| Introduction:It is well known that the dysfunction of intestinal mucosal barrier is the key to initiating bacterial translocation,MODS and enterogenous hypermetabolism after severe burn injury.Maintaining the structure and function of intestinal mucosa and promoting the recovery of injuried intestine can lower enterogenic infection,inflammatory response and enterogenous hypermetabolism.The hormones secreted from intestifial endocrine cells are the most important messengers that regulate intestinal function, including cytoprotection,nutrition and cell proliferation.Among these is the intestinotrophic peptide glucagon-like peptide-2(GLP-2),which plays a significant role in the adaptive regulation of bowel mass and mucosal integrity.However,the mechanism of GLP-2 has not yet been elucidated.Objectives:This study was intended to design prokaryotic expression vector,induce GLP-2 expression in E.coli BLR(DE3),and investigate the protective effects of recombinant GLP-2 on intestinal mucosa of burned rats in vivo and on hypoxic intestinal epithelial cells in vitro.The intestinal epithelial cell line stably expressing GLP-2 receptor (GLP-2R) was constructed to explore the mechanisms of enteroprotection of GLP-2 and the regulation of transmembrane signal transduction of GLP-2R by caveolin-1.Material and Methods:1.AlwN I-digested and dephosphorylated pET-31b(+) DNA was used.The second amino acid,alanine,of GLP-2 coding sequences was replaced by glycine.The new sequences were cloned as tandem repeats interspersed with single methionine residues and placed downstream of a 125aa ketosteroid isomerase(KSI) gene and upstream of a His·Tag. The recombinant vector,GLP-2/pET31b(+),was transfected into the BLR(DE3) expression cells,and the target peptide production was induced by adding IPTG to the culture media. The purified GLP-2 with affinity chromatograph and CNBr cleavage was stored after cryodrying. 2.Rats of either sex infliced with 30%TBSAⅢ°burns were randomly divided into 4 groups:normal group(N),burn control group(C),recombinant GLP-2 group(Gr,treated with recombinant GLP-2,100nM.kg-1.d-1 subcutaneously for 7 days) and synthesized GLP-2 treated group(G,treatment with synthesized GLP-2,100nM.kg-1.d-1subcutaneously for 7 days).The rats were killed on 7d postburn and the following indexes were measured, including mucosal pathological examination,mucosa permeability,the ratio of mucosa wet weight and bowel mass or carcase weight,and the content of intestinal mucosal protein. For in vitro study,the GLP-2R/PCDNA3.1(+) was transfected into Caco-2 with Lipofectamine2000,and the stably transfected cells were selected and maintained by growth in G418.Individual cell clones were obtained by limited dilution cloning,verified by determining the GLP-2R protein expression with Western blot method,expanded for further studies,and named Caco2/GLP-2R(+).The cells were divided into 4 groups: normal group(N),hypoxia control group(C),recombinant GLP-2 group(Gr.treatment with recombinant GLP-2 after hypoxia for 8h,1000nM for 30min,4,24,72h) and synthesized GLP-2 treated group(G,treatment with synthesized GLP-2 after hypoxia for 8h,1000nM for 30min,4,24,72h),The following indexes were measured including cell proliferation. activities of lactate dehydrogenase and sucrase,the protein expressions of CDK4 and ZO1.3.The caveolin-1/pEGFPN2 plasmid was constructed.The small interference RNA of caveolin-1 obtained from an in vitro transcription reaction was transiently transfected into Caco2/GLP-2R(+).The cells were divided into 5 groups:normal group(AN),hypoxia control group(AC),GLP-2 group(AG,treatment with synthesis GLP-2 after hypoxia 8h, 1000nM for 30min,24h),caveolin-1 up-regulation group(CG,treatment with synthesized GLP-2 after hypoxia 8h,1000nM for 30min,24h);caveolin-1 down-regulation group(EG, treatment with synthesized GLP-2 after hypoxia for 8h,1000nM for 30min,24h).The kinase inhibitors including U0126,LY294002,SB203580 were uesed to explore the GLP-2 signal transduction pathway.The cAMP content and cell proliferation were detected.The phosphorylation of c-Raf,MEK1/2,ERK1/2,Akt and p90RSK were analysized to investigate the mechanism of enteroprotection of GLP-2 and regulation of transmembrane signal transduction of GLP-2 receptor by caveolin-1. Results:1.The sequence of GLP-2/pET31b(+) is confirmed by DNA sequencing,and the expressed GLP-2 peptide was verified by Western Blot.Approximate 100mg of the GLP-2 monomer were obtained in the current expression system.2.Histologically,the structure of intestinal mucosa was damaged as evidenced by the thinning of mucosa,shortening of villus,and decreasing of villus surface area in burn group on 7d postburn,while both recombinant GLP-2 and synthesis GLP-2 could alleviate intestinal mucosa injury obviously.Compared with those in burn group,the mucosa permeability significantly decreased in both GLP-2 groups.The ratio of mucosa wet weight and bowel mass or carcase weight,the content of intestinal mucosal protein were more significantly increased in both GLP-2 groups than those in burn control group.The in vitro results showed that GLP-2 directly stimulated proliferation of the hypoxic Caco2/GLP-2R(+) cells at 24,72h after GLP-2 treatment.There was no significant difference between recombinant GLP-2 and synthesized GLP-2.The activities of lactate dehydrogenase and sucrase had no significant changes at 72h after GLP-2 treatment.The protein expressions of CDK4 and ZO1 were increased in hypoxic Caco2/GLP-2R(+) cells at 72h after GLP-2 treatment.3.The data also showed that the cAMP content were increased and maintained at high level in cells treated with GLP-2.The phosphorylated GRK2 level increased after treated with 1000nM GLP-2.The effect of GLP-2 on stimulating cells proliferation was blocked by the kinase inhibitors U0126 and LY294002,but not SB203580.Western blot analysis of hypoxic Caco2/GLP-2R(+) cell treated with GLP-2 showed that ERK1/2 was activated,the expressions of phosphorylated c-Raf,MEK1/2,ERK1/2 and p90RSK of cellular nucleus were increased.The expression of phosphorylated Akt was enhanced too. Our results showed that caveolin-1 ehanced the phosphorylation of ERK and PI3 pathway and promoted cell proliferation.Meanwhile,the phosphorylation of cell signal transduction was weakened after knocking down the caveolin-1 by transfecting caveolin-1 siRNA into Caco2/GLP-2R(+) cell Conlusions:1.The prokaryotic expression is a new approach to product the GLP-2. 2.Both the recombinant GLP-2 and synthesized GLP-2 could alleviate intestinal mucosa injury in burn rats and promote proliferation of the hypoxic Caco2/GLP-2R(+) cells3.The GLP-2 stimulate proliferation of the hypoxic Caco2/GLP-2R(+) cells by activating ERK/PI3- but not P38-dependent pathway.4.The transduction of transmembrane signal of GLP-2 receptor is regulated by caveolin-1...
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