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. 2011 Aug;54(2):687-96.
doi: 10.1002/hep.24419. Epub 2011 Jun 26.

"V体育2025版" Inhibition of glycogen synthase kinase 3 beta ameliorates liver ischemia reperfusion injury by way of an interleukin-10-mediated immune regulatory mechanism

Feng Ren  1 , Zhongping Duan, Qiao Cheng, Xiuda Shen, Feng Gao, Li Bai, Jun Liu, Ronald W Busuttil, Jerzy W Kupiec-Weglinski, Yuan Zhai
Affiliations

Inhibition of glycogen synthase kinase 3 beta ameliorates liver ischemia reperfusion injury by way of an interleukin-10-mediated immune regulatory mechanism

Feng Ren et al. Hepatology. 2011 Aug.

Abstract

The ubiquitous serine/threonine kinase glycogen synthase kinase 3 beta (Gsk3β) differentially regulates macrophage Toll-like receptor (TLR)-triggered pro- and anti-inflammatory cytokine programs. This study was designed to determine the in vivo role and therapeutic potential of Gsk3β modulation in tissue inflammation and injury in a murine model of liver partial warm ischemia/reperfusion injury (IRI). As a constitutively activated liver kinase, Gsk3β became quickly inactivated (phosphorylated) following IR. The active Gsk3β, however, was essential for the development of IRI pathology, as administration of its specific inhibitor, SB216763, ameliorated the hepatocellular damage, evidenced by reduced serum alanine aminotransferase (sALT) levels and well-preserved liver architecture compared with controls. The liver protective effect of Gsk3β inhibition was dependent on an immune regulatory mechanism, rather than direct cytoprotection via mitochondria permeability transition pores (MPTP). Indeed: (1) coadministration of SB216763 and atractyloside (MPTP opener) failed to abrogate a local cytoprotective Gsk3β inhibition effect; (2) SB216763 selectively inhibited IR-triggered liver pro-inflammatory, but spared interleukin (IL)-10, gene induction programs; and (3) IL-10 neutralization restored liver inflammation and IRI in SB216763-treated mice. Gsk3β inactivation by IR was a self-regulatory mechanism in liver homeostasis, critically dependent on phosphoinositide 3 (PI3)-kinase activation, as administration of a PI3 kinase inhibitor, wortmannin, reduced Gsk3 phosphorylation and augmented liver damage. In vitro, IL-10 was critical for the suppression of pro-inflammatory gene programs by Gsk3 inhibition in bone marrow-derived macrophages in response to TLR4 stimulation VSports手机版. .

Conclusion: Our novel findings document the key immune regulatory function of Gsk3β signaling in the pathophysiology of liver IRI, and provide a rationale to target Gsk3β as a refined therapeutic strategy to ameliorate liver IRI V体育安卓版. .

