Tanshinone IIA protects intestinal epithelial cells from ferroptosis through the upregulation of GPX4 and SLC7A11

Background:Inflammatory bowel disease(IBD)is a chronic inflammatory disease of the gastrointestinal tract.The destruction of the intestinal epithelial barrier is one of the major pathological processes in IBD pathology.Growing evidence indicated that epithelial cell ferroptosis is linked to IBD and is considered a target process.Methods:RAS-selective lethal 3(RSL3)was used to induce ferroptosis in intestinal epithelial cell line No.6(IEC-6)cells,and cell ferroptosis and the effects of tanshinone IIA(Tan IIA)were determined by cell counting kit-8(CCK-8),reactive oxygen species(ROS)staining,Giemsa staining and transmission electron microscope(TEM).The cell viability of natural product library compounds was determined by CCK-8.The expression of ferroptosis-related genes were detected by real-time quantitative polymerase chain reaction(RT-qPCR)and western blot.Results:Treatment of IEC-6 cells results in the accumulation of ROS and typical morphological characteristics of ferroptosis.RSL3 treatment caused rapid cellular cytotoxicity which could be reversed by ferrostatin-1(Fer-1)in IEC-6 cells.Natural product library screening revealed that Tan IIA is a potent inhibitor of IEC-6 cell ferroptosis.Tan IIA could significantly protect the RSL3-induced ferroptosis of IEC-6 cells.Furthermore,the ferroptosis suppressors,glutathione peroxidase 4(GPX4),solute carrier family 7 member 11(SLC7A11),and miR-17-92 were found to be early response genes in RSL3-treated cells.Treatment of IEC-6 cells with Tan IIA resulted in upregulation of GPX4,SLC7A11,and miR-17-92.Conclusion:Our study demonstrated that Tan IIA protects IEC-6 cells from ferroptosis through the upregulation of GPX4,SLC7A11,and miR-17-92.The findings might provide a theoretical grounding for the future application of Tan IIA to treat or prevent IBD.

Intestinal epithelial cells in inflammatory bowel diseases

The pathogenesis of inflammatory bowel diseases (IBDs) seems to involve a primary defect in one or more of the elements responsible for the maintenance of intestinal homeostasis and oral tolerance. The most important element is represented by the intestinal barrier, a complex system formed mostly by intestinal epithelial cells (IECs). IECs have an active role in producing mucus and regulating its composition; they provide a physical barrier capable of controlling antigen traff ic through the intestinal mucosa. At the same time, they are able to play the role of non-professional antigen presenting cells, by processing and presenting antigens directly to the cells of the intestinal immune system. On the other hand, immune cells regulate epithelial growth and differentiation, producing a continuous bi-directional cross-talk within the barrier. Several alterations of the barrier function have been identif ied in IBD, starting from mucus features up to its components, from epithelial junctions up to the Toll-like receptors, and altered immune responses. It remains to be understood whether these defects are primary causes of epithelial damage or secondary effects. We review the possible role of the epithelial barrier and particularly describe the role of IECs in the pathogenesis of IBD.

A Comparative Study on Rat Intestinal Epithelial Cells and Resident Gut Bacteria (ii) Effect of Arsenite

Objective In order to use facultative gut bacteria as an alternate to animals for the initial gastrointestinal toxicity screening of heavy metals, a comparative study on rat intestinal epithelial cells and resident gut bacteria was undertaken. Methods in vitro growth rate of four gut bacteria, dehydrogenase （DHA） and esterase （EA） activity test, intestinal epithelial and bacterial cell membrane enzymes and in situ effect of arsenite were analysed. Results Growth profile of mixed resident population of gut bacteria and pure isolates of Escherichia coli, Pseudomonas sp., Lactobacillus sp., and Staphylococcus sp. revealed an arsenite （2-20 ppm） concentration-dependent inhibition. The viability pattern of epithelial cells also showed similar changes. DHA and EA tests revealed significant inhibition （40%-72%） with arsenite exposure of 5 and 10 ppm in isolated gut bacteria and epithelial cells. Decrease in membrane alkaline phosphatase and Ca^2＋-Mg^2＋-ATPase activities was in the range of 33%-55% in four bacteria at the arsenite exposure of 10 ppm, whereas it was 60%-65% in intestinal epithelial villus cells, in situ incubation of arsenite using intestinal loops also showed more or less similar changes in membrane enzymes of resident gut bacterial population and epithelial cells. Conclusion The results indicate that facultative gut bacteria can be used as suitable in vitro model for the preliminary screening of arsenical gastrointestinal cytotoxic effects.

