Background:Enterotoxigenic Escherichia coli(ETEC)K88 commonly colonize in the small intestine and keep releasing enterotoxins to impair the intestinal barrier function and trigger inflammatory reaction.Although Lactob...Background:Enterotoxigenic Escherichia coli(ETEC)K88 commonly colonize in the small intestine and keep releasing enterotoxins to impair the intestinal barrier function and trigger inflammatory reaction.Although Lactobacillus salivarius(L.salivarius)has been reported to enhance intestinal health,it remains to be seen whether there is a functional role of L.salivarius in intestinal inflammatory response in intestinal porcine epithelial cell line(IPEC-J2)when stimulated with ETEC K88.In the present study,IPEC-J2 cells were first treated with L.salivarius followed by the stimulation of ETEC K88 for distinct time period.ETEC K88 adherent status,pattern recognition receptors(PRRs)mRNA,mitogen-activated protein kinase(MAPK)and nuclear factor-κB(NF-κB)activation,the release of pro-inflammation cytokines and cell integrity were examined.Results:Aside from an inhibited adhesion of ETEC K88 to IPEC-J2 cells,L.salivarius was capable of remarkably attenuating the expression levels of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),IL-8,Toll-like receptor(TLR)4,nucleotide-binding oligomerization domain(NOD)-like receptor pyrin domain-containing protein(NLRP)3 and NLRP6.This alternation was accompanied by a significantly decreased phosphorylation of p38 MAPK and p65 NF-κB during ETEC K88 infection with L.salivarius pretreatment.Western blot analysis revealed that L.salivarius increased the expression levels of zona occludens 1(ZO-1)and occludin(P<0.05)in ETEC K88-infected IPEC-J2 cells.Compared with ETEC K88-infected groups,the addition of L.salivarius as well as extra inhibitors for MAPKs and NF-κB to ETEC K88-infected IPEC-J2 cells had the capability to reduce pro-inflammatory cytokines.Conclusions:Collectively,our results suggest that L.salivarius might reduce inflammation-related cytokines through attenuating phosphorylation of p38 MAPK and blocking the NF-κB signaling pathways.Besides,L.salivarius displayed a potency in the enhancement of IPEC-J2 cell integrity.展开更多
MicroRNAs play important roles in various cellular processes, including differentiation, proliferation and survival. Using a pig model, this study sought to identify the miRNAs responsible for crypt-villus axis renewa...MicroRNAs play important roles in various cellular processes, including differentiation, proliferation and survival. Using a pig model, this study sought to identify the miRNAs responsible for crypt-villus axis renewal of the small intestinal epithelium.Compared to the villus upper cells, there were 15 up-regulated and 41 down-regulated miRNAs in the crypt cells of the jejunum.Notably, we found that miR-100 was expressed more in the villus upper cells than in the crypt cells, suggesting an effect on intestinal epithelium differentiation. Overexpression of miR-100 increased the activity of alkaline phosphatase, confirming that miR-100 promoted IPEC-J2 cell differentiation. MiR-100 can inhibit cell proliferation as evidenced by CCK-8 and cell cycle assay results. We also showed that miR-100 significantly inhibited the migration of IPEC-J2 cells and promoted cell apoptosis through caspase-3-dependent cleavage of Bcl-2. Furthermore, FGFR3 was identified as a potential target of miR-100 by bioinformatics analysis. We confirmed that overexpression of miR-100 suppressed FGFR3 expression in IPEC-J2 cells by directly targeting the FGFR3 3′-UTR. This is the first report of miRNAs acting on the renewal of the intestinal crypt-villus axis.Our results also showed that miR-100 promotes the differentiation and apoptosis, and inhibits the proliferation and migration of enterocytes of pigs.展开更多
