Sodium Butyrate Ameliorates Oxidative Stress-Induced Intestinal Epithelium Barrier Injury and Mitochondrial Damage through AMPK-Mitophagy Pathway

Sodium butyrate has gained increasing attention for its vast beneficial effects. However, whether sodium butyrate could alleviate oxidative stress-induced intestinal dysfunction and mitochondrial damage of piglets and its underlying mechanism remains unclear. The present study used a hydrogen peroxide- (H2O2-) induced oxidative stress model to study whether sodium butyrate could alleviate oxidative stress, intestinal epithelium injury, and mitochondrial dysfunction of porcine intestinal epithelial cells (IPEC-J2) in AMPK-mitophagy-dependent pathway. The results indicated that sodium butyrate alleviated the H2O2-induced oxidative stress, decreased the level of reactive oxygen species (ROS), increased mitochondrial membrane potential (MMP), mitochondrial DNA (mtDNA), and mRNA expression of genes related to mitochondrial function, and inhibited the release of mitochondrial cytochrome c (Cyt c). Sodium butyrate reduced the protein expression of recombinant NLR family, pyrin domain-containing protein 3 (NLRP3) and fluorescein isothiocyanate dextran 4 kDa (FD4) permeability and increased transepithelial resistance (TER) and the protein expression of tight junction. Sodium butyrate increased the expression of light-chain-associated protein B (LC3B) and Beclin-1, reduced the expression of P62, and enhanced mitophagy. However, the use of AMPK inhibitor or mitophagy inhibitor weakened the protective effect of sodium butyrate on mitochondrial function and intestinal epithelium barrier function and suppressed the induction effect of sodium butyrate on mitophagy. In addition, we also found that after interference with AMPKα, the protective effect of sodium butyrate on IPEC-J2 cells treated with H2O2 was suppressed, indicating that AMPKα is necessary for sodium butyrate to exert its protective effect. In summary, these results revealed that sodium butyrate induced mitophagy by activating AMPK, thereby alleviating oxidative stress, intestinal epithelium barrier injury, and mitochondrial dysfunction induced by H2O2.


Introduction
The intestinal epithelium barrier is known as the important barrier to prevent the invasion of toxins or antigens [1]. Studies have found that oxidative stress is involved in the intestinal barrier impairment of piglets, which mainly refers to the imbalance of reactive oxygen species (ROS) and antioxidant system [2,3]. Under oxidative stress, the overproduction of ROS can cause lipid peroxidation, protein, and DNA damage [4]. Multiple studies have shown that dietary sodium butyrate can protect the intestinal barrier of piglets by providing energy for intestinal epithelial cells, antiinflammation, histone deacetylation, immune regulation [5][6][7], etc. The in vivo rat experiments and in vitro intestinal epithelial cell experiments have demonstrated that the protective effects of intestinal barrier function by sodium butyrate are related to its antioxidant capacity [8][9][10]. Since mitochondria are the main resource of ROS, the antioxidant effect of sodium butyrate may result from the targeting on mitochondria.
The gut needs a lot of energy to maintain homeostasis, which is mainly supplied by intracellular mitochondria [9,10]. However, under oxidative stress, mitochondria are not only the main resource of ROS but also an important attack target of it [11]. Impaired mitochondria induced by oxidative stress can produce more than ten times more ROS than the normal mitochondria, which further aggravated mitochondrial damage [12]. In order to block this vicious cycle, the body will selectively remove damaged mitochondria via lysosome degradation, a process called mitophagy [13]. A recent study reported that sodium butyrate can induce mitophagy in Chinese hamster ovary cells [14]. We speculate that the antioxidant effect of sodium butyrate is related to decreasing mitochondrial ROS production and mitophagy.
Adenosine monophosphate-activated protein kinase (AMPK) is a key energy sensor, as well as an important oxidative stress sensor, which exerts an important role in mitochondrial function and mitophagy [15,16]. AMPKα is the catalytic core of AMPK, and Toyama et al. found that AMPK is activated in cells under energy-deficient situation, as indicated by that AMPK promoted the further recruitment of multiple proteins related to mitochondrial division by phosphorylating the downstream protein mitochondrial fission factor (MFF), thereby causing mitochondrial division [17,18], which links the energy receptor AMPK with mitochondrial division. It is well known that sodium butyrate is an important energy substance for intestinal epithelial cells and has the functions of alleviating oxidative stress and protecting the intestinal barrier [9,19]. Mollica et al. have reported that feeding sodium butyrate to insulin-resistant obese mice can increase liver AMPK activity, reduce ROS production, and improve liver mitochondrial function [20]. However, whether sodium butyrate can alleviate oxidative stress and improve mitochondrial function through AMPK remains unclear. Further research is needed to study the mechanism through which sodium butyrate alleviates oxidative stress and improves intestinal barrier function. Therefore, this experiment utilized an IPEC-J2 cell oxidative stress model by H 2 O 2 to study the effects of sodium butyrate on intestinal barrier injury, mitochondrial function, mitophagy, and the underlying molecular mechanisms.
2.3. Cell Viability Experiment. IPEC-J2 cells in logarithmic phase were seeded in a 96-well plate, 10 μl of CCK-8 solution was added to each well, incubated at 37°C for 4 h, and the absorbance at 450 nm was measured with a microplate reader (FLx800, Bio-Tek Instruments Inc., Winooski, USA). The cell viability is normalized based on the absorbance of cells in the control group.
2.4. Transepithelial Electrical Resistance (TER) and FD4 Flux of IPEC-J2 Cells in the Transwell System. According to the previous study, transwell (Corning, NT, USA) is premoistened with DMEM-F12 medium in the incubator for more than 30 minutes before use [21]. After trypsinization of the cells, stop the reaction with fresh medium, centrifuge at 1500 rpm for 5 min, discard the solution and add a certain volume of fresh medium, adjust the cell concentration to 1 × 10 6 cells/ml, add 150 μl cells to the upper chamber suspension, add 1.5 ml of fresh medium to the lower chamber,  [23]. In brief, the cells in the 2 ml centrifuge were incubated with 1 ml serum-free diluted DCFH-DA (1 : 1000) at 37°C for 30 min in the dark. After washed twice with PBS and resuspended, flow cytometry (BD, USA) was used to detect the cellular ROS. The data was calculated by FlowJo software 10.4 (V 7.6.1).