Effect of Dachengqi Tang(大承气汤)Granule on Proliferation ofIntestinal Epithelial Cells in Rats with ExperimentalIntestinal Obstruction

Objective: To study the effects of Dachengqi Tang (DCQT) granule on the proliferation of the intestinal epithelial cells in rats with experimental intestinal obstruction. Methods: Experimental intestinal obstruction models were established in rats and autoradiography with 3 H-TdR was used to determine 3H-TdR labeling counts of intestinal epithelial cells in rats. Results: DCQT granule had no effects on 3H-TdR labeling counts of intestinal epithelial cells in normal rats. DCQT granule obviously increases the rate of renovation in intestinal epithelial cells of the intestinal obstruction rats. Conclusion: DCQT granule could reinforce the intestinal mucosa's defensive function by means of increasing the proliferation of intestinal epithelial cells.

In Vitro Evaluation of Effects of Mg-6Zn Alloy Extracts on Apoptosis of Intestinal Epithelial Cells

We assessed the in vitro cytotoxicity of Mg-6Zn alloy and analyzed the cell apoptosis rate and the expression of caspase-3 to evaluate the effects of Mg-6Zn alloy extracts on apoptosis of intestinal epithelial cells（IEC）-6. IEC-6 cells were cultured in different concentrations of Mg-6Zn alloy extracts（40%, 20%） and in the control group. The indirect effects of Mg-6Zn alloy on IEC-6 cells were studied by calculating the cell relative growth rate（RGR）, measuring the apoptosis of IEC-6 cells through flow cytometry, and investigating the expression of caspase-3 using real-time polymerase chain reaction. The experimental results show that the cytotoxicity of these extracts is Grade 0-1. The level of apoptosis in IEC-6 cells cultured in 40% Mg-6Zn alloy extracts is significantly higher than that in cells treated with 20% extract and the control group. The expression of caspase-3 is found to be up-regulated in the 40% extract as compared to 20% extract and the control group. Taken together, the data show that the Mg-6Zn alloy in 40% and 20% concentration extracts proves noncytotoxicity. But the 40% concentration of Mg-6Zn alloy extract can induce the apoptosis and the related caspase-3 expression in vitro.

Cinnamicaldehyde regulates the expression of tight junction proteins and amino acid transporters in intestinal porcine epithelial cells

Background： Cinnamicaldehyde（CA） is a key flavor compound in cinnamon essential oil possessing various bioactivities. Tight junction（TJ） proteins are vital for the maintenance of intestinal epithelial barrier function,transport, absorption and utilization of dietary amino acids and other nutrients. In this study, we tested the hypothesis that CA may regulate the expression of TJ proteins and amino acid transporters in intestinal porcine epithelial cells（IPEC-1） isolated from neonatal pigs.Results： Compared with the control, cells incubated with 25 μmol/L CA had increased transepithelial electrical resistance（TEER） and decreased paracellular intestinal permeability. The beneficial effect of CA on mucosal barrier function was associated with enhanced protein abundance for claudin-4, zonula occludens（ZO）-1, ZO-2, and ZO-3. Immunofluorescence staining showed that 25 μmol/L CA promoted the localization of claudin-1 and claudin-3 to the plasma membrane without affecting the localization of other TJ proteins, including claudin-4, occludin,ZO-1, ZO-2, and ZO-3, compared with the control cells. Moreover, protein abundances for rBAT, xCT and LAT2 in IPEC-1 cells were enhanced by 25 μmol/L CA, while that for EAAT3 was not affected.Conclusions： CA improves intestinal mucosal barrier function by regulating the distribution of claudin-1 and claudin-3 in enterocytes, as well as enhancing protein abundance for amino acid transporters rBAT, xCT and LAT2 in enterocytes. Supplementation with CA may provide an effective nutritional strategy to improve intestinal integrity and amino acid transport and absorption in piglets.

