Influence Of HSPA12B On Pulmonary Vascular Permeability And Its Mechanisms In Sepsis-induced Acute Lung Injury

As a common infectious syndrome which threatens people’s lives horribly, sepsis owns a very high morbidity and a terrifying mortality in clinical research. Its progressive pathological deterioration leads to septic shock and multiple organ dysfounctions(MODS). ALI is one of the most common complications of sepsis. The pulmonary endothelial barrier dysfunction, which causes pulmonary vascular hyperpermeability, plays an important role in the occurrence and development of ALI. The increased pulmonary vascular permeability is a critical pathophysiological characteristic. Thus, protecting pulmonary vascular endothelial cells and maintaining the function of pulmonary endothelial barrier has the vital clinical significance in therapeutic interventions against ALI.Heat shock protein A12B(HSPA12B) is one of the HSP70 superfamily members and is different from other members. It is mainly expressed in vascular endothelial cells as a specific marker, involving in the generation and stabilization of vascellum. The upregulation of HSPA12 B compromises the heart dysfunction induced by sepsis, and relieves the injury of brain tissue in cerebral ischemiare perfusion. However, the exact role of HSPA12 B in the acute lung injury induced by sepsis and its potential mechanisms has remained unclear.By in-vitro and in-vivo experiments, our research investigated the effect of HSPA12 B on the pulmonary vascular permeability and its potential mechanisms. During in-vitro assay, the expression of HSPA12 B in HUVEC stimulated by LPS was studied; we also investigated the role of HSPA12 B high-expression in the pulmonary vascular permeability using an artificial highly-efficient expression plasmid. In vivo, the effect of HSPA12 B down-regulation on the pulmonary vascular permeability in CLP mice was observed.Part 1 The expression of HSPA12 B in HUVEC induced by LPS and its possible mechanismsits possible mechanisms. Objective Investigate the expression changes of HSPA12 B in HUVEC treated by LPS andMethods 1. HUVEC were stimulated with a final concentration of 1μg/ml LPS, then collect the cells at 0h, 3h, 6h, 9h, 12 h, 24 h respectively. The m RNA and protein levels of HSPA12 B were detected by RT-PCR and Western blot methods. 2. The cultured HUVEC were pretreated with the NF-kappa B, p38 inhibitor(NF-kappa B inhibitor, SB203580) and their control reagent DMSO respectively. And then, the protein level of HSPA12 B in HUVEC collected at 12 h following LPS stimulation was detected by Western blot method.Results 1. After incuced by LPS, the m RNA and protein level of HSPA12 B in HUVEC increased gradually over 0-12 h, up to the peak at 12 h and then decreased. 2. Compared with DMSO, NF-kappa B inhibitor and SB203580 can reduce the expression level of HSPA12 B in HUVEC induced by LPS.Conclusion The expression of HSPA12 B increases in HUVEC induced by LPS. Its possible mechanisms are associated with the NF-kappa B and p38 signal pathway.Part 2 Influence of HSPA12 B on permeability of HUVEC induced by LPSObjective Research the effect of HSPA12 B on permeability of HUVEC induced by LPS.Methods The HUVEC cultured in vitro were divided into four groups: control group(without any treatment); LPS group(1μg/ml LPS); LPS+HSPA12B group(theHSPA12B gene overexpression plasmid was transiently transfected into HUVEC, and then LPS of 1μg/ml was added); LPS+Flag group(the negative control plasmid was transiently transfected into HUVEC, and then LPS of 1μg/ml was added). The trans-endothelial electrical resistance(TEER) value of HUVEC was measured by MERSST×01 Electrode at 12 h.Results The TEER value of control group, LPS group, LPS+HSPA12B group, LPS+Flag group was 36±2?, 25±1?, 28±1?, 45±2? respectively. Compared with LPS group and LPS+Flag group, the TEER value of LPS+HSPA12B group increased significantly(P < 0.001).Conclusion HSPA12 B can reduce the permeability of HUVEC stimulated by LPS. So HSPA12 B may play a protection role in vascular endothelial barrier function.Part 3 Possible mechanisms of HSPA12 B affecting permeability of HUVEC stimulated by LPSObjective Explore the possible mechanisms of HSPA12 B affecting permeability of HUVEC induced by LPS.Methods The HUVEC cultured in vitro were divided into four groups: control group; LPS group; LPS+HSPA12B group; LPS+Flag group. LPS-treated cells were collected at 12 h, Western blot was applied to detect expression level of adhesion molecules VE-cadherin, ICAM-1, E-selectin, and expression level of permeability releated kinase MLC, SRC, cdc42. In addition, the expression level of VE-cadherin was examined by immunofluorescence.Results Compared with LPS+Flag group, the expression levels of VE-cadherin in LPS+HSPA12B group increased significantly at 12 h. The expression of the kinases MLC, SRC, cdc42 was also increased, without change of their active form P-MLC, P-SRC, P-cdc42. Immunofluorescence result showed that the expression level of VE-cadherin in LPS+HSPA12B group was higher than in LPS+Flag group.Conclusion The possible mechanisms of HSPA12 B affecting permeability of HUVEC stimulated by LPS are related to increased expression of VE-cadherin, MLC, SRC, cdc42.Part 4 Influence of HSPA12 B on lung permeability of mice in sepsis-induced acute lung injuryObjective Study the impact of HSPA12 B on lung permeability of mice in sepsis-induced acute lung injury.Methods The mice were randomly divided into four groups: sham operation group, CLP group, NC-CLP group, HSP12B-si RNA-CLP group. Mice were performed CLP surgery followed by nasal inhalation of nano-polymer mediated si RNA. Mice were further injected with Evans Blue via the jμgular vein at 30 h after CLP. Lungs were harvested to detect the content of EB dye at 2h after injection.Results Compared with NC-CLP group, mice lung tissue in HSPA12B-si RNA-CLP group became bluer obviously in general view. The Evans Blue dye content of mice lung tissue in sham operation group, CLP group, NC-CLP group,HSP12B-si RNA-CLP group, was 10.28±0.72μg/g 、 12.22±0.37μg/g 、11.97±0.79μg/g 、 15.65±2.36μg/g respectively. The Evans Blue dye content in HSP12B-si RNA-CLP group increased significantly comparing with NC-CLP group(P < 0.001).Conclusion Down-regulating the expression of HSPA12 B can increase permeability of mice lung obviously in sepsis-induced ALI. Conversely, if the expression of HSPA12 B was up-regulated, the lung permeability can be reduced and the pulmonary blood barrier function can be protected.