Measurement of Mitochondrial Membrane Potential
(MMP). The mitochondrial membrane potential was measured by mitochondrial membrane potential assay kit (Beyotime Biotechnology, China) according to the previous study [24]. JC-1 monomers (green) can form aggregates (red fluorescence) in the mitochondria with high △Ψm, which cannot form aggregates in the mitochondria with low △Ψm. Briefly, the cells were incubated with JC-1 working solution 37°C for 25 minutes. After washed for three times with PBS, flow cytometry (BD, USA) or a confocal laser microscope (FV1000, Olympus, Japan) was used to detect mitochondrial membrane potential (MMP).

mRNA Expression Level Analysis by Quantitative Real-
Time PCR (RT-qPCR). The total RNA extraction was conducted by Trizol according to the manufacturer's instruction (Takara, Japan). After detecting RNA concentration and purity, it is used for cDNA synthesis (Vazyme, Nanjing, China) and then for RT-qPCR according to the manufacturer's instructions (qPCR, master mix, Vazyme, Nanjing, China) as previously described [25]. The primer sequences are shown in supplementary Table 1. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as an internal reference gene to calculate the relative expression of each gene, and the data was normalized using the 2 -ΔΔCt method.
2.9. Western Blot Experiment. The western blot experiment was conducted according to the previous study [26]. Total protein extraction and quantification were performed through the RIPA lysis buffer and BCA protein Assay Kit (Beyotime Biotechnology, Shanghai, China). Proteins from the jejunum mucosa were run and isolated through SDS-PAGE. Then, proteins were transferred to a polyvinylidene difluoride membrane. After blocking with 5% defat milk powder at room temperature for one hour, blots were incubated with specific primary antibodies overnight and then with horseradish peroxidase-(HRP-) conjugated secondary antibodies. 2.12. Immunofluorescence Analysis. The IPEC-J2 cells were seeded in a laser confocal culture dish. After the indicated treatment, the cells were washed twice with precooled PBS, and go through the steps of fixation, permeabilization (Tritox X-100, Beyotime Biotechnology, Shanghai, China), blocking, primary antibody incubation (Claudin-1, LC3B, 3 Oxidative Medicine and Cellular Longevity and PINK1), secondary antibody incubation (Dylight-conjugated, Earthox, USA), nuclear staining (DAPI), and sealing. The samples were observed and photographed under a confocal laser microscope (FV1000, Olympus, Japan).
2.13. siRNA and Cell Transfection. IPEC-J2 cells were seeded into 6-well plates to grow about 80% confluent. The next day, individual targeted siRNA and plasmid were mixed with Lipofectamine RNAiMAX or Lipofectamine 2000 (Invitrogen, USA). The siRNA sequence is as follows: siAMPKα (5 ′ -GCT GCACCAGAAGTAATTTTT-3 ′ ) and siControl (5 ′ -UUCUCCGAACGUGUCACG UTT-3 ′ ). The RNAiMAX/siRNA mixture was added to IPEC-J2 cells in antibiotic-free medium and cultured for 4-6 h. Medium containing siRNA was refreshed with the general medium. Sodium butyrate and H 2 O 2 were added to the indicated group according to the experimental design. Then, the samples were collected for subsequent experiments.