Qingyi decoction attenuates intestinal epithelial cell injury via the calcineurin/nuclear factor of activated T-cells pathway

BACKGROUND Recent studies have demonstrated that dysfunction of the intestinal barrier is a significant contributing factor to the development of severe acute pancreatitis(SAP).A stable intestinal mucosa barrier functions as a major anatomic and functional barrier,owing to the balance between intestinal epithelial cell(IEC)proliferation and apoptosis.There is some evidence that calcium overload may trigger IEC apoptosis and that calcineurin(CaN)/nuclear factor of activated Tcells(NFAT)signaling might play an important role in calcium-mediated apoptosis.AIM To investigate the potential mechanisms underlying the therapeutic effect of Qingyi decoction(QYD)in SAP.METHODS A rat model of SAP was created via retrograde infusion of sodium deoxycholate.Serum levels of amylase,tumor necrosis factor(TNF-α),interleukin(IL)-6,D-lactic acid,and diamine oxidase(DAO);histological changes;and apoptosis of IECs were examined in rats with or without QYD treatment.The expression of the two subunits of CaN and NFAT in intestinal tissue was measured via quantitative realtime polymerase chain reaction and western blotting.For in vitro studies,Caco-2 cells were treated with lipopolysaccharide(LPS)and QYD serum,and then cell viability and intracellular calcium levels were detected.RESULTS Retrograde infusion of sodium deoxycholate increased the severity of pancreatic and intestinal pathology and the levels of serum amylase,TNF-α,and IL-6.Both the indicators of intestinal mucosa damage(D-lactic acid and DAO)and the levels of IEC apoptosis were elevated in the SAP group.QYD treatment reduced the serum levels of amylase,TNF-α,IL-6,D-lactic acid,and DAO and attenuated the histological findings.IEC apoptosis associated with SAP was ameliorated under QYD treatment.In addition,the protein expression levels of the two subunits of CaN were remarkably elevated in the SAP group,and the NFATc3 gene was significantly upregulated at both the transcript and protein levels in the SAP group compared with the control group.QYD significantly restrained CaN and NFATc3 gene expression in the intestine,which was upregulated in the SAP group.Furthermore,QYD serum significantly decreased the LPS-induced elevation in intracellular free Ca^(2+)levels and inhibited cell death.CONCLUSION QYD can exert protective effects against intestinal mucosa damage caused by SAP and the protective effects are mediated,at least partially,by restraining IEC apoptosis via the CaN/NFATc3 pathway.

Adipose-derived mesenchymal stem cells alleviate TNBS-induced colitis in rats by influencing intestinal epithelial cell regeneration, Wnt signaling, and T cell immunity

