Journal of Integrative Agriculture 2017, 16(9): Available online at ScienceDirect

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1 Journl of Integrtive Agriculture 2017, 16(9): Avilble online t ScienceDirect RESEARCH ARTICLE Mngnese enhnces the expression of the mngnese superoxide dismutse in cultured primry chick embryonic myocrdil cells QIN Shi-zhen 1, 2, LIAO Xiu-dong 1, LU Lin 1, ZHANG Li-yng 1, XI Lin 3, GUO Yn-li 2, LUO Xu-gng 1 1 Minerl Nutrition Reserch Division, Institute of Animl Sciences, Chinese Acdemy of Agriculturl Sciences, Beijing , P.R.Chin 2 College of Animl Science nd Technology, Gnsu Agriculturl University, Lnzhou , P.R.Chin 3 Deprtment of Animl Science, North Crolin Stte University, Rleigh, NC 27695, USA Abstrct In the present study, the effect of mngnese (Mn) on ntioxidnt sttus nd the expression of the mngnese superoxide dismutse (MnSOD) gene in cultured primry myocrdil cells collected from the chick embryos ws investigted. The hypothesis tht Mn supplementtion would enhnce the expression of MnSOD in cultured primry myocrdil cells of chick embryos ws tested. Eggs collected from Mn-depleted Arbor Acres lying breeder hens were incubted for 10 dys nd then myocrdil cells were isolted nd cultivted for 8 dys. The embryonic myocrdil cells on dy 6 were treted with Mn in the cell culture medium t different time points when the proportion of cells showing spontneous contrction ws over 95% fter the 3-dy primry culture. A completely rndomized design involving 3 Mn levels (0, 0.5 nd 1.0 mmol L 1 ) 3 incubtion time points (12, 24 nd 48 h) fctoril rrngement of tretments (n=6) ws used in the current experiment. The results showed tht MnSOD ctivity nd mrna expression level were induced by Mn nd incresed with incubtion time, which supported the hypothesis tht Mn would enhnce the expression of the MnSOD gene, nd thus might protect myocrdil cells from oxidtive stress during the chick embryonic development. Keywords: mngnese, MnSOD, expressions, cultured primry myocrdil cells, chick embryos 1. Introduction Mny cellulr processes, such s the embryonic development, involve reduction nd oxidtion (redox) rections. These redox processes occur in mny forms, from simple Received 3 November, 2016 Accepted 13 December, 2016 QIN Shi-zhen, E-mil: qinshizhen@163.com; Correspondence LUO Xu-gng, Tel: , E-mil: wlysz@263.net 2017, CAAS. Publishing services by Elsevier B.V. All rights reserved. doi: /S (16) electron trnsfer rections to rdicl processes nd thiol/disulfide exchnges (Winyrd et l. 2005). Mny reserchers hve reported tht the chick embryos were more susceptible to oxidtive stress thn the mmmlin fetus during the embryonic development (Suri 1999, b, 2000; Jdhv nd Kengr 2016). This is becuse the embryos obtin oxygen from n ir-cell within the egg-spce nd is therefore in direct contct with the externl environment (Strck 1998). As result, the chick embryos re subjected to high oxidtive stress s they grow nd must rely on n effective ntioxidnt system for protection. Mngnese (Mn) is n essentil trce element required in smll mounts for norml growth nd development of the vin embryos (Svge 1968). It is lso crucil component

