脱氧雪腐镰刀菌烯醇对IPEC-J2细胞增殖、凋亡和屏障功能的影响及其自噬机制
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摘要
[目的]本试验旨在研究脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)对体外培养的仔猪空肠上皮细胞(IPEC-J2)增殖、凋亡和屏障功能的影响,探讨DON的肠道毒性作用及相关机制。[方法]以不同浓度(0、0.5、1、2、4、8、和16μmol·L~(-1))DON处理IPEC-J2细胞24 h后,采用MTT、Hoechst 33258、Western blot、Millipore-ERS和IFA法检测DON对IPEC-J2细胞增殖、凋亡、屏障功能和自噬水平的影响;在4μmol·L~(-1) DON处理组添加自噬激活剂雷帕霉素(Rapa)来提高细胞的自噬水平,用上述方法检测Rapa对DON引起的IPEC-J2细胞增殖毒性、凋亡和屏障功能的影响。[结果]与对照组相比,当DON浓度为4、8和16μmol·L~(-1)时,细胞活性显著(P<0.05)或极显著(P<0.01)下降。4和8μmol·L~(-1) DON处理凋亡细胞数量和凋亡相关蛋白Cleaved-Caspase3表达显著增加(P<0.01),细胞跨膜电阻(TEER)值和紧密连接蛋白Occludin表达显著下降(P<0.05),自噬相关蛋白LC3-Ⅱ和Atg5表达显著下降(P<0.05)。添加Rapa可以显著缓解DON引起的细胞毒性、凋亡和屏障功能障碍。[结论]DON通过抑制自噬引起IPEC-J2细胞增殖下降、凋亡增加和屏障功能障碍。
[Objectives]The purpose of this study was to investigate the effects of deoxynivalenol(DON)on proliferation,apoptosis and barrier function of intestinal porcine epithelial cells(IPEC-J2)in vitro,and its autophagy mechanisms. [Methods]IPEC-J2 cells were treated with different concentrations of DON(0,0.5,1,2,4,8 and 16 μmol·L~(-1))for 24 h. Cell proliferation,apoptosis,barrier damage and autophgay were detected by thiazolyl blue tetrazolium bromide(MTT),Hoechst 33258,Western blot,Millipore-ERS and immunofluorescence(IFA). After autophagy activator rapamycin(Rapa)inducing an increased level of autophagy in 4 μmol·L~(-1) DON group,the effects of DON on cell proliferation,apoptosis and barrier damage of IPEC-J2 cells were assayed. [Results]The results showed that DON(4,8 and 16 μmol·L~(-1))significantly reduced the cell viability(P<0.05 or P<0.01). DON(4 and 8 μmol·L~(-1))increased the number of apoptotic cells and the expression of apoptosis-related protein Cleaved-Caspase3(P<0.01),and decreased cell transmembrane resistance(TEER)value and the expression of tight junction protein Occludin(P<0.05). DON(4 and 8 μmol·L~(-1))also significantly decreased the expressions of autophagy-related protein LC3-Ⅱ and Atg5(P<0.05). However,the Rapa could significantly alleviate the cell toxicity,apoptosis and barrier disorder induced by DON. [Conclusions]In conclusion,DON can decrease cell proliferation and barrier dysfunction,and increase apoptosis by inhibiting autophagy in IPEC-J2 cells.
[Objectives]The purpose of this study was to investigate the effects of deoxynivalenol(DON)on proliferation,apoptosis and barrier function of intestinal porcine epithelial cells(IPEC-J2)in vitro,and its autophagy mechanisms. [Methods]IPEC-J2 cells were treated with different concentrations of DON(0,0.5,1,2,4,8 and 16 μmol·L~(-1))for 24 h. Cell proliferation,apoptosis,barrier damage and autophgay were detected by thiazolyl blue tetrazolium bromide(MTT),Hoechst 33258,Western blot,Millipore-ERS and immunofluorescence(IFA). After autophagy activator rapamycin(Rapa)inducing an increased level of autophagy in 4 μmol·L~(-1) DON group,the effects of DON on cell proliferation,apoptosis and barrier damage of IPEC-J2 cells were assayed. [Results]The results showed that DON(4,8 and 16 μmol·L~(-1))significantly reduced the cell viability(P<0.05 or P<0.01). DON(4 and 8 μmol·L~(-1))increased the number of apoptotic cells and the expression of apoptosis-related protein Cleaved-Caspase3(P<0.01),and decreased cell transmembrane resistance(TEER)value and the expression of tight junction protein Occludin(P<0.05). DON(4 and 8 μmol·L~(-1))also significantly decreased the expressions of autophagy-related protein LC3-Ⅱ and Atg5(P<0.05). However,the Rapa could significantly alleviate the cell toxicity,apoptosis and barrier disorder induced by DON. [Conclusions]In conclusion,DON can decrease cell proliferation and barrier dysfunction,and increase apoptosis by inhibiting autophagy in IPEC-J2 cells.
引文
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[18] Awad W A,Aschenbach J R,Zentek J.Cytotoxicity and metabolic stress induced by deoxynivalenol in the porcine intestinal IPEC-J2 cell line[J].J Anim Physiol Anim Nutr,2012,96(4):709-716.
[19] Hamilton R M,Thompson B K,Trenholm H L,et al.Effects of feeding white Leghorn hens diets that contain deoxynivalenol(vomitoxin)-contaminated wheat[J].Poult Sci,1985,64(10):1840-1852.
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[21] Suzuki T.Regulation of intestinal epithelial permeability by tight junctions[J].Cell Mol Life Sci,2013,70(4):631-659.
