The ciliary protein Spef2 stimulates acinar Ampkα/Sirt1 signaling and ameliorates acute pancreatitis and associated lung injury

Abstract

Background: Pancreatic acinar cells are susceptible to nuclear factor kappa B (NF-κB)-mediated inflammation and resulting cell necrosis during early acute pancreatitis. As adenosine monophosphate-activated protein kinase alpha (Ampkα)/sirtuin 1 (Sirt1) pathway activity attenuates NF-κB activity, we examined whether the Ampkα/Sirt1 axis affects the progression of acute pancreatitis and associated lung injury in vivo. Furthermore, we explored the role of the ciliary protein sperm flagellar 2 (Spef2, Kpl2) in regulating Ampkα/Sirt1 activity in vitro and in vivo.

Methods: Pancreatic injury, oxidative stress, acinar cell necrosis and apoptosis, acinar levels of Ampkα/Sirt1/NF-κB signaling activity, NF-kB-mediated inflammatory markers, and markers of associated lung injury were measured in rat models of acute pancreatitis following pharmacological Ampkα activation with A769662 or self-complementary recombinant adeno-associated virus serotype 6 (scAAV6)-mediated Spef2 overexpression. Additional in vivo rescue studies involving Ampkα silencing and/or constitutively active (CA)-Sirt1 overexpression were performed in acute pancreatitis rats. In vitro immunoblotting and Ampkα activity assays were conducted in the pancreatic acinar cell line AR42J.

Results: Pharmacological Ampkα activation or Spef2 overexpression reduced acute pancreatitis severity, oxidative stress, necrosis, apoptosis, NF-kB-mediated inflammatory markers, and the degree of associated lung injury. Spef2 overexpression in AR42J cells in vitro promoted Ampkα^Thr172 phosphorylation and Ampkα activity. In vivo rescue studies revealed that Spef2's suppressive effect on acute pancreatitis and associated lung injury is mediated via the Ampkα/Sirt1 axis.

Conclusions: This study established the existence of a Spef2/Ampkα/Sirt1 axis in pancreatic acinar cells that is involved in the regulation of NF-κB-mediated acinar cell inflammation and resulting cell necrosis during acute pancreatitis.

Keywords: Acute pancreatitis; Ampkα; NF-κB; Sirt1; Spef2.

Figure 1

Enhancing Ampkα activity attenuates acute pancreatitis and associated lung injury in vivo. Pancreatic and lung tissues and peripheral blood samples were harvested from sham-operated control (SO Ctrl), acute pancreatitis + DMSO vehicle (AP + Veh), and acute pancreatitis + Ampkα agonist A769662 (AP + A76) rats 24 hours post-induction. (A) Representative H&E staining of pancreatic tissues. (B) Pathological scoring of pancreatic injury. (C) Pancreatic LDH, MDA, LPO, and MPO activity levels. (D) Pancreatic ATP levels. (E) Pancreatic necrosis levels. (F) Representative immunoblots of pancreatic Ampkα/Sirt1/NF-κB signaling proteins. (G) Serum CRP, TNF-α, IL-1β, IL-6, and IL-18 levels from peripheral blood samples. (H) H&E staining of lung tissue showing extensive edema, alveolar congestion, and immune cell infiltration (scale bar =500 μm). (I) Pathological scoring of lung tissue injury. (J) Lung W/D ratio. (K) Lung MPO activity. (L) BALF TNF-α and IL-1β levels and (M) BALF protein content. Data represented as means ± SDs. N=6 rats per cohort. *P<0.05, **P<0.01 (one-way ANOVA with Bonferroni post-hoc). DMSO, dimethyl sulfoxide; Ampkα, adenosine monophosphate-activated protein kinase alpha; H&E, hematoxylin and eosin; LDH, lactate dehydrogenase; MDA, malondialdehyde; LPO, lipid peroxide; MPO, myeloperoxidase; ATP, adenosine triphosphate; Sirt1, sirtuin 1; NF-κB, nuclear factor kappa B; CRP, C-reactive protein; TNF-α, tumor necrosis factor alpha; IL-1β, interleukin-1 beta; W/D, wet-to-dry; BALF, bronchoalveolar lavage fluid; SD, standard deviation; ANOVA, analysis of variance.