2 QIN Shi-zhen et l. Journl of Integrtive Agriculture 2017, 16(9): of the metlloenzyme, Mn superoxide dismutse (MnSOD). This enzyme is primry mitochondril ntioxidnt enzyme for detoxifiction of rective oxygen species (ROS), in prticulr, superoxide free rdicls, generted s by-products of oxidtive phosphoryltion or under oxidtive stress (Oberley nd Buettner 1979). The MnSOD ctlyzes the conversion of two molecules of superoxide nion (O 2. ) into hydrogen peroxide (H 2 O 2 ) tht is further oxidized to wter (McCord et l Cells lcking MnSOD re oxygen intolernt (Fridovich 1995). The MnSOD hs been shown to protect cells from oxidtive injury (Iwmoto nd Tked 1990; Lindu-Sheprd et l. 1994; Ishiym et l. 1995; Stephnz et l. 1996) to increse resistnce to tumor necrosis fctor (TNF) (Wong et l. 1989) nd to protect cells ginst irrdition (Fridovich 1978; Estgte et l. 1993; Wong 1995; Zhong et l. 1996). Other ntioxidnts, such s glutthione (Arechig et l. 1995) or vitmin E (Cederberg et l. 200, cn further reduce the deleterious effects of oxidtive dmge nd support development of embryos s mesured during in vitro culture. The use of synthetic SOD (SOD mimetic), or incresed MnSOD expression by trnsfection/infection, hs lso been studied s method to suppress the mlignnt phenotype or to protect cells from oxidtive stress injury (Wd et l. 1994; Ksten et l. 1995; Roberfroid nd Clderon 1995). The MnSOD is highly expressed in differentited orgns tht contin lrge number of mitochondri such s the hert, liver, nd kidneys (Mrklund 1980). Luo et l. (1991, 1992) demonstrted tht the hert ws the most sensitive trget tissue for MnSOD ctivity compred to other tissues, nd the MnSOD ctivity in hert ws sensitive biomrker for estimting the dietry Mn requirement of broilers. Lter in vivo studies in our lbortory hve lso clerly demonstrted tht MnSOD mrna nd protein expression levels, s well s the enzymtic ctivity in the broiler hert were ll very sensitive in response to dietry Mn supplementtion (Li et l. 2004, 2005, 2008, 2011; Luo et l. 2007). Supplementtion of Mn in vivo t 15 mg Mn d 1 significntly incresed lymphocyte MnSOD expression (Dvis nd Greger 1992), nd Mn lso induced MnSOD expression in dose- nd time-dependent mnners in humn brest cncer Hs578T (Thongphsuk et l. 1999). Our previous study indicted tht Mn supplementtion elevted MnSOD mrna nd protein levels nd enzymtic ctivity in the broiler myocrdil cells in dose- nd time-dependent mnners (Go et l These observtions highlight the importnt role of MnSOD in norml hert tissue for protection from oxidtive stress. However, embryonic development conditions nd physiologicl chrcteristics re completely different from those of chicks, nd it is not known if the model used by Go et l. (201 is pproprite for embryonic myocrdil cells in vitro or whether results from their study re pplicble to embryonic myocrdil cells. Therefore, we investigted the effect of Mn on the expression of MnSOD in cultured primry chick embryonic myocrdil cells in vitro. We hypothesized tht Mn supplementtion would enhnce the expression of MnSOD in cultured primry chick embryonic myocrdil cells. This enhncement my protect the chick embryos from oxidtive dmge. 2. Mterils nd methods 2.1. Animls nd diets All experimentl procedures were pproved by Animl Welfre Committee of the Institute of Animl Sciences, Chinese Acdemy of Agriculturl Sciences. A totl of forty 18-week-old femle broiler breeders (Arbor Acres; Hudu Broiler Compny, Beijing, Chin) were rndomly llocted to stinless steel cges coted with plstics (2 birds per cge). All broiler breeders were fed conventionl corn-soyben mel diet contining 133 mg Mn kg 1 by