Protective Effects of Nano-Zno on the Primary Culture Mice Intestinal Epithelial Cells in in vitro Against Oxidative Injury

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1 World Journal of Agricultural Sciences 6 (): 495, ISSN IDOSI Publications, Protective Effects of Nano-Zno on the Primary Culture Mice Intestinal Epithelial Cells in in vitro Against Oxidative Injury AI Dawei, Wang Zhisheng and Zhou Anguo,,, Animal Nutrition Institute, Sichuan Agricultural University, Ya an 654, China Engineering Research Center of Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Ya an 654, China stract: Nano-ZnO is a new products which may used to be feed resources. The aim of this study was to exam whether nano-zno is le to protect cell integrity from potential damage by free-radical oxidative injury. A factorial experiment was used to investigate the interaction between particle diameter (5, 5 and nm) and concentration (.4,.6 and 6.4 µg ml ) of the primary culture mice intestinal epithelium cell (IEC) in in vitro. The results indicated that the all the nano-zno groups MTT OD value were significantly higher than the perhydrol group (P <.5) and LDH decreased, but not significant (P>.5). The MDA content in all the nano-zno groups were decreased significant (P<.). The SOD and CAT in all nano-zno groups were higher than the perhydrol group(p>.5). The results showed that nano-zno released the IEC oxidative injury. Key words: Intestinal epithelium cell, Nano-ZnO, Antioxidant, Antioxidant enzymes, Integrity INTRODUCTION nano-zno is le to protect membrane integrity from potential damage by free-radical oxidative injury remains The role of zinc in the protective effect in resisting to be elucidated. Therefore, to understand the effect intestinal disease is well known[,]. In particular, several of anti-oxidative activity of nano-zno, we designed studies conducted on piglets showed that to examine the primary culture mice intestinal epithelium pharmacological concentrations of zinc (from ZnO) cell (IEC) the effects of different diameter and levels on prevent or alleviate diarrhea, which is mainly caused by IEC survival rate. Moreover, we determined the L-lactate oxidative stress [,4]. Zinc plays an important role in dehydrogenase (LDH), malondialdehyde (MDA), maintaining membrane function and stility. Some superoxide dismutase (SOD) and catalase (CAT) in researchers suggest that the mechanism of this activity response to the different treatments. involves the stilization of the membrane structure [5] or the displacement of redox-active metals to prevent MATERIALS AND METHODS free-radical oxidative damage[6]. Nano-ZnO is a new product whose particle diameter is between to nm. Experimental Animals: 7-day old mice were obtained Recently, nano-zno attracted much attention gradually in from the National Experimental Teaching Demonstration the area of animal science. Its strong energy restriction Center of Animal Science, Ya'an, China. All animal effect and interface effect, which lead to a higher electron protocols were approved by the Institutional Animal Care conductivity, transparency and transmission ility and Use Committee of Sichuan Agricultural University than the bulk ones and other metallic oxide materials[7], (Ya'an, China). will open more opportunities to make use of this nano-material. Experimental Design: The study used a -test design The integrity of the intestinal barrier is fundamental that tested for factors: the particle diameter 5nm to the proper functioning of the epithelial cells [8]. In nano-zno group(,- and - group), the 5nm many published studies, zinc plays a role in maintaining nano-zno group(, - and - group) and the nm epithelial barrier integrity and function [9,]. Whether nano-zno group(, - and - group). The levels Corresponding Author: Prof. Wang Zhisheng, Institute of Animal Nutrition, Sichuan Agricultural University, 654,Ya an, Sichuan, China 49

