Resveratrol protects against L-arginine-induced acute necrotizing pancreatitis in mice by enhancing SIRT1-mediated deacetylation of p53 and heat shock factor 1

Abstract

Acute necrotizing pancreatitis (ANP) is a common severe critical illness with a high mortality rate. Resveratrol, a polyphenol compound derived from various plants such as grape skin, peanut, berry and veratrum, exhibits multiple biological activities, especially potent anti‑inflammatory activity, but its effect on ANP has not yet been fully elucidated. The present study aimed to investigate the effects of resveratrol on L-arginine-induced ANP and the possible mechanisms. A mouse model of ANP was established by 2 hourly intraperitoneal injections of 8% L-arginine (4 g/kg). Then the mice were treated by intragastric administration of resveratrol (80 mg/kg) every 12 h immediately after the second injection of L-arginine. Mice with ANP showed increased apoptosis of pancreatic acinar cells, pancreatic myeloperoxidase activity, serum lactate dehydrogenase activity, amylase, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) levels as well as decreased serum IL-10 level, pancreatic expression of heat shock factor 1 (HSF1), sirtuin 1 (SIRT1) and p53, but the ratio of acetylated HSF1 and p53 was markedly increased. Resveratrol enhanced the survival rate of mice with ANP from 47.8 to 71.4% and obviously restored the changes in mice with ANP as mentioned above. Additionally, interactions between SIRT1 and p53 and between SIRT1 and HSF1 in the pancreas of the mice were confirmed by co-immunoprecipitation. These data suggest that resveratrol protects against L-arginine-induced ANP, which may be related to the enhancement of SIRT1-mediated deacetylation of p53 and HSF1.

Figure 1

Effect of resveratrol on the survival of mice with L-arginine-induced acute necrotizing pancreatitis (ANP). The mice were randomly divided into three groups: Control group (Con, n=20), L-arginine exposure group [acute pancreatitis (AP), n=46], L-arginine and resveratrol treatment group (AP+RSV, n=49). Mice in the AP+RSV group were treated with intragastric administration of 80 mg/kg resveratrol every 12 h immediately after the second injection of L-arginine. The mice in the Con group and AP group were treated with the same volume of 0.5% sodium carboxymethyl cellulose. The mice were kept and observed for 7 days after the first injection of L-arginine. Kaplan-Meyer method was used to compare the differences in the 7-day survival rate between groups. ^*p<0.01 vs. Con; ^#p<0.05 vs. AP.

Figure 2

Effect of resveratrol on the pancreatic damage of mice with L-arginine-induced acute necrotizing pancreatitis (ANP). (A) General morphology of the pancreas. (B) H&E-stained pancreatic sections (magnification, ×40) and (C) pathological score of the pancreas at 48 and 72 h after the second injection of L-arginine. ^*p<0.05 vs. Con; ^#p<0.05 vs. acute pancreatitis (AP) (n=5). Con, control group; AP, L-arginine exposure group; AP+RSV, L-arginine and resveratrol treatment group.

Figure 2

Effect of resveratrol on the pancreatic damage of mice with L-arginine-induced acute necrotizing pancreatitis (ANP). (A) General morphology of the pancreas. (B) H&E-stained pancreatic sections (magnification, ×40) and (C) pathological score of the pancreas at 48 and 72 h after the second injection of L-arginine. ^*p<0.05 vs. Con; ^#p<0.05 vs. acute pancreatitis (AP) (n=5). Con, control group; AP, L-arginine exposure group; AP+RSV, L-arginine and resveratrol treatment group.

Figure 2

Effect of resveratrol on the pancreatic damage of mice with L-arginine-induced acute necrotizing pancreatitis (ANP). (A) General morphology of the pancreas. (B) H&E-stained pancreatic sections (magnification, ×40) and (C) pathological score of the pancreas at 48 and 72 h after the second injection of L-arginine. ^*p<0.05 vs. Con; ^#p<0.05 vs. acute pancreatitis (AP) (n=5). Con, control group; AP, L-arginine exposure group; AP+RSV, L-arginine and resveratrol treatment group.