nlysis during dption from 18 to 29 weeks ccording to Arbor Acres breeder mngement guidelines. After the dpttion period, ll broiler breeders were fed the corn-soyben mel bsl diet with no Mn ddition (contining mg Mn kg 1 by nlysis, Tble from 30 to 32 wk to deplete the body Mn storge nd obtin the low-mn eggs. This bsl diet ws formulted to meet or exceed the Ntionl Reserch Council (NRC 1994) requirements for other nutrients except for Mn for lying broiler breeders. During the 32 wk of ge, eggs with similr weights ((65±5) g egg 1 ) were selected nd then incubted in the sme incubtor (9TDJ-A; Lntinjio Electronic Technology Co., Ltd., Beijing, Chin) t 37.8 C nd reltive humidity of 55 to 60% for 10 dys. The Mn contents in the yolks of the eggs collected from the broiler breeders fed the Mn-unsupplemented corn-soyben mel bsl diet nd the conventionl corn-soyben mel diet during the 32 weeks of ge were nlyzed to be 0.68 nd 1.27 μg Mn g 1 egg yolk, respectively Isoltion nd cultivtion of primry chick embryonic myocrdil cells Embryonic myocrdil cells were cquired from the 10-dyold chick embryos using the methods described by Wu et l. (2013). Briefly, the embryonic hert ws removed under sterile conditions nd blood cells were wshed using phosphte-buffered sline (PBS, Ct no , Life Technologies, Crlsbd, CA). The myocrdil tissue ws minced nd digested with 0.12% collgense II (Ct no , Life Technologies) for 8 min t 37 C. To collect enough cells, digestion ws repeted three times. The digested mixtures were centrifuged t g for 4 min

3 2040 QIN Shi-zhen et l. Journl of Integrtive Agriculture 2017, 16(9): Tble 1 Composition nd nutrient levels of the bsl diet (sfed bsis) Lying period Item (from 30 to 32 weeks of ge) Ingredients (%) Ground yellow corn Soyben mel Soyben oil 1.26 CCO CHPO NCl 0.30 DL-Methionine (99%) 0.18 Micronutrients 2) 0.35 Nutrient composition (%) Metbolizble energy (MJ kg 1 ) Crude protein (CP) 3) Lysine 0.77 Methionine 0.40 Methionine+Cysteine 0.63 C 3) 3.29 Nonphytte phosphorus 0.33 Mn (mg kg 1 ) 3) Regent grde. 2) Provided per kilogrm of diet: vitmin A (s ll-trns retinol cette), IU; choleclciferol, 4500 IU; vitmin E (s ll-rcα-tocopherol cette), 36 IU; vitmin K (s mendione sodium bisulfte), 3.9 mg; thimin (s thimin mononitrte), 4.5 mg; riboflvin, 10.5 mg; vitmin B 6, 4.5 mg; vitmin B 12, mg; nicin, 39 mg; clcium pntothente, 18 mg; folic cid, 0.18 mg; choline (s choline chloride), mg; Cu (CuSO 4 5H 2 O), 10 mg; Fe (FeSO 4 7H 2 O), 50 mg; Zn (ZnSO 4 7H 2 O), 100 mg; I (KI), 2.00 mg; Se (N 2 SeO 3 ), 0.30 mg. 3) Anlysed vlues bsed on triplicte determintions. to recover cells. These cells were then re-suspended nd cultured in the DMEM/F-12 medium (Ct no , Life Technologies) supplemented with 10% fetl bovine serum (Ct no , Life Technologies), 200 µmol L 1 L- glutmine (Ct no , Life Technologies), 100 U ml 1 penicillin nd 100 µg ml 1 streptomycin (Ct no , Life Technologies) t 37 C in humidified incubtor (Model 3110 series, Thermo Electron Corportion, USA) with 5% CO 2 nd 95% ir. The medium ws then chnged every dy. After 72 h in the culture medium, the proportion of cells showing spontneous contrction ws over 95%, nd then these cells were used for the following trils A preliminry tril to evlute the vibility of chick embryonic myocrdil cells Enzyme ctivities were mesured in preliminry method vlidtion tril to evlute the vibility of cultured primry chick embryonic