| Intestinal ischemia reperfusion(II/R)injury is a common pathophysiological process,which can be secondary to a variety of clinical diseases,such as major surgery,organ transplantation,shock,trauma,and so on.The effect of systemic state has far exceeded the local intestinal damage and destruction,is believed to be the important initial factor of systemic inflammatory response syndrome(SIRS)and multiple organ dysfunction syndrome(MODS).The II/R has a high mortality rate in clinic.The damage of mucosal barrier function and immune function is a leading cause of serious complications and high mortality in the patients with II/R injury,and repair of intestinal mucosa barrier function after II/R injury is very important.The mechanism failed to repair the damaged mucous barrier function will lead to the II/R patients with high mortality in clinic.Therefore,how to effectively improve the regeneration ability of intestine after II/R injury has important clinical significance.To study the mechanism of intestinal mucosal regeneration after II/R injury and therapeutic interventions of the mechanism is very important for prognosis of II/R.Nuclear receptor associated factor 1(Nurr1)is a transcription factor,belonging to the nuclear receptor superfamily.As a gene transcriptional regulatory protein,Nurr1 participate many physiological and pathological processes,such as dopaminergic neuronal development and survival in the midbrain,long-term memory,liver regeneration and inflammation,etc.The qPCR method was used to detect the high expression of Nurr1 mRNA in the gastrointestinal tract of mice,but the role of Nurr1 in the IIR and its regulation mechanism were not related.p21 gene is a member of the CIP family,and it is a cyclin dependent kinase inhibitor.p21 constitutes the cell cycle checkpoint of G1/S,which can block the cell cycle in the G1 phase.p21 plays an important role in the differentiation and proliferation of intestinal epithelial cells.p21 can be regulated by p53-dependent and p53-independent.In the II/R injury,p53 has no significant changed,and p21 mainly play a role through the p53-independent manner.The study has been showed that the expression of p21 could be inhibited by the overexpression of Nurr1.Therefore,we propose the following hypothesis:Nurr1/p21 signaling pathway may exist in intestinal regeneration after intestinal ischemia and reperfusion injury.In this pathway,Nurrl may be to promote regeneration after intestinal ischemia/reperfusion injury by inhibiting the expression of p21;and regulating the pathway could effectively improve the proliferation of intestinal epithelial cell and promote the intestinal regeneration after ischemia/reperfusion injury.Therefore,we propose the following hypothesis:Nurr1 could be induced in the regeneration after intestinal ischemia-reperfusion injury.Nurr1 might promote regeneration after intestinal ischemia-reperfusion injury by inhibiting the expression of p21,and inhibition of p21 expression by Nurr1 may be p53-independent.It could effectively improve the intestinal epithelial cell proliferation and promote the intestinal regeneration after ischemia/reperfusion injury by regulating the Nurr1/p21 pathway.In this study,the wild type Nurrl rat II/R injury experimental model,hypoxia/reoxygenation oxygen(H/R)model of IEC-6 cell line and some human ischemic intestinal tissue were used.Using advanced molecular biological methods,such as overexpression or interference the expression of Nurr1,immunoprecipitation,real-time quantity PCR,luciferase reporter gene and immunofluorescence,to detect the expressions of Nurrl and p21.To evaluate and detect the changes of morphology and function of small intestinal tissue and IEC-6 cell line in vitro and clarify the mechanism of p21 regulated by Nurr1 in the regeneration after II/R injury,also explore the regulatory role of Nurr1/p21 pathway,which provides a new theoretical basis and target for the intestinal regeneration after II/R injury.Part ?Nurr1 can be induced by intestinal regeneration after ischemia/reperfusion injury and proliferation induced hypoxia/reperfusion in IEC-6 cellsObjective:To investigate the expression of Nurr1 intestinal regeneration after