Activation of the NF-κB and MAPK Signaling Pathways Contributes to the Inflammatory Responses, but Not Cell Injury, in IPEC-1 Cells Challenged with Hydrogen Peroxide

Oxidative stress can lead to intestinal cell injury as well as the induction of inflammation. It is not clear whether inflammation is an important factor leading to cell injury caused by oxidative stress. The purpose of this study was to investigate the role of inflammation in intestinal injury caused by hydrogen peroxide (H2O2). Our results revealed that H2O2 stimulation significantly decreased the viability of intestinal porcine epithelial cells (IPEC-1), increased lactate dehydrogenase (LDH) activity, and disrupted the distribution of the tight junction protein claudin-1. H2O2 significantly increased the mRNA expression of interleukin-6 (IL-6), IL-8, and tumor necrosis factor-α (TNF-α). H2O2 stimulation also led to increased phosphorylation of p38 and jun N-terminal kinase (JNK), and p65 NF-κB protein translocation into the nucleus of IPEC-1 cells. Cells treated with the NF-κB inhibitor (BAY11-7082), the p38 inhibitor (SB202190), or the JNK inhibitor (PD98059) significantly decreased mRNA and protein expression of IL-6, IL-8, and TNF-α. However, treatment with mitogen-activated protein kinase (MAPK) or NF-κB inhibitors did not prevent the damage effect on cell viability, LDH activity, or the distribution of claudin-1 in cells challenged with H2O2. In summary, our data demonstrate that activation of the NF-κB and MAPK signaling pathways can contribute to the inflammatory response, but not cell injury, in IPEC-1 cells challenged with H2O2.


Introduction
The intestinal barrier is the first line of defense inside the body against harmful antigens and pathogens, within the intestinal lumen [1,2]. It is a single layer of cells lining the gut, consisting of enterocyte membranes and tight junctions between enterocytes. The integrity of the intestinal barrier is essential for the digestion and absorption of nutrients, both in humans and animals [3][4][5]. Intestinal epithelial cells establish a barrier between the hostile external environment and the internal milieu. However, numerous factors such as infection, inflammation, and oxidative stress can result in intestinal epithelia injury, and dysfunction [1].
Hydrogen peroxide (H 2 O 2 ) is a highly reactive oxygen specie and serves as a signaling molecule in the regulation of a wide variety of biological processes [6][7][8]. Excessive H 2 O 2 is capable of diffusing throughout mitochondria and across cell membranes, where it can cause multiple forms of cellular oxidative injury [9,10]. Oxidative stress can lead to intestinal cell injury by various means [11,12]. It can cause cell damage by free radicals or by induction of inflammation [13]. Oxidative stress appears to be an important driving force for an enhanced cytokine production in intestinal epithelial cells, resulting in gut mucosal inflammation [14]. However, it is not clear whether inflammation is an important factor leading to cell injury caused by oxidative stress. Mitogen-activated protein kinase (MAPK) and NF-κB pathways are common inflammatory signaling pathways. Activation of MAPKs and NF-κB pathways, caused proinflammatory cytokines such as interleukin-6 (IL-6), IL-8, and tumor necrosis factor-α (TNF-α) to be released, which could lead to the inflammatory response [15]. Traditionally, it is thought that cell death is the most extreme form of injury produced by inflammation [16]. Inflammation and cell death are distinct but highly interconnected and mutually regulated host-mediated mechanisms, which are important for maintaining homeostasis in organisms [17].
Therefore, the aim of the present study was to investigate the role of inflammation in intestinal injury caused by H 2 O 2 . It was found that H 2 O 2 induced cell injury, upregulated proinflammatory cytokine-related gene expression, and activated NF-κB, and MAPK signaling pathways in intestinal porcine epithelial cells (IPEC-1). In addition, the inhibition of p38, JNK, and NF-κB with selective inhibitors downregulated the inflammatory response but did not prevent cell injury in IPEC-1 cells. Our data demonstrated that activation of the NF-κB and MAPK signaling pathways could contribute to the inflammatory responses but not cell injury in IPEC-1 cells challenged with H 2 O 2 .