myocrdil cells. From the third dy to the ninth dy of cultivtion, the superntnts of the myocrdil cell cultures were collected dily, nd stored t 20 C. The ctivities of sprtte minotrnsferse (AST), lctte dehydrogense (LDH), nd cretine kinse (CK) were mesured ccording to the instructions in the commercil kits (C010-2, A020-2 nd A032, Nnjing Jincheng Bioengineering Institute, Chin). Ech mesurement t ech time point (dy) ws repeted 6 times (n=6) Design nd tretments Bsed on the mesurements mde from the bove preliminry experiment, 6-dy-old cultured primry chick embryonic myocrdil cells were used in subsequent experiment. In this tril, we used completely rndomized design involving 3 Mn levels in the culture medium (no Mn supplementtion (C), 0.5 mmol L 1 or 1.0 mmol L 1 of Mn s the inorgnic MnCl 2 (I, regent grde, Beijing Biochemicl Regent Compny, Beijing, Chin)) 3 incubtion time points (12, 24 nd 48 h) fctoril rrngement of tretments (n=6) Tretments of myocrdil cells, smple collections nd preprtions The bove cultured primry chick embryonic myocrdil cells in 6-well pltes (Ct no. 3516, Corning Life Sciences, USA) on dy 6 were treted with both different Mn levels nd incubtion time s described bove when the proportion of cells showing spontneous contrction ws over 95% fter the 3-dy primry culture. There were 6 replictes of 6-well cells for ech tretment. At the end of either Mn or incubtion time tretment, the medium ws removed nd the cell monolyer ws wshed with ice-cold PBS three times. The cells in the first 2 wells for ech replicte were scrped in ice-cold sline, nd then sonicted t 4 C for 2 min (2 s with 10 s intervls). Lystes were centrifuged t g for 10 min t 4 C to hrvest the superntnts for MnSOD ctivity nlysis. The cells in the second 2 wells for ech replicte were scrped in TRIzol regent (Ct no , Life Technologies) nd frozen ( 80 C) for MnSOD mrna expression nlysis. The cells in the third 2 wells for ech replicte were lysed in ice-cold lysis buffer (50 mmol L 1 Tris, ph 7.4, contining 150 mmol L 1 NCl, 1 mmol L 1 PMSF, 1 mmol L 1 EDTA, 1% Triton X-100, 1% sodium deoxycholte, nd 0.1% SDS), sonicted t 4 C for 2 min (2 s with 10 s intervls), nd then incubted on ice for 30 min. Lystes were centrifuged t g for 5 min t 4 C to obtin the superntnts, nd then the superntnts were frozen ( 80 C) for MnSOD protein expression nlysis Anlyses of dietry Mn, clcium (C), crude protein (CP) contents, nd Mn content in egg yolks The diet smples were collected for nlyses of dietry Mn, C nd CP contents, nd the eggs collected from lying broiler breeders were seprted to get egg yolks for

4 QIN Shi-zhen et l. Journl of Integrtive Agriculture 2017, 16(9): nlysis of egg yolk Mn content. The Mn nd C contents were mesured using n inductively coupled plsm emission spectroscope (model IRIS Intrepid II; Thermo Jrrell Ash, USA) fter wet digestions with HNO 3 nd HClO 4, s described by Zhu et l. (2015). Vlidtion of the minerl nlysis ws conducted using bovine liver powder (GBW (E) ; Ntionl Institute of Stndrds nd Technology, Chin) s stndrd reference mteril. Dietry CP content ws determined using Assocition of Officil Anlyticl Chemists methods (AOAC 1990) Assy of MnSOD ctivity The MnSOD ctivity ws mesured by the nitrite method s described by Li et l. (2016). The protein concentrtions in superntnts were determined by BCA Protein Assy Kit (Ct no , Thermo Scientific Pierce), nd the Mn- SOD