2.14. Statistical Analysis. The experimental data was statistically analyzed using the SPSS 20.0 software. One-way ANOVA was used for test analysis. Turkey method was used for multiple comparisons, and the GraphPad Prism 7.01 software was used for graphing. P < 0:05 indicates significant difference.  Figure 1(a), it was found that H 2 O 2 of 600, 800, and 1000 μmol/L significantly reduced cell viability (P < 0:05). Cells were treated with 0.25, 0.5, 1, 2, 4, 8, and 16 μmol/L sodium butyrate for 24 hours, and it was found that 0.25-2 mmol/L sodium butyrate had no significant effect on cell viability, and more than 4 mmol/L would significantly inhibit cell growth (Figure 1(b)). In addition, the cells were treated with 0.25, 0.5, 1, 2, and 4 mmol//L sodium butyrate, and it was found that 1 mmol/L sodium butyrate could effectively inhibit the decrease in cell viability induced by H 2 O 2 (P < 0:05) (Figure 1(c)). Therefore, the follow-up experiments used 600 μmol/L H 2 O 2 -induced oxidative stress model and 1 mmol/L sodium butyrate pretreatment. As shown in Figures 1(d)-1(f), compared with the control group, H 2 O 2 treatment significantly reduced the SOD and GSH activities of IPEC-J2 cells (P < 0:05) and increased the MDA level (P < 0:05); meanwhile, compared with the H 2 O 2 group, sodium butyrate significantly inhibited the decreased activity of SOD and GHS induced by H 2 O 2 , alleviated the increase of MDA level (P < 0:05). Similarly, flow cytometry data showed that, in comparison with the control group, sodium butyrate pretreatment significantly inhibited the increase in ROS levels induced by H 2 O 2 (P < 0:05) (Figure 1(g)).

Effect of Sodium Butyrate on the Mitochondrial Function of IPEC-J2 Cells Treated with H 2 O 2 .
Flow cytometry data showed that compared with the control group, H 2 O 2 significantly increased the proportion of depolarized cells (P < 0:05), while sodium butyrate pretreatment reversed the increase in the proportion of depolarized cells induced by H 2 O 2 (P < 0:05) (Figure 2(a)). In addition, under laser confocal microscope, we found that compared with the control group, H 2 O 2 reduced the ratio of JC-1 aggregates to monomer (P < 0:05), indicating that the mitochondrial membrane potential was reduced, and the sodium butyrate treatment alleviated the decrease of cell mitochondrial membrane potential induced by H 2 O 2 (P < 0:05) (Figure 2(b)). By detecting the amount of mitochondrial DNA (mtDNA) (Figure 2(c)) and the mRNA expression level of mitochondrial function-related genes ( Figure 2(d)), it was found that compared with the control group, H 2 O 2 treatment significantly reduced the amount of mtDNA and the mRNA expression of mitochondrial function-related gene mitochondrial transcription factor A (TFAM), nuclear respiratory factor-1 (NRF-1), and peroxisomal proliferatoractivated receptor-g coactivator-1α (PGC-1α) (P < 0:05); sodium butyrate significantly alleviated the reduction of mtDNA and mRNA level of mitochondrial functionrelated genes induced by H 2 O 2 (P < 0:05). Similarly, under the transmission electron microscope, it was observed ( Figure 2(e)) that the cells in the H 2 O 2 group showed obvious mitochondrial swelling, mitochondrial crista breakage, and mitochondrial vacuolization; sodium butyrate significantly improved the ultrastructure of mitochondria. As shown in Figure 2(f), compared with the control group, H 2 O 2 increased the level of cytochrome c (Cyt c) in the cytoplasm (P < 0:05) and decreased the level of Cyt c in the mitochondria (P < 0:05), suggesting that H 2 O 2 increased the permeability of the inner and outer mitochondrial membranes and resulted in the release of mitochondrial Cyt c into the cytoplasm, indicating that the mitochondrial function is damaged; compared with the H 2 O 2 group, sodium butyrate alleviated the release of mitochondrial Cyt c caused by H 2 O 2 (P < 0:05), indicating that sodium butyrate treatment can help improve mitochondrial function under oxidative stress.

