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Objective To investigate the regulation of miR-7 on intestinal trefoil factor (TFF3) and its effect on the proliferation and migration of intestinal epithelial cells. Methods miR-7 mimics and miR-7 inhibitor were transfected into LS174T cells respectively. The experiment was divided into 5 groups,including blank control group,miR-7 mimic negative control group,miR-7 mimic group,miR-7 inhibitor negative con-trol group,miR-7 inhibitor group. MTT assay was used to detect cell proliferation. Cell scratch assay was used to detect the effect of miR-7 on cell migration. Western blot was used to detect the change of TFF3 protein in each group. Results Compared with the blank control group,the miR-7 mimic negative control group and the miR-7 inhibitor negative control group,the OD value of the miR-7 mimic group decreased significantly, the difference was statistically significant(P<0. 05); the cell scratch interval increased,the cell migration rate decreased,the difference was statistically significant( P<0. 05); and the TFF3 protein expression was accompanied(P<0. 05). The OD value of the miR-7 inhibitor group significantly increased,the difference was statistically significant(P<0. 05); the cell scratch gap decreased,the cell migration rate was enhanced, the difference was statistically significant( P <0. 05); and the expression of TFF3 protein increased ( P <0. 05). Conclusion miR-7 can regulate the expression of TFF3 and further inhibit the proliferation and migration of LS174T cells.
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ObjectiveTo compare the values of serum citrulline, intestinal fatty acid binding protein (IFABP) and intes-tinal trefoil factor (ITF) in diagnosis of acute gastrointestinal injury in critically ill children.MethodsA total of 84 critically ill children were enrolled. The serum citrullin, IFABP, and ITF were measured by high performance liquid chromatography and enzyme-linked immunosorbent assay. The testing results and clinical data were analyzed.ResultsCompared with non gastroin-testinal injury group, the serum citrulline level was signiifcantly lower and IFABP and ITF levels were signiifcantly higher in gas-trointestinal injury group (allP<0.05). In critically ill children, the serum citrulline level was negatively correlated with C-reactive protein, procalcitonin and hospitalization time (r=-0.36 to -0.31,P<0.01).ConclusionsThe levels of citrulline, IFABP and ITF have diagnostic values for acute gastrointestinal injury in critically ill children. The level of citrulline may relfect the degree of acute gastrointestinal injury in critically ill children.
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Intestinal trefoil factor (ITF or TFF3) is a member of trefoil factor family which is most studied , mainly in the in-testinal tract .TFF3 plays an important role in gastrointestinal mucosal protection and epithelial repair .Action mechanism includes in-teraction with mucin, migration, anti-apoptosis, angiogenesis, immune regulation, interaction with its receptor, etc.Recombinant TFF3 has been approved as a national drug and its therapeutic indications for repair mucosa , prevention and treatment of gastrointesti-nal ulcers and inflammation .Furthermore, the classic TFF3 receptor and signaling pathway is highly valuable to make TFF 3 become an effective treatment for gastrointestinal injury disease .
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Objective To explore the effects of intestinal trefoil factor ( ITF) on gastric mucosal epithelial cell proliferation and its possible molecular mechanism . Methods The cultured GES-1 cells were treated with ITF in the concentration of 100 ng/mL and 500 ng/mL in vitro, and then were observed using microscope for the morphological analysis .The Cell Counting Kit-8 ( CCK-8) was used to detect the proliferation activity of GES-1.The cultured GES-1 cells were treated with 100 ng/mL ITF and the specific inhibitor of PI3K/Akt signaling pathway LY294002 (15μmol/L) in vitro, and then were observed using microscope for the morphological analysis . The proliferation activity of treated GES-1cells was detected using CCK-8 and the expressions of p-Akt and Akt of PI3K/Akt signaling pathway were determined by Western blot . Results Compared with the control group , the proliferation activity of GES-1 cells in-creased after being treated with ITF and the higher concentration of ITF induced the higher proliferation activity .LY294002 inhibited the increased proliferation activity of GES-1induced by ITF.The data of Western blot indicated that ITF induced the expression of p -Akt and activated the P3IK/Akt signaling pathway to modulate the proliferation activity of GES -1 cells.However, LY294002 inhibited the PI3K/Akt signaling pathway and then decreased the proliferation activity of GES -1 cells. Conclusion ITF could promote the proliferation ac-tivity of GES-1 cells by activating PI3K/Akt signaling pathway .