Cell Viability Assays.
Cell viability was detected using the Cell Counting Kit-8 detection kit (CCK-8, Beyotime Institute of Biotechnology, Wuhan, CH). Cells were cultured in 96well plates. When they were cultured to 70%-80% confluency, medium was removed and after washing two times with phosphate-buffered saline (PBS), cells were treated with different concentrations of H 2 O 2 (0, 0.2, and 0.5 mM) for 3 h, or pretreated with different inhibitors (Bay11-7082, SB202190, and PD98059) for 1 h, in the presence or absence of 0.5 mM H 2 O 2 for 3 h. Dulbecco's Modified Eagle's Medium-F12 medium was added to the control group. After a 3 h incubation, media was removed and 10% CCK-8 reagent (100 μL per well) was added and incubated at 37°C for 1 h according to the manufacturer's instructions. The absorbance was quickly measured at a wavelength of 450 nm using a microplate reader (Bio-Rad, CA, USA). The viability of the cells was recorded as the percentage relative to untreated controls. All assays were performed in triplicate and in at least three independent experiments.
2.3. Lactate Dehydrogenase (LDH) Activity Measurement. The concentration of LDH released into the culture medium through damaged cell membranes was measured spectrophotometrically using an LDH assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). IPEC-1 cells were cultured in 12-well plates until 80% confluent. Cells were then exposed to H 2 O 2 (0, 0.2, and 0.5 mM) or pretreated with different inhibitors (Bay11-7082, SB202190, or PD98059) for 1 h, in the presence or absence of 0.5 mM H 2 O 2 for 3 h. Subsequently, cell supernatants were collected for LDH measurement according to the manufacturer's protocol. The absorbance was read at a wavelength of 450 nm using an automated microplate reader (Bio-Rad, CA, USA).

Quantitative Real-Time PCR.
To investigate the effects of H 2 O 2 or the inhibitors on proinflammatory cytokine expression, we used 0.5 mM H 2 O 2 to treat the cells or cells were pretreated with different inhibitors (Bay11-7082, SB202190, or PD98059) for 1 h, in the presence or absence of 0.5 mM H 2 O 2 for 3 h; cells were then collected for mRNA expression analysis. Messenger RNA levels were determined by realtime PCR as described by [16]. After washing IPEC-1 cells two times with ice-cold PBS, total RNA was extracted using the RNAiso Plus Kit (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) following the manufacturer's instructions. After purification and quantitation, reverse transcription was performed using the PrimeScript® RT Reagent Kit (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) following the manufacturer's instructions. Quantitative analysis of PCR was carried out on a ABI 7500 Real-Time PCR System (Applied Biosystems, Life Technologies) using a SYBR® Premix Ex Taq™ qPCR Kit (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China). The primer pairs used are listed in Table 1. Results were analyzed by the 2 −ΔΔCT method with GAPDH as the housekeeping gene as GAPDH displayed no variation among all groups [16]. All samples were run in triplicate. Relative mRNA abundance of each target gene was normalized to the control group.
2.5. Cytokine Production Measurement. IPEC-1 cells were seeded in 12-well plates and treated with different concentrations of H 2 O 2 (0 and 0.5 mM) for 3 h or pretreated with different inhibitors (Bay11-7082, SB202190, and PD98059) for 1 h, in the presence or absence of 0.5 mM H 2 O 2 for 3 h. Subsequently, cell supernatants were collected for cytokine IL-6, IL-8, and TNF-α measurement by ELISA using commercial kits (4A Biotech, Bejing, China) according to the manufacturer's protocol.