2 World J. Agric. Sci., 6 (): 495, were.4,.6 and 6.4 µg ml, respectively. in The collection of cells and culture solution in every addition, There were a perhydrol group(pg) and a normal 4-well plates well in.5ml centrifuge tubes, centrifuged group(ng). For each level of 8 to repeat and repeated at r/min for min, the collection of the supernatant each of well. and sediment saved at - C for the test LDH, MDA, SOD and CAT assays All the test using kits which were Cell Culture and the Determination of Indicators: purchased from Nanjing Jiancheng Bioengineering The method of cell isolation for mice intestinal epithelial Institute, Nanjing, China. cell (IEC) was according to Evans et al. []. Cells were 5 diluted to ml and seeded in 96-well and 4-well Statistical Analysis: All data were expressed as plates with DMEM supplemented with mm L glucose, means±sd. A -way analysis of variance (ANOVA) was, U L penicillin,, µg L streptomycin sulfate used to perform comparisons using SPSS (version 4.; and 5 % fetal bovine serum and placed into an incubator SPSS Inc,Chicago, Ill). Group differences resulting in P with 7 C, 5% CO (BB56UV type, Heraeus, Germany). values of less than.5(.) were considered to be The medium was changed every 48 h and the cells statistically significant(very significant). were left for 7 to d to allow differentiation. And then different concentrations of particle diameter RESULTS nano-zno (final concentration were.4,.6 and 6.4µg ml, respectively.) were added for 4h, to which HO The influence of different particle diameter and level (final concentration of µm L ) was added except the of nano-zno on cell MTT OD value is shown on Fig.. normal group and placed into an incubator with 7 C, 5% All the nano-zno groups OD value were significantly CO for 4h. higher than the perhydrol group (P <.5). The 5nm.4, After the incubation, each well of the 96-well plates.6 µg ml groups OD value were very significantly by adding Thiazolyl blue (MTT) solution (5g L ) µl, higher than the other nano-zno groups. And the continued to train for 4 h. Centrifuge to the supernatant, differences among the 5nm.4,.6 µg ml and NG adding dimethyl sulfoxide µl and was fully shocked groups was not significant (P>.5). after min. The Colorimetric detection of the optical LDH, MDA, SOD and CAT content are presented in density value at 57nm wavelength was analyzed using a Fig.. to Fig.5. The addition of nano-zno decreased LDH, fully automatic microplate reader (Wellsan MK type, but not significant (P>.5). The MDA content in all the Thermo, U.S.A). nano-zno groups were decreased significant (P<.), BCc c BCc BCc Cd PG NG Fig. : The influence of nano-zno on the MTT OD value of mice intestinal epithelium cell in vitro culture Note: The difference between data with different capital letters was significant among groups (P.) and the difference between data with different small letters was significant among groups (P.5) and the same letters was not significant (P ¾.5) 5

3 World J. Agric. Sci., 6 (): 495, 4 5 b a PG NG Fig. : The influence of nano-zno on the LDH release of mice intestinal epithelium cell in vitro culture b b b b Bc.5 Cd PG NG Fig. : The influence of nano-zno on the MDA release of mice intestinal epithelium cell in vitro culture b b b b b b b Cd 4 Bc PG NG Fig. 4: The influence of nano-zno on the SOD of mice intestinal epithelium cell in vitro culture 5

4 World J. Agric. Sci., 6 (): 495,.5 Cc ACa PG NG Fig. 5: The influence of nano-zno on the CAT of mice intestinal epithelium cell in vitro culture compared to the perhydrol group. The 5nm when exposured a high concentration ROS nano-zno group seemed lower than any other environment. MDA is the byproducts of cell membrane diameter group. As for the antioxidase activities, lipid peroxidation. In our study, either small- or large- SOD and CAT, All the nano-zno groups were higher diameter nano-zno decrese LDH (P>.5) and MDA than the perhydrol group. The trend of influence of the (P<.), compared to that of perhydrol group. The results nano-zno groups on SOD activities was in an downward indicated that nano-zno released lipid peroxidation in trend along with the particle diameter augmentation. mitochondria and cell membranes, so that stilized the However, the influence among different particle diameters cell membrane structure. This is in close agreement with and levels of nano-zno on CAT were not significant previous studies[5,6]. It was in our expectation that (P>.5). cells supplemented with nano-zno had the better performance and higher antioxidant enzymes activities, DISCUSSION which may due to the enhance of cell producing more SOD and CAT enzymes. The formations of superoxide Zn plays an essential role in cell membrane and peroxide are inextricly linked, as superoxide is integrity and is a component of more than different converted to peroxide and oxygen, a reaction catalyzed enzymes that function in many aspects of cellular by SOD. SOD plays a role in modulating ROS metolism, involving metolism of proteins, lipids and is sensitive to Zn[5]. The cytotoxic effects of and carbohydrates[,]. However, the mechanism hydrogen peroxide (HO ) are limited by its degradation of the nano-zno protective effect is largely unknown. by CAT. CAT, localised in peroxisomes, exerts a We hypothesized that nano-zno could release the function: decomposition of HO to yield HO and O.[7] IEC oxidative injury so as to gain a better performance Santon et al. [8] have shown that the maintenance on of cells. Previous research has found that ZnO can basal levels of the antioxidant enzymes activities support enhance the reparation of epithelial cells[4]. In our the hypothesis that Zn can retard oxidative mechanisms, study, all the nano-zno groups OD value were suggesting a role in modulating ROS. significantly higher than the perhydrol group (P <.5), indicated that nano-zno enhanced the number of live CONCLUSION cells. It seems that the effect of small diameter better than the effect of large one. The present study showed that nano-zno The generation of reactive oxygen species (ROS), released the IEC oxidative injury and enhance SOD and which derive from electron transfer reactions CAT enzymes activities. However further studies are in mitochondria and endoplasmic reticulum, is inherent needed in order to find the mechanism of the nano-zno in aerobic cell metolism[5]. The cell would be injury protective effect. 5

