Curcumin Prevents Acute Necrotizing Pancreatitis In An Experimental Mouse Model

Objectives:Acute necrotizing pancreatitis (ANP) is an inflammatory condition of the pancreas characterized clinically by abdominal pain. It progresses rapidly and is difficult to treat. NF-κB is usually elevated in the pancreas during ANP, and activation of NF-κB is a pivotal step that leads to the inflammatory cascade in ANP, therefore suppressing NF-κB activity may reduce the release of pro-inflammatory factors and alleviates inflammatory responses in the pancreas. Curcumin is a phenolic pigment extracted from the rhizomes of tumeric herbs. Curcumin have anti-inflammatory effects, which are thought to be mediated through disruption of NF-κB transcriptional activity and inhibit the release of inflammatory mediators, and this effect may be related with the upregulation of PPARy. The aims of this study were to investigate the preventative effects of curcumin against caerulein-induced acute necrotizing pancreatitis in mice and try to find the possible mechanism involving NF-κB and PPARy pathway through further research in vivo and in vitro.Methods:In vivo experiment:A total of 80 healthy Kun Ming mice were randomly apportioned into four groups. Mice in the ANP group were only injected with caerulein. Mice in the curcumin group were intraperitoneally injected with 50 mg/kg curcumin for 6 consecutive days and subsequently with caerulein to induce ANP. In the control group, caerulein was replaced with normal saline. An additional group of mice was intravenously injected with 0.3 mg/kg GW9662 (a specific antagonist of PPARy) along with curcumin injection. Ten hours after ANP was successfully established, mice were sacrificed and blood samples were collected for assessment of serum amylase, transaminase activity and levels of TNF-a and IL-6. Mice pancreases were analyzed histologically for pathology.In vitro experiment:Murine macrophage RAW264.7 cells were first treated with 100μmol/ml curcumin at 37℃for 2 hrs or not and then stimulated with 0.1μg/ml LPS. An enzyme-linked immunosorbent assay (ELISA) was used to detect TNF-a and IL-6 levels in the plasma and cell culture media. RT-PCR was used to analyze the expression of PPARy mRNA in the pancreas and in RAW264.7 cells. Western Blot analysis was used to detect protein levels of NF-KB-p65 and PPARy in the pancreas and in RAW264.7 cells.Results:1,Effect of curcumin on serum amylase and transaminase activitiesTen hours after induction of ANP, the serum amylase, ALT and AST activities of mice in the ANP group were increased in comparison with those in the control group (P<0.01). However, curcumin treatment significantly reversed the elevation of serum amylase, ALT and AST activities in ANP mice (P<0.05).2,Pancreatic water contentTen hours after induction of ANP, the pancreatic water content in the ANP group was increased in comparison with those in the control group (P<0.01). However, curcumin treatment significantly reduced the pancreatic water content in ANP mice (P<0.05).3,Pathological examination of mice pancreasesCompared with those in the control group (histopathological scoring:1.5±0.2), the pancreases of mice in the ANP group (histopathological scoring:13.2±1.7) showed obvious hemorrhage and exudation:the pancreatic acinuses were diffusely expended, the pancreatic cells underwent necrosis, and the inter-lobular and perivascular regions were infiltrated with multiple leukocytes. The necrosis rate and inflammatory cell infiltration were significantly decreased in the pancreases of mice in the curcumin group (histopathological scoring:6.9±0.8) as compared to the ANP group.4,Effect of curcumin on the expression and release of TNF-αIn the in vivo experiment, mice serum TNF-a levels were increased in the ANP group at 10 hrs after induction of ANP, whereas curcumin treatment significantly reduced the serum TNF-a levels in ANP mice. Consistent with the in vivo experiment, TNF-a levels in the culture media of RAW264.7 cells were significantly elevated after LPS stimulation, while being significantly reduced in the presence of 100μmol/ml curcumin.5,Effect of curcumin on the expression and release of IL-6In the in vivo experiment, mice serum IL-6 levels were increased in the ANP group at 10 hrs after induction of ANP, whereas curcumin treatment significantly reduced the serum IL-6 levels in ANP mice. Consistent with the in vivo experiment, IL-6 levels in the culture media of RAW264.7 cells were significantly elevated after LPS stimulation, while being significantly reduced in the presence of 100μmol/ml curcumin.6,Effect of curcumin on NF-κB activationWestern blotting revealed that NF-KB-p65 was barely expressed in the nucleus of mice pancreases in the control group but was markedly elevated in ANP mice. Curcumin treatment inhibited the activation of NF-κB in ANP mice. After LPS stimulation, NF-κB-p65 protein level was significantly increased in the nucleus of RAW264.7 cells and considerably decreased in the presence of 100μmol/ml curcumin.7,Effect of curcumin on PPARy expressionPPARγexpression was reduced at both the mRNA and protein levels in ANP mice pancreases, and this effect was significantly reversed by curcumin treatment. Similarly, after LPS stimulation, PPARγexpression was decreased at both the mRNA and protein levels in RAW264.7 cells and was significantly increased at both levels in the presence of 100μmol/ml curcumin.8,Effect of the PPARγantagonist GW9662In order to demonstrate that the preventative effect of curcumin against ANP is associated with activation of the PPARγpathway, GW9662, a specific antagonist of PPARγ, was used. GW9662 was intravenously injected into ANP mice at the same time that curcumin was administered. GW9662 partially counteracted the effects of curcumin on serum amylase, ALT, AST, TNF-αand IL-6 levels in ANP mice. A western blot analysis of NF-κB expression showed that curcumin-mediated downregulation of nuclear NF-κB-p65 protein was significantly suppressed by GW9662.Conclusion:1,Curcumin may prevent pancreatic and hepatic injury in experimental ANP mice.2,Curcumin may inhibit the activation of NF-κB and the release of inflammatory factors thereafter, and this effect may involve PPARγactivation.