Western Blot.
To evaluate the effects of H 2 O 2 on MAPK signaling activation, we used the 0.5 mM concentrations of H 2 O 2 to treat IPEC-1 cells. After incubation for 3 h, we collected the cells for Western blot analysis. Cells were then lysed and centrifuged, and the supernatants were collected for western blot and protein assay according to our previous study [16]. After transmembrane electrophoresis, blots were incubated with primary antibodies against ERK, p38, JNK, phospho-ERK (p-ERK), phospho-p38 (p-p38), and phospho-JNK1/2 (p-JNK1/2) (Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C and then incubated with a secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 25°C for 2 h. The blots were visualized with an enhanced Chemiluminescence Western Blot Kit (Amersham Biosciences) and processed with the Quantity One® software (Bio-Rad, CA, USA). The results were expressed as the abundance of p-ERK, p-p38, and p-JNK1/2 relative to the total protein content of ERK, p38, and JNK, respectively. 2.7. Immunofluorescence. IPEC-1 cells were seeded onto 12well glass coverslips (Corning, MA, USA). Cells were then exposed to H 2 O 2 (0 or 0.5 mM) for 3 h when the cells reached 70%-80% confluency. After fixing and blocking, the cells were then incubated with primary rabbit anti-NF-κB p65 antibody overnight at 4°C, followed by incubation with the secondary Cy3-labelled antibody in a humidified chamber for 1 h at room temperature. Subsequently, the cells were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride solution. Each of the steps above was followed by washing three times for 5 mins each in cold PBS. Finally, the IPEC-1 cells on coverslips were preserved in antifade mounting medium. The activation and nuclear translocation of NF-κB were observed using a fluorescent microscope (Olympus, Tokyo, Japan).

Confocal
Laser Scanning Microscope Analysis. IPEC-1 cells were seeded onto glass microscope coverslips (Corning, MA, USA). To assess the effect of inhibitors on H 2 O 2induced barrier function, cells were pretreated with SB202190, PD98059, or Bay11-7082 for 1 h and then challenged with H 2 O 2 when cells reached 70%-80% confluency. All cells were fixed with 4% paraformaldehyde for 10 min at room temperature (RT) and permeabilized with 0.2% Triton X-100 in PBS at RT for 10 min. The cells were blocked with 1% bovine serum albumin in PBS for 30 min at RT and then incubated with anti-claudin-1 antibody overnight. After being washed five times in PBS, the cells were incubated for 2 h at RT with a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (Invitrogen, CA, USA), followed by counterstaining with 4,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO, USA). The coverslips were mounted onto glass microscope slides using mounting buffer, and the stained cells were visualized using a fluorescent     For the inhibitor trial, the effects of inhibitors and H 2 O 2 were analyzed as a 2 × 2 factorial arrangement using the general linear model (GLM) procedures. All data were presented as means with standard errors of means. The statistical model included the effects of inhibitors, H 2 O 2 , and their interactions. When significant inhibitor × H 2 O 2 interaction or a trend for inhibitor × H 2 O 2 interaction occurred, multiple comparison tests were performed using ANOVA. All data were represented by at least three independent experiments. Differences were considered significant for values of P ≤ 0:05.  Means within a row differ significantly (P ≤ 0:05). 1 The 2 −ΔΔCt method was used to analyze the relative gene expression (fold changes), which was calculated relative to the values in samples from the control group.  (Figure 1(a)).

LDH.
This enzyme is found in virtually all living cells, is released extracellularly when cells are damaged, and is commonly used as a marker of cell injury. In our study, compared with control cells, 0.5 mM H 2 O 2 stimulation significantly increased the LDH activity in the cell supernatant ( Figure 1(b)). This data, in combination with a cell viability indicator, led us to use 0.  (Figure 3(c)). Our data also demonstrated that H 2 O 2 treatment increased NF-κB p65 protein entering the nucleus of IPEC-1 cells (Figures 4(a)  and 4(b)). These data demonstrated that H 2 O 2 activated MAPK (p38 and JNK phosphorylation) and NF-κB p65 signaling pathways in IPEC-1 cells after 3 h incubation.

Tight Junction
Protein. Next, we sought to identify the role of the p38, JNK, and NF-κB signaling pathways in cell barrier function by using confocal microscopic analysis. As presented in Figures 6, 7, 8, and 9, H 2 O 2 treatment significantly destroyed the normal distribution of claudin-1 protein in the cells. Pretreating cells with inhibitors (SB202190, PD98059, or Bay11-7082) 1 h prior to the H 2 O 2 incubation had no effect on the distribution of claudin-1 after H 2 O 2 challenge. This suggested that inhibition of the MAPKs (p38 and JNK) and NF-κB signaling pathways did not prevent barrier function injury.