ctivities were determined with superoxide dismutse typed ssy kit by hydroxylmine method (Ct no. A001-2, Nnjing Jincheng Bioengineering Institute) ccording to the mnufcturer s instructions. The MnSOD ctivity ws expressed s nitrite units (NU) per milligrm of protein (NU mg 1 protein), nd one NU ws defined s the mount of enzyme needed to obtin 50% inhibition of nitrite formtion Determintion of MnSOD mrna expression levels The MnSOD mrna level ws determined s described by Zhu et l. (2015) with slight modifictions. Briefly, totl of 1 μg of RNA ws used to obtin complementry DNA (cdna) by reverse trnscription using the QuntiTect Reverse Trnscription Kit (Ct no , Qigen). The smples were treted with RNse-free DNse nd reverse trnscribed ccording to the mnufcturer s instructions. Rel-time PCR rections were performed on n ABI 7500 Rel-Time PCR System using SYBR-Green PCR Mster Mix (Applied Biosystems, Foster City, CA, USA). According to the sequences of the genes published in GenBnk, the primer sequences for MnSOD, β-ctin nd glycerldehyde-3-phosphte dehydrogense (GAPDH) mrna were listed in Tble 2. The PCR rections were performed for 10 min t 95 C, 40 cycles of 95 C for 15 s nd 60 C for 1 min. The 2 ΔΔCt ws used to clculte the mrna expression level of the trget gene, nd the geometric men of internl references, β-ctin nd GAPDH, ws used to normlize the expression of the trget gene (Xie et l. 2014) Anlysis of MnSOD protein expression levels The superntnt ws subjected to Western blotting nlysis s described by Zhu et l. (2015). Briefly, totl protein concentrtion ws determined using BCA Protein Assy Kit. Protein extrct (30 µg) from ech smple ws then loded onto 10% SDS-PAGE gels, nd the seprted proteins were trnsferred onto nitrocellulose membrnes (Ct no. IPVH00010; Merck-Millipore, Germn). After the trnsfer, membrnes were blocked for 1 h t room temperture in blocking buffer with 5% skimmed milk nd then incubted overnight t 4 C with the following primry ntibodies purchsed from Abcm: MnSOD (Ct no. b13533, Abcm, Englnd, 1:3 000) nd GAPDH (Ct no. b22555, Abcm 1:5 000). After four wshes for 10 min ech with Trisbuffered sline contining Tween, membrnes were incubted with got nti-rbbit horserdish peroxidse-conjugted secondry ntibodies (1:5 000, Ct no. CW0103A; ComWin Biotech, Chin) for 1 h t room temperture. After 4 wshes for 10 min ech, bnds were visulised by enhnced chemiluminescence using SuperSignl West Pico Tril Kit (Ct no ; Pierce, USA). The signls were recorded with n ImgeQunt LAS 4000 scnner (GE Helthcre Life Sciences, USA) nd nlysed with the TotlLb Qunt Softwre (TotlLb, UK). The GAPDH protein ws used to normlize the expression level of the trget protein Sttisticl nlyses The dt of AST, LDH, nd CK ctivities in the superntnt of cultured myocrdil cells were nlyzed by one-wy ANOVA using the generl liner model (GLM) procedure of the SAS 9.2 (SAS Inst. Inc., Cry, NC). The dt of MnSOD ctivity, mrna nd protein expression levels were Tble 2 Primer sequences for rel-time PCR mplifiction Gene GenBnk ID 2) PCR products (bp) Primer sequences 3) β-ctin NM F: 5 -ACCTGAGCGCAAGTACTCTGTCT-3 R: 5 -CATCGTACTCCTGCTTGCTGAT-3 GAPDH NM F: 5 -CTTTGGCATTGTGGAGGGTC-3 R: 5 -ACGCTGGGATGATGTTCTGG-3 MnSOD NM F: 5 -TTCCTGACCTGCCTTACGACTAT-3 R: 5 -CCAGCGCCTCTTTGTATTTCT-3 GAPDH, glycerldehyde-3-phosphte dehydrogense; MnSOD, mngnese superoxide dismutse. 2) ID, identity. 3) F, forwrd; R, reverse.

