the neonatal small intestine is particularly vulnerable to damage induced by endotoxin , and this damage may be involved in plasma and intracellular production of reactive oxygen species ( ros ) , resulting in cell apoptosis , reducing antioxidative capacity and mitochondrial dysfunction [ 13 ] . the intestinal epithelium , the border between the body and the environment , is the main place to transport the nutrient . and the enterocyte is the main target of harmful factors and stress , for example , toxin and ros . moreover , a large of evidence suggests that oxidant derivatives and ros are produced in excess by the inflamed mucosa and may be pathogenic factors in some intestinal diseases [ 5 , 6 ] . oxidative stress generated by an imbalance between ros and antioxidants contributes to the pathogenesis of arthritis , cancer , cardiovascular , liver , and respiratory diseases . ros is generic and includes a wide variety of molecules , free radicals , or ions derived from molecular oxygen , for instance , singlet oxygen ( o2 ) , superoxide anion radical ( o2 ) , hydrogen peroxide ( h2o2 ) , and hydroxyl radical ( ho ) . low doses of ros are mitogenic and promote cell proliferation , while intermediate doses of ros induce temporary or permanent growth arrest , and high doses of ros cause cell death . h2o2 is an abundant and stable form of ros , responding to inflammation , cellular dysfunction , and apoptosis , which ultimately lead to tissue and organ damage . mitochondrion is the main target of intracellular oxidative stress and is regarded as the main source for endogenous ros . previous studies showed that an acute , noncytotoxic dose of h2o2 caused a delay fragmentation of the mitochondrial reticulum and depressed the mitochondrial membrane potential and maximal respiratory rate . n - acetylcysteine ( nac ) , the precursor of l - cysteine , is known as an antioxidant that acts as a source of thiols and functions in glutathione synthesis , glutathione peroxidase ( gpx ) activity , and detoxification and acts directly on reactive oxidant radicals as a superoxide scavenger which interacts with ros such as ho and h2o2 . the previous study showed that weaning increased the concentrations of no and h2o2 in the serum in postweaning piglets , and feeding antioxidant - containing diets could prevent the ros - induced damage and suppress oxidative stress . there is growing evidence that nac might be a promising agent to improve intestinal health in piglets . nac supplementation could alleviate the mucosal damage and improve the absorptive function of the small intestine in lipopolysaccharide- ( lps- ) challenged piglets . nac regulates antioxidative responses , cell apoptosis , and epidermal growth factor gene expression under acetic acid challenges . however , the mechanisms by which nac exerts protective effects in intestinal damage are incompletely understood . we hypothesize that nac enhances cell growth and mitochondrial bioenergetics and decreases cell apoptosis on h2o2-induced oxidative damage in intestinal cells . the present study was designed to test this hypothesis using a model of h2o2-induced damage of intestinal porcine epithelial cells ( ipec - j2 ) . high - glucose ( 25  mm ) dulbecco 's modified eagle 's ( dmem - h ) , fetal bovine serum ( fbs ) , and antibiotics were procured from invitrogen ( grand island , ny , usa ) . unless indicated , all other chemicals were purchased from sigma - aldrich ( st . louis , mo , usa ) . ipec - j2 cells were seeded and cultured with dmem - h medium containing 10% fbs , 5  mm l - glutamine , 100  u / ml penicillin , and 100  g / ml streptomycin at 37c in a 5% co2 incubator . after an overnight incubation , the cells were changed to culture in basal medium containing 0 or 800  m nac . the following day , 0 or 100  m h2o2 was added for 4 hours and then the mediums were changed as before . the cells were collected for further research after 2-day incubation . about 1  10 cells per well of ipec - j2 cells were seeded in 96-well plates and grown as usual . after incubation in 0 , 500 , 650 , 800 , or 1000  m nac medium for 24  h , then 100  m h2o2 were added for 4  h. the wells were washed and fresh basal medium was replaced . cell counting kit-8 ( cck-8 ) was added to each well , incubated for 2  h , and read on the spectrophotometer at 450  nm ; the measured absorbance is proportional to the number of viable cells . ipec - j2 cells ( 1  10 ) were seeded in 96-well plates and cultured for a 2-day period . dna synthesis during cell proliferation in all treatment groups was quantified using 5-ethynyl-2-deoxyuridine ( edu ; invitrogen ) incorporation using cell - light edu kit ( rui bo biotechnology limited company , guangzhou , china ) , as described in our previous studies . briefly , ipec - j2 cells were cultured in dmem - h mediums containing 50  m edu for 1  h. an olympus bx51 microscope ( olympus , japan ) was used to observe edu - positive cells . images of the apoll 567 hoechst 33342 were captured . the percentage of edu - positive cells