BACKGROUND Conventional Crohn’s disease(CD)treatments are supportive rather than curative and have serious side effects.Adipose-derived mesenchymal stem cells(ADSCs)have been gradually applied to treat various diseases.The therapeutic effect and underlying mechanism of ADSCs on CD are still not clear.AIM To investigate the effect of ADSC administration on CD and explore the potential mechanisms.METHODS Wistar rats were administered with 2,4,6-trinitrobenzene sulfonic acid(TNBS)to establish a rat model of CD,followed by tail injections of green fluorescent protein(GFP)-modified ADSCs.Flow cytometry,qRT-PCR,and Western blot were used to detect changes in the Wnt signaling pathway,T cell subtypes,and their related cytokines.RESULTS The isolated cells showed the characteristics of ADSCs,including spindle-shaped morphology,high expression of CD29,CD44,and CD90,low expression of CD34 and CD45,and osteogenic/adipogenic ability.ADSC therapy markedly reduced disease activity index and ameliorated colitis severity in the TNBS-induced rat model of CD.Furthermore,serum anti-sacchromyces cerevisiae antibody and panti-neutrophil cytoplasmic antibody levels were significantly reduced in ADSCtreated rats.Mechanistically,the GFP-ADSCs were colocalized with intestinal epithelial cells(IECs)in the CD rat model.GFP-ADSC delivery significantly antagonized TNBS-induced increased canonical Wnt pathway expression,decreased noncanonical Wnt signaling pathway expression,and increased apoptosis rates and protein level of cleaved caspase-3 in rats.In addition,ADSCs attenuated TNBS-induced abnormal inflammatory cytokine production,disturbed T cell subtypes,and their related markers in rats.CONCLUSION Successfully isolated ADSCs show therapeutic effects in CD by regulating IEC proliferation,the Wnt signaling pathway,and T cell immunity.

Effect of EPEC endotoxin and bifidobacteria on intestinal barrier function through modulation of toll-like receptor 2 and toll-like receptor 4 expression in intestinal epithelial cell-18

AIM To investigate toll-like receptor 2(TLR2) and TLR4 expression, following bifidobacteria and low-dose EPEC endotoxin treatment, and intestinal barrier function in rat intestinal epithelial cell-18(IEC-18).METHODS Six experimental groups were established-normal control, EPEC, Bifidobacteria infantis(B. infantis), B. longum, B. bifidum, and B. youth groups. Optimal EPEC endotoxin concentration, bifidobacteria fold dilution, and treatment duration were determined. Quantitative real-time polymerase chain reaction and western blot, respectively, were conducted to detect TLR2 and TLR4 m RNA and protein expression in IEC-18 cells. Transepithelial electrical resistance(TEER) was measured by the EVOM chopstick voltohmmeter in each group. All experiments were conducted in triplicate and data were analyzed on SPSS 16.RESULTS TLR2 and TLR4 m RNA and protein expression in the EPEC group were significantly higher than in the control group(P < 0.05). TLR2 m RNA and protein expression in the B. infantis, B. longum and B. youth groups were significantly lower than in the normal control group(P < 0.05). TLR4 m RNA and protein expression in the B. bifidum and B. youth groups were significantly lower than in normal controls(P < 0.05). In addition, the TEER in B. infantis, B. longum, B. bifidum, and B. youth groups were decreased by 19%, 18%, 23% and 23%, respectively, after 120 min of intervention, as compared to the control group. However, the TEER in the EPEC group was significantly decreased by 67% in comparison to the normal control group(P < 0.05).CONCLUSION Bifidobacteria protect IEC-18 cells against injury by down-regulating TLR2 and TLR4 expression and enhance intestinal barrier function to protect the intestinal epithelial cells from pathogenic invasion.

Monogenic deficiency in murine intestinal Cdc42 leads to mucosal inflammation that induces crypt dysplasia