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Objective: To investigate the effect ofintestinal trefoil factor (ITF) on the transcriptional activity of ITF promoter and to explore the regulatory mechanismofJanus kinase/signal transducersand activators of transcription(JAK/STAT) on ITF promoter. Methods: The 5' flanking sequence of the ITF gene was cloned from human whole blood genomic DNA by PCR. ITF promoter fragment was cloned and inserted into the pGL3-Basic vector to construct recombinant vector. ITF promoter vector was stimulated with ITF at various concentrations and the luciferase activity was measured. The JAK-STAT3 signal transductionpathway was then blocked by a speciifc inhibitor AG490 to determine the signal pathway involved in ITF promoter activity. Results: Restriction endonuclease analysis and DNA sequencing confirmed that the recombinant plasmid, containing ITF promoter, was constructed successfully. After transient transfection, the activity of ITF promoter was increased signiifcantly in the presence of ITF (P<0.05). Blockage of the JAK-STAT3 signal transduction pathway with AG490 signiifcantly reduced the ITF promoter activity (P<0.05). Conclusion: ITF increases the transcriptional activity of ITF promoter via the JAK-STAT3 signal transduction pathway.
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OBJECTIVE: To construct an optimal human intestinal trefoil factor (hITF) gene delivery system.
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Objective To observe the expression of ITF in colon of mice infected by mouse cytomegalovirus (MCMV) and the in-volvement of ganciclovir(GCV) .Methods Forty-eight BALB/c young mice were randomly divided into blank group ,virus group and GCV group ,each with 8 mice .Mice in virus group and GCV group received injection of 100 μL MCMV virus suspension (TCID50105 .31 /mL) ,and GCV group was given intraperitoneal injection of GCV once a day at the dose of 50 mg/kg from day 0 (24 hours after vaccination of virus ) ,for 14 days .At the same time the virus group and blank group were given the same dose of normal saline as controls .Murine cytomegalovirus loads in livers and colons were measured by PCR .The expression levels of ITF in mRNA in colon were detected by RT-PCR .Results After MCMV injection ,mice in virus group manifested aggressively apparent poor appetite ,less activity ,fur laxly ,unresponsiveness to stimuli ,growth retardation ,body weight not increased .All liver and colon tissue MCMV-DNA PCR electrophoresis of virus group had positive strip ,while the blank group did not .GCV group also showed less bright positive strip when compared with the virus group .Expression level of ITF mRNA was significant higher in GCV group than virus group ,there was statistically significant difference(P<0 .05) .Expression of ITF mRNA in virus group were higher than that in blank group ,there was statistical difference(P<0 .05) .Conclusion ITF is regarded as a fast reaction peptide in the course of mucosa impairments ,so ITF plays a protective role in delayed healing process after acute MCMV infection .
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BACKGROUND:The intestine is not only the main target attacked by sepsis but also the vital organ which mediated sepsis. The recovery of the damaged intestinal barrier structure and function is related to the occurrence and outcome of multiple organ dysfunction syndrome (MODS). How to protect and reduce the damage of the intestinal mucosa and how to promote the reconstruction of the intestinal mucosa have been the important topics in sepsis for many years. This study aimed to investigate the influential factors of intestinal mucosal reconstruction after intestinal epithelial injuryin vivo in a mouse model of sepsis.METHODS:Mice were subjected to cecal ligation and puncture (CLP) for induction of sepsis to assess intestinal mucosal damage, epithelial cell apoptosis, and transformed number of goblet cells, and to detect the concentration of TNF-α, IL-1 and TGF-β1 and TFF3 (trefoil factor 3) expression in the small intestinal mucosa. All above were performed by HE staining, western blot, ELISA and immunohistochemistry respectively. The experimental animals were divided into a sepsis group and a sham-operation group. The animals with sepsis were separately killed at 6 (7 animals), 24 (7 animals) and 48 hours (7 animals) after CLP.RESULTS:Injured intestinal mucosa was observed in the 3 groups under a light microscope, in which damage scores in the 24-hour and 48-hour groups were higher than in the 6-hour group and no difference was found between the two groups. Moreover, less of goblet cells or other epithelial cells adjacent to the injured surface migrated into the wound to cover the denuded area. The number of goblet cells was substantially decreased in the three CLP groups compared with the sham-operation group. Protein levels of IL-1 and TNF-α were significantly increased by 3-4 fold at all time points when compared with the sham-operation group, and cleaved caspase-3 by 4 fold. Although TFF3 expression was modestly increased for 6 hours after the onset of CLP, it appeared to decline at 24 hours and 48 hours as shown by Western blot. A similar tendency was observed upon TGF-β1, i.e. the protein level was not elevated at 24 hours and 48 hours, but increased modestly at 6 hours.CONCLUSIONS:Sepsis from CLP shows less restitution on the surface of injured intestinal mucosa. There is evidence that both constant inflammatory reaction and epithelial cell apoptosis may affect mucosal reestablishment of the intestine at the onset of sepsis. Mucosa after severe sepsis showed the state of high inflammation, and declined goblet cell function and mucosal reconstruction, which affected the repair of damaged intestinal barrier. Constant inflammatory reaction, and declined goblet cell function and mucosal reconstruction ability may affect the reestablishment of intestinal mucosa at the onset of sepsis.