5 2042 QIN Shi-zhen et l. Journl of Integrtive Agriculture 2017, 16(9): nlyzed by two-wy ANOVA using the bove SAS system, nd the model included the Mn level, incubtion time, nd their interction. The replicte served s the experimentl unit. If the vrinces were significnt, differences mong mens were tested by the LSD method, nd P<0.05 ws considered to be sttisticlly significnt. Min effects were not tested if there ws significnt Mn time interction. 3. Results The AST, LDH, nd CK ctivities in the superntnts from the incubted primry myocrdil cells re shown in Fig. 1. The AST ctivities in the superntnts of myocrdil cells were significntly (P<0.05) influenced by incubtion time nd incresed with incubtion time nd reched the mximum on the 9th d. The LDH nd CK ctivities displyed the similr tendencies nd decresed significntly (P<0.05) on the 6th d nd then incresed grdully. Vlues of CK (U L 1 ) Vlues of LDH (U L 1 ) Vlues of AST (U L 1 ) Incubtion time (d) Fig. 1 Activity levels of enzymes in the superntnts of cultured primry chick embryonic myocrdil cells. AST, sprtte minotrnsferse; LDH, lctte dehydrogense; CK, cretine kinse. Mens with different letters t different incubtion time points (d) for ech enzyme re different (P<0.05). Vlues re reported s mens±sd of 6 replictes (n=6). b b c bc Incubtion time (d) Incubtion time (d) d c c c b The dt on the effects of Mn level, incubtion time nd their interction on MnSOD enzymtic ctivity, mrna nd protein expression levels in cultured primry chick embryonic myocrdil cells re shown in Tble 3. As for the MnSOD ctivity, n interction (P<0.05) between Mn level nd incubtion time ws observed. At 12 h, there were no differences (P>0.05) mong Mn levels. At 24 h, however, compred with the control with no Mn ddition nd 0.5 mmol L 1 Mn level, the ddition of 1.0 mmol L 1 Mn significntly incresed (P<0.05) MnSOD ctivity with no difference (P>0.05) between the control nd 0.5 mmol L 1 Mn level, nd the similr results were observed t 48 h. For the MnSOD mrna expression level, no interction (P>0.05) between Mn level nd incubtion time ws observed, but both Mn level (P<0.05) nd incubtion time (P<0.05) hd significnt effects on MnSOD expression. Compred with the control, the dditions of 0.5 nd 1.0 mmol L 1 Mn levels significntly incresed (P<0.05) the MnSOD mrna expression levels with no difference (P>0.05) between 0.5 nd 1.0 mmol L 1 Mn levels. The MnSOD mrna expression ws significntly greter t 48 h thn t 12 or 24 h. There ws no difference (P>0.05) in MnSOD mrna levels between the 12 nd 24 h. The MnSOD protein level ws not ffected (P>0.05) by Mn level, incubtion time nd their interction. 4. Discussion The ctivities of AST, LDH, nd CK re indictors of cellulr integrity nd crdic function, nd re used to evlute the dmge of the hert in humns nd livestock (Georgopoulos nd Welch 1993; Mitchell et l. 1995; Lin et l. 2001; Buyukokuroglu et l. 2004; Bo et l. 2008; Srvnn et l. 2011; Zhng et l. 2012). Increses in enzymtic ctivities re consequence of disruption in cell membrne function nd permebility. In the present study, the results of AST, LDH, nd CK ctivities indicte tht the cells hd the best vitlity on dy 6, nd thus the dy 6 ws chosen s the Mn tretment time in the subsequent experiment. The results from the present study support our hypothesis tht Mn would enhnce the expression of MnSOD in cultured primry chick embryonic myocrdil cells. We observed n ssocition between Mn nd the MnSOD expression in the chick embryonic myocrdil cells. Mn supplementtion elevted the MnSOD mrna expression in dose- nd time-dependent mnners. Other studies with the yest Scchromyces cerevisie (Cyrne et l. 2003), humn fibroblsts (Akshi et l. 1995), pulmonry rtery nd microvsculr endothelil cells (Visner et l. 1991, 1992) nd intestinl epithelil cell lines (Vlentine nd Nick 1992) hve lso shown dose- nd time-dependent increses in mrna levels of MnSOD in response to rnge of fctors, including cellulr redox stte, irrdition, nd vrious

