Dopamine D2 receptor signalling controls inflammation in acute pancreatitis via a PP2A-dependent Akt/NF-κB signalling pathway

Abstract

Background and purpose: Dopamine has multiple anti-inflammatory effects, but its role and molecular mechanism in acute pancreatitis (AP) are unclear. We investigated the role of dopamine signalling in the inflammatory response in AP.

Experimental approach: Changes in pancreatic dopaminergic system and effects of dopamine, antagonists and agonists of D₁ and D₂ dopamine receptors were analysed in wild-type and pancreas-specific Drd2^-/- mice with AP (induced by caerulein and LPS or L-arginine) and pancreatic acinar cells with or without cholecystokinin (CCK) stimulation. The severity of pancreatitis was assessed by measuring serum amylase and lipase and histological assessments. The NF-κB signalling pathway was evaluated, and macrophage and neutrophil migration assessed by Transwell assay.

Key results: Pancreatic dopamine synthetase and metabolic enzyme levels were increased, whereas D₁ and D₂ receptors were decreased in AP. Dopamine reduced inflammation in CCK-stimulated pancreatic acinar cells by inhibiting the NF-κB pathway. Moreover, the protective effects of dopamine were blocked by a D₂ antagonist, but not a D₁ antagonist. A D₂ agonist reduced pancreatic damage and levels of p-IκBα, p-NF-κBp65, TNFα, IL-1β and IL-6 in AP. Pancreas-specific Drd2^-/- aggravated AP. Also, the D₂ agonist activated PP2A and inhibited the phosphorylation of Akt, IKK, IκBα and NF-κB and production of inflammatory cytokines and chemokines. Furthermore, it inhibited the migration of macrophages and neutrophils by reducing the expression of CCL2 and CXCL2. A PP2A inhibitor attenuated these protective effects of the D₂ agonist.

Conclusions and implications: D₂ receptors control pancreatic inflammation in AP by inhibiting NF-κB activation via a PP2A-dependent Akt signalling pathway.

Figure 1

Changes in the dopaminergic system in the pancreas during AP. (A) Comparison of dopamine (DA) levels in serum of healthy controls (NC, n = 21), mild acute pancreatitis (MAP, n = 76), moderately severe acute pancreatitis (MSAP, n = 10) and severe acute pancreatitis (SAP, n = 10) patients. (B) ELISA of dopamine in serum of caerulein‐ and LPS‐induced model of pancreatitis. (C, D) Representative micrographs of H&E‐stained pancreatic sections (200×). (C) caerulein‐ and LPS‐induced AP, (D) L‐Arg‐induced AP. (E) Immunoblot analysis of TH, DDC, DBH and MAO proteins of pancreas (left) in mice treated with caerulein and LPS or PACs (right) with CCK stimulation. (F) Immunoblot analysis of D₁ (DRD1) and D₂ (DRD2) receptors of pancreatic tissue in two models of AP‐ or CCK‐treated PACs. n = 6 per group. Cae, caerulein; L‐Arg, L‐arginine; NC, normal control. Scale bar = 100 μm. *P < 0.05 versus NC, ^# P < 0.05 versus MAP and ⁺ P < 0.05 between groups.

Figure 2

Dopamine (DA) signalling inhibits NF‐κB activation in AP and can be blocked by a D₂ antagonist. (A, B) PACs were isolated from the pancreas of Balb/C mice. PACs were stimulated by 200 nM CCK 8 and treated with dopamine (250, 500 and 750 μM) at the same time. (A) Immunoblot analysis of IκBα, NF‐κBp65 phosphorylation level (at 1 h after CCK stimulation) and TNFα, IL‐1β and IL‐6 levels (at 4 h after CCK stimulation) in CCK‐stimulated PACs. (B) qRT‐PCR of mRNA levels of Tnfα, Il1β and Il6 in CCK‐stimulated PACs (at 4 h after CCK stimulation). (C–F) AP was induced by injection of caerulein (100 μg·kg⁻¹) and LPS (5 mg·kg⁻¹) in Balb/C mice, and the D₁ antagonist SCH (10 mg·kg⁻¹, i.p., 0.5 h before the first caerulein injection) or D₂ antagonist eticlopride (10 mg·kg⁻¹, i.p., 0.5 h before the first caerulein injection) was used in combination with dopamine (50 mg·kg⁻¹, i.p., at the same time as caerulein). Mice were killed at 12 h after the first caerulein injection. (C) The time axis of AP model building and drug intervention. (D) Representative micrographs of H&E‐stained pancreatic sections (200×). (E) Histological score was determined as described in Methods. (F) Change in serum activity of amylase (left) and lipase (right). *n = 6 per group. Cae, caerulein; NC, normal control. Scale bar = 100 μm. *P < 0.05 versus NC, ^# P < 0.05 versus AP or CCK and ⁺ P < 0.05 versus dopamine.