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Twenty-nine thyroid tissue samples were collected from patients with thyroid nodules.The total RNA were extracted,the gene expressions of TFF3,HMGA2,C1orf24,and DDIT3 were detected by RT-PCR.In comparison with normal thyroid tissues,the expression of C1orf24 mRNA was decreased in the follicular thyroid adenoma (FTA) group,but increased in the follicular thyroid carcinomas (FTC) group.The expressions of DDIT3 and HMGA2 mRNA were increased in both FTA and FTC groups,and were even higher in the latter.The expressions of TFF3 mRNA level were decreased in FTA and FTC.The data suggested that molecular markers C1ort24,DDIT3,HMGA2,and TFF3 in thyroid tissue seem to be helpful in the differential diagnosis between follicular adenomas and carcinomas.
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Objective To investigate the relationship of intestinal trefoil factor(HF) for regulating of nuclear factor(NF)-κB and tumor necrosis factor(TNF)-α and the protective effect of intestinal injury.Methods Twenty-four 10-day Wistar rats were divided into 3 groups:the control group(NS group,n =8),given 9 g/L sodium injection intraperitoneally,1 mL/kg; the lipopolysaccharide (LPS) group (n =8),given LPS (5 g/L),5 mg/kg intraperitoreally; ITF group(n=8),given 1TF(5 g/L) 0.1 mL/each plus LPS 5 mg/kg intraperitoreally.Rats were sacrificed 3 hours after injection.A segment of distal ileum was dissected.The pathologic changes of small intestine were observed under the optical microscope(hematoxylin-eosin staining).The expressions of NF-κB mRNA and protein were detected by reverse transcription polymerase chain reaction(RT-PCR),immunohistochemistry,respectively.TNF-α level in intestinal tissues was measured by enzyme linked immunosorbent assay.Results The structure of small intestine in the control group remained normal.The inflammatory cells infiltration and the edema of interstitial substance and epithelium were observed in LPS group and ITF group,while the change in the ITF group was significantly lower than that in LPS group.The expressions of NF-κB mRNA and protein in ITF group were significantly lower than those in LPS group(all P ＜ 0.01).The secretion of TNF-α in the rTF group was significantly lower than that in LPS group(P ＜ 0.01).Conclusion Protective effects of ITF on intestinal injury are related to the down regulation of NF-κB mRNA and protein expressions and the reduction of the secrete of mediators of inflammation TNF-α.