was expressed as the ration of red nuclei cells to blue nuclei cells in at least five different microscopic fields randomly selected for counting at 200-fold magnification . ipec - j2 cells ( 50  10 ) were seeded in 10  cm dishes for determination of total antioxidant capacity ( t - aoc ) and lactate dehydrogenase ( ldh ) using their corresponding assay kits ( nanjing jiancheng , nanjing , china ) according to the manufacturer 's instructions . all samples were measured by uv / visible spectrophotometer - uv-2450 ( shimadzu , kyoto , japan ) to get the results . ipec - j2 cells ( 10  10 ) were seeded in 6-well cell culture plates for flow cytometry analysis . after a 2-day period of culture in dmem - h medium containing 0 or 800  m nac and 0 or 100  m h2o2 , medium and cells were collected separately . about 1  10 cells were pelleted at 16  000  g for 5  min . the supernatant was removed and 1  ml of 70% cold ethanol was slowly added during vigorous mixing . cells were washed once with ice - cold pbs and resuspended in 1  ml of staining reagent containing 50  mg / ml pi and 100  mg / ml rnase for 30  min in the dark . to assess apoptosis , harvested cells were stained with pi / annexin - v - fitc ( keygen , nanjing , china ) according to the manufacturer 's instructions . cell cycle arrest and apoptosis were analyzed by flow cytometry ( bd facscalibur , usa ) . fluorescence of pi and annexin - v - fitc was monitored at 630  nm and 525  nm , respectively . the xf-24 extracellular flux analyzer and cell mito stress test kit from seahorse biosciences were used to examine the effects of nac treatment on mitochondrial respiration in h2o2-induced cells as described by tan et al . . after a 2-day period of culture , the basal medium was changed prior to the bioenergetic measurements to serum - free unbuffered ( without sodium bicarbonate ) dmem medium base supplemented with 2  mm l - glutamine , 25  mm d - glucose , and 1  mm sodium pyruvate , at ph 7.4  0.1 at 37c . to measure indices of mitochondrial function , oligomycin , carbonyl cyanide - p - trifluoromethoxyphenylhydrazone ( fccp ) , and rotenone and antimycin a were injected sequentially at the final concentrations of 0.5 , 1 , and 1  m , respectively . this allowed for an estimation of the contribution of non - atp  linked oxygen consumption ( proton leak ) and atp - linked mitochondrial oxygen consumption ( atp production ) . the spare respiratory capacity was represented by the maximal respiratory capacity subtracted from the baseline oxygen consumption rate ( ocr ) . the residual oxygen consumption that occurred after addition of rotenone and antimycin a was ascribed to nonmitochondrial respiration and was subtracted from all measured values in the analysis . owing to the effects of nac on ipec - j2 proliferation , ipec - j2 cells ( 50  10 ) were seeded in 10  cm dishes for gc - ms analysis as described by morita et al . . and then cells were collected and pelleted at 1000  g for 5  min . after being quenched using 500  l of prechilled 50% ( v / v ) methanol , cells were centrifuged at 1000  g for 5  min and then removed and added 500  l of prechilled 100% ( v / v ) methanol . cells were measured by an agilent 7890b-5977a gc - ms equipped with hp-5ms ( 30  m  250  m  0.25  m ) capillary column ( agilent j&w , santa clara , ca , usa ) . cells were rinsed twice using pbs , harvested , pelleted by centrifugation , and lysed in ripa buffer ( 150  mm nacl , 1% triton x-100 , 0.5% sodium deoxycholate , 0.1% sds , 50  mm tris - hcl at ph 7.4 ) , plus a protease inhibitor cocktail and phosphatase inhibitors . protein concentrations of cell homogenates were measured using the bca method and bovine serum albumin as standard , as described by our previous studies . soluble proteins were subjected to sds - page and transferred to pvdf membranes , blocked with 5% nonfat milk in tbs - with 0.05% tween-20 for 1  h , and incubated overnight with the following primary antibodies overnight at 4c with gentle rocking : cytochrome c ( 1  :  1,000 ; cell signaling technology ) , bax ( 1  :  1,000 ; cell signaling technology ) , caspase-3 ( 1  :  1,000 ; cell signaling technology ) , bcl - xl ( 1  :  400 ; santa cruz biotechnology , dallas , tx ) , cleaved caspase-3 ( 1  :  400 ; santa cruz biotechnology , dallas , tx ) , or -actin ( 1  :  400 ; santa cruz biotechnology , dallas , tx ) , followed by horseradish peroxidase - linked secondary antibodies . the density of the protein bands was determined using the alpha imager 2200 software ( alpha innotech corporation ) and normalized the data with inner control . the statistical analysis was performed by one - way anova using spss 17.0 ( spss inc . , viability assay of ipec - j2 cells was performed by firstly treating the cells with different concentrations of nac ( 0 , 500 , 650 , 800 , and 1000  m , resp . ) for one day and then with 100  m h2o2 for 4  h. the results indicated that 100  m h2o2 decreased ipec - j2 cell viability , while addition of nac enhanced cell viability of h2o2-treated ipec - j2 