Figure 3

Dopamine signalling inhibits NF‐κB activation via D₂ receptors in AP. (A, B) AP was induced by injection of caerulein (Cae; 100 μg·kg⁻¹) and LPS (5 mg·kg⁻¹) in Balb/C mice, and the D₁ antagonist SCH (10 mg·kg⁻¹, i.p., 0.5 h before the first caerulein injection) or D₂ antagonist eticlopride (10 mg·kg⁻¹, i.p., 0.5 h before the first caerulein injection) was used in combination with dopamine (DA; 50 mg·kg⁻¹, i.p., at the same time as caerulein). Mice were killed at 12 h after the first caerulein injection. (A) Immunoblot analysis of IκBα, NF‐κBp65 phosphorylation levels and TNFα, IL‐1β and IL‐6 expression levels in pancreatic tissue in mice. (B) qRT‐PCR of mRNA levels of Tnfα, Il1β and Il6 in pancreatic tissue. (C, D) PACs were isolated from the pancreas of Balb/C mice. A D₁ antagonist SCH (10 μM) or D₂ antagonist eticlopride (10 μM) was used in combination with dopamine (500 μM) at the time of CCK (200 nM) stimulation. (C) Immunoblot analysis of IκBα, NF‐κBp65 phosphorylation levels (at 1 h after CCK stimulation) and TNFα, IL‐1β and IL‐6 levels (at 4 h after CCK stimulation) in CCK‐stimulated PACs. (D) qRT‐PCR of mRNA levels of Tnfα, Il1β and Il6 in CCK‐stimulated PACs (at 4 h after CCK stimulation). n = 6 per group. *P < 0.05 versus normal control, ^# P < 0.05 versus AP or CCK and ⁺ P < 0.05 versus dopamine.

Figure 4

D₂ receptor signalling inhibits NF‐κB activation in AP. In vivo, AP was induced by injection of caerulein (100 μg·kg⁻¹) and LPS (5 mg·kg⁻¹) in Balb/C mice, and a D₂ agonist quinpirole (10 mg·kg⁻¹, i.p., 0.5 h before the first caerulein injection) was used instead of dopamine (50 mg·kg⁻¹, i.p., at the same time as caerulein). Mice were killed at 12 h after the first caerulein injection. In vitro, PACs were isolated from the pancreas of Balb/C mice. A D₂ agonist quinpirole (5 μM) was used instead of dopamine (500 μM) at the time of CCK (200 nM) stimulation. (A) Representative micrographs of H&E‐stained pancreatic sections (200×). (B) Histological score was determined as described in Methods. (C) Change in serum activity of amylase (left) and lipase (right). (D) Immunoblot analysis of IκBα, NF‐κBp65 phosphorylation levels and TNFα, IL‐1β and IL‐6 expression levels of pancreatic tissue in mice. (E) qRT‐PCR of mRNA levels of Tnfα, Il1β and Il6 in pancreatic tissue. (F) Immunoblot analysis of IκBα, NF‐κBp65 phosphorylation levels (at 1 h after CCK stimulation) and TNFα, IL‐1β and IL‐6 levels (at 4 h after CCK stimulation) in CCK‐stimulated PACs. (G) qRT‐PCR of mRNA levels of Tnfα, Il1β and Il6 in CCK‐stimulated PACs (at 4 h after CCK stimulation). (H) EMSA analysis of NF‐κB binding ability. n = 6 per group. Cae, caerulein; NC, normal control. Scale bar = 100 μm. *P < 0.05 versus NC; ^# P < 0.05 versus AP or CCK.