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Objective To investigate the potential mechanism of intestinal trefoil factor(ITF)against methotrexate (MTX)- induced injury in intestinal mucosa. Methods Cultured IEC-6 cells were divided into groups as follows: blank group, MTX treated group, ITF treated group and experimental group treated with gradient concentrations of ITF plus MTX. Expression of E-cadherin mRNA was determined by Real-Time polymerase chain reaciton (RT- PCR). The activity of matrix metalloproteinase(MMP)-2 and MMP-9 was measured by gelatin zymogramphy. Caspases-3 activity was measured by colorimetric assay. Cell proliferation was assessed by cell counting kit-8 (CCK-8)assay. Migration of IEC-6 in vitro was observed using modified Boyden chamber assay. Results The expression of E-cadherin mRNA in experimental group (treated with 0.1 mg/ml or 1 mg/ml of ITF) was significantly down-regulated (0. 538±0. 109 or 0. 528±0. 132, respectively) in comparison with MTX treated group (0. 763±0. 139) with significant difference (P=0. 021 or P=0. 025, respectively). There was no significant difference in activity of MMP-2 and MMP-9 among groups (P＞0. 05). When compared with MTX treated group (0. 090 ±0. 011 ), the activity of Caspase3 in experimental group (treated with 0. 1 mg/ml or 1 mg/ml of ITF) was significantly decreased (0. 077±0. 009, P=0. 032 or 0. 044±0. 009,P=0. 005, respectively). There was no statistical difference in cell proliferation between experimental group (treated with 1 μg/ml, 0.01 mg/ml, 0. 1 mg/ml or 1.0 mg/ml of ITF) and MTX treated group (P=0. 132,0. 150,0. 114 or 0. 367, respectivley). More migratory cells attached to the bottom surface of the membrane in experiment group (treated with 0. 1 mg/ml or 1 mg/ml of ITF) in comparison with MTX treated group (P ＜0. 001 ). Moreover, more migratory cells were found in experimental group treated with 1.0 mg/ml of ITF than those in group treated with 0. 1 mg/ml of ITF (P＜0. 001). Conclusions Without cell proliferation, the protective effect of ITF is related to its functions of promoting cell migration and inhibiting cell apoptosis, which may down-regulate expression of E-cadherin mRNA.
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ObjectiveTo investigate the unfavorable factors of intestinal mucosa repair after the intestinal epithelial injury in vivo in a mouse model of sepsis. MethodsThe method of cecal ligature and puncture (CLP) was used to induce sepsis and then the intestinal mucosa damage, epithelial cell apoptosis and the number of transformed goblet cells were observed, and the concentrations of serum TNF-αt, IL-1 and TGF-β1 and TFF3 ( trefoil factor 3) in small intestinal mucosa were determined. All above various laboratory examinations were made by different assays including H-E staining, western blot, ELISA and immunohistochemistry respectively. The experimental mice were divided into sepsis group and sham operation control group. The mice with sepsis were separately sacrificed 6 hours ( n = 7 ), 24 hours ( n = 7) and 48 hours ( n = 7) after CLP. Results In septic mice group, the injured intestinal mucosa was found 6 hours after CLP. The damage scores in mice 24 h and 48 h after CLP were higher than those 6 h after CLP, but there was no significant difference between those 24 h and 48 h after CLP. Moreover, a few goblet cells or other epithelial cells adjacent to the injured surface migrated onto the wound to cover the denuded area. The number of goblet cells was substantially decreased in mice of sepsis group 6 hours after CLP compared with sham operation control group. Compared with sham operation control group, levels of IL-1 and TNF-α significantly increased 3-4 times in mice of sepsis group at all intervals, and the phosphorylated caspase-3 increased 4 times. Although TFF3 assayed by using Western blot showed modest increase 6 h after CLP and it declined 24 h and 48 h later. A similar change was found in TGF-β1, it modestly increased 6h after CLP, but it didn't elevate 24 h and 48 h later. ConclusionsSevere sepsis keeps on the inflammatory reaction and epithelial cell apoptosis, preventing the repair of intestinal mucosa from injury.