Effect of Sodium Butyrate on Inflammasome and Inflammatory Factors of IPEC-J2 Cells Treated with H 2 O 2 . As shown in Figures 3(a)-3(c)
, compared with the control group, H 2 O 2 significantly increased the mRNA level and protein level of NLRP3, Caspase-1, and IL-1β in IPEC-J2 cells (P < 0:05) and increased the content of IL-1β (P < 0:05); compared with the H 2 O 2 group, sodium butyrate significantly reduced the mRNA level and protein expression of NLRP3, Caspase-1, and IL-1β (P < 0:05) and reduced the content of IL-1β (P < 0:05).                 , compared with the NaB+H 2 O 2 group, Mdivi-1 or Compound C treatment significantly reduced Beclin-1 levels (P < 0:05) and increased P62 levels (P < 0:05 ). Similarly, compared with the NaB+H 2 O 2 group, Mdivi-1 or Compound C treatment also reduced the expression levels of mitophagy proteins PINK1 and Parkin (P < 0:05). Immunofluorescence result showed that inhibition of mitophagy or AMPKα reduced the colocalization of LC3B and mitochondria and reduced the colocalization of PINK1 and LC3B (Figures 7(c) and 7(d)). These results indicate that inhibition of mitophagy and AMPKα suppresses the induction of mitophagy by sodium butyrate.    [27]. Excessive production of ROS would affect the electronic respiration chain of mitochondria, open mitochondrial permeability transition pores, and lead to depolarization of mitochondrial membranes [28]. We found that sodium butyrate alleviated H 2 O 2 -induced decrease in mitochondrial membrane potential (MMP), as indicated by the decreased proportion of depolarized cells and the increased ratio of red (JC-1 polymer)/green (JC-1 polymer) fluorescence intensity when compared with the H 2 O 2 group. Consistent with our research, Li et al. reported that sodium butyrate inhibited LPS-induced MMP loss of bovine mammary epithelial cells [29]. Increased ROS production in mitochondria and decreased MMP in mitochondria can lead to mitochondrial DNA damage, which could be related to abnormal mitochondrial biogenesis [28]. We found that sodium butyrate alleviated mitochondrial dysfunction by increasing mtDNA and mRNA expression levels of mitochondrial functionrelated genes TFAM, NRF-1, and PGC-1α. Similarly, a previous study found that sodium butyrate alleviated the oxidative damage of HepG2 cells, as indicated by increasing mtDNA copy number and the mRNA expression of PGC-1α and TFAM [30]. Changes in mitochondrial function will be accompanied by changes in mitochondrial ultrastructure.

