Propofol Attenuates Small Intestinal Ischemia Reperfusion Injury through Inhibiting NADPH Oxidase Mediated Mast Cell Activation

Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2015, Article ID 167014, 15 pages http://dx.doi.org/10.1155/2015/167014 Research Article Propofol Attenuates Small Intestinal Ischemia Reperfusion Injury through Inhibiting NADPH Oxidase Mediated Mast Cell Activation Xiaoliang Gan,1,2 Dandan Xing,1 Guangjie Su,1 Shun Li,1 Chenfang Luo,1 Michael G. Irwin,3 Zhengyuan Xia,3 Haobo Li,3 and Ziqing Hei1 1 Department of Anesthesiology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China Zhongshan Ophthalmic Center, Department of Anesthesiology, Sun Yat-sen University, Guangzhou 510060, China 3 Department of Anesthesiology, University of Hong Kong, Hong Kong 2 Correspondence should be addressed to Haobo Li; and Ziqing Hei; Received 18 July 2014; Accepted 7 September 2014 Academic Editor: Mengzhou Xue Copyright © 2015 Xiaoliang Gan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Both oxidative stress and mast cell (MC) degranulation participate in the process of small intestinal ischemia reperfusion (IIR) injury, and oxidative stress induces MC degranulation. Propofol, an anesthetic with antioxidant property, can attenuate IIR injury. We postulated that propofol can protect against IIR injury by inhibiting oxidative stress subsequent from NADPH oxidase mediated MC activation. Cultured RBL-2H3 cells were pretreated with antioxidant N-acetylcysteine (NAC) or propofol and subjected to hydrogen peroxide (H2 O2 ) stimulation without or with MC degranulator compound 48/80 (CP). H2 O2 significantly increased cells degranulation, which was abolished by NAC or propofol. MC degranulation by CP further aggravated H2 O2 induced cell degranulation of small intestinal epithelial cell, IEC-6 cells, stimulated by tryptase. Rats subjected to IIR showed significant increases in cellular injury and elevations of NADPH oxidase subunits p47phox and gp91phox protein expression, increases of the specific lipid peroxidation product 15-F2t -Isoprostane and interleukin-6, and reductions in superoxide dismutase activity with concomitant enhancements in tryptase and 𝛽-hexosaminidase. MC degranulation by CP further aggravated IIR injury. And all these changes were attenuated by NAC or propofol pretreatment, which also abrogated CP-mediated exacerbation of IIR injury. It is concluded that pretreatment of propofol confers protection against IIR injury by suppressing NADPH oxidase mediated MC activation. 1. Introduction Small intestinal ischemia reperfusion (IIR) injury has been emerged in many pathophysiological settings, including septic and hemorrhagic shock induced hypoperfusion [1], as well as acute mesenteric ischemia [2] and small intestine transplantation or liver resection [3]. IIR remains to be a critical problem associated with high mortality [4]. During IIR, oxidative stress is increased due to burst production of reactive oxygen species (ROS), which is a major mechanism of IIR injury [5]. Numerous studies have revealed that increased ROS production resulted from overactivation of the prooxidant enzyme NADPH oxidase, which abundantly exists in intestine tissue [6] and plays critical roles in mediating tissue injury related to a range of inflammatory diseases [7], including ischemia reperfusion injury [8, 9]. Inhibition of NADPH oxidase by N-acetylcysteine (NAC), a scavenger of oxygen radicals, has been shown to greatly attenuate myocardial ischemia reperfusion injury [10, 11]. However, the mechanism governing ROS production, specifically, from overactivation of NADPH oxidase, during IIR is yet to be explored. Activation of NADPH oxidase can lead to mast cells (MC, cells that widely present throughout small intestine) degranulation/activation [12], wherever MCs activation has been demonstrated to be a key mediator in the pathogenesis of IIR and contributing to many disorders as a result of increased release a diverse range of mediators including histamine, tryptase, and inflammatory cytokines such as interleukin-6 (IL-6) [13] and that inhibition of MCs from degranulation 2 can alleviate IIR injury [14, 15]. We have previously found that ROS production was significantly increased after IIR which is parallel with the enhancements in MC degranulation [16], but it is unknown whether a causal relationship exists between increased ROS production and MC degranulation in the setting of IIR in vivo. These findings [6, 17] together with reports showing that MC can be activated in a ROSdependent pathway [18], both in vitro [18] and in vivo [16], prompted us to postulate that during IIR increased ROS production initiates and/or exacerbates IIR injury primarily via activating MC and that NADPH oxidase activation is increased during IIR which may be a major source of ROS overproduction during IIR. Propofol, an intravenous anesthetic with antioxidant property that we widely used in intensive care unit and operation theatre, has been shown to dose-dependently attenuate myocardial ischemia reperfusion injury in patients [19]. Propofol has also been shown to inhibit mast cell exocytosis in a dose-dependent manner in vitro [20]. A most recent study shows that propofol attenuates brain trauma induced cerebral injury through inhibiting NADPH oxidase activation [21]. We, therefore, hypothesized that inhibition of ROS mediated MC activation subsequent to attenuation of intestinal NADPH oxidase activation may represent a major mechanism by which propofol attenuates IIR injury. This hypothesis was tested in a rat model of mesenteric ischemia reperfusion in vivo and a rat cell line of mast cell exposed to ROS in vitro. 2. Materials and Methods 2.1. Cell Culture. Rat mast cell line (RBL-2H3) was purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China) and was cultured in Eagle’s minimum essential medium containing 10% fetal bovine serum, supplemented with 100 U/mL penicillin and 100 𝜇g/mL streptomycin at 37∘ C in a humidified atmosphere of 5% CO2 as described [22]. A rat small intestinal epithelial cell line (IEC-6) was also obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China). This cell line was cultured in 1640 RPMI medium with 10% fetal bovine serum at 37∘ C in a 5% CO2 incubator. 2.2. Measurement of Cell Degranulation. Degranulation of RBL-2H3 cells was measured by determining the activity of released 𝛽-hexosaminidase in the culture supernatants and cell lysates [23]. The RBL-2H3 cells were incubated with different concentrations of NAC [24] or propofol [20] for 12 h; after the cells were washed with 1 × PBS for 3 times, RBL-2H3 cells were incubated in a 24-well plate (1 × 106 cells/well) at 37∘ C overnight. The above cells were washed with 1 × PBS and then incubated with different concentrations of lipopolysaccharides (LPS) [25] or (...truncated)