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Down-regulation of nuclear HMGB1 reduces ischemia-induced HMGB1 translocation and release and protects against liver ischemia-reperfusion injury

View Article: PubMed Central - PubMed

ABSTRACT

Hepatocyte-specific HMGB1 deletion has been found to worsen the injury and inflammation in liver ischemia-reperfusion injury (IRI), highlighting a role for intracellular HMGB1 in cellular protection. Down-regulation of nuclear HMGB1 by small interfering RNA (siRNA) might not only decrease its injurious extracellular role by reducing its release but also serve to maintain its beneficial intracellular role, thus protecting against IRI. We established a non-lethal liver IRI model in mice via segmental hepatic warm ischemia for 1 h and reperfusion for 6 h. HMGB1-siRNA achieved a reduction of ~60–70% in the nuclear HMGB1 expression in the liver at 48 h post-treatment. Knockdown of nuclear HMGB1 expression dramatically reduced both the degree of nuclear-cytoplasmic translocation of HMGB1 during hepatic ischemia and of HMGB1 release after hepatic reperfusion, resulting in significant preservation of liver function and a marked reduction in pathological damage. Also, HMGB1-siRNA pretreatment markedly inhibited the increases in hepatic expression of TLR4, TLR2, RAGE, TNF-α, IL-1β, IL-6, MCP-1, iNOS, and COX-2 seen in control mice after hepatic reperfusion. We demonstrated for the first time that down-regulation of nuclear HMGB1 reduces ischemia-induced HMGB1 release and protects against liver IRI, which is helpful for better understanding the role of HMGB1 in organ IRI.

No MeSH data available.


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Knockdown of nuclear HMGB1 expression reduces the degree of HMGB1 nucleocytoplasmic shuttling during hepatic ischemia.Mouse livers were subjected to 60 min of ischemia without reperfusion. (A) Immunohistochemical staining of HMGB1 from sections of livers harvested from the unclamped sham-operated mice (sham) or from mice clamped and pretreated with PBS, scrambled siRNA (scramble), or HMGB1-siRNA (si-HMGB1) (original magnification, x400 and x1000). Images are representative liver sections from 9 mice per group. (B) Western blot analysis for HMGB1 was performed using cytoplasmic protein samples from sham-operated livers or the ischemic livers from PBS-, scrambled siRNA- or HMGB1-siRNA-pretreated mice, with each lane representing a separate animal (a). The blots shown are representative of three experiments with similar results. The corresponding densitometric analyses are shown as bar graphs (b). Means ± SEM are reported (*P < 0.05, n = 3 per group).
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f4: Knockdown of nuclear HMGB1 expression reduces the degree of HMGB1 nucleocytoplasmic shuttling during hepatic ischemia.Mouse livers were subjected to 60 min of ischemia without reperfusion. (A) Immunohistochemical staining of HMGB1 from sections of livers harvested from the unclamped sham-operated mice (sham) or from mice clamped and pretreated with PBS, scrambled siRNA (scramble), or HMGB1-siRNA (si-HMGB1) (original magnification, x400 and x1000). Images are representative liver sections from 9 mice per group. (B) Western blot analysis for HMGB1 was performed using cytoplasmic protein samples from sham-operated livers or the ischemic livers from PBS-, scrambled siRNA- or HMGB1-siRNA-pretreated mice, with each lane representing a separate animal (a). The blots shown are representative of three experiments with similar results. The corresponding densitometric analyses are shown as bar graphs (b). Means ± SEM are reported (*P < 0.05, n = 3 per group).

Mentions: To determine whether the gene silencing of HMGB1 could reduce the degree of early nuclear-cytoplasmic trafficking of HMGB1 in clamped ischemic hepatic lobes without reperfusion, immunohistochemical studies were performed to detect changes in the localization of HMGB1. In sham-operated livers, HMGB1 was predominantly confined to the nuclei of hepatic parenchymal cells (Fig. 4A). However, after 60 min of warm ischemia without reperfusion, cytoplasmic HMGB1 expression was seen to be significantly increased in hepatocytes of both PBS-treated and scrambled siRNA-treated control mice (Fig. 4A). In contrast, knockdown of nuclear HMGB1 expression by pretreatment with HMGB1-siRNA markedly reduced the degree of ischemia-induced translocation of nuclear HMGB1 to the cytoplasm (Fig. 4A). Western blot analysis of hepatic cytoplasmic proteins further confirmed the HMGB1 translocation following ischemia. HMGB1-siRNA-treated livers that had undergone 60 min of ischemia had only a slightly higher amount of HMGB1 in the cytoplasm than did the sham-operated livers (Fig. 4B). In contrast, a significant amount of cytoplasmic HMGB1 was detected in both PBS-treated and scrambled siRNA-treated control animals when the livers were subjected to 60 min of warm ischemia.