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Oxidative Medicine and Cellular Longevity vacuolation while sodium butyrate pretreatment significantly improved the ultrastructure of mitochondria. The changes in the ultrastructure of mitochondria are not only the structural basis of mitochondrial oxidative damage but also lead to the release of Cyt c from mitochondria into the cytoplasm. We found that H 2 O 2 increased Cyt c level in the cytoplasm and decreased Cyt c level in mitochondria, suggesting that H 2 O 2 increased the permeability of the inner and outer mitochondrial membranes, causing the release of mitochondrial Cyt c into the cytoplasm. Meanwhile, sodium butyrate alleviated the release of mitochondrial Cyt c induced by H 2 O 2 , indicating that sodium butyrate treatment could help improve mitochondrial function under oxidative stress. The above results revealed that sodium butyrate could effectively alleviate H 2 O 2 -induced redox status imbalance and mitochondrial impairment. The intestinal barrier is an important line of defense for the body to prevent harmful substances from invading the body, and maintaining its integrity is vital to the health of the body [31,32]. We found that sodium butyrate significantly alleviated H 2 O 2 -intestinal epithelium barrier injury of IPEC-J2 cells, as indicated by increasing intestinal epithelium TER and decreasing FD4 permeability. Similarly, Valenzano et al. and Wang et al. found that treatment of Caco-2 cells with a certain concentration of butyric acid could effectively improve the intestinal epithelium barrier [33,34]. Sodium butyrate also significantly inhibited the decrease of Claudin-1, Occludin, and ZO-1 in IPEC-J2 cells induced by H 2 O 2 . Consistent with our research, Yan and Ajuwon found that sodium butyrate increased the protein level of Claudin-3 and Claudin-4, thereby alleviating the damage of LPS to the integrity of IPEC-J2 monolayer cells [35]. The integrity of intestinal epithelial cells is not only related to the expression level of tight junction protein but also affected by its distribution area. In our study, we found that after H 2 O 2 treatment, the normal distribution and expression of Claudin-1 were destroyed and sodium butyrate prevented the disorder of Claudin-1 distribution and the decrease of Claudin-1 expression caused by H 2 O 2 . Similarly, studies found that when Caco-2 cells were adversely stimulated, the Occludin and ZO-1 proteins originally expressed on the cell membrane would be transferred to the cytoplasm, thereby increasing cell permeability; however, butyric acid promoted tight junction protein redistribute and increased the expression level of tight junction [9,36]. These results indicated that sodium butyrate could effectively alleviate the intestinal epithelium barrier damage and the disorder of tight junction protein expression and distribution induced by H 2 O 2 in IPEC-J2 cells.
Oxidative stress could cause intestinal inflammation, as proved by that excessive ROS production in the intestines of patients with ulcerative colitis and Crohn disease weakened antioxidant capacity and aggravated oxidative damage [27]. According to our results, H 2 O 2 increased the mRNA    Mitophagy is a self-protection mechanism to remove dysfunctional mitochondria [39]. However, there were no reports about the effect of sodium butyrate on mitophagy of IPEC-J2 cells. We found that sodium butyrate enhanced mitophagy of IPEC-J2 cells, as indicated by increasing mRNA and protein level of mitophagy protein and promoting the colocalization of LC3B and mitochondria as     [40]. Wang et al. found that sodium butyrate activated the PINK1-Parkin pathway and induced mitophagy [41]. J. S. Lee and G. M. Lee also found that sodium butyrate could induce mitophagy in Chinese hamster ovary (CHO) cells, and mitophagy protein Parkin was recruited to mitochondria, suggesting that sodium butyrate induced mitophagy to remove damaged mitochondria [14]. According to the results above, it was reasonable to assume that sodium butyrate could regulate PINK1 and Parkin and trigger mitophagy to obliterate the damaged mitochondria caused by H 2 O 2 , which could prevent the intestine from metabolism disorders.
In the present experiment, we found that sodium butyrate protected IPEC-J2 cells from H 2 O 2 -induced oxidative damage, intestinal epithelium barrier damage, and mitochondrial impairment and enhanced mitophagy, but its specific molecular mechanism remained to be further studied. AMPK is a key energy sensor that can regulate cell energy metabolism [15]. Mollica et al. have reported that feeding sodium butyrate to insulin-resistant obese mice can increase liver AMPK activity, reduce ROS production, and improve liver mitochondrial function [20]. However, whether sodium butyrate could alleviate oxidative stress of IPEC-J2 cells through AMPK-dependent mitophagy remained to be further explored. Therefore, in the following experiments, we used AMPK inhibitor and mitophagy inhibitor to explore the specific role of AMPK signaling pathway and mitophagy in the protective effects of sodium butyrate on intestinal epithelium barrier. We found that sodium butyrate inhibited the decrease of SOD activity and the increase of MDA level induced by H 2 O 2 . However, the use of mitophagy inhibitor Mdivi-1 or AMPK inhibitor Compound C increased cellular MDA levels and reduced SOD activity. These results indicated that inhibition of mitophagy and AMPK could reduce