Discussion
The intestinal epithelium performs a dual function as it can absorb dietary nutrients and it can form a physical barrier against noxious stimuli. As the first line of defense, intestinal epithelial cells also participate in multiple physiological activities including immune response and tissue renewal. Oxidative stress has been identified as a key factor in the breakdown of intestinal function in pigs [18]. H 2 O 2 is a highly reactive oxygen species and serves as a signaling molecule in the regulation of a wide variety of biological processes [6][7][8]. H 2 O 2 is often used in in vitro studies of redoxregulated processes because it is relatively stable in vivo compared to other reactive oxygen species.
Our results indicated that H 2 O 2 stimulation significantly decreased cell viability and increased LDH activity in the IPEC-1 cell supernatant, demonstrating a role for H 2 O 2 in cell injury. In agreement with our study, Zheng et al. found that incubation with H 2 O 2 caused significant damage to nucleus pulposus cells, as indicated by an increase in apoptotic cells [19]. Similarly, H 2 O 2 has been reported to decrease the cell viability and increase cell apoptosis in human umbilical vein endothelial cells [20]. H 2 O 2 can also cause high death rates and induce chronic intestinal injury in mice, as demonstrated by increased apoptotic intestinal epithelial cells and damaged intestinal morphology [21]. The intestinal barrier is composed of a layer of columnar epithelium and intraepithelial tight junctions. Tight junction proteins, such as claudins, occludins, and zonula occludins, form the tight junction, work as a rate-limiting step in the paracellular pathway, and form a selectively permeable barrier [22]. We determined the distribution of tight junction claudin-1 and found   [24]. It is known that H 2 O 2 could cause the production of reactive oxygen species, resulting in oxidative damage of the intestinal barrier and cell apoptosis and necrosis [25,26]. Therefore, it is possible that stimulation destroyed the IPEC-1 cell structure and then disrupted the distribution of the tight junction protein in the cell membrane.
Oxidative stress is an important driving force for an enhanced proinflammatory cytokine production in intestinal epithelial cells, causing gut mucosal inflammation. Several cytokines are now known to contribute to tissue injury and cell death, including TNF-α, interferon-γ, IL-1β, and IL-18 [27]. In the present study, we found that 0.5 mM H 2 O 2 significantly increased the mRNA and protein expression of IL-6, IL-8, and TNF-α. Previous studies have reported that acute oxidative stress affected IL-8 and TNF-α expression in IPEC-J2 porcine epithelial cells [28]. It has been found that short-term H 2 O 2 and TNF-α incubation induced IL-8 secretion in Caco-2 and HT-29 cells [29].
We further explored the signaling pathways involved in proinflammatory cytokine regulation. It is well known that inflammation can result in tissue injury through multiple signaling pathways. NF-κB signaling is a key pathway whose activation can lead to proinflammatory cytokine production. MAPKs are a family of serine threonine kinases and are able to transduce signals from a diverse array of extracellular stimuli including oxidative stress and cytotoxic factors [30]. The extracellular regulated kinases (ERK1/2), p38 MAPK, 9 Oxidative Medicine and Cellular Longevity and JNK represent the three primary MAPK signaling pathways. We found that H 2 O 2 stimulation significantly enhanced NF-κB pathway activation and increased its translocation to the nucleus. As well as canonical NF-κB pathways, MAPK pathways have been reported to be involved in proinflammatory cytokine regulation [31]. We also detected MAPK activation in this study. Our results demonstrated that H 2 O 2 stimulation activated the phosphorylation of p38 and JNK in IPEC-1 cells but had no significant effect on phosphorylation of ERK. In agreement with our results, Zhou et al. [32] reported that H 2 O 2 stimulation activated the p38 signaling pathway in mouse intestinal epithelial cells, potentially causing apoptosis. H 2 O 2 stimulation has been reported to activate JNK and nuclear NF-κB phosphorylation in human HepG2 cells [33]. The majority of studies have demonstrated that JNK activation ultimately leads to cellular damage. Therefore, it may be possible that activation of MAPKs and NF-κB signaling pathways contribute to the cell damage associated with H 2 O 2 .
To further explore the underlying mechanism of cell injury induced by H 2 O 2 , we used MAPK and NF-κB inhibitors to block their signaling pathways in order to evaluate their roles in cell injury. As expected, pretreatment with the p38 inhibitor SB202190, JNK inhibitor PD98059, and NF-κB inhibitor (Bay11-7082) significantly decreased IL-6, IL-8, and TNF-α gene expression. Similarly, other studies have demonstrated that p38 and JNK signaling pathways could activate proinflammatory cytokine release in numerous cell types [34][35][36][37]. It was found by Kim and Lee that inhibition of p38 and JNK signaling reduced the lipopolysaccharideinduced production of inflammatory mediators in keratinocytes [37]. It was also reported that inhibition of the p38 signaling pathway could suppress apoptosis and expression of proinflammatory cytokines in human chondrocytes [38].