6 QIN Shi-zhen et l. Journl of Integrtive Agriculture 2017, 16(9): Tble 3 Effects of mngnese level nd incubtion time on the expressions of mngnese superoxide dismutse (MnSOD) in cultured primry chick embryonic myocrdil cells Item Mn level (mmol L 1 ) MnSOD ctivity (NU mg 1 protein) 4) MnSOD mrna (RQ) 5) MnSOD protein (AU) 6) Incubtion time (h) cde cd bc de de b e de SEM Mn level (mmol L 1 ) 2) b SEM Incubtion time (h) 3) b b SEM P-vlue Time Mn level < < Time Mn Vlues represent mens of 6 replictes (n=6). 2) Vlues represent the mens of 18 replictes (n=18). 3) Vlues represent the mens of 18 replictes (n=18). 4) One NU ws defined s the mount of enzyme needed to obtin 50% inhibition of nitrite formtion. 5) The MnSOD mrna bundnces were clculted s the reltive quntities (RQ) of the MnSOD mrna to the geometric men of internl reference genes β-ctin nd GAPDH mrna; RQ=2 ΔΔCt (C t =threshold cycle). 6) The GAPDH ws used to normlize the expression level of the MnSOD protein s n rbitrry unit. Mens with different lettes within the sme column differ (P<0.05). inflmmtory meditors (interleukin-1β, interleukin-6, nd lipolyscchride). We lso observed tht MnSOD mrna level reched pek t 24 h fter 1.0 mmol L 1 Mn ws dded, but the enzymtic ctivity did not increse to the sme extent s MnSOD mrna did. Furthermore, intrcellulr MnSOD protein expression level did not respond to incresing Mn levels. These results re not different from those of Go et l. (201, which reported tht Mn elevted MnSOD mrna nd protein levels s well s enzymtic ctivity in the primry broiler myocrdil cells in dose- nd timedependent mnners. The inconsistency might be due to the difference in chick developmentl stges. The results my lso suggest tht the Mn-induced MnSOD gene expression in the primry chick embryonic myocrdil cells could be modulted t trnscriptionl nd post-trnsltionl levels, or through modified stbility of synthesized mrna nd protein. Numerous studies hve shown tht the MnSOD gene expression is induced by vrious stimuli ssocited with oxidtive stress, such s 12-O-tetrdecnoylphorbol-3-cette (TPA) (Whitsett et l. 1992), TNF-α (Fujii nd Tniguchi 199, nd irrdition (Qdri et l. 2004). However, Thongphsuk et l. (1999) reported tht the MnCl 2 -induced MnSOD protein expression ws not inhibited by superoxide scvenger 5,5-dimethyl-1-pyrroline-1-oxide or ROS scvenger N-cetylcysteine (NAC) in humn brest cncer Hs578T cells (Thongphsuk et l. 1999). A recent study from our lbortory lso showed tht the MnSOD mrna nd protein levels of Mn-treted cells were not ffected by NAC, suggesting tht Mn-induced MnSOD mrna nd protein expressions or their stbilities might be independent of ROS (Li et l. 2016). We do not know how Mn induced MnSOD expression in chick embryonic myocrdil cells, nd the relted signling pthwys re still uncler. Some studies indicted tht the mitogen-ctivted protein kinses (MAPKs) re group of protein serine/threonine kinses tht re ctivted in response to vriety of extrcellulr stimuli nd medite signl trnsduction from the cell surfce to the nucleus. In the MAPKs pthwys, either p38 mitogen-ctivted protein kinse (p38 MAPK) or c-jun N-terminl kinse (JNK) induced the regultion of MnSOD expression by heptitis C virus (Qdri et l. 2004) nd rchidonic cid (Binchi

7 2044 QIN Shi-zhen et l. Journl of Integrtive Agriculture 2017, 16(9): et l. 2002). However, this did not occur in the induction of MnSOD expression by TNF-α (Rogers et l Rective oxygen intermedites cuse n increse in protein tyrosine kinse (PTK) ctivity nd decresed synthesis of MnSOD protein (Brumell et l. 1996). Li et l. (2016) demonstrted tht the phosphoryltions of the three MAPKs were not ctivted by the Mn sources, suggesting tht p38mapk, JNK or ERK signling pthwys might be not involved in the Mn-induced MnSOD trnscriptionl modultion in the primry broiler myocrdil cells. And they lso found tht the dditions of MAPKs inhibitors resulted in the decreses in MnSOD mrna levels in Mn-treted cells. The highly integrted nd efficient modern poultry industry produces wholesome protein source for growing world popultion. Intensive genetic selection for high producing birds nd nutritionl reserch for optiml rtions hve