Role Of Vasodilator Stimulated Phosphoprotein Phosphorylation In Intestinal Barrier Dysfunction Following Hemorrhagic Shock In Rats

Intestinal epithelial permeability is increased in victims of severe trauma or hemorrhagic shock. By permitting bacteria or microbial products to cross from the lumen into the systemic compartment, intestinal barrier dysfunction might contribute to the development of systemic inflammatory respone syndrome(SIRS) and multiple organ dysfunction syndrome(MODS). Based on the basic research, tight junction which selectively regulate the passage of molecules and ions via the paracellular pathway play an important regulatory role in epithelial paracellular permeability and barrier function. The function of tight junction is mainly determined by an array of protein complexes, and the intestinal barrier dysfunction after hemorrhagic shock is related with the decrease of ZO-1 protein expression and disassociation of these protein complexes. High-fat enteral nutrition prevente loss of tight junction structure protein zonula occludens-1(ZO-1) and preserve intestinal barrier function. In human microvascular endothelial cells, ZO-1 immunoprecipitate for the existence of a phosphorylated proteins as targets for barrier regulation, this protein was identified as vasodilator-stimulated phosphoprotein(VASP), which was originally discovered as a focal adhesion protein in blood disc, also localizes to endothelial junctional complexes and colocalizes with ZO-1, and plays a central role in the regulation of barrier function via changing the bounding with ZO-1 after phosphorylated by PKA at Ser-157. Since VASP also distributes in the intestinal epithelial cell(IEC), does VASP phosphorylation participates in the regulation of intestinal barrier dysfunction after hemorrhagic shock? If it does, will it does that through ZO-1? So with hemorrhagic shock model of rats, we observe the role of VASP phosphorylation in intestinal barrier dysfunction after hemorrhagic shock, and its possible mechanism. Methods:The experiments were conducted in two parts.Part I. Role of VASP phosphorylation in intestinal barrier dysfunction after hemorrhagic shock1. With the hemorrhagic shock rats of different time(1h, 2h, 4h), the changes of intestinal barrier function, reflected by plasma diamine oxidase(DAO) activity, hematoplasma D-lactate content and mucous membrane of small intestine histomorphology,and that of VASP and phospho-VASP(Ser-157) expression in small intestine mucous membrane were observed to analyze the relationship between these protein expression and intestinal barrier function.2. With the 2h shock rats, the effect of PKA agonist cAMP, which phosphrolates VASP at Ser-157, on intestinal barrier dysfunction after hemorrhagic shock were observed to illuminate the role of VASP phosphorylation in intestinal barrier dysfunction after that.Part II. Mechanism through which VASP phosphorylation participate in the regulation of intestinal barrier dysfunction after hemorrhagic shockThe change of protein expression of ZO-1 after hemorrhagic shock(1h, 2h, 4h), and the effect of PKA agonist cAMP on the protein expression of ZO-1 after 2h shock were observed to explain the possible mechanism.Results:1. Compared with normal control, plasma DAO activity and hematoplasma D-lactate content increased after hemorrhagic shock, lightly after 1h shock, and seriously after 2h and 4h shock(P<0.01), the mucous membrane of small intestine histomorphology showed a significant injury after shock especilly since 2h shock. VASP expression decreased gradually after shock(P<0.01), while phospho-VASP(Ser-157) increased obviously (P<0.01), but the amplification decreased since 2h shock. cAMP could significantly increase the protein expression of phospho-VASP(Ser-157) (P<0.01), and decrease plasma DAO activity and hematoplasma D-lactate content after 2h shock(P<0.01), and lighten the injury in the mucous membrane of small intestine histomorphology after 2h shock.2. Compared with normal control, the protein expression of ZO-1 decreased gradually after shock(P<0.01), especially after 2h and 4h shock, and cAMP could increase the protein expression of ZO-1 as compared with 2h shock group(P<0.01). Conclusions: VASP phosphorylation is an important relative factor which contributes to intestinal barrier dysfunction after hemorrhagic shock in rats, which participates into the regulation of intestinal barrier dysfunction after hemorrhagic shock possibly through ZO-1.