Down-regulation of nuclear HMGB1 reduces ischemia-induced HMGB1 translocation and release and protects against liver ischemia-reperfusion injury
Knockdown of nuclear HMGB1 expression reduces the degree of HMGB1 nucleocytoplasmic shuttling during hepatic ischemia.Mouse livers were subjected to 60 min of ischemia without reperfusion. (A) Immunohistochemical staining of HMGB1 from sections of livers harvested from the unclamped sham-operated mice (sham) or from mice clamped and pretreated with PBS, scrambled siRNA (scramble), or HMGB1-siRNA (si-HMGB1) (original magnification, x400 and x1000). Images are representative liver sections from 9 mice per group. (B) Western blot analysis for HMGB1 was performed using cytoplasmic protein samples from sham-operated livers or the ischemic livers from PBS-, scrambled siRNA- or HMGB1-siRNA-pretreated mice, with each lane representing a separate animal (a). The blots shown are representative of three experiments with similar results. The corresponding densitometric analyses are shown as bar graphs (b). Means ± SEM are reported (*P < 0.05, n = 3 per group).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: Knockdown of nuclear HMGB1 expression reduces the degree of HMGB1 nucleocytoplasmic shuttling during hepatic ischemia.Mouse livers were subjected to 60 min of ischemia without reperfusion. (A) Immunohistochemical staining of HMGB1 from sections of livers harvested from the unclamped sham-operated mice (sham) or from mice clamped and pretreated with PBS, scrambled siRNA (scramble), or HMGB1-siRNA (si-HMGB1) (original magnification, x400 and x1000). Images are representative liver sections from 9 mice per group. (B) Western blot analysis for HMGB1 was performed using cytoplasmic protein samples from sham-operated livers or the ischemic livers from PBS-, scrambled siRNA- or HMGB1-siRNA-pretreated mice, with each lane representing a separate animal (a). The blots shown are representative of three experiments with similar results. The corresponding densitometric analyses are shown as bar graphs (b). Means ± SEM are reported (*P < 0.05, n = 3 per group).
Mentions: To determine whether the gene silencing of HMGB1 could reduce the degree of early nuclear-cytoplasmic trafficking of HMGB1 in clamped ischemic hepatic lobes without reperfusion, immunohistochemical studies were performed to detect changes in the localization of HMGB1. In sham-operated livers, HMGB1 was predominantly confined to the nuclei of hepatic parenchymal cells (Fig. 4A). However, after 60 min of warm ischemia without reperfusion, cytoplasmic HMGB1 expression was seen to be significantly increased in hepatocytes of both PBS-treated and scrambled siRNA-treated control mice (Fig. 4A). In contrast, knockdown of nuclear HMGB1 expression by pretreatment with HMGB1-siRNA markedly reduced the degree of ischemia-induced translocation of nuclear HMGB1 to the cytoplasm (Fig. 4A). Western blot analysis of hepatic cytoplasmic proteins further confirmed the HMGB1 translocation following ischemia. HMGB1-siRNA-treated livers that had undergone 60 min of ischemia had only a slightly higher amount of HMGB1 in the cytoplasm than did the sham-operated livers (Fig. 4B). In contrast, a significant amount of cytoplasmic HMGB1 was detected in both PBS-treated and scrambled siRNA-treated control animals when the livers were subjected to 60 min of warm ischemia.

View Article: PubMed Central - PubMed

ABSTRACT

Hepatocyte-specific HMGB1 deletion has been found to worsen the injury and inflammation in liver ischemia-reperfusion injury (IRI), highlighting a role for intracellular HMGB1 in cellular protection. Down-regulation of nuclear HMGB1 by small interfering RNA (siRNA) might not only decrease its injurious extracellular role by reducing its release but also serve to maintain its beneficial intracellular role, thus protecting against IRI. We established a non-lethal liver IRI model in mice via segmental hepatic warm ischemia for 1&thinsp;h and reperfusion for 6&thinsp;h. HMGB1-siRNA achieved a reduction of ~60&ndash;70% in the nuclear HMGB1 expression in the liver at 48&thinsp;h post-treatment. Knockdown of nuclear HMGB1 expression dramatically reduced both the degree of nuclear-cytoplasmic translocation of HMGB1 during hepatic ischemia and of HMGB1 release after hepatic reperfusion, resulting in significant preservation of liver function and a marked reduction in pathological damage. Also, HMGB1-siRNA pretreatment markedly inhibited the increases in hepatic expression of TLR4, TLR2, RAGE, TNF-&alpha;, IL-1&beta;, IL-6, MCP-1, iNOS, and COX-2 seen in control mice after hepatic reperfusion. We demonstrated for the first time that down-regulation of nuclear HMGB1 reduces ischemia-induced HMGB1 release and protects against liver IRI, which is helpful for better understanding the role of HMGB1 in organ IRI.

No MeSH data available.


Related in: MedlinePlus