Oxidative Medicine and Cellular Longevity
Our results demonstrated that inhibition of MAPKs and NF-κB signaling pathways could prevent the cell inflammatory response induced by H 2 O 2 stimulation.
To further determine whether p38, JNK, and NF-κB signaling pathways are important for the induction of the cell injury after H 2 O 2 stimulation, we measured cell injury after treatment with MAPK and NF-κB inhibitors. Unexpectedly, we found that inhibiting MAPK and NF-κB signaling pathways did not attenuate the cell injury as indicated by decreased cell viability and increased LDH in the cell supernatant. Bay11-7082 further decreased the cell viability after H 2 O 2 stimulation. That is because Bay11-7082 can inhibit the cell viability and promote apoptosis. These data suggested that p38, JNK, and NF-κB signaling pathways were not the principle pathways mediating cell injury in IPEC-1 cells. In fact, cell death in vivo can trigger an inflammatory response. Damaged cells release "danger signals" that alert the host to cell death. Some of these molecules are recognized by cellular receptors that can stimulate the generation of proinflammatory mediators [39]. Dead cells can also release danger signals activating dendritic cells and promoting the generation of an immune response to antigens in and around the dying cells. For example, factor-related apoptosis suicide (FAS) is a prototypical death receptor which stimulates cells to undergo apoptosis when activated [40]. Injection of an agonistic form of an anti-FAS antibody into mice causes hepatocytes (which express Fas) to undergo apoptosis and generate a very strong inflammatory response [37]. If apoptosis is blocked, however, this inflammatory response is inhibited [41]. It is possible that the stressful stimuli (oxidative stress) firstly lead to cell damage and then induce an inflammatory response. Our results led us to speculate that proinflammatory cytokines were not the factors leading to cell injury in IPEC-1 cells after H 2 O 2 stimulation. It is also known that dead cells can induce inflammation by releasing proinflammatory signal(s). Taking the inflammation index into consideration, it is possible that, in our study, H 2 O 2 first induced cell death and then induced a secondary release of proinflammatory cytokines.
From our results, H 2 O 2 caused a cell inflammatory response through the activation of MAPK and NF-κB pathways in IPEC-1 cells. However, MAPK and NF-κB pathways were not the specific pathways involved in the mediation of cell injury. Many other pathways such as those involved in apoptosis, necrosis, and autophagy may be involved in the cell damage induced by H 2 O 2 [42]. There is a need for further research to determine the contribution of cell death in the regulation of inflammation and to improve our ability to distinguish these cytotoxic mechanisms from other pathways by which death-related molecules regulate inflammation. Such progress will provide us with better tools with which to clarify the role of cell death in inflammation. It will also pave the way for the development of new therapies whereby cell death can be targeted and manipulated, ultimately modulating the course of inflammation.

Conclusion
In conclusion, the present study demonstrates that H 2 O 2 stimulation can cause IPEC-1 cell injury and alter the distri-bution of tight junction proteins, leading to the inflammatory response and activation of p38, JNK, and NF-κB signaling pathways. Inhibiting p38, JNK, and NF-κB signaling pathways prevents the cell inflammatory response but does not alleviate the cell injury. Therefore, it is suggested that H 2 O 2 stimulation firstly leads to cell injury, followed by an inflammatory response in IPEC-1 cells.

Data Availability
The data used to support the findings of this study are included within the article.

Disclosure
Kan Xiao and Congcong Liu are co-first authors.