provided broilers tht cn rech mrket weight in excess of 6 pounds in only 42 d. The 21-d incubtion period for chicken eggs mkes up one 3rd of bird s entire life cycle, nd the percentge of htched helthy chicks for grow-out (htchbility) is n importnt fctor to the industry. The high metbolic het production nd insufficient exchnge of oxygen enhnce the formtion of ROS with n induction of intrcellulr oxidtive stress during the embryonic development. Although oxidtive stress is centrl topic in biochemicl nd medicl reserches, the number of reports on the chick embryonic development is still low. Thus, n effective resolution is needed to solve the problem in the relistic production. Mternl dietry composition is mjor determinnt of ntioxidnt system development in the chicks during embryogenesis nd in erly postntl development (Suri 1999, b). Biologicl trce element is effectively trnsferred from food into egg yolk (Suri 1998). Mngnese is crucil component of the metlloenzyme MnSOD (Luo et l. 1992), which is the most dominnt dismutse functioning s free rdicl scvenger in the mitochondri (Umok et l. 1992). Gllup nd Norris (1939) reported tht Mn deficiency in lying hens cused decresed htchbility nd incresed mortlity of embryos. The recent study from our lbortory hs demonstrted tht mternl dietry supplementtion with Mn could improve htchbility s well s ntioxidnt bility to protect offspring chick embryos ginst het chllenge during incubtion from the 10th to 18th dys of incubtion (Zhu et l. 2015b). Therefore, supplementtion with Mn in lying hen diets cn be n lterntive wy to protect cells from oxidtive stress during the chick embryonic development. 5. Conclusion In the present study, Mn enhnced the MnSOD mrna expression nd its ctivity, nd thus might protect cells from oxidtive stress during the chick embryonic development. However, Mn did not significntly ffect the MnSOD protein expression. This suggests tht the regultion of Mn for the MnSOD expressions might be not only t trnscriptionl level but lso t post-trnsltionl level. The exct mechnisms need to be further investigted to include signl conduction pthwys of Mn for regulting MnSOD expression. Acknowledgements The present study ws supported by the Key Interntionl Coopertion Progrm of the Ntionl Nturl Science Foundtion of Chin ( ), the Ntionl Nturl Science Foundtion of Chin ( ), the Agriculturl Science nd Technology Innovtion Progrm, Chin (ASTIP-IAS08) nd the Chin Agriculture Reserch System (CARS-42). In ddition, we would highly pprecite Dr. Dvid Msters in the University of Western Austrli for reviewing nd correcting this mnuscript. References Akshi M, Hchiy M, Pquette R L, Osw Y, Shimizu S, Suzuki G Irrdition increses mngnese superoxide dismutse mrna levels in humn fibroblsts. Possible mechnisms for its ccumultion. Journl of Biologicl Chemistry, 270, AOAC (Assocition of Officil Anlyticl Chemists) Officil Methods of Anlysis. 9th ed. Assocition of Offiil Anlyticl Chemists, Arlington, Virgini, USA. Arechig C F, Ely A D, Hnsen P J Evidence tht glutthione is involved in thermotolernce of preimplnttion murine embryos. Biology of Reproduction, 52, Bo E, Sultn K R, Nowk B, Hrtung J Expression nd distribution of het shock proteins in the hert of trnsported pigs. Cell Stress Chperones, 13, Binchi A, Becuwe P, Frnck P, Duc M Induction of MnSOD gene by rchidonic cid is medited by rective oxygen species nd p38 MAPK signling pthwy in humn HepG2 heptom cells. Free Rdicl Biology nd Medicine, 32, Brumell J H, Burkhrdt A L, Bolen J B, Grinstein S Endogenous rective oxygen intermedites ctivte tyrosine kinses in humn neutrophils. Journl of Biologicl Chemistry, 271, Buyukokuroglu M E, Tysi S, Buyukvci M, Bkn E Prevention of cute drimycin crdiotoxicity by dntrolene in rts. Humn & Experimentl Toxicology, 23, Cederberg J, Simn C M, Eriksson U J Combined tretment with vitmin E nd vitmin C decreses oxidtive stress nd improves fetl outcome in experimentl dibetic pregnncy. Peditric Reserch, 49, Cyrne L, Mrtins L, Fernndes L, Mrinho H S Regultion

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