ischemia/reperfusion injury and proliferation induced hypoxia/reperfusion in IEC-6 cells.Methods:Sixty healthy male Sprague-Dawley rats were randomly divided into sham operation group(sham group),intestinal ischemia and 3h of reperfusion group(I/R 3h group),ischemia and 6 h of reperfusion group(I/R 6 h group),ischemia and 12h of reperfusion group(I/R 12h group),intestinal ischemia and 24h of reperfusion group(I/R 24h group)and intestinal ischemia and 72h of reperfusion group(I/R 72h group).The small intestine I/R model was constructed by Megison method,and the superior mesenteric artery was clipped for 1 hours and then reperfusion.To detect the pathological changes of small intestine and the expression levels of Ki-67 and phosphorylated histone H3(pH3)in intestinal epithelial cells.Western blot and RT-PCR method were used to detect the changes of Nurr1 protein and mRNA levels in small intestinal tissue,and the correlation between Nurr1 and epithelial cell proliferation was analyzed.IEC-6 cells H/R model was used to detect the proliferation of 3h,6h,12h and 24h reoxygenation and H/R after 6h hypoxia by CCK-8 and immunofluorescence,and the changes of Nurr1 protein and mRNA levels in the cells were also investigated by western blot and RT-PCR.Results:1.In I/R 3h group,Ki-67,pH3 positive cells in intestinal tissue were less than sham group and further reduced in 6 hours of reperfusion group.In I/R 12h group,Ki-67,pH3 positive cells were increased,a further increase in I/R 24h group,and in I/R 72h group,the number of positive cells of reduced to the level of sham group.Compared to the small intestine Nurr1 protein and mRNA levels in the sham group,Nurrl mRNA and protein expression levels in I/R 3h group and I/R 6h group were significantly reduced(P<0.01).However,Nurr1 protein and mRNA levels rebounded in I/R 12h group,I/R 24h group and reached the highest expression at 24 h.In I/R 72h group,Nurr1 protein and mRNA expression was restored to sham group level.The expression of Nurr1 was positively correlated with the expression of pH3 and Ki-67 during the regeneration of intestinal mucosa.2.In the model of IEC-6 cells 6h hypoxia after reoxygenation,Nurr1 mRNA and protein expression levels in 3h and 6h group were significantly reduced compared to the control group(P<0.01).Nurr1 protein and mRNA levels increased in12h group,24h group.Nurr1 protein and mRNA in 24h group expression was restored to the level of the control group.The number of Ki-67 positive cells in IEC-6 cells 6h hypoxia and 3 hours of reoxygenation was decreased compared with normal group,and the amount of positive cells decreased further in 6h reoxygenation group.But the number of Ki-67 positive cells in 12h reoxygenation group increased,a further increase in the 24h reoxygenation group,and there was no significant difference compared to control group(P>0.05).Conclusion:1.Intestinal ischemia-reperfusion injury can lead to severe intestinal injury and activate regeneration mechanism of intestinal mucosa.2.Nurrl can be induced in intestinal regeneration after ischemia-reperfusion injury.3.The expression of Nurr1 was positively correlated with the proliferation of intestinal epithelium after intestinal ischemia/reperfusion injury.Part ?Nurr1 promote proliferation of IEC-6 cells after hypoxia/reoxygenation injury by inhibiting p21 by p53-independent mannerObjective:To establish the IEC-6 cells H/R model and explore the role of Nurr1 in H/R injury proliferation,and investigate the exact mechanism of Nurr1 effect on IEC-6 cells proliferation in H/R injury by overexpression or inhibition of Nurr1 expression,and further elucidate the effect of Nurr1/p21 pathway on cells proliferation in H/R injury.Methods:1.The effects of si-Nurr1 on cell proliferation and cell cycle:the cells were randomly divided into 3 groups:negative control group(N-control group),empty transfection group(Control group)and si-Nurr1 group.Western blot and RT-PCR methods were used to detect the changes of Nurr1 protein and mRNA levels.The cell proliferation and cell cycle were detected by CCK-8 and flow cytometry.2.Effects of si-Nurr1 cells on the level of p21 expression in IEC-6 cells:the cells were randomly divided