Figure 5

Pancreas‐specific Drd2 knockout mice promotes Akt/NFkB inflammatory signalling pathways in experimental AP. AP was induced by injection of caerulein (100 μg·kg⁻¹) and LPS (5 mg·kg⁻¹) in WT C57BL6/J and in pancreas‐specific Drd2 ^−/− on C57BL6/J background mice, and the blood samples and pancreatic tissue were collected at 12 h after the first caerulein injection. (A) Representative micrographs of H&E‐stained pancreatic sections (200×) and histological scores. (B) Immunoblot analysis of PI3K, Akt, IKK, IκBα and NF‐κBp65 phosphorylation levels of pancreatic tissue in WT and Drd2 ^−/− mice. (C) Immunoblot analysis of TNFα, IL‐1β and IL‐6 expression levels of pancreatic tissue in WT or Drd2^−/− mice. (D) qRT‐PCR of mRNA levels of Tnfα, Il1β, Il6, Ccl2 and Cxcl2 of pancreatic tissue in WT or Drd2 ^−/− mice. (E) ELISA of serum TNFα, IL‐1β, IL‐6, CCL2 and CXCL2 levels in WT or Drd2 ^−/− mice. (F) Representative micrographs of macrophage marker F4/80 and neutrophil marker Ly6G immunohistochemical analyses in pancreas (200×). (G) Representative micrographs of H&E‐stained lung sections and neutrophil marker Ly6G immunohistochemical analyses in lung (200×). n = 6 per group. NC, normal control. Scale bar = 100 μm. *P < 0.05 versus WT‐NC, ^# P < 0.05 versus KO‐NC and ⁺ P < 0.05 versus WT‐AP.

Figure 6

D₂ receptor signalling inhibits NF‐κB activation via PP2A‐dependent Akt signalling pathway in CCK‐stimulated PACs. (A) Immunoblot analysis of PPP2R2C and PP2A‐C of quinpirole (5 μM)‐treated PACs at the indicated time point in vitro (left). Immunoblot analysis of PPP2R2C and PP2A‐C of pancreatic tissues in mice treated with quinpirole at the indicated time point in vivo (right). (B–E) PACs were stimulated by 200 nM CCK and treated with 5 μM quinpirole (at the time of CCK stimulation) with or without 2 nM okadaic acid (15 min before CCK stimulation) for 4 h. (B) Immunoblot analysis of Akt, IKK phosphorylation levels (at 15 min after CCK stimulation) and IκBα, NF‐κBp65 phosphorylation levels (at 1 h after CCK stimulation) of PACs. (C) Immunoblot analysis of TNFα, IL‐1β and IL‐6 levels (at 4 h after CCK stimulation) of PACs. (D) qRT‐PCR of mRNA levels of Tnfα, Il1β and Il6 (at 4 h after CCK stimulation) of PACs. (E) EMSA analysis of NF‐κB binding ability (at 1 h after CCK stimulation) of PACs. n = 6 per group. *P < 0.05 versus normal control (NC), ^# P < 0.05 versus AP and ⁺ P < 0.05 versus quinpirole.

Figure 7

D₂ receptor signalling inhibits inflammation in AP via PP2A in vivo. Two models of AP were induced in Balb/C mice in vivo (see Methods section). Mice were pretreated with quinpirole (10 mg·kg⁻¹, i.p., 0.5 h before the first caerulein injection) with or without okadaic acid (100 ng per mouse, i.p., 0.5 h before quinpirole) intervenion. Mice were killed at 12 h after the first caerulein injection and at day 3 after the second L‐Arg injection. (A, B) The time axis of drug intervention and caerulein plus LPS AP model (A) or L‐Arg induced AP (B). (C) Representative micrographs of H&E‐stained pancreatic sections (200×) and histological scores from caerulein and LPS‐induced AP and L‐Arg‐induced AP. (D) Immunoblot analysis of PPP2R2C and PP2A‐C of CCK‐treated PACs or pancreatic tissue in WT or Drd2 ^−/− mice of both models of AP. (E) Representative micrographs of macrophage marker F4/80 (up) and neutrophil marker Ly6G (down) immunohistochemical analyses in pancreas (200×). (F) Representative micrographs of H&E‐stained lung sections and neutrophil marker Ly6G immunohistochemical analyses in lung (200×). n = 6 per group. Cae, caerulein; L‐Arg, L‐arginine. Scale bar = 100 μm. *P < 0.05 versus NC, ^# P < 0.05 versus AP and ⁺ P < 0.05 versus quinpirole.