Background:Lentinan(LNT)may regulate many important physiological functions of human and animals.This study aimed to verify whether LNT administration could relieve diarrhea via improving gut immunity in rotavirus(RV)...Background:Lentinan(LNT)may regulate many important physiological functions of human and animals.This study aimed to verify whether LNT administration could relieve diarrhea via improving gut immunity in rotavirus(RV)-challenged weaned pigs.Methods:Twenty-eight weaned pigs were randomly fed 2 diets containing 0 or 84 mg/kg LNT product for 19 d(n=14).RV infection was executed on d 15.After extracting polysaccharides from LNT product,its major monosaccharides were analyzed.Then,LNT polysaccharide was used to administrate RV-infected IPEC-J2 cells.Results:Dietary LNT supplementation supported normal function of piglets even when infected with RV,as reflected by reduced growth performance loss and diarrhea prevalence,and maintained gut immunity(P<0.05).The polysaccharide was isolated from LNT product,which molecular weight was 5303 Da,and major monosaccharides included glucose,arabinose and galactose.In RV-infected IPEC-J2 cells,this polysaccharide significantly increased cell viability(P<0.05),and significantly increased anti-virus immunity via regulating pattern recognition receptors and host defense peptides(P<0.05).Conclusion:Those results suggest that LNT administration increases the piglets’resistance to RV-induced stress,likely by supporting intestinal immunity.展开更多
Background:Oxidative stress is a main cause of piglet gut damage and diarrhea.Pyrroloquinoline quinone(PQQ),is a novel redox cofactor with antioxidant properties.However,the effect and mechanism that PQQ supplementati...Background:Oxidative stress is a main cause of piglet gut damage and diarrhea.Pyrroloquinoline quinone(PQQ),is a novel redox cofactor with antioxidant properties.However,the effect and mechanism that PQQ supplementation decreases oxidative injury in weaned pigs is not understood.Therefore,the aim of this study is to confirm the effect of PQQ on regulating redox status in weaned pigs and the mechanism for antioxidant function by porcine intestinal epithelial cell line(IPEC-J2)challenged with H_(2)O_(2).Results:Experiment 1,144 Duroc×Landrace×Yorkshire pigs(weaned at 28 d)were allocated to four groups:received a basal diet(control)and diets supplemented with 0.15%,0.30%and 0.45%PQQ,respectively.On d 28,growth performance,diarrhea incidence and redox factors were measured.Experiment 2,IPEC-J2 were treated with or without PQQ in the presence or absence of H_(2)O_(2)for indicated time points.Experiment 3,IPEC-J2 were transfected with or without Nrf2 siRNA,then treated according to Experiment 2.The cell viability,redox factors,protein of tight junctions and Nrf2 pathway were determined.In vivo,PQQ supplementation demonstrated dose-related improvements in average daily gain,and gain to feed ratio(Linear P<0.05).During d 0–28,compared to controls,0.45%PQQ supplementation for pigs decreased diarrhea incidence and MDA content in liver and jejunum,and increased concentration of SOD in liver;0.3%PQQ supplementation decreased ileal and liver MDA concentration;and 0.15%PQQ supplementation decreased ileal MDA concentration(P<0.05).In vitro,compared to cells cultured with H_(2)O_(2),pre-treatment with PQQ increased cell viability,tight junction proteins expression including ZO-1,ZO-2,Occludin and Claudin-1;and decreased ROS concentration and level of Caspase-3(P<0.05);as well as upregulated the ratio of Bcl-2 to Bax and protein expression of nuclear Nrf2,HO-1.Notably,Nrf2 knockdown by transfection with Nrf2 siRNA largely abrogated the positive effects of PQQ pretreatment on H_(2)O_(2)-induced intracellular changes.Conclusion展开更多
旨在利用CRISPR/Cas9基因组编辑技术制备CD13基因敲除的IPEC-J2(猪空肠上皮细胞系)细胞,揭示细胞表面分化抗原13(cluster of differentiation 13,CD13)在肠道病原微生物感染肠道细胞中所起的作用。本研究在猪CD13基因第2外显子区域设计...旨在利用CRISPR/Cas9基因组编辑技术制备CD13基因敲除的IPEC-J2(猪空肠上皮细胞系)细胞,揭示细胞表面分化抗原13(cluster of differentiation 13,CD13)在肠道病原微生物感染肠道细胞中所起的作用。本研究在猪CD13基因第2外显子区域设计2个向导RNA(guide RNA,gRNA),命名为g3、g5,然后分别将g3和g5克隆到pX330-GFP和pX330-RFP载体骨架上,电转染IPEC-J2细胞,48 h后通过流式细胞术分选具有双荧光标记的细胞,培养并获得单克隆细胞,PCR扩增、测序,从而获得CD13基因纯合敲除的阳性细胞系。对获得的20个细胞单克隆进行PCR,并挑取部分克隆PCR产物测序,发现共有7个克隆发生了基因片段缺失,其中1个克隆为单等位基因片段缺失,3个克隆为双等位基因杂合片段缺失,3个克隆为双等位基因纯合片段敲除。本研究检测双等位基因纯合片段敲除细胞的蛋白表达水平,其结果显示,在该纯合敲除细胞中CD13蛋白几乎不表达,表明成功构建了CD13敲除的IPEC-J2细胞系。本研究获得的CD13基因敲除细胞系为探究CD13在猪肠道病原微生物感染肠道细胞中所起的作用奠定了基础。展开更多