CDC42 controls intestinal epithelial(IEC)stem cell(IESC)division.How aberrant CDC42 initiates intestinal inflammation or neoplasia is unclear.We utilized models of inflam-matory bowel diseases(IBD),colorectal cancer,aging,and IESC injury to determine the loss of intestinal Cdc42 upon inflammation and neoplasia.Intestinal specimens were collected to determine the levels of CDC42 in IBD or colorectal cancer.Cdc42 floxed mice were crossed with Villin-Cre,Villin-CreERT2 and/or Lgr5-eGFP-IRES-CreERT2,or Bmi1-CreERT2 mice to generate Cdc42 deficient mice.Irradiation,colitis,aging,and intestinal organoid were used to evaluate CDC42 upon mucosal inflammation,IESC/progenitor regenerative capacity,and IEC repair.Our studies revealed that increased CDC42 in colorectal cancer correlated with lower survival;in contrast,lower levels of CDC42 were found in the inflamed IBD colon.Colonic Cdc42 depletion significantly reduced Lgr5+IEsCs,increased progenitors'hyperplasia,and induced mucosal inflammation,which led to crypt dysplasia.Colonic Cdc42 depletion markedly enhanced irra-diation-or chemical-induced colitis.Depletion or inhibition of Cdc42 reduced colonic Lgr5+IESC regeneration.In conclusion,depletion of Cdc42 reduces the IESC regeneration and IEC repair,leading to prolonged mucosal inflammation.Constitutive monogenic loss of Cdc42 in-duces mucosal inflammation,which could result in intestinal neoplasia in the context of aging.

Ubiquitin-specific protease 47 regulates intestinal inflammation through deubiquitination of TRAF6 in epithelial cells

Deubiquitinates(DUBs) alter the stabilities, localizations or activities of substrates by removing their ubiquitin conjugates,which are closely related to the development of inflammatory response. Here, we show that ubiquitin-specific protease 47(USP47) prevents inflammation development in inflammatory bowel disease(IBD). Compared with wild-type mice, Usp47 knockout mice are more susceptible to dextran sodium sulfate(DSS)-induced acute and chronic colitis with higher inflammatory cytokines expression and severe intestinal tissue damage. Chimeric mouse experiments suggest that non-hematopoietic cells mainly contribute to the phenotype. And, DSS-induced colitis of the Usp47 knockout mice depends on commensal bacteria.Mechanistically, down-regulation of USP47 aggravates the activation of NF-κB signaling pathway by increasing the K63-linked poly-ubiquitination of tumor necrosis factor receptor-associated factor 6(TRAF6) in intestinal epithelial cells. Furthermore, the expression of USP47, negatively correlated with the degree of inflammation, is lower at colonic inflammatory lesions than that non-inflammatory sites from the intestine from ulcerative colitis(UC) and Crohn's disease(CD) patients. These data, taken together, indicate that USP47 regulates intestinal inflammation through de-ubiquitination of K63-linked poly-ubiquitination TRAF6 in intestinal epithelial cells.

Anthrax lethal toxin and tumor necrosis factor-a synergize on intestinal epithelia to induce mouse death

Bacillus anthracis lethal toxin(LT)is a determinant of lethal anthrax.Its function in myeloid cells is required for bacterial dissemination,and LT itself can directly trigger dysfunction of the cardiovascular system.The interplay between LT and the host responses is important in the pathogenesis,but our knowledge on this interplay remains limited.Tumor necrosis factor-a(TNF-a)is a pleiotropic pro-inflammatory cytokine induced by bacterial infections.Since LT accumulates and cytokines,predominantly TNF,amass during B.anthracis infection,co-treatment of TNF+LT in mice was used to mimic in vivo conditions for LT to function in inflamed hosts.Bone marrow transplantation and genetically engineered mice showed unexpectedly that the death of intestinal epithelial cells(IECs)rather than that of hematopoietic cells led to LT+TNF-induced lethality.Inhibition of p38a mitogen-activated protein kinase(MAPK)signaling by LT in IECs promoted TNF-induced apoptosis and necroptosis of IECs,leading to intestinal damage and mouse death.Consistently,p38a inhibition by LT enhanced TNF-mediated cell death in human colon epithelial HT-29 cells.As intestinal damage is one of the leading causes of lethality in anthrax patients,the IEC damage caused by LT+TNF would most likely be a mechanism underneath this clinical manifestation and could be a target for interventions.