cells in a dose - dependent manner , and 800 and 1000  m nac addition showed the best promotion effects compared with the 0 and 500  m nac treatment in h2o2-treated cells ( p < 0.05 ) ( figure 1 ) . the results of edu incorporation illustrated in figure 2 have showed that the percentages of edu - positive cells were significantly decreased in response to h2o2 treatment ( p < 0.05 ) , while addition of nac to cells showed a tendency to increase the percentages of edu - positive cells compared with nc group . the results of mitochondrial respiration in ipec - j2 cells are shown in figure 3 . addition of 100  m h2o2 gradually decreased ( p < 0.05 ) individual parameters for basal respiration , proton leak , maximal respiration , nonmitochondrial respiration , and atp production in cells while addition of nac elevated the rate of mitochondrial respiration in 100  m h2o2-treated cells ( p < 0.05 ) but not in normal cells . the relative content of pyruvic acid , lactic acid , and tca cycle intermediates ( citric acid , alpha - ketoglutarate , succinic acid , fumaric acid , and malic acid ) of ipec - j2 cells are illustrated in figure 4 . addition of 100  m h2o2 significantly decreased lactic acid , and tca cycle intermediates compared with the nc treatment ( p < 0.05 ) . compared to nc treatment , addition of 800  m nac significantly decreased the content of pyruvic acid and lactic acid ( p < 0.05 ) , while there were no differences in the contents of pyruvic acid , lactic acid , succinic acid , fumaric acid , malic acid and -ketoglutaric acid between nac and nac + h2o2 treatments ( p > 0.05 ) . compared with the nc group , 100  m h2o2 significantly decreased the concentrations of t - aoc but increased the concentrations of ldh ( p < 0.05 ) . however , in h2o2-treated cells , addition of nac markedly increased the concentrations of t - aoc and decreased ldh leakage into the culture medium ( p < 0.05 ) . cell apoptosis was analyzed by annexin - v - fitc / pi staining , the results showed that compared with the nc group , 100  m h2o2 significantly increased the percentage of both early and late apoptosis of cells , and 800  m nac addition also increased the apoptosis rate ( p < 0.05 ) . however , in h2o2-treated cells , addition of 800  m nac significantly decreased the percentage of early and late apoptosis ( p < 0.05 ) ( figure 6 ) . the relative expression levels of cytochrome c , bax , b - cell lymphoma / leukaemia - xl ( bcl - xl ) , cleaved caspase-3 , and total caspase-3 proteins are shown in figure 7 . addition of 100  m h2o2 significantly increased protein levels for cytochrome c , bcl - xl , cleaved caspase-3 , and total caspase-3 proteins ( p < 0.05 ) , while addition of 800  m nac significantly decreased the above parameters in 100  m h2o2-treated cells ( p < 0.05 ) . nac has promising effects in different diseases , including cancer , liver toxicity , cardiovascular diseases , and metal toxicity , due to its role in attenuating pathophysiological processes including oxidative stress , apoptosis , and mitochondrial dysfunction . in addition , nac could attenuate inflammation in the liver of lps - treated mice . similarly , supplement of nac has been reported to improve growth performance and energy status , reduce inflammation , and ameliorate tissue damage . recently , yi et al . have found that nac could stimulate protein synthesis and inhibit proteolysis in ipec-1 cells . in the present study , we found that nac could not only ameliorate h2o2-induced cell growth inhibition , but also attenuate mitochondrial dysfunction in h2o2-treated cells . furthermore , nac downregulated the mitochondria - depended apoptosis in h2o2-treated cells . therefore , our data suggest that nac might repair intestinal damage through improving the mitochondrial function . intragastric or intraperitoneal administration of h2o2 could decrease growth performance and caused oxidative stress [ 2123 ] . furthermore , previous studies showed that addition of h2o2 ( 300  m ) to chicken intestinal epithelial cells for 24  h significantly decreased cell survival and sod activity . similarly , the results indicated that h2o2 at 100  m for 4  h decreased the growth of ipec - j2 . additionally , the percentages of edu - positive cells were decreased in response to 100  m h2o2 treatment . in addition , the reports show that excess intracellular ros level could cause oxidative damage to lipids , dna , and proteins via apoptosis . in the present study , mitochondrial function was destroyed in h2o2 treatment , which is in accordance with results from fan et al . . these data indicate that h2o2 induces mitochondrial ros production and then leads to dna damage in ipec - j2 cells . the report by yi et al . indicated that nac increased the growth of ipec-1 cells and suggested that nac at low concentrations ( < 1  mm ) could stimulate cell growth . in line with these results , this experiment showed that addition of nac at 5001000  m to 100  m h2o2 treatment increased the cell viability . and adding nac at 800  m to 100  