuk National University Medical School, Jeonbuk, Republic of Korea.

ABSTRACT
Hepatic ischemia/reperfusion (I/R) injury leads to oxidative stress and acute inflammatory responses that cause liver damage and have a considerable impact on the postoperative outcome. Much research has been performed to develop possible protective techniques. We aimed to investigate the efficacy of SPA0355, a synthetic thiourea analog, in an animal model of hepatic I/R injury. Male C57BL/6 mice underwent normothermic partial liver ischemia for 45 min followed by varying periods of reperfusion. The animals were divided into three groups: sham operated, I/R and SPA0355 pretreated. Pretreatment with SPA0355 protected against hepatic I/R injury, as indicated by the decreased levels of serum aminotransferase and reduced parenchymal necrosis and apoptosis. Liver synthetic function was also restored by SPA0355 as reflected by the prolonged prothrombin time. To gain insight into the mechanism involved in this protection, we measured the activity of nuclear factor-κB (NF-κB), which revealed that SPA0355 suppressed the nuclear translocation and DNA binding of NF-κB subunits. Concomitantly, the expression of NF-κB target genes such as IL-1β, IL-6, TNF-α and iNOS was significantly downregulated. Lastly, the liver antioxidant enzymes superoxide dismutase, catalase and glutathione were upregulated by SPA0355 treatment, which correlated with the reduction in serum malondialdehyde. Our results suggest that SPA0355 pretreatment prior to I/R injury could be an effective method to reduce liver damage.

Show MeSH
Related in: MedlinePlus