Background:Enterotoxigenic Escherichia coli(ETEC)F4 commonly colonizes the small intestine and releases enterotoxins that impair the intestinal barrier function and trigger inflammatory responses.Although Bacillus lic...Background:Enterotoxigenic Escherichia coli(ETEC)F4 commonly colonizes the small intestine and releases enterotoxins that impair the intestinal barrier function and trigger inflammatory responses.Although Bacillus licheniformis(B.licheniformis)has been reported to enhance intestinal health,it remains to be seen whether there is a functional role of B.licheniformis in intestinal inflammatory response in intestinal porcine epithelial cell line(IPEC-J2)when stimulated with ETEC F4.Methods:In the present study,the effects of B.licheniformis PF9 on the release of pro-inflammation cytokines,cell integrity and nuclear factor-κB(NF-κB)activation were evaluated in ETEC F4-induced IPEC-J2 cells.Results:B.licheniformis PF9 treatment was capable of remarkably attenuating the expression levels of inflammation cytokines tumor necrosis factor-α(TNF-α),interleukin(IL)-8,and IL-6 during ETEC F4 infection.Furthermore,the gene expression of Toll-like receptor 4(TLR4)-mediated upstream related genes of NF-κB signaling pathway has been significantly inhibited.These changes were accompanied by significantly decreased phosphorylation of p65 NF-κB during ETEC F4 infection with B.licheniformis PF9 treatment.The immunofluorescence and western blotting analysis revealed that B.licheniformis PF9 increased the expression levels of zona occludens 1(ZO-1)and occludin(OCLN)in ETEC F4-infected IPEC-J2 cells.Meanwhile,the B.licheniformis PF9 could alleviate the injury of epithelial barrier function assessed by the trans-epithelial electrical resistance(TEER)and cell permeability assay.Interestingly,B.licheniformis PF9 protect IPEC-J2 cells against ETEC F4 infection by decreasing the gene expressions of virulence-related factors(including luxS,estA,estB,and elt)in ETEC F4.Conclusions:Collectively,our results suggest that B.licheniformis PF9 might reduce inflammation-related cytokines through blocking the NF-κB signaling pathways.Besides,B.licheniformis PF9 displayed a significant role in the enhancement of IPEC-J2 cell integrity.展开更多
基金funded by the Tianjin Natural Science Foundation(18JCYBJC30000)the National Natural Science Foundation of China(31702147)the Tianjin“131”Innovative Talents Team(20180338).
文摘Background:Enterotoxigenic Escherichia coli(ETEC)K88 commonly colonize in the small intestine and keep releasing enterotoxins to impair the intestinal barrier function and trigger inflammatory reaction.Although Lactobacillus salivarius(L.salivarius)has been reported to enhance intestinal health,it remains to be seen whether there is a functional role of L.salivarius in intestinal inflammatory response in intestinal porcine epithelial cell line(IPEC-J2)when stimulated with ETEC K88.In the present study,IPEC-J2 cells were first treated with L.salivarius followed by the stimulation of ETEC K88 for distinct time period.ETEC K88 adherent status,pattern recognition receptors(PRRs)mRNA,mitogen-activated protein kinase(MAPK)and nuclear factor-κB(NF-κB)activation,the release of pro-inflammation cytokines and cell integrity were examined.Results:Aside from an inhibited adhesion of ETEC K88 to IPEC-J2 cells,L.salivarius was capable of remarkably attenuating the expression levels of interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),IL-8,Toll-like receptor(TLR)4,nucleotide-binding oligomerization domain(NOD)-like receptor pyrin domain-containing protein(NLRP)3 and NLRP6.This alternation was accompanied by a significantly decreased phosphorylation of p38 MAPK and p65 NF-κB during ETEC K88 infection with L.salivarius pretreatment.Western blot analysis revealed that L.salivarius increased the expression levels of zona occludens 1(ZO-1)and occludin(P<0.05)in ETEC K88-infected IPEC-J2 cells.Compared with ETEC K88-infected groups,the addition of L.salivarius as well as extra inhibitors for MAPKs and NF-κB to ETEC K88-infected IPEC-J2 cells had the capability to reduce pro-inflammatory cytokines.Conclusions:Collectively,our results suggest that L.salivarius might reduce inflammation-related cytokines through attenuating phosphorylation of p38 MAPK and blocking the NF-κB signaling pathways.Besides,L.salivarius displayed a potency in the enhancement of IPEC-J2 cell integrity.
