Protective Role Of Heat Shock Protein 70 In Intestinal Mucosal Injury After Severe Burn

Multiple organ dysfunction still remains as a major cause of burn-related deaths, hypoxic/ischemic insult is increasingly recognized as a primary effector of organ damage after severe burn injury. Hypoxia/ ischemia may result in the dysfunction of cellular energy metabolism, with consequent trigger the inflammatory responses, which ultimately result in cell necrosis and apoptosis. As one of the most sensitive organ to hypoxia, the intestinal hypoxic/ischemic insult has been shown in the course of severe burn injury. There is evidence to suggest that the intestinal mucosal barrier function might be breakdown after serious trauma, burn injury, etc. This breakdown has been blamed for the translocation of indigenous gut bacteria and the aetiological factor of septic complications and multiple organ system failure following severe burn. These facts suggest that preservation of the intestinal mucosal barrier is, therefore, of paramount clinical importance for the outcome of burn victims.Heat shock proteins (HSPs) are a large family of highly conserved proteins that contribute to cell survival after various pathological and physiological stresses. HSPs induction is associated with remarkably enhanced tolerance to subsequent non-thermal stress. Following heat shock or other stresses, Hsps are produced after transactivation of the genes by a family of DNA-binding proteins called the HSF. HSF binds to a specific consensus heat shock regulatory element (HSE) in the heat shock gene promoter to exert the transcriptional activation. One of the most conserved and best characterized inducible class of HSPs is the 70-kDa heat shock protein 70(HSP70). Recent studies have reported the importance of several endogenous cellular factors that protect intestinal epithelial cells from the effects of stress and injury. Of these, inducible HSP70 have been shown to increase survival of intestinal epithelial cells against oxidant and heat-induced stress.The mechanism of this protective effect of HSP against hypoxic/ischemic injury has not been elucidated fully, and the protective potential of HSP70 against intestinal mucosal injury inburns has not been investigated, though various studies have shown the involvement of HSP70 in conferring protection against cellular damage.Therefore, the present study was designed to investigate the expression of HSP70 in intestinal mucosal after severe burn injury and to evaluate the protective role of HSP70 in intestinal mucosal injury after severe burn in rats, The possible mechanism for this protective effect is discussed.Material and MethodsFirstly, with model of 30% TBSA of full-thickess burned rats, the expression and contribution of HSP70 and HSF1 in intestinal mucosal were detected at various time points post-burn.Secondly, sodium arsenite(SA) was used to induce the expression of HSP70 in intestinal mucosa. To confirm the effect of HSP70 further, quercetin was used to block the induction of HSP70 by injection with SA. Rats were inflicted with 30% TBSA of full-thickness burn injury.the expression of heat shock protein 70 and Bcl-2 in intestinal mucosa were determine. Plasma endotoxin, DAO, D-lactic acid content and MDA, SOD content in intestinal mucosa were determined, histological assessment of muscosal injury and apoptosis of intestinal epithelial cells was detected.Thirdly, whole long HSP70 gene was cloned into the cosmid vector pAxCAwt, co-transfected with adenovirus DNA-TPC into 293 cells. The recombinant adenovirus (Ad-HSP70) was constructed successfully by the clones selection. Ad-HSP70 was transfected into the intestinal epithelial cell line (IEC-6) in vitro and then suffered from 90 min of hypoxia followed by 60 min of reoxygenation, The LDH leaking, cell viability, ATP level, apoptosis and dead cells ratio, Intracellular calcium concentration, mitochondria membrane potential (mt) were evaluated. The expression of HSP70, Bcl-2, pro-Caspase-3 and cytochrome C were detected, and the contribution of HSP70 and cytochrome C in IEC-6 were investigated.Results1. The expression of HSP...