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Objective To investigate the change of the intestinal permeability,the expression level of intestinal trefoil factor (ITF) mRNA and the relationship between them after total body irradiation (TBI),and explore the effect of TBI on the development of intestinal permeability and the expression level of ITF mRNA.Methods Twenty two BALB/c mice were randomly divided into 4 equal groups: 3 groups at 4,8 and 12 d after TBI with the total dose of 8.0 Gy and the dose rate of 1.0 Gy/min respectively,and a control group.Lactulose (L) and mannitol (M) were perfused into the esophagus before the experiment and urine samples were collected.Liquid chromatography was used to measure the L/M excretion ratio in the urine samples collected 4,8,and 12 days after the TBI.And then the mice were killed with their intestine were taken out.The expression of ITF mRNA in the jejunum tissue was detected by real-time fluorescence quantitative PCR.Results The urine L/M ratio levels of the groups 4,8 and 12 days after TBI were (0.5092 ± 0.0352),(0.7174 ± 0.0116),and (0.7295 ± 0.0533) respectively,all significantly higher than that of the control group [(0.2908 ± 0.0533),F = 321.47,P ＜ 0.05].The ITF mRNA expression levels of groups 4,8 and 12 days after TBI were (0.78612 ±0.1428),(0.2521 ±0.1223),and (0.2306 + 0.0221 ) respectively,all significantly lower than that of the control group [( 1.3498 + 0.0476),F = 235.71 ,P ＜ 0.05].The urine L/M ratio was significantly negatively correlated with the expression of ITF mRNA in all TBI groups (r = - 0.985,P ＜ 0.01 ).Conclusions The intestinal permeability increases and the expression level of ITF mRNA decreases after TBI.The urine L/M ratio is negatively correlated with the expression level of ITF mRNA after TBI.ITF is involved in protection against intestinal permeability induced by TBI.
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Intestinal trefoil factor(ITF)is a low molecular weight polypeptide expressed in intestine. ITF is also found in the brain,respiratory,eyes and so on.To study recombinant ITF will have an important impact for the treatment of gastrointestinal disease, respiratory tract disease, eyes disease and so on.
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Objective To analyze the influence of intestinal trefoil factor(ITF) on Bim and Bcl-xl gene expression in neonatal rats with necrotizing enterocolitis(NEC),and to discuss the protective machanism of ITF on NEC.Methods Thirty neonatal rats were divided randomly into control group,NEC group and ITF group.NEC group were given intraperitoneal injection of saline 0.2 ml after NEC model of neonatal rats were established.ITF group were given intraperitoneal injection ITF 0.2mg after NEC model of neonatal rats were established.On the 4th day,all the subjects were put to death.We made HE stainting of the slice and made a histopathological examination and immunohistochemical method to detect Bim and Bc1-xl genes expression,and make image analysis.Results The pathological lesions indicated that intestinal tissue necrosis was severe in NEC group,which median was 3 point,but obviously lessen in ITF group,which median was 1 point,with ITF interfering.Image analysis showed the NEC group Bim gene expression (7.87 ± 0.14) higher than those in the control group (2.15±0.28) and ITF group (3.27±0.34),there were significant differences between 3 groups(P<0.05).Bcl-xl gene expression(11.23±0.22)in ITF group was higher than that in control group(1.89±0.28) and NEC group(2.51±0.13),there were significant differences between 3 groups(P<0.05).Conclusions Intestinal injury was ameliorated after ITF was injected intraperitoneally,ITF may protect the intestinal injury of neonatal rats with NEC by changing the Bim gene and Bc1-xl gene expresstion ratio.
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Objective To investigate the effect of recombinant intestinal trefoil factor(rITF)on the intestinal mucosa of acute necrotizing pancreatitis(ANP)rats and explore the mechanism.Methods 60 male SD rats were randomly divided into control group(n=20),ANP group(n=20),rITF group(n=20).ANP was induced by retrograde injection of 5%sodium taurocholate(100μl/100 g)into biliopancreatic duct.Rats in rITF group were injected with rITF(0.5 mg/100 g)before and after ANP induction.The rats in ANP group and control group received same amount of normal sailne.Each group were sacrificed 12 h or 24 h later.respectively.Blood sample was taken to determine the serum level of amylase.Terminal ileum was resected to observe the pathologic changes;immunohistochemistry method was applied to detect the activity of NF-κB;the expression of TNF-α mRNA,ICAM-1 mRNA in intestinal mucosa was measured by RT-PCR.Results Intestinal injury score of ANP group was higher than that ofcontrol group(P＜0.05),intestinal injury score of ANP 12 h group was higher than that of rITF 12 h group(P＜0.05),but there was no significant difference between ANP 24 h group and rITF 24 h group.The number of positive NF-κB cells was 26±4,55±8,49±4,respectively;the relatively expression of TNF-α mRNA in terminal ileum was 0.050±0.005,1.040±0.031 and 0.792±0.0256,respectively;the relatively expression of ICAM-1 mBNA was 0.045±0.010,0.795±0.037 and 0.400±0.031,respectively,in control group,ANP group and rITF 12 h group.The corresponding values in the ANP group were significantly increased compared with those values in the control group (P＜0.05 or P＜0.01).Conclusions rITF may protect the intestinal mucosa.the mechanism may include inhibit NF-κB activation,down-regulate TiNF-α mBNA,ICAM-1 mRNA expression.