Figure 8

D₂ receptor signalling inhibits NF‐κB activation via a PP2A‐dependent Akt signalling pathway in AP in vivo. Two models of AP were induced in Balb/C mice in vivo (see Methods section). Mice were pretreated with quinpirole (10 mg·kg⁻¹, i.p., 0.5 h before the first caerulein injection) with or without okadaic acid (100 ng per mouse, i.p., 0.5 h before quinpirole). Mice were killed at 12 h after the first caerulein injection and at day 3 after the second L‐Arg injection. (A) Immunoblot analysis of Akt, IKK, IκBα and NF‐κBp65 phosphorylation levels of pancreatic tissue in mice of caerulein‐ and LPS‐induced AP. (B) Immunoblot analysis of TNFα, IL‐1β and IL‐6 levels of pancreatic tissue in mice of caerulein‐ and LPS‐induced AP. (C) qRT‐PCR of mRNA levels of Tnfα, Il1β and Il6 of pancreatic tissue in mice of caerulein‐ and LPS‐induced AP. (D) ELISA of serum TNFα, IL‐1β and IL‐6 levels in mice of caerulein‐ and LPS‐induced AP. (E) Immunoblot analysis of Akt, IKK, IκBα and NF‐κBp65 phosphorylation levels in pancreatic tissue in mice of L‐Arg‐induced AP. (F) Immunoblot analysis of TNFα, IL‐1β and IL‐6 levels in pancreatic tissue in mice of L‐Arg‐induced AP. (G) qRT‐PCR of mRNA levels of Tnfα, Il1β and Il6 in pancreatic tissue in mice of L‐Arg‐induced AP. (H) ELISA of serum TNFα, IL‐1β and IL‐6 levels in mice of L‐Arg‐induced AP. n = 6 per group. Cae, caerulein; L‐Arg, L‐Arginine. *P < 0.05 versus NC, ^# P < 0.05 versus AP and ⁺ P < 0.05 versus quinpirole.

Figure 9

D₂ receptor signalling inhibits macrophage and neutrophil migration by reducing CCL2 and CXCL2 expression. Bone marrow‐derived macrophages and neutrophils were collected from Balb/C mice, and their migration was assessed by Transwell assay. (A) ELISA of CCL2 in supernatants of CCK‐stimulated PACs. (B) qRT‐PCR of mRNA levels of Ccl2 in CCK‐stimulated PACs. (C) Flow cytometry (FCM) of macrophage purity at day 8. (D) Quantitative analysis of the number of migrated macrophages. (E) Representative micrographs (200×) of macrophage migration after staining with crystal violet. (F) ELISA of CXCL2 in supernatants of CCK‐stimulated PACs. (G) qRT‐PCR of mRNA level of Cxcl2 in CCK‐stimulated PACs. (H) FCM of neutrophil purity. (I) Quantitative analysis of the number of migrated neutrophils. (J) Representative micrographs (200×) of neutrophil migration after staining with crystal violet and in the lower chamber (lower graphs). Scale bar =100 μm. *P < 0.05 versus normal control (NC) and ^# P < 0.05 versus CCK.

Figure 10

D₂ receptor signalling inhibits macrophage and neutrophil migration by activating PP2A. Bone marrow‐derived macrophages and neutrophils were collected from Balb/C mice, and their migration was assessed by Transwell assay. (A) ELISA of serum CCL2 and qRT‐PCR of Ccl2 mRNA in caerulein‐challenged mice treated with 10 mg·kg⁻¹ quinpirole with or without 100 ng okadaic acid. (B) ELISA of CCL2 in supernatants and qRT‐PCR of Ccl2 mRNA from CCK‐stimulated PACs treated with 5 μM quinpirole with or without 2 nM okadaic acid for 6 h. (C) Representative micrographs (200×) of macrophage migration after stained with crystal violet and quantitative analysis of the number of migrated macrophages. (D) ELISA of serum CXCL2 and qRT‐PCR of Cxcl2 mRNA in caerulein‐ challenged mice treated with 10 mg·kg⁻¹ quinpirole with or without 100 ng okadaic acid. (E) ELISA of CXCL2 in supernatants and qRT‐PCR of Cxcl2 mRNA from CCK‐stimulated PACs treated with 5 μM quinpirole with or without 2 nM okadaic acid for 6 h. (F) Representative micrographs (200×) of neutrophil migration after staining with crystal violet and in the lower chamber (lower graphs) and quantitative analysis of the number of migrated neutrophils, including the adherent cells in the membrane and suspension of cells in the lower chamber. (G) Schematic diagram summarizing the mechanisms by which D₂ receptor (DRD2) signalling controls inflammation in experimental pancreatitis. The protective effects of the D₂ receptor signalling on AP are exerted at least in part through Akt dephosphorylation via PP2A. n = 6 per group. Cae, caerulein; NC, normal control. Scale bar = 100 μm. *P < 0.05 versus NC, ^# P < 0.05 versus AP or CCK and ⁺ P < 0.05 versus quinpirole.