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Biogenic nano-selenium particles effectively attenuate oxidative stress-induced intestinal epithelial barrier injury by activating the Nrf2 antioxidant pathway Deguang Song, Yuanzhi Cheng, Xiaoxiao Li, Fengqin Wang, Zeqing Lu, Xiao Xiao, and Yizhen Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b03377 • Publication Date (Web): 13 Apr 2017 Downloaded from http://pubs.acs.org on April 15, 2017
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Biogenic nano-selenium particles effectively attenuate oxidative stress-induced intestinal epithelial barrier injury by activating the Nrf2 antioxidant pathway Deguang Song, Yuanzhi Cheng, Xiaoxiao Li, Fengqin Wang, Zeqing Lu, Xiao Xiao, Yizhen Wang* National Engineering Laboratory of Biological Feed Safety and Pollution Prevention and Control, Key laboratory of Molecular Animal Nutrition, Ministry of Education, Institute of Feed Science, Zhejiang University, 866 Yuhang Tang Road, Hangzhou 310058, China ABSTRACT In the present study, a new form of selenium nanoparticle (biogenic nano-selenium (BNS) particles) was synthesized using bacteria. The protection of BNS particles against oxidative stress-induced intestinal barrier dysfunction and the inherent mechanisms of this process were investigated, and selenomethionine (SeMet) and chemically synthesized nano-selenium (Nano-Se) particles were used for comparison. Characterization of BNS particles revealed that they were monodisperse and homogeneous spheres with an average size of 139.43 ± 7.44 nm. In the mouse model of intestinal oxidative stress, BNS particles were found to protect the mouse intestinal barrier function and preserve intestinal redox homeostasis more efficiently than SeMet and Nano-Se. In vitro experiments with porcine jejunum epithelial (IPEC-J2) cells verified the stronger epithelial barrier-protecting effect of BNS particles against oxidative stress with reduced cell apoptosis and an improved cell redox state. BNS activated nuclear factor (erythroid-derived-2)-like 2 (Nrf2) and increased the expression of its downstream genes, including thioredoxin reductase (TXNRD)-1, NADPH dehydrogenase (NQO)-1, heme oxygenase (HO)-1 and thioredoxin (Trx), in dose- and time-dependent manners. In contrast, SeMet and Nano-Se merely enhanced the activity of the selenoenzymes TXNRD-1 and glutathione peroxidase (GPx)-1, indicating the role of selenium donors. Moreover, knockdown of Nrf2 significantly blocked the antioxidative effect of BNS, confirming that BNS protects the intestinal barrier from oxidative stress-induced damage by activating Nrf2 and its downstream genes. Our results suggest that BNS is a promising selenium species with potential application in treating oxidative stress related intestinal diseases. Keywords: biogenic nano-selenium particles; antioxidant; oxidative stress; intestinal barrier; apoptosis; Nrf2 INTRODUCTION Oxidative stress has been implicated in a wide range of human diseases. In fact, it is difficult to find a pathology in which oxidative stress does not play a role 1. Oxidative stress occurs when reactive oxygen species (ROS) production exceeds the antioxidant capacity of cells, thus leading to the induction of protein modification and lipid peroxidation and subsequent cellular dysfunction and diseases 2. Among all of the fully differentiated organs, the intestine is more vulnerable to oxidative stress than others because of frequent enterocyte renewal and continuous exposure to exogenous agents, including oxidants 3-4. Increasing evidence suggests that various intestinal diseases, even their pathogeneses, are related to oxidative stress; such is the case for inflammatory bowel disease, ischemic-reperfusion disorders, and intestinal cancers5-7. Nuclear factor (erythroid-derived-2)-like 2 (Nrf2), the master cellular sensor of oxidative stress, remains in the cytoplasm bound to kelch-like ECH-associated protein 1 (Keap1) in its inactive state 8-9. When cells are under oxidative 1
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stress, cysteine residues, the active sites of Keap1, are oxidized, preventing Keap1 from interacting with Nrf2. As Nrf2 accumulates in the cytoplasm, Nrf2 moves to the nucleus, where it binds to the small Maf protein and antioxidant response element (ARE), leading to the activation of its downstream genes, including antioxidant enzymes10-11. Thus, drugs that effectively activate the Nrf2 pathway are thought to be promising therapies for diseases related to the delicate balance of oxidative species of cells12. Selenium in its antioxidant role, notably as glutathione peroxidase (GPx) and thioredoxin reductase (TXNRD), can reduce hydrogen peroxide, lipid and phospholipid hydroperoxides, thereby dampening the propagation of ROS and free radicals13. Selenium deficiency results in the profound reduction of GPx activity, causing oxidative stress14-15. In fact, selenium is toxic at a level only slightly higher than the beneficial requirement, which is now mainly thought to be due to its pro-oxidant ability16. The generally considered safe selenium-adding form, selenomethionine (SeMet), may lead to accumulated toxicity due to its nonspecific incorporation into proteins in place of methionine17. Elemental selenium powder in the redox state of 0 is not soluble and is generally considered to be biologically inert. However, nano-sized elemental selenium particles were found to possess prominent bioactivity and biosafety properties, which are attracting