Figure 2

Pancreatic acinar cell-specific Spef2 overexpression attenuates acute pancreatitis and associated lung injury in vivo. Pancreatic and lung tissues and peripheral blood samples were harvested from sham-operated control (SO Ctrl), acute pancreatitis + DMSO vehicle (AP + Veh), and acute pancreatitis + scAAV6-delivered Spef2 (AP + scAAV6.Spef2) rats 24 hours post-induction. (A) Representative H&E staining of pancreatic tissues. (B) Pathological scoring of pancreatic injury. (C) Pancreatic LDH, MDA, LPO, and MPO activity levels. (D) Pancreatic ATP levels. (E) Pancreatic necrosis levels. (F) Representative immunoblots of pancreatic Spef2 and Ampkα/Sirt1/NF-κB signaling proteins. (G) Serum CRP, TNF-α, IL-1β, IL-6, and IL-18 levels from peripheral blood samples. (H) H&E staining of lung tissue showing extensive edema, alveolar congestion, and immune cell infiltration (scale bar =500 μm). (I) Pathological scoring of lung tissue injury. (J) Lung W/D ratio. (K) Lung MPO activity. (L) BALF TNF-α and IL-1β levels and (M) BALF protein content. Data represented as means ± SDs. N=6 rats per cohort. *P<0.05, **P<0.01 (one-way ANOVA with Bonferroni post-hoc). Spef2, sperm flagellar 2; DMSO, dimethyl sulfoxide; H&E, hematoxylin and eosin; LDH, lactate dehydrogenase; MDA, malondialdehyde; LPO, lipid peroxide; MPO, myeloperoxidase; ATP, adenosine triphosphate; Ampkα, adenosine monophosphate-activated protein kinase alpha; Sirt1, sirtuin 1; NF-κB, nuclear factor kappa B; CRP, C-reactive protein; TNF-α, tumor necrosis factor alpha; IL-1β, interleukin-1 beta; W/D, wet-to-dry; BALF, bronchoalveolar lavage fluid; SD, standard deviation; ANOVA, analysis of variance.

Figure 3

Spef2’s suppressive effect on in vitro pancreatic acinar cell necrosis and inflammation mediated via the Ampkα/Sirt1 axis. 72 hours following scAAV6.NC, scAAV6.Spef2, or scAAV6.Spef2 + CA-Sirt1 infection and 12 hours following siCtrl or siAmpkα transfection, AR42J cells were incubated with cerulein to establish an in vitro model of acute pancreatitis. (A) Representative immunoblots of pancreatic Spef2 and Ampkα/Sirt1/NF-κB signaling proteins. (B) LDH levels. (C) ATP levels. (D) Cell necrosis levels. (E) Representative TUNEL staining images and (F) associated quantitative measurements of apoptosis. (G) Representative CC-3 fluorescent staining images. CC-3 staining in green and DAPI nuclei staining in blue (scale bar =200 μm). (H) IOD quantitation of fluorescent CC-3 staining. (I) Cell supernatant TNF-α and IL-1β levels. Data represented as means ± SDs. N=3 biological replicates × 2 technical replicates. *P<0.05, **P<0.01 (one-way ANOVA with Bonferroni post-hoc). Spef2, sperm flagellar 2; Ampkα, adenosine monophosphate-activated protein kinase alpha; Sirt1, sirtuin 1; CA, constitutively active; NF-κB, nuclear factor kappa B; LDH, lactate dehydrogenase; ATP, adenosine triphosphate; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; CC-3, cleaved caspase-3; DAPI, 4',6-diamidino-2-phenylindole; TNF-α, tumor necrosis factor alpha; IL-1β, interleukin-1 beta; SD, standard deviation; ANOVA, analysis of variance; IOD, integrated optical density.

Figure 4

Spef2’s suppressive effect on in vivo acute pancreatitis and associated lung injury mediated via the Ampkα/Sirt1 axis. Pancreatic and lung tissues and peripheral blood samples were harvested from sham-operated control (SO Ctrl), acute pancreatitis + DMSO vehicle (AP + Veh), acute pancreatitis + scAAV6-delivered Spef2 + control siRNA (AP + scAAV6.Spef2 + siCtrl), acute pancreatitis + scAAV6-delivered Spef2 + Ampkα siRNA (AP + scAAV6.Spef2 + siAmpkα), and acute pancreatitis + scAAV6-delivered Spef2 + Ampkα siRNA + scAAV6-delivered CA-Sirt1 (AP + scAAV6.Spef2 + CA-Sirt1 + siAmpkα) rats 24 hours post-induction. (A) Representative H&E staining of pancreatic tissues. (B) Pathological scoring of pancreatic injury. (C) Pancreatic LDH, MDA, LPO, and MPO activity levels. (D) Pancreatic ATP levels. (E) Pancreatic necrosis levels. (F) Representative immunoblots of pancreatic Spef2 and Ampkα/Sirt1/NF-κB signaling proteins. (G) Serum CRP, TNF-α, IL-1β, IL-6, and IL-18 levels from peripheral blood samples. (H) H&E staining of lung tissue showing extensive edema, alveolar congestion, and immune cell infiltration (scale bar =500 μm). (I) Pathological scoring of lung tissue injury. (J) Lung W/D ratio. (K) Lung MPO activity. (L) BALF TNF-α and IL-1β levels and (M) BALF protein content. Data represented as means ± SDs. N=6 rats per cohort. *P<0.05, **P<0.01 (one-way ANOVA with Bonferroni post-hoc). Spef2, sperm flagellar 2; Ampkα, adenosine monophosphate-activated protein kinase alpha; Sirt1, sirtuin 1; DMSO, dimethyl sulfoxide; siRNA, small-interfering RNA; CA, constitutively active; H&E, hematoxylin and eosin; LDH, lactate dehydrogenase; MDA, malondialdehyde; LPO, lipid peroxide; MPO, myeloperoxidase; ATP, adenosine triphosphate; NF-κB, nuclear factor kappa B; CRP, C-reactive protein; TNF-α, tumor necrosis factor alpha; IL-1β, interleukin-1 beta; W/D, wet-to-dry; BALF, bronchoalveolar lavage fluid; SD, standard deviation; ANOVA, analysis of variance.