Diosmetin ameliorates the severity of cerulein-induced acute pancreatitis in mice by inhibiting the activation of the nuclear factor-κB

Abstract

Diosmetin (3', 5, 7-trihydroxy-4'-methoxyflavone), the aglycone part of the flavonoid glycosides diosmin occurs naturally in citrus fruit, was considered to exhibit anti-inflammatory and antioxidant properties. Our study aimed to investigate the effect of diosmetin in a murine model of cerulein-induced acute pancreatitis (AP). Experimental AP was induced in mice by seven intraperitoneal injection of cerulein (50 ug/kg) at hourly intervals. Diosmetin (100 mg/kg) or vehicle was pretreated 2 h before the first cerulein injection. After 6 h, 9 h, 12 h of the first cerulein injection, the severity of acute pancreatitis was evaluated biochemically and morphologically. Pretreatment with diosmetin significantly reduced serum levels of amylase and lipase; the histological injury; the secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6; myeloperoxidase (MPO) activity, trypsinogen activation peptide (TAP) level, the expression of inducible nitric oxide synthase (iNOS); and the nuclear factor (NF)-κB activation in cerulein-induced AP. This study showed that administration of diosmetin demonstrated a beneficial effect on the course of cerulein-induced AP in mice. Therefore, diosmetin may become a new therapeutic agent in future clinical trials for treatment of AP.

Keywords: Acute pancreatitis; NF-κB; cytokine; diosmetin; iNOS; inflammation.

Figure 1

Effect of diosmetin on pancreas pathology in cerulein induced AP. Mice (n=8 for each group) were given seven hourly injection of cerulein (50 ug/kg). Diosmetin (25 mg/kg, 50 mg/kg, 100 mg/kg; p.o.) or vehicle (p.o.) was administrated 2 h before the first cerulein injection. The control group was given saline (0.9% NaCl) solution intraperitoneally instead cerulein. Mice were sacrificed 12 h after the first injection of cerulein. Changes in serum amylase level (A) and lipase level (B). Data are represented as mean ± SD. #P < 0.05 vs control group, $P < 0.05 vs vehicle-treated group, &P < 0.05 vs 25 mg/kg group, *P < 0.05 vs 50 mg/kg group. (C) Effect of diosmetin on pancreas histology in cerulein-induced AP. Mice (n=24 for each group) were given seven hourly injection of cerulein (50 ug/kg). Diosmetin (100 mg/kg; p.o.) or vehicle (p.o.) was administrated 2 h before the first cerulein injection. The control group was given saline (0.9% NaCl) solution intraperitoneally instead cerulein. Mice were sacrificed 6 h, 9 h, 12 h after the first injection of cerulein, five mice at every time point in each group. Representative hematoxylin and eosin-stained sections of pancreas are shown. Original magnifications: 200×.

Figure 2

Effect of diosmetin on AP-induced enzyme production and trypsinogen activation. Changes in serum amylase level (A) and lipase level (B), and pancreatic tissue TAP level (C) in all groups. Data are represented as m ean ± SD. #P < 0.05 vs controlgroup at the same time point, $P < 0.05 vs vehicle-treated group at the same time point.

Figure 3

Anti-inflammatory effect of diosmetin in cerulein-induced AP. Serum proinflammatory cytokine such as TNF-α, IL-β and IL-6 were measured by ELISA (A-C). Changes in pancreatic tissue MPO activity (D). Data are represented as mean ± SD. #P < 0.05 vs control group at the same time point, $P < 0.05 vs vehicle-treated group at the same time point.

Figure 4

Antioxidant effect of diosmetin in caerulein-induced AP. Pancreatic tissue expression level of iNOS protein was detected by western blot (A, B). β-actin was used as the internal reference for total tissue proteins. Data are presented as mean ± SD. #P < 0.05 vs control group at the same time point, $P < 0.05 vs vehicle-treated group at the same time point. The results were similar in three additional experiments.

Figure 5

Diosmetin inhibits nuclear translocation of NF-κB. Pancreatic tissue expression level of NF-κB p65 protein in nucleus were detected by western blot (A, B). Histone H1 was used as the internal reference for nuclear proteins. Data are presented as mean ± SD. #P < 0.05 vs control group at the same time point, $P < 0.05 vs vehicle-treated group at the same time point. (C) Immunohistochemical analysis of NF-κB p65 in mouse pancreas at 6 h after induction of AP. Original magnifications: 400×.