Heat Shock Protein A12B Protects Vascular Endothelial Cells Against Sepsis-Induced Acute Lung Injury in Mice.

BACKGROUND: Pulmonary endothelial injury is a critical process in the pathogenesis of acute lung injury (ALI) during sepsis. Heat shock protein A12B (HSPA12B) is mainly expressed in endothelial cells and protects against several harmful factors. However, the effects of HSPA12B in sepsis-induced ALI and its potential mechanisms of action remain unclear. METHODS: For in vivo experiments, C57BL/6 mice were randomly divided into four groups (n=15): a sham operation group, a cecal ligation and puncture (CLP) group, a HSPA12B siRNA-CLP group and a negative control (NC) siRNA-CLP group. The mice were treated by nasal inhalation of 2-OMe-modified HSPA12B siRNA or NC siRNA. Sepsis was induced by CLP. Samples were harvested 24 and 48 hours post-CLP surgery. Pathological changes and scoring of lung tissue samples were monitored using hematoxylin and eosin staining. Levels of pro-inflammatory cytokines (e.g., interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and IL-6) and myeloperoxidase activity in bronchoalveolar lavage fluid were analyzed by ELISA. Pulmonary edema was assessed using a wet-to-dry weight ratio. Neutrophils and alveolar macrophages were counted using flow cytometry. Pulmonary endothelial cell apoptosis was detected by TUNEL staining. Expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blot analysis. In addition, 7-day survival was recorded. For in vitro experiments, human umbilical vein endothelial cells were pre-transfected with HSPA12B siRNA or pIRES2-EGFP-HSPA12B-Flag plasmid and treated with lipopolysaccharide; subsequently, the expression levels of MAPK family signaling molecules and caspase-3 were measured by Western blotting. RESULTS: Nasal inhalation of nano-polymer-encapsulated HSPA12B siRNA specifically downregulated mRNA and protein expression levels of HSPA12B in lung tissues. The administration of HSPA12B siRNA aggravated lung pathological injury, upregulated pro-inflammatory cytokine (e.g., IL-1ß, TNF-α, and IL-6) expression, and increased myeloperoxidase activity, neutrophil infiltration, pulmonary edema, and pulmonary endothelial cell apoptosis. Additionally, HSPA12B knockdown worsened survival after CLP surgery. The potential protective mechanisms of HSPA12B may involve the inhibition of ERK phosphorylation and caspase-3 activation in vivo and in vitro. CONCLUSION: HSPA12B protected against sepsis-induced ALI. The potential mechanism may be partly due to the inhibition of ERK phosphorylation and caspase-3 activation. These findings provide a potential therapeutic target for treating sepsis.

Heat shock protein A12B protects against sepsis-induced impairment in vascular endothelial permeability.

BACKGROUND: As a common and life-threatening infectious syndrome, sepsis contributes significantly to morbidity and mortality in clinical settings. Vascular endothelial injury and hyperpermeability play an important role in the development of sepsis-induced organ dysfunction. Heat shock protein A12B (HSPA12B) is one of the HSP70 superfamily members and is mainly expressed in vascular endothelial cells. The present study was performed to investigate the role of HSPA12B in endothelial barrier dysfunction during sepsis. METHODS: Human umbilical vein endothelial cells (HUVECs) were stimulated with 1 µg/mL of lipopolysaccharide (LPS) and harvested at 0, 3, 6, 9, 12, and 24 h. The messenger RNA and protein levels of HSPA12B were detected by Real Time-polymerase chain reaction and Western blot. Upregulation of HSPA12B was induced by transfection of pIRES2-EGFP plasmid carrying the HSPA12B complementary DNA. The in vitro effect of HSPA12B overexpression on endothelial permeability was manifested by the transendothelial electrical resistance value, expression of the adhesion molecules VE-cadherin, and the level of permeability-related kinase myosin light chain, SRC, and CDC42. Mice received cecal ligation and puncture surgery followed by nasal inhalation of nano-polymer-mediated siRNA. Lung endothelial permeability was assessed via intrajugular vein injection of Evans Blue 30 h after cecal ligation and puncture. RESULTS: After LPS induction, the messenger RNA and protein level of HSPA12B in HUVECs increased and peaked at 12 h, whereas they returned to the baseline level at 24 h. Overexpression of HSPA12B can reduce the permeability of HUVEC stimulated by LPS in vitro, while increasing the expression of VE-Cadherin, myosin light chain, and CDC42. On the other hand, downregulating the expression of HSPA12B can significantly increase lung permeability in mice with sepsis-induced vascular injury. CONCLUSIONS: HSPA12B plays a protective role in vascular endothelial barrier dysfunction by preserving the endothelial permeability during sepsis.

Ultrasound-guided medial branch of the superior laryngeal nerve block to reduce peri-operative opioids dosage and accelerate patient recovery.

