A nuclear import inhibitory peptide ameliorates the severity of cholecystokinin-induced acute pancreatitis

Abstract

Aim: To assess the effect of our novel cell-permeable nuclear factor-kappaB (NF-kappaB) inhibitor peptide PN50 in an experimental model of acute pancreatitis. PN50 was produced by conjugating the cell-penetrating penetratin peptide with the nuclear localization signal of the NF-kappaB p50 subunit.

Methods: Pancreatitis was induced in male Wistar rats by administering 2X100 mug/kg body weight of cholecystokinin-octapeptide (CCK) intraperitoneally (IP) at an interval of 1 h. PN50-treated animals received 1 mg/kg of PN50 IP 30 min before or after the CCK injections. The animals were sacrificed 4 h after the first injection of CCK.

Results: All the examined laboratory (the pancreatic weight/body weight ratio, serum amylase activity, pancreatic levels of TNF-alpha and IL-6, degree of lipid peroxidation, reduced glutathione levels, NF-kappaB binding activity, pancreatic and lung myeloperoxidase activity) and morphological parameters of the disease were improved before and after treatment with the PN50 peptide. According to the histological findings, PN50 protected the animals against acute pancreatitis by favoring the induction of apoptotic, as opposed to necrotic acinar cell death associated with severe acute pancreatitis.

Conclusion: Our study implies that reversible inhibitors of stress-responsive transcription factors like NF-kappaB might be clinically useful for the suppression of the severity of acute pancreatitis.

Figure 1

Experimental protocol of CCK-induced acute pancreatitis.

Figure 2

PN50 suppresses transcriptional activity of NF-κB in TNF-α-stimulated L929 fibroblasts (A) and LPS-activated RAW 264.7 macrophages (B) in vitro.

Figure 3

In vivo delivery of the NF-κB p50 NLS with penetratin into the pancreas (A) and lung (B).

Figure 4

Effect of PN50 on the pancreatic weight/body weight ratio (pw/bw) (A) and serum amylase activity (B) in CCK-induced acute pancreatitis. ^bP<0.01 vs group PS; ^dP<0.01 vs group CCK.

Figure 5

Effect of PN50 on the pancreatic (A) and lung (B) myeloperoxidase (MPO) activity and pancreatic TNF-α (C) and IL-6 (D) levels in CCK-induced acute pancreatitis. ^aP<0.05, ^bP<0.01 vs group PS; ^cP<0.05, ^dP<0.01 vs group CCK.

Figure 6

Effect of PN50 treatment on the pancreatic malondialdehyde (MDA) (A) and reduced glutathione (GSH) levels (B) in CCK-induced acute pancreatitis. ^bP<0.01 vs group PS; ^cP<0.05, ^dP<0.01 vs group CCK.

Figure 7

Effect of PN50 on pancreatic NF-κB binding activity in CCK-induced acute pancreatitis. A: a representative EMSA for pancreatic NF-κB DNA binding activity; B: the intensities of NF-κB bands. ^aP<0.05, ^bP<0.01 vs group PS; ^dP<0.01 vs group CCK.

Figure 8

Effect of PN50 on pancreatic morphological damage in CCK-induced pancreatitis. A: group PS (control group): normal pancreas (HE ×100); B: group CCK: marked edema, inflammatory activity, stasis (HE ×100); microfocal necroses, vacuolar degenerations in the acinar cells (insert: HE ×250); C: group PN50+CCK: mild inflammation, mild edema, fairly maintained parenchymal structure (HE ×100); mild degenerative changes in the acini (insert: HE ×250); D: group CCK+PN50: mild edema, minimal inflammatory activity (HE ×100); degenerative changes with apoptotic cells (arrows) in the acini (insert: HE ×250).