Chang'an Ⅱ Decoction(肠安Ⅱ号方)-Containing Serum Ameliorates Tumor Necrosis Factor-α-Induced Intestinal Epithelial Barrier Dysfunction via MLCK-MLC Signaling Pathway in Rats

Objective To investigate the effect of Chang’an Ⅱ Decoction(肠安Ⅱ号方))-containing serum on intestinal epithelial barrier dysfunction in rats.Methods Tumor necrosis factor(TNF)-α-induced injury of Caco-2 monolayers were established as an inflammatory model of human intestinal epithelium.Caco-2 monolayers were treated with blank serum and Chang’an Ⅱ Decoction-containing serum that obtained from the rats which were treated with distilled water and Chang’an Ⅱ Decoction intragastrically at doses of 0.49,0.98,1.96 g/(kg·d)for 1 week,respectively.After preparation of containing serum,cells were divided into the normal group,the model group,the Chang’an Ⅱ-H,M,and L groups(treated with 30 ng/mL TNF-αand medium plus 10%high,middle-,and low-doses Chang’an Ⅱ serum,respectively).Epithelial barrier function was assessed by transepithelial electrical resistance(TER)and permeability of fluorescein isothiocyanate(FITC)-labeled dextran.Transmission electron microscopy was used to observe the ultrastructure of tight junctions(TJs).Immunofluorescence of zonula occludens-1(ZO-1),claudin-1 and nuclear transcription factor-kappa p65(NF-κBp65)were measured to determine the protein distribution.The mRNA expression of myosin light chain kinase(MLCK)was measured by real-time polymerase chain reaction.The expression levels of MLCK,myosin light chain(MLC)and p-MLC were determined by Western blot.Results Chang’an Ⅱ Decoction-containing serum significantly attenuated the TER and paracellular permeability induced by TNF-α.It alleviated TNF-α-induced morphological alterations in TJ proteins.The increases in MLCK mRNA and MLCK,MLC and p-MLC protein expressions induced by TNF-αwere significantly inhibited in the Chang’an Ⅱ-H group.Additionally,Chang’an Ⅱ Decoction significantly attenuated translocation of NF-κBp65 into the nucleus.Conclusion High-dose Chang’an Ⅱ-containing serum attenuates TNF-α-induced intestinal barrier dysfunction.The underlying mechanism may be involved in inhibiting the MLCK-MLC phosphorylation signaling pathway mediated by NF-κBp65.

Phytoglycoprotein isolated from Dioscorea batatas Decne promotes intestinal epithelial wound healing

Dioscorea batatas Decne(DBD)has been used to heal various illnesses of the kidney and intestine as an herbal medicine in Asia.As a source of therapeutic agents,many glycoproteins have been isolated from mushrooms and plants,but the functional role of glycoprotein in intestinal epithelial wound healing has not been understood yet.In the present study,we investigated the wound healing potentials of the 30 kDa glycoprotein(DBD glycoprotein)isolated from DBD in human intestinal epithelial(INT-407)cells.We found that DBD glycoprotein(100μg·mL^-1)significantly increased the motility of INT-407 cells for 24 h by activating protein kinase C(PKC).DBD glycoprotein stimulated the activation of p38 mitogen-activated protein kinase(MAPK),which is responsible for the phosphorylation of NF-κB inhibitorα(IκBα).DBD glycoprotein increased the level of profilin-1(PFN1),α-actinin and F-actin expression via activation of transcription factor,nuclear factor-kappa B(NF-κB)during its promotion of cell migration.Experimental mouse colitis was induced by adding dextran sulfate sodium(DSS)to the drinking water at a concentration of 4%(W/V)for 7 days.We figured out that administration of DBD glycoprotein(10 and 20 mg·kg-1)lowers the levels of disease activity index and histological inflammation in DSS-treated ICR mice.In this regard,we suggest that DBD glycoprotein has ability to promote the F-actin-related migration signaling events via activation of PKC and NF-κB in intestinal epithelial cells and prevent inflammatory bowel disease.

