| Endoplasmic Reticulum(ER) is an important organelle in eukaryocyte. It’s verysensitive to the changes of internal and external environment, so that oxidative stress,ischemic injury,calcium homeostasis and overexpression of normal and abnormal foldedproteins would cause it dysfunctions. This process and response are called Endoplasmic. Inthe early state, ERS is a state for overexpression of Glucose-regulated proteins78(GRP78),Glucose-regulated proteins94(GRP94), calprotectin and CCAT/enhancer-bindingproterin-homologous protein(CHOP). Current studies showed that ERS mainly participatesin regulation of cell-differentiation and apoptosis. But it’s not clear that whether ERSparticipates in the functional regulation of cell. There are some evidences that suggestGRP78and CHOP could participate in regulation of skeleton reconstruction of vascularendothelial cell(VEC)after septic shock and cause the changes of vascular permeability byregulating the activity of IP3R and affecting the release of Ca2+in ER. Nevertheless, thereneeds further studies to prove that whether the changes of vascular permeability after septicshock are related to ERS and whether GRP78and CHOP play effects via regulation IP3Rand release of intracellular cacium.Methods:Experiments are divided into in vivo and in vitro experimentsIn vivo experiment: septic shock models of rats was established by Cecal Ligation andPuncture(CLP). Experiments were devided into seven groups: CLP1h, CLP2h, CLP4h,CLP6h, CLP8h, CLP6h+taurine and control group. spectrophotometer was used to measurethe fluorescein that leaked out to pulmonary tissue to reflect the changes of vascularpermeability. The expression of protein GRP78, CHOP and IP3R was determined bywestern blot. Intracellular calcium concentration was detected by calcuim imagingtechnology. In vitro experiment:4healthy and adult rats were used to take the pulmonary arteryand vascular endothelial cell(VEC) for primary culture.2μmol/L of thapsigargin was usedto induce ERS in VEC. The vascular permeability was determined by Transwell method.the formation of strss fiber was determined by laser confocal microscopy. The expression ofprotein GRP78, CHOP and IP3R was determined by western blot. Intracellular caruimconcentration was detected by calcuim imaging technology. Statistical methods were usedto analyze the protein expression\intracellular free calcuim concentration and vascularpermeability to discuss whether the expression of GRP78and CHOP regulate intracellularCa2+level and pulmonary vascular permeability through the IP3receptor pathway.Results:1. in vivo experiment:(1) Changes of vascular permeability and expression of ERS proterin in rats afterseptic shock:1hour after CLP, fluorescent permeability rate began to rise in each group.As the time went on, fluorescent permeability was gradually increased. The expressions ofprotein GRP78and CHOP was began to rise1hour after CLP. As the time extended, otherexpressions continued to increase. Statistical analysis displays they were significantlypositively correlated with the changes of fluorescent permeability rate.(2) Changes of stress fiber in VEC:1hour after CLP, F-actin was mainly distributedaround cell membrane in VEC from main pulmonary artery of septic shock rats. It swelledswelling outward as burr shape and mainly seen in cytoplasm. Cellular morphologychanged as septic shock prolonged. As stress fiber was increased the arrangement was moreirregular and burr outward was more and more obvious. In control group, F-actin was notobvious and the cell morphology is more regular.(3) Expression of IP3Receptor: The protein expression of IP3R was increased as thetime extended after spetic shock in the lung tissue.1hour after CLP, the protein expressionof IP3R began to increase. As the time extended, it continued to rise. Statistical analysisdisplayed they were positively correlated with the expressions of GRP78and CHOP.(4) Changes of calcium concentration in VEC after septic shock:1hour after CLP,calcuim concentration in VEC was increased in rats after septic shock. As the time prolonged, it was continously increased. Statistical analysis displayed the expression ofGRP78and CHOP were positively correlated with changes of caluim concentration. Andthe expression of IP3R.(5) Beneficial effect of taurine: Compared to group CLP6h，200mg/kg of taurinecould significantly decrease the vascular permeability, meanwhile, after administration oftaurine, the expressions of GRP78, CHOP and IP3R, and calcuim concentration in VECwere significantly decreased.2. in vitro experiment:(1). Changes of vascular permeability and expression of GRP78, CHOP and IP3R:2μ mol/L of β-Thapsigargin significantly decreased the fluorescent permeability ofcultured VEC. As the time of incubation prolonged, the permeability of VEC wassignificantly increased, meanwhile, the expressions of protein GRP78,CHOP and IP3Rincreased as the time extended. Statistical analysis displayed the permeability of VEC wassignificantly positively correlated with the expression of protein GRP78and CHOP.(2) Changes of stress fiber and calcium concentration in VEC: β-Thapsigargintreatment significantly increased the expression of F-actin and the concentration of calcuimin VEC.(3) Beneficial effect of taurine: As compared to6h TA treatment group，200mg/Ltaurine treatment could significantly decrease. The permeability of VEC and decrease theexpressions of GRP78, CHOP and IP3R, meanwhile, the calcuim concentration of VEC wasalso significantly decreased after taurine treatment.Conclusion:1. Septic shock can induce endoplasmic reticulum stress in vascular endothelial cells.ERS participats in the occurrence of vascular permeability increasing. Protein GRP78andCHOP are the important regulatory molecule, in vascular permeability after shock.2. Protein GRP78and CHOP may regulate the formation of stress fiber andvascular permeability via regulation of IP3R and the concentration of Ca2+. |