BACKGROUND: To explore whether the medial branch block of superior laryngeal nerve can reduce the stress response of patients undergoing intubation and further reduce the dosage of opioids. METHODS: 80 patients undergoing gynecological laparoscopic surgery were selected, and randomly divided into 4 groups. All patients in the experimental groups received bilateral internal branch of superior laryngeal nerve block and transversus abdominis plane block. But the dosage of sufentanil used for anesthesia induction in the group A, B, and C was 0.4, 0.2, and 0µg/kg, respectively. Group D do not underwent supralaryngeal nerve block and the dosage of sufentanil was 0.4µg/kg. The heart rate (HR) and mean arterial pressure(MAP) were recorded at the time of entering the operating room(T1), before intubation after induction(T2), immediately after intubation(T3), 5min after intubation(T4), before extubation(T5), immediately after extubation(T6), 5min after extubation(T7). We also recorded the stay time in the recovery room, the number of cases of postoperative sore throat, the number of cases of nausea and vomiting, the first intestinal exhaust time, the length of hospital stay after operation. RESULTS: The HR of group A, C and D at T3 was significantly higher than that at T2(P < 0.01), while the HR of group B had no significant change. The HR of group A, C and D at T4 was lower than that at T3(P < 0.01), while the HR of group B had no obvious change. The HR of group C and D at T3 was significantly higher than that at T1 (P < 0.01). The MAP of group A and D at T4 was significantly lower than that at T1 (P<0.001). The first postoperative intestinal exhaust time in group A, B and C was significantly shorter than that in group D. The length of hospital stay after operation in group B and C was shorter than that in group D. CONCLUSIONS: Ultrasound-guided superior laryngeal nerve block combined with 0.2µg/kg sufentanil can reduce the intubation reaction, have better hemodynamic stability, reduce the first postoperative intestinal exhaust time and postoperative hospital stay, thereby accelerating the postoperative recovery of patients.

MiR-4505 aggravates lipopolysaccharide-induced vascular endothelial injury by targeting heat shock protein A12B.

Heat shock protein family A member 12B (HSPA12B) is a heat shock protein primarily expressed in endothelial cells. Our previous study showed that it was protective against endothelial injury induced by lipopolysaccharide (LPS). The present study was performed to investigate whether micro (mi)RNA was involved in HSPA12B expression in endothelial cells challenged by LPS. We first screened the miRNA candidates potentially related to HSPA12B by bioinformatics analysis. Then the mimics of the miRNA candidates were transfected into human umbilical vein endothelial cells (HUVECs) to investigate the miRNAs that negatively regulated HSPA12B expression. The miRNA expression was also determined in LPS­stimulated HUVECs. Dual luciferase activity assay was performed to confirm the relationship between the candidate miRNA and HSPA12B. Role of nuclear factor (NF)­κB in the miRNA expression was investigated by using its inhibitor. Finally, the role of the miRNA on LPS induced injury was investigated. Eleven miRNAs were screened by bioinformatics analysis and 4 of them could inhibit HSPA12B expression at both mRNA and protein levels. Among the 4 miRNA candidates, only miR­4505 was highly expressed in HUVECs stimulated by LPS. Luciferase analysis showed that miR­4505 directly interacted with the 3'untranslated region of HSPA12B. LPS­induced upregulation of miR­4505 was blocked by NF­κB inhibitor. Transfection with miR­4505 mimics reduced the transendothelial electrical resistance and vascular endothelial­cadherin expression. The scratch test demonstrated that miR­4505 inhibited endothelial migration capacity. In conclusion, miR­4505 downregulates the expression of HSPA12B and aggravates the LPS­induced vascular endothelial cell injury.

Plasma HSPA12B is a potential predictor for poor outcome in severe sepsis.

INTRODUCTION: Endothelium-derived molecules may be predictive to organ injury. Heat shock protein (HSP) A12B is mainly located in endothelial cells, which can be detected in the plasma of septic patients. Whether it is correlated with prognosis of sepsis remains unclear. METHODS: Extracellular HSPA12B (eHSPA12B) was determined in plasma of septic mice at 6 h, 12 h, 24 h and 48 h after cecal ligation and puncture (CLP). It was also detected in plasma of patients with severe sepsis, sepsis, systemic inflammatory response syndrome and healthy volunteers. The predictive value for prognosis of severe sepsis was assessed by receiver operating curve (ROC) and Cox regression analyses. RESULTS: eHSPA12B was elevated in plasma of CLP mice at 6 h and peaked at 24 h after surgery. A total of 118 subjects were included in the clinical section, including 66 patients with severe sepsis, 21 patients with sepsis, 16 patients with SIRS and 15 volunteers. Plasma eHSPA12B was significantly higher in patients with severe sepsis than in patients with sepsis, SIRS and volunteers. The level of eHSPA12B was also higher in non-survivals than survivals with severe sepsis. The area under the curve (AUC) of eHSPA12B in predicting death among patients with severe sepsis was 0.782 (0.654-0.909) in ROC analysis, much higher than that of IL-6 and IL-10. Cox regression analysis showed that cardiovascular diseases, IL-6 and eHSPA12B were risk factors for mortality in patients with severe sepsis. Survival curve demonstrated a strikingly significant difference between 28-day survival rates of patients with an eHSPA12B lower or not lower than 1.466 ng/ml. CONCLUSIONS: Plasma eHSPA12B is elevated in both septic mice and patients. It may be a good predictor for poor outcome in patients with severe sepsis.