into 3 groups:negative control group(N-control group),empty transfection group(Control group)and si-Nurr1 group.p21 protein and mRNA levels were detected by blot western and RT-PCR methods.The cell proliferation and cell cycle were detected by CCK-8 and flow cytometry after si-Nurr1 and/or siRNA-p21.And then study the effect of Nurr1/p21 pathway on cell proliferation.3.The inhibition of Nurr1 on p21 expression levels and the relationship with p53:after si-Nurr1 and/or si-p53,the expression level of p21 protein was detected by western blot and RT-PCR methods.The cell proliferation and cell cycle were detected by CCK-8 and flow cytometry.And then study the effect of Nurr1 on the inhibition of p21 and the relationship with p53.4.The effects of Nurr1 overexpression plasmid transfection on cell proliferation and cell cycle:IEC-6 cells were divided for three groups:negative control group(N-control group),empty transfection group(control group)and plasmid transfection of Nurr1 expression cells group(IEC6-Nurr1 group).Protein and mRNA levels of Nurr1,p21 and p53 were detected by blot western and RT-PCR methods.Cell proliferation and cell cycle were detected by CCK-8 and flow cytometry.Results:1.The H/R model was carried out after si-Nurr1 was transfected into IEC-6 cells.The proliferation in the siNurr1 cells after H/R injury appeared significantly inhibition of proliferation compared to the normal IEC-6 cells(P<0.01),and there was significant statistical difference between the two groups(P<0.01).The proportion of G1 phase cells in si-Nurr1 IEC-6 cells after 6h hypoxia and reoxygenation 24 h was significantly increased than in normal IEC-6 cells(P<0.01).2.The expression levels of p21 protein and mRNA in IEC-6 cells transfected with si-Nurr1 were significantly higher than those in the control group(P<0.01).Compared with H/R group,cell proliferation of si-Nurr1 group was significantly reduced(P<0.01).Cell proliferation of si-p21 group was significantly increased(P<0.01).Cell proliferation of si-p21 and si-Nurrl co-transfected was significantly increased in si-Nurrl group(P<0.01),and there was no statistical significance compared with H/R group(P>0.05).Compared with H/R group,the percentage of cells in G1 phase increased significantly in si-Nurrl group(P<0.01),the percentage of cells in G1 phase was significantly reduced in si-p21 group(P<0.01);and G1 phase cell cycle in si-p21 and si-Nurrl co-transfected group was significantly reduced compared with si-Nurrl group,and there was no statistical significance compared with H/R group(P>0.05).3.Compared w:ith the control group,the expression of p53 in Si-Nurrl cells was not significant change.After si-Nurrl and si-p53 were co-transfected into IEC-6 cells,the expressions of p53 and Nurrl in IEC-6 cells were significantly down-regulated,and the expression of Nurrl induced by p21 knockdown was not significantly inhibited by p53.Under the condition of H/R,the expression of Nurrl was inhibited,then knockdown of p21 expression could promote cell proliferation,whereas knockdown of p53 expression did not significantly increase cell proliferation,the differences between the two groups were significant(P<0.01).When the expression of Nurrl was significantly inhibited,then knockdown of p21 expression can significantly reduce the percentage of cells in G1 phase,and knockdown of p53 protein expression did not significantly reduce the percentage of cells in G1 phase and the difference between the two groups were significant(P<0.01).4.In Nurrl overexpression plasmid transfect into IEC-6 cells,Nurrl mRNA and protein appeared significantly increased,and the increase of Nurrl mRNA and protein significantly statistical difference compared to the normal IEC-6 cells(P<0.01).Overexpression of Nurrl could significantly inhibit the protein and mRNA expression levels of p21,while the expression level of p53 protein and mRNA were not significantly inhibited.After transfection with Nurrl overexpression plasmid into IEC-6 cells,the H/R model was carried out on IEC-6 cells,the time of hypoxia was 6h,and the reoxygenation was 12 and 24h.In Nurrl overexpression plasmid transfection of IEC-6 cells after H/R injury appears to accelerate