TLR4-mediated NF-κB signaling pathway mediates HMGB1-induced pancreatic injury in mice with severe acute pancreatitis

Abstract

Severe acute pancreatitis (SAP) is an extremely dangerous acute abdominal disorder which causes multiple complications and has a high mortality rate. Previous research has suggested that high-mobility group box 1 (HMGB1) plays an important role in the pathogenesis of SAP; however, the mechanisms underlying this strong correlation remain unclear. In this study, to further investigate whether HMGB1 acts as a stimulating factor, and whether Toll-like receptor 4 (TLR4) acts as its major mediator in the development of pancreatic injury during SAP, recombinant human HMGB1 (rhHMGB1) and TLR4-deficient mice were used. We found that HMGB1 and TLR4 were highly expressed, and nuclear factor-κB (NF-κB) was activated in our mouse model of SAP. We noted that the rhHMGB1 pancreas-targeted injection activated the TLR4-mediated NF-κB signaling pathway and induced pancreatic injury in wild-type mice. In TLR4-deficient mice, the rhHMGB1-induced activation of NF-κB and pathological changes in the pancreas were less evident than in wild-type mice. Therefore, this study provides evidence that HMGB1 promotes the pathogenesis of pancreatitis, and its downstream TLR4-mediated NF-κB signaling pathway is a potential important mediator in the development of this form of pancreatic injury.

Figure 1

Model of severe acute pancreatitis (SAP). (A) Histopathological changes of the pancreas: representative H&E-stained micrographs (original magnification, ×200) from the (a) sham and (b) SAP groups are presented. The pancreatic tissues in the SAP group exhibited marked edema and infiltration of inflammatory cells; a mass of necrotic acinar cells and the disappearance of normal structure in pancreas lobes were also observed. (B) Histological scores of the pancreas in the sham and SAP groups. (C) Changes in serum amylase and lipase levels in sham and SAP groups. Data are expressed as the means ± SD (n=6/group). ^*P<0.01 compared to the sham injection group (control group). Sham, sham injection.

Figure 2

Expression of high-mobility group box 1 (HMGB1) and Toll-like receptor 4 (TLR4) and activation of nuclear factor-κB (NF-κB) in pancreatic tissue from mice after L-arginine administration or sham injection. (A) Serum HMGB1 concentrations [(a) ELISA] were assessed in the sham and severe acute pancreatitis (SAP) groups. The expression of HMGB1 was assessed using the mRNA [(b) qPCR] and protein [(c) western blot analysis] levels in the pancreas tissue from the sham and SAP groups. (B) The expression of TLR4 was assessed with the mRNA [(a) qPCR] and protein [(b) western blot analysis] levels in the pancreatic tissue from the sham and SAP groups. (C) The activation of NF-κB was assessed from the nuclear p65 [(a) western blot analysis] and its downstream, relative levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) [(b) ELISA] in the pancreatic tissue from the sham and SAP groups. ^*P<0.01 compared to the sham group (control group). The relative levels of HMGB1 and TLR4 protein and mRNA compared to the control group are normalized to β-actin. The relative levels of nuclear p65 protein compared to the control group are normalized to histone H3. The relative levels of TNF-α and IL-1β compared to the control group are normalized to the total protein concentration of each sample. Data are expressed as the means ± SD (n=6/group). Blots shown are from a representative experiment that was repeated 3 times with similar results (n=6/group). Sham, sham injection.

Figure 3

Pancreatic injury induced by recombinant human high-mobility group box 1 (rhHMGB1). (A) Histopathological changes of the pancreas in response to rhHMGB1 administration in mice at 48 h. Representative H&E-stained micrographs (original magnification, ×200) from the (a) sham, (b) low dose of HMGB1 (HM-LD) and (c) high dose of HMGB1 (HM-HD) groups. The pancreatic tissues in the HM-LD group exhibited marked edema, neutrophil infiltration and acinar cell necrosis; more intense accumulation of neutrophils and a mass of necrotic acinar cells were also observed in the HM-HD group. (B) Histological scores of the pancreas in the sham, HM-LD and HM-HD groups. (C) Serum amylase and lipase changes in the sham, HM-LD and HM-HD groups. Data are expressed as the means ± SD (n=6/group). ^*P<0.01 compared to the sham group (control group); ^#P<0.05 compared to the HM-LD group. Sham, sham injection.

Figure 4

Toll-like receptor 4 (TLR4) expression and nuclear factor-κB (NF-κB) activation in pancreatic tissue after recombinant human high-mobility group box 1 (rhHMGB1) administration. (A) The expression of TLR4 was assessed using the mRNA [(a) qPCR] and protein [(b) western blot analysis] levels in the pancreatic tissue from the sham, low-dose of HMGB1 (HM-LD) and high-dose of HMGB1 (HM-HD) groups at 48 h after rhHMGB1 administration. (B) The activation of NF-κB was assessed from the nuclear p65 [(a) western blot analysis] and its downstream, relative levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) [(b) ELISA] in the pancreatic tissue of the sham, HM-LD and HM-HD groups. ^*P<0.01 compared to the sham group (control group); ^#P<0.05 compared to the HM-LD group. The relative levels of the TLR4 protein and mRNA compared to the control group are normalized to β-actin. The relative levels of nuclear p65 protein compared to the control group are normalized to histone H3. The relative levels of TNF-α and IL-1β concentrations compared to the control group are normalized to the total protein concentration of each sample. Data are expressed as the means ± SD (n=6/group). Blots shown are from a representative experiment that was repeated 3 times with similar results (n=6/group). Sham, sham injection.

Figure 5

Response of Toll-like receptor 4 (TLR4)-deficient mice to recombinant human high-mobility group box 1 (rhHMGB1) administration. (A) Pancreatic histopathological changes in wild-type (WT) and TLR4-deficient mice 48 h after the administration of a high dose of rhHMGB1. Representative H&E-stained micrographs (original magnification, ×200) from the (a) WT and (b) TLR4^−/− groups. The pancreatic tissues in the wild-type group exhibited marked edema, infiltration of inflammatory cells, a mass of necrotic acinar cells and the disappearance of the normal lobe structure in the pancreas; there was a significant reduction in the TLR4-deficient group. (B-a) Histological scores of the pancreas and (b) serum amylase and lipase changes in the WT and TLR4^−/− groups. (C) The activation of nuclear factor-κB (NF-κB) was assessed from the nuclear p65 [(a) western blot analysis] and its downstream, relative levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) [(b) ELISA] in the pancreatic tissue from the WT and TLR4^−/− groups. ^*P<0.01 compared to the WT group. The relative levels of nuclear p65 protein compared to the control group are normalized to histone 3 (H3). The relative levels of TNF-α and IL-1β compared to the control group are normalized to the total protein concentration of each sample. Data are expressed as the means ± SD (n=6/group). Blots shown are from a representative experiment that was repeated 3 times with similar results (n=6/group). WT, WT mice given a high dose of rhHMGB1; TLR4^−/−, TLR4-deficient mice administered a high dose of rhHMGB1.