Effect of Echinacea Polysaccharide on Secretion of IkB-αmRNA by IEC-6 Injured by LPS

[ Objective] This study was conducted to investigate the mechanism of Echinacea polysaccharide （EPS） in treatment of various bacterial infection and reduction of inflammation, so as to provide a theoretical basis for clinic application of EPS. [ Method ] Nuclear protein extracted from six groups, the normal control group, the simple lipopolysaccharide （LPS） group and the EPS （ with concentrations of 50,100,200 and 500 μg/ml, respectively） ＋ LPS groups was subjected to SDS-PAGE electrophoresis, and pIkB-α protein contents in the extracts were analyzed by Coomassie brilliant blue （CBB） staining and Western-Blot method. [ Result] The simple LPS group showed the highest pIkB-α protein level, and in the EPS concentration range of 0 -200 μg/ml, the expression level of pIkB-α pro- tein was improved with the increase of EPS concentration. [ Conclusion The expression level of pIkB-α protein was improved under the simulation of IEC-6 by LPS, while EPS could effectively inhibit the expression of pIkB-α protein. The expression level of pIkB-α was the lowest in the LPS ＋500 μg/ml EPS group.

The developmental changes in intestinal epithelial cell proliferation,differentiation,and shedding in weaning piglets

Intestinal epithelial homeostasis plays an important role in intestinal morphology and function.However,the developmental changes in intestinal epithelial cell turnover in piglets during early weaning are unknown so far.Thus,the aim of this work was to detect changes in piglet gut development from weaning to post-weaning d 14.Accordingly,40 piglets were used in the present study,and 8 piglets were randomly selected for sampling at d 0,1,3,7 and 14 post-weaning,respectively.The results showed that weaning stress significantly affected small intestinal morphological architecture,and this impact was the worst on d 3,and then returned to normal on d 14.Furthermore,the number of the marker of proliferation Ki-67(Ki67)positive cells was decreased on d 1 and 3,and then recovered on d 14(P<0.001).Also,weaning strikingly increased jejunal epithelial cell shedding on d 1 to 7 compared on d 0(P<0.05).Moreover,weaning remarkably affected the number of small intestinal enterocytes,goblets and endocrine cells(P<0.05),and there were also significant differences in genes expression related to proliferation and differentiation(P<0.05).Additionally,the mechanistic target of rapamycin(mTOR)phosphorylation level was higher on d 3(P<0.05).However,the Wingless/Int1(WNT)/b-catenin pathway was not influenced by post-weaning days.Taken together,weaning induced noteworthy changes in intestinal epithelial cell proliferation,differentiation and shedding,and the mTOR signaling pathway was involved in this process.Our findings provide a cellular mechanism for intestinal developmental changes during weaning periods.This may provide nutritionists with better insight into designing efficient in-feed alternatives for preventing the unfavorable gut development in weaning piglets.

Effect of Echinacea Polysaccharide on Secretion of IL-1αmRNA by IEC-6 Injured by LPS

[ Objective ] This study was conducted to investigate Echinacea polysaccharide （EPS） on expression of tumor necrosis factor （TNF） under injury of intestinal epithelial cells （IEC-6） by lipopolysaccharide （LPS）, so as to discuss the action mechanism of EPS to injured cells. [Method] Total DNA was extracted with TRIzon reagent, TNF-α mRNA was amplified, and the amplification products were subjected to agarose gel electrophoresis and imaging. [Result] 50 μg/ml EPS could partially inhibited the production of IL-α mRNA by IEC-6 under the stimulation of LPS, while the inhibition of 2（10 and 500 μg/ml EPS on the level of IL-1α mRNA gradually increased with the concentration increasing; and IEC-6 cells pretreated with 50,100,200 and 500 μg/ml EPS for 24 h and then stimulated by 10 μml IPS for 1 and 4 h were analyzed by RT-PCR method, and it was found that the expression of IL-α mRNA induced by LPS could be effectively inhibited by EPS, and the inhibition rate at 4 h was higher than that at 1 h. [ Conclusion] EPS could play its role of protecting intestinal mucosa by inhibiting the secretion of IL-1α mRNA by cells under the stimulation of LPS, and such inhibition effects of EPS had concentration dependency and time dependency.