[22] Liao P,Liao M,Li L,et al.Effect of deoxynivalenol on apoptosis,barrier function,and expression levels of genes involved in nutrient transport,mitochondrial biogenesis and function in IPEC-J2 cells[J].Toxicol Res,2017,6(6):866-877.
[23] Wirawan E,Berghe V T,Lippens S,et al.Autophagy:for better or for worse[J].Cell Res,2012,22(1):43-61.
[24] Ge D,Gao J,Han L,et al.Novel effects of sphingosylphosphorylcholine on the apoptosis of breast cancer via autophagy/AKT/p38 and JNK signaling[J].J Cell Physiol,2019,234(7):11451-11462.
[2] Wang S,Yang J,Zhang B,et al.Deoxynivalenol impairs porcine intestinal host defense peptide expression in weaned piglets and IPEC-J2 cells[J].Toxins(Basel),2018,10(12):541-556.
[3] Pierron A,Alassane-Kpembi I,Oswald I P.Impact of two mycotoxins deoxynivalenol and fumonisin on pig intestinal health[J].Porcine Health Manag,2016,2:21-28.
[4] Lucke A,Bohm J,Zebeli Q,et al.Dietary deoxynivalenol and oral lipopolysaccharide challenge differently affect intestinal innate immune response and barrier function in broiler chickens[J].J Anim Sci,2018,96(12):5134-5143.
[5] Vancamelbeke M,Vermeire S.The intestinal barrier:a fundamental role in health and disease[J].Expert Rev Gastroenterol Hepatol,2017,11(9):821-834.
[6] Luissint A C,Parkos C A,Nusrat A.Inflammation and the intestinal barrier:leukocyte-epithelial cell interactions,cell junction remodeling,and mucosal repair[J].Gastroenterology,2016,151(4):616-632.
[7] Kim H J,Lee S,Jung J U.When autophagy meets viruses:a double-edged sword with functions in defense and offense[J].Semin Immunopathol,2010,32(4):323-341.
[8] Sun Y,Yu S,Ding N,et al.Autophagy benefits the replication of Newcastle disease virus in chicken cells and tissues[J].J Virol,2014,88(1):525-537.
[9] Levine B,Kroemer G.Biological functions of autophagy genes:a disease perspective[J].Cell,2019,176(1/2):11-42.
[10] Chen X,Xu J,Liu D,et al.The aggravating effect of selenium deficiency on T-2 toxin-induced damage on primary cardiomyocyte results from a reduction of protective autophagy[J].Chem Biol Interact,2019,300:27-34.
[11] Cadwell K.Crosstalk between autophagy and inflammatory signalling pathways:balancing defence and homeostasis[J].Nat Rev Immunol,2016,16(11):661-675.
[12] Matsuzawa-Ishimoto Y,Shono Y,Gomez L E,et al.Autophagy protein ATG16L1 prevents necroptosis in the intestinal epithelium[J].J Exp Med,2017,214(12):3687-3705.
[13] Ghareeb K,Awad W A,Bohm J,et al.Impacts of the feed contaminant deoxynivalenol on the intestine of monogastric animals:poultry and swine[J].J Appl Toxicol,2015,35(4):327-337.
[14] Meurens F,Summerfield A,Nauwynck H,et al.The pig:a model for human infectious diseases[J].Trends Microbiol,2012,20(1):50-57.
[15] Patterson J K,Lei X G,Miller D D.The pig as an experimental model for elucidating the mechanisms governing dietary influence on mineral absorption[J].Exp Biol Med,2008,233(6):651-664.
[16] Adesso S,Autore G,Quaroni A,et al.The food contaminants nivalenol and deoxynivalenol induce inflammation in intestinal epithelial cells by regulating reactive oxygen species release[J].Nutrients,2017,9(12):1343-1359.
[17] Pierron A,Mimoun S,Murate L S,et al.Intestinal toxicity of the masked mycotoxin deoxynivalenol-3-beta-D-glucoside[J].Arch Toxicol,2016,90(8):2037-2046.
[18] Awad W A,Aschenbach J R,Zentek J.Cytotoxicity and metabolic stress induced by deoxynivalenol in the porcine intestinal IPEC-J2 cell line[J].J Anim Physiol Anim Nutr,2012,96(4):709-716.
[19] Hamilton R M,Thompson B K,Trenholm H L,et al.Effects of feeding white Leghorn hens diets that contain deoxynivalenol(vomitoxin)-contaminated wheat[J].Poult Sci,1985,64(10):1840-1852.
[20] Capaldo C T,Powell D N,Kalman D.Layered defense:how mucus and tight junctions seal the intestinal barrier[J].J Mol Med,2017,95(9):927-934.
[21] Suzuki T.Regulation of intestinal epithelial permeability by tight junctions[J].Cell Mol Life Sci,2013,70(4):631-659.
[22] Liao P,Liao M,Li L,et al.Effect of deoxynivalenol on apoptosis,barrier function,and expression levels of genes involved in nutrient transport,mitochondrial biogenesis and function in IPEC-J2 cells[J].Toxicol Res,2017,6(6):866-877.
[23] Wirawan E,Berghe V T,Lippens S,et al.Autophagy:for better or for worse[J].Cell Res,2012,22(1):43-61.
[24] Ge D,Gao J,Han L,et al.Novel effects of sphingosylphosphorylcholine on the apoptosis of breast cancer via autophagy/AKT/p38 and JNK signaling[J].J Cell Physiol,2019,234(7):11451-11462.