the proliferation,and accelerate the proliferation had significant statistical difference compared with normal IEC-6 cells(P<0.01).The proportion of cells in G1 phase in Nurrl overexpression plasmid transfected into IEC-6 cells was significantly reduced compared with normal IEC-6 cells.Conclusion:1.Nurr1 can significantly promote the proliferation of IEC-6 cells after H/R injury,the promotion of cell proliferation is achieved by inhibiting the expression of p21 which can induced cell cycle arrest in the G1 phase.2.Inhibition of p21 expression by Nurr1 was p53-independent.Part ?The inhibition of p21 transcription by Nurr1 is achieved by directly binding the p21 promoter.Objective:To further explore the way of inhibiting the expression of p21 gene by Nurr1 via bioinformatics,CHIP and luciferase reporter gene.Methods:1.p21 gene promoter sequence and the Nurr1 binding sequence in the promoter region of p21 gene were detected by the method of bioinformatics.2.The application of chromatin immunoprecipitation(CHIP):Using CHIP assay,to detect the binding site of Nurr1 from the prediction of bioinformatics.3.To detect the effects of Nurr1 on the expression of p21 gene by Luciferase Report Gene method:Nurr1 binding sequence in p21 gene promoter region mutations were designed from the result of CHIP experiments,and cells were divided three groups according to the sequence:negative control plasmid group(control),p21 promoter wild type(WT)plasmid group and Nurr1 binding sites mutant plasmid(MUTANT)group.The plasmids were transfected to Nurr1 overexpression IEC-6 cells respectively.For detecting the fluorescence intensity,dual luciferase report gene detection reagent kit was used.Results:1.Bioinformatics predict the three most possible Nurrl binding sequence NBRE-like motifs(NLs),NL1(from-274 to-281),NL2(from-424 to-431),NL3(from-1784 to-1791)in the p21 gene promoter sequence.2.CHIP experimental results confirmed that the NL2 sequence design primers could be amplified by PCR method.3.The negative control plasmid group(control),p21 promoter wild type(WT)plasmid and Nurr1 binding sites NL2 mutant plasmid(MUTANT)respectively transfected to Nurrl overexpression IEC-6 cells.Using dual luciferase report gene detection reagent,the determination of fluorescence intensity results such as:fluorescence intensity in WT group was significantly increased than in the control group(P<0.01),and compared with WT group,the fluorescence intensity of MUTANT group was significantly enhanced(P<0.01).Conclusions:1.Nurrl can directly bind the Nurrl specific binding sites in the p21 promoter region.2.Nurrl inhibited the p21 gene transcription by binding Nurrl specific binding site in the p21 promoter region directly.Part IVExpressions of Nurrl,p53 and p21 in human ischemic intestineObjective:To study the expressions of Nurrl,p53 and p21 and its relationship with the proliferation in human ischemic intestine.Methods:1.Proliferation was detected by fluorescence examination of human normal and ischemic intestine tissues.2.The expressions of p21,p53 and Nurrl were used by western blot and RT-PCR in human normal and ischemic intestine tissues.Results:1.In normal mucosa of the intestine,villi arranged in neat rows and epithelial gap without expansion;and in ischemic intestine,villi disarranged and almost complete disruption and loss,epithelial gap widened or shedding,capillary congestion.The number of Ki-67 positive cells in the ischemic intestinal tissue was significantly decreased compared with the normal intestinal tissue,and the difference was statistically significant(P<0.05).2.Compared with normal intestinal tissue,the expression of Nurrl in the ischemic intestinal tissue was significantly decreased(P<0.01)5 p21 expression was significantly increased(P<0.01),while p53 expression was not significantly different(P>0.05).Conclusions:1.In the human ischemic intestine,the expression of Nurrl is inhibited,and the expression of p21 is induced,while the expression of p53 has no significant difference.2.The expression of Nurr1 was consistent with the proliferation of intestinal epithelial cells in human ischemic intestine. |
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