基金supported by the Key Programs of Frontier Scientific Research of the Chinese Academy of Sciences (QYZDYSSW-SMC008)National Program on Key Basic Research Project (2016YFD0500504)+5 种基金National Nature Science Foundation of China (31330075 31572420)Hunan Provincial Natural Science Foundation of China (2018JJ3094 2018JJ1028)Research Foundation of Education Bureau of Hunan Province, China (18B476)Hunan Provincial Innovation Foundation for Postgraduate (CX2016B172)
文摘MicroRNAs play important roles in various cellular processes, including differentiation, proliferation and survival. Using a pig model, this study sought to identify the miRNAs responsible for crypt-villus axis renewal of the small intestinal epithelium.Compared to the villus upper cells, there were 15 up-regulated and 41 down-regulated miRNAs in the crypt cells of the jejunum.Notably, we found that miR-100 was expressed more in the villus upper cells than in the crypt cells, suggesting an effect on intestinal epithelium differentiation. Overexpression of miR-100 increased the activity of alkaline phosphatase, confirming that miR-100 promoted IPEC-J2 cell differentiation. MiR-100 can inhibit cell proliferation as evidenced by CCK-8 and cell cycle assay results. We also showed that miR-100 significantly inhibited the migration of IPEC-J2 cells and promoted cell apoptosis through caspase-3-dependent cleavage of Bcl-2. Furthermore, FGFR3 was identified as a potential target of miR-100 by bioinformatics analysis. We confirmed that overexpression of miR-100 suppressed FGFR3 expression in IPEC-J2 cells by directly targeting the FGFR3 3′-UTR. This is the first report of miRNAs acting on the renewal of the intestinal crypt-villus axis.Our results also showed that miR-100 promotes the differentiation and apoptosis, and inhibits the proliferation and migration of enterocytes of pigs.
基金This study was financially supported by the grant from the earmarked fund for the China Agriculture Research System(CARS-35)the fund from the Project of SAU-Adisseo Centre of Research on Nutrition and Healththe fund from Agricultural Science and Technology Achievement Transformation Fund Project of Sichuan Province(2016NYZ0052 and 20NZZH0057).
文摘Background:Lentinan(LNT)may regulate many important physiological functions of human and animals.This study aimed to verify whether LNT administration could relieve diarrhea via improving gut immunity in rotavirus(RV)-challenged weaned pigs.Methods:Twenty-eight weaned pigs were randomly fed 2 diets containing 0 or 84 mg/kg LNT product for 19 d(n=14).RV infection was executed on d 15.After extracting polysaccharides from LNT product,its major monosaccharides were analyzed.Then,LNT polysaccharide was used to administrate RV-infected IPEC-J2 cells.Results:Dietary LNT supplementation supported normal function of piglets even when infected with RV,as reflected by reduced growth performance loss and diarrhea prevalence,and maintained gut immunity(P<0.05).The polysaccharide was isolated from LNT product,which molecular weight was 5303 Da,and major monosaccharides included glucose,arabinose and galactose.In RV-infected IPEC-J2 cells,this polysaccharide significantly increased cell viability(P<0.05),and significantly increased anti-virus immunity via regulating pattern recognition receptors and host defense peptides(P<0.05).Conclusion:Those results suggest that LNT administration increases the piglets’resistance to RV-induced stress,likely by supporting intestinal immunity.
基金supported by the National Natural Science Foundation of China(Grant No.32072772,31672459,31372317 and 30871808).