Synthesis of quaternary 8-(1-acylethene-1-yl)-13-methylcoptisine chlorides and their selective growth inhibitory activity between human cancer cell lines and normal intestinal epithelial cell-6

In this paper, quaternary 8-（1-acylethene-l-yl）-13-methylcoptisine chlorides targeting thioredoxin reductases （TrxRs） were designed to test the growth inhibitory activity against human cancer cell lines and the effect on viability of the normal intestinal epithelial cell-6 （IEC-6） in vitro and to evaluate structure-activity relationship （SAR）. The introduced α, β-unsaturated ketone groups at C-8 consisting of n-alkanoyls possessing five to ten carbons or aroyls or cyclohexylcarbonyl increased the tested activity against the target cancer cell lines. By and large, this type of improvement was increasingly graced by the elongation of the aliphatic chain of the n-alkanoyls in the range of less than ten carbon atoms. The relatively more polar l-acylethene-l-yls displayed no effect on improving the activity. All the explored aroyls showed significant effect on improving the activity of the target compounds against the tested cancer cell lines with no SAR being observed, The findings of this study suggested that oil]water partition coefficient of the test compounds was one of the key factors impacting the target activity against the tested cancer cell lines. At the concentration of 10 μmol/L, except for the compounds with n-all（anoyls possessing seven or more carbons or with α-naphthoyl, none of the other compounds displayed obvious cytotoxicity on normal IEC-6 cell when co-incubated. The survival rate of IEC-6 cell ranged from 75% to 100% for the noncytotoxic compounds.

Microbial sensing in the intestine

The gut microbiota plays a key role in host health and disease,particularly through their interactions with the immune system.Intestinal homeostasis is dependent on the symbiotic relationships between the host and the diverse gut microbiota,which is influenced by the highly co-evolved immune-microbiota interactions.The first step of the interaction between the host and the gut microbiota is the sensing of the gut microbes by the host immune system.In this review,we describe the cells of the host immune system and the proteins that sense the components and metabolites of the gut microbes.We further highlight the essential roles of pattern recognition receptors(PRRs),the G protein-coupled receptors(GPCRs),aryl hydrocarbon receptor(AHR)and the nuclear receptors expressed in the intestinal epithelial cells(IECs)and the intestine-resident immune cells.We also discuss the mechanisms by which the disruption of microbial sensing because of genetic or environmental factors causes human diseases such as the inflammatory bowel disease(IBD).

Mannose metabolism normalizes gut homeostasis by blocking the TNF-α-mediated proinflammatory circuit

Mannose is a naturally occurring sugar widely consumed in the daily diet;however,mechanistic insights into how mannose metabolism affects intestinal inflammation remain lacking.Herein,we reported that mannose supplementation ameliorated colitis development and promoted colitis recovery.Macrophage-secreted inflammatory cytokines,particularly TNF-α,induced pathological endoplasmic reticulum stress(ERS)in intestinal epithelial cells(IECs),which was prevented by mannose via normalization of protein N-glycosylation.By preserving epithelial integrity,mannose reduced the inflammatory activation of colonic macrophages.On the other hand,mannose directly suppressed macrophage TNF-αproduction translationally by reducing the glyceraldehyde 3-phosphate level,thus promoting GAPDH binding to TNF-αmRNA.Additionally,we found dysregulated mannose metabolism in the colonic mucosa of patients with inflammatory bowel disease.Finally,we revealed that activating PMM2 activity with epalrestat,a clinically approved drug for the treatment of diabetic neuropathy,elicited further sensitization to the therapeutic effect of mannose.Therefore,mannose metabolism prevents TNF-α-mediated pathogenic crosstalk between IECs and intestinal macrophages,thereby normalizing aberrant immunometabolism in the gut.