Figure 3

Effect of resveratrol on the serum amylase level, lactate dehydrogenase (LDH) activity and pancreatic myeloperoxidase (MPO) activity in mice with L-arginine-induced acute necrotizing pancreatitis (ANP). Serum amylase level (A), LDH activity (B) and pancreatic MPO activity (C) at 48 and 72 h after the second injection of L-arginine. ^*p<0.05 vs. Con; ^#p<0.05 vs. acute pancreatitis (AP) (n=5). Con, control group; AP, L-arginine exposure group; AP+RSV, L-arginine and resveratrol treatment group.

Figure 4

Effect of resveratrol on the apoptosis of pancreatic acinar cells in mice with L-arginine-induced acute necrotizing pancreatitis (ANP). Apoptotic pancreatic acinar cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. TUNEL-positive cells are indicated by brown staining. Representative photomicrographs demonstrating TUNEL staining of pancreatic sections (A) and quantitative analysis of TUNEL-positive cells as the apoptotic index (B) are shown. ^*p<0.05 vs. Con; ^#p<0.05 vs. acute pancreatitis (AP) (n=5). Con, control group; AP, L-arginine exposure group; AP+RSV, L-arginine and resveratrol treatment group.

Figure 5

Effect of resveratrol on the pancreatic interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-10 mRNA expression levels and serum IL-6, TNF-α, IL-10 levels in mice with L-arginine-induced acute necrotizing pancreatitis (ANP). Pancreatic IL-6 (A), TNF-α (B), IL-10 (C) mRNA expression levels were detected at 48 h and 72 h after the second injection of L-arginine by real-time PCR. Serum IL-6 (D), TNF-α (E) and IL-10 (F) levels were detected at 48 h and 72 h after the second injection of L-arginine by ELISA. The values are shown as means ± SD. ^*p<0.05 vs. Con; ^#p<0.05 vs. acute pancreatitis (AP) (n=5). Con, control group; AP, L-arginine exposure group; AP+RSV, L-arginine and resveratrol treatment group.

Figure 6

Effect of resveratrol on the sirtuin 1 (SIRT1) protein expression in the pancreas of mice with L-arginine-induced acute necrotizing pancreatitis (ANP). Western blotting was performed to detect the SIRT1 protein expression in the pancreas of mice with L-arginine-induced acute pancreatitis at 48 h (A and B), 72 h (C and D) after the second injection of L-arginine. ^*p<0.05 vs. Con; ^#p<0.05 vs. acute pancreatitis (AP) (n=3). Con, control group; AP, L-arginine exposure group; AP+RSV, L-arginine and resveratrol treatment group.

Figure 7

Effect of resveratrol on the p53 and acetylated p53 expression in the pancreas of mice with L-arginine-induced acute necrotizing pancreatitis (ANP). Interaction between sirtuin 1 (SIRT1) and p53 in the pancreas was detected by co-immunoprecipitation at 48 h (A), 72 h (B) after the second injection of L-arginine. Expression levels of p53 and acetylated p53 in the pancreas were detected by immunoblotting at 48 h (C and E), 72 h (D and F) after the second injection of L-arginine. ^*p<0.05 vs. Con; ^#p<0.05 vs. acute pancreatitis (AP) (n=3). Con, control group; AP, L-arginine exposure group; AP+RSV, L-arginine and resveratrol treatment group.

Figure 8

Effect of resveratrol on the expression and acetylation of heat shock factor 1 (HSF1) in the pancreas of mice with L-arginine-induced acute necrotizing pancreatitis (ANP). Interaction between sirtuin 1 (SIRT1) and HSF1 in the pancreas was detected by co-immunoprecipitation at 48 h (A) and 72 h (E) after the second injection of L-arginine. Expression of HSF1 and acetylated HSF1 in the pancreas was detected by immunoblotting at 48 h (B–D) and 72 h (F–H) after the second injection of L-arginine. ^*p<0. vs. Con; ^#p<0.05 vs. acute pancreatitis (AP) (n=3). Con, control group; AP, L-arginine exposure group; AP+RSV, L-arginine and resveratrol treatment group.