m h2o2 increased the cell proliferation , indicating that nac might improve the h2o2-induced cell growth damage . there is increasing evidence supporting that nac improve redox status and directly react with oxidative metabolites [ 12 , 26 , 27 ] . nac protects cells against oxidative stress through reducing glutathione ( gsh ) and interacting with ros . in this study , h2o2 exposure induced oxidative stress evidenced by decreased cell viability , inhibited t - aoc , and increased leakage of ldh , while nac treatment markedly improved antioxidant system . mitochondria are the powerhouses of the cell , producing a considerable share of cellular atp and playing a central role in cellular function and metabolism . our previous report showed that mitochondrial dysfunction was observed with decrease in the basal respiration , maximal respiration , and nonmitochondrial respiration after lps treatment . the present data also demonstrated that mitochondrial function damage induced by h2o2 was observed , showing decrease in basal respiration , proton leak , maximal respiration , spare respiratory capacity , nonmitochondrial respiration , and atp production . in vivo the previous studies have reported that nac improved oxygen delivery   and systemic oxygen consumption   and regulated mitochondrial tca cycle metabolism by stimulation of carbon flux through pyruvate dehydrogenase , a key enzyme for hepatocellular mitochondrial energy metabolism by acetyl - coa supply [ 31 , 32 ] . the results showed that nac influenced metabolism of cellular pyruvic acid , lactic acid , succinic acid , malic acid , and citric acid , which contribute to mitochondrial redox balancing and are transported into mitochondria to affect atp production by oxidative phosphorylation [ 33 , 34 ] . reports showed that ros and mitochondrial dysfunction could mediate apoptosis , indicating that ros are important in cellular apoptosis . based on studies from various cell types , it is increasingly clear that nac could inhibit the cell apoptosis [ 12 , 36 , 37 ] . flow cytometry analysis showed that 100  m h2o2 significantly induced cell apoptosis , while nac could attenuate this effect of h2o2 by inhibiting cell apoptosis at both early and late stages , which is consistent with mayer and noble and shen et al . unbelievably , this inhibition of nac on cell apoptosis is only observed in h2o2 treated cells and nac induced cell apoptosis in normal cells , which require further research . cytochrome c is released from mitochondria due to formation of a channel , the mitochondrial apoptosis - induced channel , in the outer mitochondrial membrane , and serves a regulatory function as it precedes morphological change associated with apoptosis . loss of mitochondrial membrane potential increased release of cytochrome c in cytosol and activated some proapoptotic molecules ( bax , cleaved caspase-9 , caspase-3 , and so on ) and caused downregulation of bcl-2 which happened in a dose - dependent manner . the previous study has found that supplementation with nac attenuated caspase-3 protein expression in the small intestine of lps - challenged pigs . in the current study , we noted that the relative proteins expressions of cell apoptosis were elevated in ipec - j2 cells after h2o2 treatment but were reduced when nac was added into ipec - j2 cells pretreated with h2o2 . thus , the beneficial effects of nac may be associated with attenuating cell apoptosis . in summary , h2o2 induced mitochondrial dysfunction and cell apoptosis , while nac promoted dna synthesis , mitochondrial bioenergetics , and mitochondria - depended apoptosis in intestinal epithelial cells . possible mechanisms for the cytoprotective effect of nac on h2o2-induced damage in ipec - j2 cells scavenged the h2o2 and then improved cell proliferation , tca cycle , and mitochondria function and reduced cell apoptosis and death . results from these studies have important implications for the use of nac in the clinical management of oxidative damage in the neonatal pigs .
the neonatal small intestine is susceptible to damage caused by oxidative stress . 
 this study aimed to evaluate the protective role of antioxidant n - acetylcysteine ( nac ) in intestinal epithelial cells against oxidative damage induced by h2o2 . 
 ipec - j2 cells were cultured in dmem - h with nac and h2o2 . 
 after 2-day incubation , ipec - j2 cells were collected for analysis of dna synthesis , antioxidation capacity , mitochondrial respiration , and cell apoptosis . 
 the results showed that h2o2 significantly decreased ( p < 0.05 ) proliferation rate , mitochondrial respiration , and antioxidation capacity and increased cell apoptosis and the abundance of associated proteins , including cytochrome c , bcl - xl , cleaved caspase-3 , and total caspase-3 . 
 nac supplementation remarkably increased ( p < 0.05 ) proliferation rate , antioxidation capacity , and mitochondrial bioenergetics but decreased cell apoptosis . 
 these findings indicate that nac might rescue the intestinal injury induced by h2o2 .