Study On CELF1 In The Pathogenesis Of Parenteral Nutrition-Associated Intestinal Atrophy

Aims:Intestinal atrophy is one of the severe manifestations of parenteral nutrition(PN),which is mainly associated with decline in proliferation and excessive apoptosis of intestinal epithelial cells(IECs).CELF1(CUGBP,Elav-like family 1)is one of the RNA binding proteins that modulate the expression of target m RNA molecules by regulating its translation and degradation,and has been shown to be involved in the pathogenesis of a variety of diseases.In this study,intestinal atrophy along with the increased expression of CELF1 was observed in a rodent model of PN.Besides,substantial evidences form in vitro experiments also revealed that CELF1 may play pivotal roles in modulating the proliferation and apoptosis of IECs.This study is divided into two sections.In section I(IEC proliferation),p53 was identified as one of the downstream genes of CELF1.In PN rats,decline in IEC proliferation was observed,associated with aberrant expression of CELF1 and p53,but their causal relationship needs to be further addressed.In section II(IEC apoptosis),apoptosis inducing factor(AIF)was identified as one of the downstream genes of CELF1.We found that olive oil-supplemented lipid emulsion(OOLE)-induced IEC apoptosis was mediated via activation of CELF1/AIF pathway,whereas supplementation of choline in OOLE could alleviate IEC apoptosis via suppression of CELF1/AIF pathway,but the underlying mechanisms need to be fully addressed.Methods:Section I: an SD-rat model of PN was established.Intestinal atrophy was examined by HE staining,and the proliferation of IECs was evaluated by Brd U incorporation;the expression of CELF1 and p53 was analyzed by Q-PCR,westernblot and IHC;the association of CELF1 and p53 m RNA was examined by RNP-IP.Caco-2,HCT-116 and IEC-6 cell lines were used as in vitro models,and the expression level of CELF1 was either up-or down-regulated by over-expression plasmids or si RNAs.The effects of CELF1 on the expression of p53,on the cell cycle and on the proliferation were studied. Moreover,the underlying mechanism by which CELF1 regulates p53 was analyzed by m RNA degradation assay,polysomal profile analysis and nascent protein synthesis.Section II:Caco-2 cells were treated with olive oil-supplemented lipid emulsion(OOLE)as an IEC model in vitro.Changes in the apoptosis and mitochondria membrane potential were evaluated by Annexin V/PI staining and JC-1 probes,respectively.The expression of CELF1 and apoptosis inducing factor(AIF)was assessed by Q-PCR,westernblot and IF.Protein stability,polysomal profile analysis and nascent protein synthesis were examined to explore the mechanism for CELF1 regulation.Moreover,a rodent model of PN(OOLE as lipid emulsion)with supplementary choline was established.The intestinal expression of CELF1 was assessed by westernblot and IHC;IEC apoptosis was determined using TUNEL assay.Caco-2 cells were treted with OOLE and supplementary choline,as an IEC model in vitro.Changes in the expression of CELF1 and AIF by supplementary choline were evaluated as described above.RNP-IP assay was performed to study the mechanism for CELF1 regulation by choline.Results:Section I: PN induced significant intestinal atrophy in SD rats,characterized by decreased villus height,decreased crypt depth and reduced Bru U incorporation.The expression of CELF1 and p53,along with the association between CELF1 and p53 m RNA was up-regulated by PN.Ectopic over-expression of CELF1 significantly suppressed the proliferation of Caco-2,HCT-116 and IEC-6 cells,whereas depletion of CELF1 by si RNA obviously promoted the cell proliferation.Moreover,G2/M arrest along with increased p53 expression was observed in the HCT-116 cells with over-expression of CELF1.Importantly,the regulation of p53 by CELF1 occurred at post-transcriptional level.The m RNA degradation assay,polysomal profile analysis and nascent protein synthesis revealed that up-regulation of p53 by CELF1 was directly mediated via stimulating its m RNA translation instead of affecting its m RNA stability.Section II: Up-regulation of CELF1 and apoptosis-inducing factor(AIF)was observed in OOLE-treated cells,suggestive of CELF1/AIF pathway in OOLE-induced IEC apoptosis.The protein expression of CELF1 was up-regulated by OOLE in a dose-and time-dependent manner,but the m RNA expression of CELF1 was unchanged.Analysis by polysomal profiling and nascent protein synthesis revealed that the regulation of CELF1 by OOLE treatment was mediated by directly stimulating its m RNA translation.In a rat model of PN,substantial reduction in apoptotic rate along with decreased expression of CELF1 was observed when supplementary choline was added to OOLE.In cultured Caco-2 cells,supplementary choline attenuated OOLE-induced apoptosis and mitochondria dysfunction.In addition,the expression of CELF1 and AIF was significantly decreased by supplementary choline compared to OOLE alone.Mechanistically,supplementary choline surpressed the expression of CELF1 by increasing the recruitment of CELF1 m RNA to processing bodies,thus leading to acceleration of m RNA degradation and suppression of m RNA translation.Conclusions:Both decline in proliferation and excessive apoptosis during PN support are closely related to the aberrant expression of CELF1.PN increases the expression of CELF1 and p53 in intestinal mucosa,leading to G2/M arrest and consequent proliferation defect.CELF1/AIF is a new pathway identified in OOLE-induced apoptosis,and importantly,supplementary choline exhibits effective protection against OOLE-induced IEC apoptosis through suppression of CELF1/AIF pathway.