the antioxidant effect of sodium butyrate. Similarly, Guo et al. found that butyric acid activated the AMPK signaling pathway through GPR109A in bovine mammary epithelial cells (BMECs) and then exerted an antioxidant effect [37]. We found that the ROS level and the percentage of depolarized cells significantly increased after using the mitophagy inhibitor Mdivi-1 or the AMPK inhibitor Compound C. Similarly, Zhao et al. reported that high concentrations of insulin in HepG2 cells could significantly reduce mitochondrial DNA and decrease the mitochondrial membrane potential, while sodium butyrate significantly increased the mitochondrial membrane potential and mitochondrial DNA, which was involved in the GPR43-β-AMPK signaling pathway [42]. We also previously reported that tributyrin activated the AMPK-mTOR signaling pathway and improved intestinal mitochondrial function in weaned piglets [25]. These results indicated that inhibition of mitophagy or AMPKα inhibited the protective effects of sodium butyrate on improving mitochondria under oxidative stress of IPEC-J2 cells. In addition, we found that the use of mitophagy inhibitor Mdivi-1 and AMPK inhibitor Compound C impaired intestinal epithelium barrier function, as indicated by the decreased TER and expression level of tight junction and the increased FD4 permeability of IPEC-J2 cells. Similarly, Elamin et al. reported that sodium butyrate alleviated the barrier dysfunction of Caco-2 cells induced by ethanol in vitro, of which the protective effects were suppressed by AMPK inhibitors or siRNA. Miao et al. found that in the Caco-2 cells, sodium butyrate activated AMPK, thereby promoting the reorganization of tight junctions [43]. Therefore, it was reasonable to assume that mitophagy and AMPK were necessary for sodium butyrate to improve the intestinal epithelium barrier under H 2 O 2 -induced oxidative stress. Moreover, we also found that the use of mitophagy inhibitor Mdivi-1 or AMPK inhibitor Compound C inhibited mitophagy, as indicated by decreasing expression level of mitophagy protein and the colocalization of PINK1 and LC3B, suggesting that inhibition of mitophagy or AMPK could suppress the mitophagy induced by sodium butyrate. In accordance with our results, Evans et al. found that butyrate induced autophagy-dependent cell apoptosis of human gingival epithelial cell Ca9-22 to alleviate periodontal disease, as indicated by activating AMPK and inducing the production of LC3B, which was attenuated by AMPK inhibition; in addition, interference with LC3B gene could also significantly inhibit butyrate-induced cell death [44]. Hence, AMPK-mediated mitophagy is necessary for sodium butyrate's protective effects against oxidative stress.
In order to further confirm the role of AMPK in sodium butyrate-induced mitophagy of IPEC-J2 cells, we then used siRNA technology to knock down AMPKα, which was known as the dominating AMPK catalytic subunit. We found that interference with AMPKα reduced the expression level of PINK1 and Parkin and increased the level of P62 when compared with the H 2 O 2 group and the NaB+H 2 O 2 group. Similarly, Luo et al. found that sodium butyrate increased the mRNA and protein expression level of LC3B and activated phosphorylated AMPK, which was suppressed by treatment with siRNA AMPK in colorectal cancer cells [45]. These results indicated that AMPKα can mediate the mitophagy induced by sodium butyrate. Next, we found that interference with AMPKα significantly increased the level of MDA and ROS production and decreased MMP, indicating that the protective effects of sodium butyrate on alleviating cellular oxidative stress and mitochondrial dysfunction were weakened. In addition, interference with AMPKα significantly increased the protein expression of NLRP3 and Caspase-1 in IPEC-J2 cells, indicating that interference with AMPKα weakened the inhibition effect of sodium butyrate on NLRP3 inflammation factors. At the same time, interference with AMPKα impaired intestinal epithelium barrier function, as indicated by decreasing TER and increasing FD4 permeability of IPEC-J2 cells, suggesting that interference AMPKα weakened the protective effects of sodium butyrate on the intestinal epithelium barrier. Our results suggested that sodium butyrate could promote mitophagy via AMPK activation, protect mitochondria, and exert a protective effect on intestinal epithelium barrier of IPEC-J2 cells under oxidative stress.

Conclusion
In conclusion, our work revealed that sodium butyrate ameliorated oxidative stress and inflammation and enhanced intestinal epithelium barrier function and mitochondrial function through AMPK-mitophagy pathway in IPEC-J2 cells.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon logical request.

Ethical Approval
All experiments were carried out in accordance with the Chinese Guidelines for Animal Welfare and Experimental Protocol and were approved by the Animal Care and Use Committee of Zhejiang University.

Conflicts of Interest
The authors declare that there is no conflict of interest.

Authors' Contributions
Caihong Hu designed the whole scheme of the experiments; Xin Li, Chunchun Wang, Jiang Zhu, Qian Lin, Minjie Yu, and Jiashu Wen conducted the experiments; Xin Li and Chunchun Wang analyzed data and prepared the initial manuscript; Jie Feng reviewed the manuscript; Caihong Hu had primary responsibility for final content. All authors read and approved the final manuscript. Chunchun Wang is cofirst author.