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Figures

Figure 1
Figure 1
Liver ischemia and reperfusion triggers Gsk3β phosphorylation. Liver samples were harvested from B6 mice that were either sham-operated or subjected to 90min of hepatic warm ischemia, followed by various length of reperfusion (0, 1, 4 h). The phosphorylated (serine 9) and total Gsk3β levels, along with phosphorylated MAP kinases including JNK, Erk and p38, and β-actin were measured by Western blots. Representative of two experiments is shown.
Figure 2
Figure 2. Gsk3 inhibition by SB216763 ameliorates liver IRI in B6 mice
(a) Mice were treated with vehicle (DMSO) or SB216763. Liver samples, harvested 24h later, were subjected to Western blot analysis of phosphorylated glycogen synthases. (b) Mice were sham-operated, or subjected to 90min warm ischemia followed by 1h or 6h reperfusion. Recipients were treated with vehicle (DMSO) or SB216763. Group averages of sALT levels at 6h were measured and plotted (n=5–12/group). (c) Representative liver histology (H/E staining at 6h). (d) Group averages of liver Suzuki scores (6h). (e) Liver MPO levels at 6h. (f) Expression levels of inflammatory TNF-α, CXCL10, IL-1β and IL-6 at 6h. (g) IL-12p40 and IL-10 at 1h and 6h of reperfusion (n=4–5/group). * indicates p<0.05.
Figure 2
Figure 2. Gsk3 inhibition by SB216763 ameliorates liver IRI in B6 mice
(a) Mice were treated with vehicle (DMSO) or SB216763. Liver samples, harvested 24h later, were subjected to Western blot analysis of phosphorylated glycogen synthases. (b) Mice were sham-operated, or subjected to 90min warm ischemia followed by 1h or 6h reperfusion. Recipients were treated with vehicle (DMSO) or SB216763. Group averages of sALT levels at 6h were measured and plotted (n=5–12/group). (c) Representative liver histology (H/E staining at 6h). (d) Group averages of liver Suzuki scores (6h). (e) Liver MPO levels at 6h. (f) Expression levels of inflammatory TNF-α, CXCL10, IL-1β and IL-6 at 6h. (g) IL-12p40 and IL-10 at 1h and 6h of reperfusion (n=4–5/group). * indicates p<0.05.
Figure 3
Figure 3. Liver protection against IRI following Gsk3 inhibition is independent of MPTP, but dependent on an IL-10 mediated immune regulation. Groups of B6 mice were treated with vehicle, or astractyloside (Atr) or SB216763 (SB), or SB + Atr or SB +anti-IL-10 Ab, and then subjected to 90min of ischemia, followed by 6h reperfusion, as described in Materials and Methods
(a, b) Group averages of sALT levels were measured and plotted (c) Representative liver histology (H/E staining) (d) Group averages of liver pro-inflammatory gene (TNF-α, CXCL10, IL-1β and IL-6) expression levels. (c), n=3–6/group, * indicates p<0.05.
Figure 4
Figure 4. PI3 kinase activation is responsible for Gsk3β phosphorylation and its inhibition deteriorates liver IRI. Groups of B6 mice were treated with SB216763 (SB), Wortmannin (WM) or both or vehicle at 1–2h prior to the liver ischemia insult, as described in Materials and Methods. Liver samples were harvested at 6h post-reperfusion
(a) Proteins were analyzed by Western blots with Abs against phosphorylated or total Gsk3β, and β-actin (b) Sixty min ischemia time was used to show the effect of PI3 kinase inhibition in liver IRI. Average sALT levels in different experimental groups were plotted. (c) Representative liver histology (H/E staining) is shown. (d) To establish the functional relationship between PI3 kinase and Gsk3β, SB was administered 2h and WM 1h prior to the ischemia insult. Serum ALT levels were measured at 6h of reperfusion.
Figure 5
Figure 5. Gsk3 inhibition regulates TLR4 response in vitro. Bone marrow-derived macrophages (BMMs) were stimulated with LPS for 1h or 6h in the absence or presence of SB216763. Gene induction profiles were measured by qRT-PCR, as described in Materials and Methods
(a) Average target gene/HPRT ratios for each experimental group were plotted. * indicates p<0.05 between LPS and SB/LPS groups. BMMs were also harvested after 15min, 30min, 1h or 2h stimulation with LPS w/ or w/o SB216763. (b) Total cell lysates were analyzed for phosphorylated Gsk3β, JNK, Erk and p38 MAP kinases, as well as total Gsk3β and β-actin protein levels by Western blots. (c) BMMs were stimulated with LPS for 6h w/ or w/o SB216763 in combination with anti-IL-10 Abs. Group averages of target gene/HPRT gene expression ratios were plotted. * indicates p<0.05 between SB/LPS and SB/αIL-10/LPS groups. Representative results of at least 2 separate experiments are shown.
Figure 5
Figure 5. Gsk3 inhibition regulates TLR4 response in vitro. Bone marrow-derived macrophages (BMMs) were stimulated with LPS for 1h or 6h in the absence or presence of SB216763. Gene induction profiles were measured by qRT-PCR, as described in Materials and Methods
(a) Average target gene/HPRT ratios for each experimental group were plotted. * indicates p<0.05 between LPS and SB/LPS groups. BMMs were also harvested after 15min, 30min, 1h or 2h stimulation with LPS w/ or w/o SB216763. (b) Total cell lysates were analyzed for phosphorylated Gsk3β, JNK, Erk and p38 MAP kinases, as well as total Gsk3β and β-actin protein levels by Western blots. (c) BMMs were stimulated with LPS for 6h w/ or w/o SB216763 in combination with anti-IL-10 Abs. Group averages of target gene/HPRT gene expression ratios were plotted. * indicates p<0.05 between SB/LPS and SB/αIL-10/LPS groups. Representative results of at least 2 separate experiments are shown.
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