文摘Background:Oxidative stress is a main cause of piglet gut damage and diarrhea.Pyrroloquinoline quinone(PQQ),is a novel redox cofactor with antioxidant properties.However,the effect and mechanism that PQQ supplementation decreases oxidative injury in weaned pigs is not understood.Therefore,the aim of this study is to confirm the effect of PQQ on regulating redox status in weaned pigs and the mechanism for antioxidant function by porcine intestinal epithelial cell line(IPEC-J2)challenged with H_(2)O_(2).Results:Experiment 1,144 Duroc×Landrace×Yorkshire pigs(weaned at 28 d)were allocated to four groups:received a basal diet(control)and diets supplemented with 0.15%,0.30%and 0.45%PQQ,respectively.On d 28,growth performance,diarrhea incidence and redox factors were measured.Experiment 2,IPEC-J2 were treated with or without PQQ in the presence or absence of H_(2)O_(2)for indicated time points.Experiment 3,IPEC-J2 were transfected with or without Nrf2 siRNA,then treated according to Experiment 2.The cell viability,redox factors,protein of tight junctions and Nrf2 pathway were determined.In vivo,PQQ supplementation demonstrated dose-related improvements in average daily gain,and gain to feed ratio(Linear P<0.05).During d 0–28,compared to controls,0.45%PQQ supplementation for pigs decreased diarrhea incidence and MDA content in liver and jejunum,and increased concentration of SOD in liver;0.3%PQQ supplementation decreased ileal and liver MDA concentration;and 0.15%PQQ supplementation decreased ileal MDA concentration(P<0.05).In vitro,compared to cells cultured with H_(2)O_(2),pre-treatment with PQQ increased cell viability,tight junction proteins expression including ZO-1,ZO-2,Occludin and Claudin-1;and decreased ROS concentration and level of Caspase-3(P<0.05);as well as upregulated the ratio of Bcl-2 to Bax and protein expression of nuclear Nrf2,HO-1.Notably,Nrf2 knockdown by transfection with Nrf2 siRNA largely abrogated the positive effects of PQQ pretreatment on H_(2)O_(2)-induced intracellular changes.Conclusion
文摘为了明确刺五加多糖(Acanthopanax senticosus polysaccharide,ASPS)处理对脂多糖(lipopolysaccharide,LPS)诱导的猪肠上皮细胞(IPEC-J2)来源的外泌体中miRNAs表达及功能的影响,试验分组培养IPEC-J2细胞,提取外泌体进行鉴定并对外泌体携带的miRNAs进行生物信息学分析。结果表明,各组提取的外泌体均呈包膜完整的圆形或椭圆形囊泡状,均能表达外泌体标志蛋白CD9。高通量测序结果显示,与LPS处理组相比,ASPS处理组共有277个差异表达的miRNAs(P<0.05),其中223个miRNAs基因表达上调,54个miRNAs基因表达下调。基因本体论(gene ontology,GO)与京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KGEE)通路富集分析发现,这些差异表达的miRNA通过转录调控靶基因可富集于细胞自噬和细胞凋亡及其相关信号通路。ASPS处理IPEC-J2细胞后再进行LPS刺激能显著影响IPEC-J2细胞外泌体中miRNAs的表达。差异表达的miRNAs在ASPS缓解LPS诱导的猪肠上皮细胞损伤中可能起重要调控作用,且这些miRNAs可能通过靶向自噬相关靶基因的3’UTR或经由相关信号通路来参与该过程的发生发展过程。
基金supported by the Agriculture and Agri-Food Canada,AAFC’s IOP project,Manitoba Pork and Swine Innovation PorcCanada Foundation for Innovation(CFI)supported by the Chinese Scholarship Council(CSC).
文摘Background:Enterotoxigenic Escherichia coli(ETEC)F4 commonly colonizes the small intestine and releases enterotoxins that impair the intestinal barrier function and trigger inflammatory responses.Although Bacillus licheniformis(B.licheniformis)has been reported to enhance intestinal health,it remains to be seen whether there is a functional role of B.licheniformis in intestinal inflammatory response in intestinal porcine epithelial cell line(IPEC-J2)when stimulated with ETEC F4.Methods:In the present study,the effects of B.licheniformis PF9 on the release of pro-inflammation cytokines,cell integrity and nuclear factor-κB(NF-κB)activation were evaluated in ETEC F4-induced IPEC-J2 cells.Results:B.licheniformis PF9 treatment was capable of remarkably attenuating the expression levels of inflammation cytokines tumor necrosis factor-α(TNF-α),interleukin(IL)-8,and IL-6 during ETEC F4 infection.Furthermore,the gene expression of Toll-like receptor 4(TLR4)-mediated upstream related genes of NF-κB signaling pathway has been significantly inhibited.These changes were accompanied by significantly decreased phosphorylation of p65 NF-κB during ETEC F4 infection with B.licheniformis PF9 treatment.The immunofluorescence and western blotting analysis revealed that B.licheniformis PF9 increased the expression levels of zona occludens 1(ZO-1)and occludin(OCLN)in ETEC F4-infected IPEC-J2 cells.Meanwhile,the B.licheniformis PF9 could alleviate the injury of epithelial barrier function assessed by the trans-epithelial electrical resistance(TEER)and cell permeability assay.Interestingly,B.licheniformis PF9 protect IPEC-J2 cells against ETEC F4 infection by decreasing the gene expressions of virulence-related factors(including luxS,estA,estB,and elt)in ETEC F4.Conclusions:Collectively,our results suggest that B.licheniformis PF9 might reduce inflammation-related cytokines through blocking the NF-κB signaling pathways.Besides,B.licheniformis PF9 displayed a significant role in the enhancement of IPEC-J2 cell integrity.