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Objective To explore the influence of intestinal trefoil factor (ITF) on interleukin-6(IL-6) in neonatal rat with necrotizing enterocolitis(NEC) ,and to discuss the protective machanism of ITF on NEC.Methods Thirty-two neonatal rats were divided randomly into four groups,group A as control group,group B as NEC group,group C as NEC+NS 0.2 mL group,group D as NEC+ITF 0.2 mg group.NEC model of neonatal rats were established.On the 4th day,all the subjects were put to death.Intestinal tissue within the boundary of ileum and cecum was obtained to observe histological changes.Other intestinal tissue was treated into homogenate.After the homogenate was centrifuged,supernates were used to test the density of IL-6.Results The density of IL-6 significantly decreased in group A,D than those in group B and C (Pa0.05).The pathological lesions indicated that intestinal tissue necrosis was severe in group B and C,which was graded as 3 points,but obviously lessen in group D,which was graded as 1 point,with ITF interfering.Conclusions Intestinal inflammation is ameliorated after ITF are injected hypodermically or intraperitoneally.ITF may provide a brand-new way for the therapy of NEC in neonatal rats.
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To develop an oral drug,ITF gene encoding ITF proein,was expressed in a live delivery vehicle lactococcus lactis.First,the ITF gene was cloned into the prokaryotic expressive vector pNICE:sec.Second,the recombinant vector pNICE:sec-ITF was transformated into Lactococcus lactis strain NZ9000 to express ITF protein.Then the recombinant ITF was induced to express and was identified by SDS-PAGE and Western blot.Rabbits are divided into blank control group,preparation group and therapeutic group which are respectively administrated wih PBS and pNICE:sec-ITF Lactococcus lactis.By grades of ulcer test whether administrated pNICE:sec-ITF Lactococcus lactis protects against HCl-induced gastric injury in rabbits.The results were described as follows.The ITF was amplified and cloned in the vector pNICE:sec successfully.The fusion protein(5.9kDa) was expressed in L.lactis by the induction of the nisin.The quantity of expression accounted for 5% of the total bacterial protein.Western bolt analysis confirmed that fusion protein could be recognized specially by Monoclonal Anti-human TFF3 Antibody.Preparation groups and therapeutic groups do good than control group.Prove that administrated pNICE:sec-ITF Lactococcus lactis is biologically active in an HCl-induced rabbit gastric mucosal injury model.
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Summary: In order to investigate the protective effects of intestinal trefoil factor (ITF) on colonic mucosa in experimental colitis of rats, ITF was detected by RT-PCR and immunohistochemistry at different time points. Three days after colitis induction, rats were treated with either 0.9 % saline solution or rhITF. Pathological changes and the expression of iNOS mRNA, NO, MDA and SOD were measured respectively. It was found that ITF was mainly located in goblet cells, significantly higher in model group than in normal group (P<0.05). rhITF could increase the iNOS mRNA expression and NO contents, and there was statistically significant difference between rhITF group and model group (P<0.05). rhITF also caused an increase of MDA and a decrease of SOD, but there was no significant difference between two groups. These results indicated that ITF has apparent therapeutic effects in ulcerative colitis, which may be associated with iNOS and NO.
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Objective To study the effect of intestinal trefoil factor(ITF) on nuclear factor-?B(NF-?B) in neonatal rats intestinal tissues with necrotizing enterocolitis(NEC),and to discuss whether ITF had protective function in NEC,and its role in the mechanism of NEC.Methods Fifty neonatal rats were randomly divided into 5 groups:group A as control group,group B as control plus ITF 0.2 mg group,group C as NEC group,group D as NEC plus normal saline(NS),group E as NEC plus ITF 0.2 mg.NEC models of neonatal rats were established.On the 4th day,all subjects were put to death.The intestinal tissue located at the boundary of ileum and cecum were obtained to observe histological changes and NF-?B level.Results The density of NF-?B(p65) increased significantly in group C and D compared with those in group A,B and E(Pa