increasing attention18-19. Several studies have discovered that the surface decoration of chemically synthesized nano-selenium (Nano-Se) particles with polysaccharides, antioxidant reagents or anticarcinogens remarkably improved the antioxidant or anticancer functions of selenium nanoparticles20-27. Therefore, selenium nanoparticles may be a more appropriate selenium-adding form to achieve antioxidative goals. Instead of synthesizing selenium nanoparticles by the chemical method, several bacteria have been found to effectively detoxify selenite by reducing it to and causing it to resemble zero-valence selenium nanoparticles28-30. However, related studies have mainly focused on how to reduce selenite pollution via microbe reduction31. To our knowledge, only one study has focused on the bioactivity of bacteria-synthesized selenium particles. The authors of that study, Rezvanfar et al., found that Klebsiella pneumoniae produced selenium particles that could effectively attenuate cisplatin-induced testis oxidative stress and sperm toxicity32; however, the mechanism of the beneficial effect of these selenium particles was not studied. Enterobacter cloacae Z0206, a bacterial strain that was previously isolated by our group, produces a large amount of extracellular polymeric substances (EPS), which functions as an enhancer of the antioxidative and immune functions of mice and livestock33-35. We recently found that E. cloacae Z0206 could transform sodium selenite to red zero-valence nano-sized selenium particles capped with proteins derived from E. cloacae Z0606, called biogenic nano-selenium (BNS) particles. In the present study, we (i) investigated the effect of BNS particles on oxidative stress-induced intestinal barrier injury in vivo and in vitro, (ii) determined the effect of BNS particles on the intestine and epithelial cell redox state, and (iii) studied the underlying mechanism of BNS particles’ antioxidative effects. SeMet and chemically synthesized Nano-Se particles were used as comparisons in this study. RESULTS AND DISCUSSION Preparation and characterization of BNS and Nano-Se particles In this study, we demonstrated a new way to synthesize selenium nanoparticles using E. cloacae Z0206, by which sodium selenite was transformed into zero-valence selenium 2
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particles coated with proteins (Fig. 1A). As shown in Fig. 1B, the culture of cells with the presence of 10 mM selenite was red, and Scanning Electronic Microscope (SEM) images exhibited plenty of BNS extracellularly, accompanied by a mass of EPS. BNS particle was synthesized intracellularly (Fig. 1C) and was secreted out of the cell. BNS particles were separated (Fig. 1D) and lyophilized, and analyzed by FT-IR (Fig. 1E). The intensity of the FT-IR peaks of BNS particles correspond to O-H stretching vibration peak at 3290 cm-1, N-H stretching vibration peaks at 3290, 1539, 1163 and 1073 cm-1, the vibration peaks of C═O at 1652 cm-1, the vibration peaks of COO- at 1455 and 1396 cm-1, and the vibration peak of CH2 and CH3 alkyls at 2924, indicating that BNS particles were capped with proteins synthesized by Z0206 cells.
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Figure. 1 Synthesis and characterization of BNS particles. (A) Schematic of hypothesis of the synthesis process of BNS particles by Z0206 cells. (B) Appearance and SEM images of Z0206 cultures with or without the presence of 10 mM selenite. (C) TEM images of Z0206 cultures with or without the presence of 10 mM selenite. (D) Appearance of the purified BNS particles. (E) FT-IR analysis of BNS particles. Fig. 2A shows the Transmission Electron Microscope (TEM) images of BNS and 4
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Nano-Se, which clearly revealed that BNS particles presented a monodispersed and homogeneous spherical structure, whereas the shape of Nano-Se particles appeared to be irregular. Elemental composition analysis of the BNS and Nano-Se particles employing Energy Dispersive X-ray Spectrum (EDX) (Fig. 2B) indicated the presence of a strong signal from the Se atoms from both particle types. According to the size distribution analysis (Fig. 2C), the size of the BNS particles ranged from 80 nm to 250 nm (average: 139.43 ± 7.44 nm), and the majority of the Nano-se particles ranged from 40 nm to 600 nm (average: 120.97 ± 10.25 nm), 1.8% ± 1.64% larger than 1,000 nm.
Figure 2. Comparison of BNS and Nano-Se particles. (A) TEM images. (B) EDX analysis. (C) Size distribution. Effect of BNS particles on oxidative stress-induced mouse intestinal injury Diquat is a bipyridyl herbicide that utilizes molecular oxygen to produce superoxide anion radical36. A number of studies have successfully established oxidative stress models by diquat37-39. Yin et al. demonstrated that diquat induced oxidative stress in piglets disrupted intestinal absorption system40. Our pilot experiment demonstrated that 25 mg/kg body weight diquat was sufficient to induce oxidative stress and that the jejunum was more sensitive than the liver and other intestinal segments (Supplemental Figs. S1 and S2). Therefore, the jejunum was chosen as the target tissue in subsequent experiments. To optimize the dose of BNS particles, we performed another pilot experiment through which a dose of 0.5 mg/kg body weight BNS was selected (Supplemental Fig. S3). As shown in Fig. 3A, diquat treatment increase serum 4kDa Fitc-Dextran (FD4) content from 4.42±0.08 µg/ml (control group) to 5.65±0.52 µg/ml (P
