Treatment with the Nrf2 activator, Dimethyl Fumarate, ameliorates acute pancreatitis in a rodent model

O RIGINAL A RTICLE Dimethyl Fumarate Ameliorates Acute Pancreatitis in Rodent Lourdes Robles, MD,* Nosratola D. Vaziri, MD,† Shiri Li, MD, PhD,* Chie Takasu, MD, PhD,* Yuichi Masuda, MD, PhD,* Kelly Vo, MS,* Seyed H. Farzaneh, MD,† Micheal J. Stamos, MD,* and Hirohito Ichii, MD, PhD* Objectives: Pancreatitis is a complex inflammatory disorder, ranging from a mild attack, to severe and potentially fatal condition. Dimethyl fumarate (DMF), a potent antioxidant and anti-inflammatory, has been used medicinally for decades. The purpose of this study was to test the hy- pothesis that treatment with DMF may ameliorate acute pancreatitis (AP) in a rodent model. Methods: Rats were treated with DMF (25 mg/kg) 24 hours prior to AP induction with L -arginine (3 g/kg). At 72 hours, the pancreas was proc- essed for histology. Serum amylase, lactate dehydrogenase, pancreatic trypsin, and lipid peroxidation product (malondialdehyde) were evaluated. Key cytokines and chemokines in the supernatant of lipopolysaccharide- stimulated splenocytes were also determined. Results: Pancreata from DMF-treated rats showed reductions in the severity of inflammatory cell infiltration, acinar damage, perilobar edema, and cell necrosis. This was associated with significantly lower amylase and malondialdehyde but not lactate dehydrogenase or trypsin levels. The apoptotic pancreatic cells (cleaved caspase 3 positive) were significantly lower in the DMF-treated rats. Lipopolysaccharide-stimulated splenocytes treated with DMF produced a significantly lower amount of key inflamma- tory mediators. Conclusion: Administration of DMF attenuates AP in rats. Key Words: dimethyl fumarate, reactive oxygen species, pancreatitis, oxidative stress, cytokine, inflammation (Pancreas 2015;44: 441–447) A cute pancreatitis (AP) is characterized by the sudden onset of local pancreatic inflammation from intrapancreatic activation of digestive enzymes. The disease carries a spectrum of severity from mild self-limiting to a highly morbid and even fatal illness. It is believed that the initial event occurs from the disruption of ac- inar cells, leading to the leakage of enzymes, resulting in auto- digestion of the pancreas. The activated enzymes cause a local inflammatory response, which if severe can result in further recruit- ment of inflammatory mediators and the development of a systemic response leading to multiorgan system failure and death. 1,2 Fortunately, because of advances in critical care, the mortal- ity rate has decreased over the last decade; however, the incidence of AP remains high worldwide. 3 Recent reports have revealed an increase in the incidence of pancreatitis in developed countries in- cluding the United States. 4,5 Injury to the pancreatic acinar cells causes a cascade of events that includes production of reactive ox- ygen species (ROS), resulting in the oxidation of lipids and pro- teins and disruption of the pancreatic membranes. 6 Increased From the Departments of *Surgery and †Medicine, University of California, Irvine, CA. Received for publication April 22, 2014; accepted August 22, 2014. Reprints: Hirohito Ichii, MD, PhD, Department of Surgery, University of California, Irvine, 333 City Blvd West Suite 1205, Orange, CA 92868 (e‐mail: hichii@uci.edu). This study was in part supported by grants from NIH-NCRR UL1 TR000153, KL2 TR000147; and the Juvenile Diabetes Research Foundation International 17-2011-609. The authors declare no conflict of interest. Copyright © 2014 Wolters Kluwer Health, Inc. All rights reserved. Pancreas • Volume 44, Number 3, April 2015 production of ROS and the resulting oxidative stress play a major role in the pathogenesis of tissue damage in AP. Under normal condition, cells respond to ROS by up-regulating expression of cytoprotective and antioxidant enzymes and related molecules such as superoxide dismutase, catalase, glutamate-cysteine ligase catalytic (GCLC), and glutathione (GSH) peroxidase, which work in concert to neutralize ROS. However, when production of ROS exceeds the antioxidant capacity, it results in oxidative stress, in- flammation, and tissue damage. 7 Currently, the treatment of AP in the United States is limited to supportive care. Although there have been numerous trials ex- ploring the efficacy of antioxidants or anti-inflammatory drugs in the treatment paradigm of AP, the results remain inconclusive. Dimethyl fumarate (BG-12) is the methyl ester of fumaric acid and was initially recognized for its anticancer effects. 8 Later, it be- came widely used in Europe for the treatment of psoriasis under the trade name Fumaderm. 9 Most recently, BG-12 received Food and Drug Administration approval in the United States for the treatment of patients with multiple sclerosis (MS). Dimethyl fu- marate is a unique and potent antioxidant and anti-inflammatory drug whose mechanism of action has yet to be elucidated. The drug has been effective for decades for the treatment of other acute and chronic inflammatory conditions; however, it has never been examined specifically in the setting of pancreatitis. The aim of the present study was to test the hypothesis that treatment with dimethyl fumarate may attenuate severity of AP in experimental animals by enhancing cellular antioxidant and anti-inflammatory machinery. MATERIALS AND METHODS In Vivo AP Rodent studies were performed in accordance with the Institu- tional Animal Care and Use Committee of University of California, Irvine (Irvine, Calif). Male Sprague-Dawley rats (control-normal rats, n = 3; L -arginine, n = 12; L -arginine + dimethyl fumarate [DMF], n = 12; n = 27) were purchased from Charles River (Wil- mington, Mass). Experimental agents used to produce AP were pur- chased from Sigma (St Louis, Mo) unless otherwise specified. Rats (250–300 g) were fed ad libitum on a standard diet with free access to water. They were maintained on a 12-hour light-dark cycle. Ex- perimental animals were given oral DMF (25 mg/kg) dissolved in methylcellulose and fed via oral gavage 24 hours prior to initiating AP and daily thereafter until the animals were killed. L -Arginine Twenty percent L -arginine was dissolved in normal saline and filtered through a syringe filter with pH adjusted to 7.0. The solu- tion was administered to nonfasted rats in 2 intraperitoneal injec- tions at a dose of 3 g/kg body weight, each injection separated by 1 hour. Animals, control and experimental, received buprenorphine (Reckitt Benkiser, Richmond, Va) pain medication (0.01 mg/kg) in- tramuscularly twice a day and regular food and water. Because of the experimental end points, anti-inflammatory medications were www.pancreasjournal.com Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.