5 World J. Agric. Sci., 6 (): 495, REFERENCES. Roselli, M., A. Finamore, I. Garaguso, M.S. Britti and E. Mengheri,. Zinc oxide protects cultured. Schell, T.C. and E.T. Kornegay, 996. Zinc enterocytes from the damage induced by Escherichia concentration in tissues and performance of coli. J Nutr., : weanling pigs fed pharmacological levels of zinc. Evans, G.S., N. Flint, A.S. Somers, B. Eyden and from ZnO, Zn methionine, Zn lysine, or ZnSO 4. C.S. Potten, 99. The development of a method for J. Anim. Sci., 74: the preparation of rat intestinal epithelial cell primary. Shankar, A.H. and A.S. Prasad, 998. Zinc and cultures. J. Cell Sci., : 9-. immune function: the biological basis of altered. Parkin, G., 4. Chemistry. Zinc zinc bonds: a new resistance to infection. Am. J. Clin. Nutr., 68: frontier. Science, 5: 78.. Powell, S.R.,. The antioxidant property of zinc. J.. McCall, K.A., C. Huang and C.A. Fierke, Nutr., : Function and Mechanism of Zinc Metalloenzymes. J. 4. Hahn, J.D. and D.H. Baker, 99. Growth and plasma Nutr., : zinc responses of young pigs fed pharmacologic 4. Lansdown, A.B., 995. Physiological and levels of zinc. J. Anim. Sci., 7: -4. toxicological changes in the skin resulting from the 5. O Dell, B.L.,. Role of zinc in plasma membrane action and interaction of metal ions. Crit. Rev. function. J. Nutr., : 446. Toxicol., 5: Canali, R., F. Vignolini, F. Nobili and E. Mengheri, 5. Stehbens, W.E.,. Oxidative stress, toxic hepatitis. Reduction of oxidative stress and cytokine- and antioxidants with particular emphasis on zinc, induced neutrophil chemoattractant (CINC) Exp. Mol. Pathol., 75: expression by red wine polyphenols in zinc 6. Tapiero, H. and K.D. Tew,. Trace elements in deficiency induced intestinal damage of rat. Free human physiology and pathology: zinc and Radic. Biol. Med., 8: metallothioneins. Biomed. Pharmacother, 57: Joshy, J. and K.M. dul,. Role of grain 7. Alessia, F., I. Paola and S. Alessandro, 7. Zinc, boundaries on the electrical conductivity of antioxidant systems and metallothionein in metal nanophase zinc oxide, Mater. Sci. Eng. A 4:8-8. mediated-apoptosis: Biochemical and cytochemical 8. Lu, L. and W.A. Walker,. Pathologic and aspects. Comparative Biochemistry and Physiology, physiologic interactions of bacteria with the Part C., 46: gastrointestinal epithelium. Am. J. Clin. Nutr., 8. Santon, A., P. Irato, V. Medici, R. D'Inca, 7: 4. V. Albergoni and G.C. Sturniolo,. Effect and 9. Bao Shenying and Daren L. Knoell, 6. Zinc possible role of Zn treatment in LEC rats, an modulates cytokine-induced lung epithelial cell animal model of Wilson's disease. Biochim. Biophys. barrier permeility. Am. J. Physiol. Lung. Cell. Mol. Acta., 67: Physiol., 9: 4. 5

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