Role Of HSF1 In Endogenous Protective Mechanism Underlying Ventilator-induced Lung Injury In Mice: Relationship With HMGB1

Objective In this study,we evaluated the role of heat shock factor 1（HSF1）in ventilator-induced lung injury（VILI）in mice and its effect on high mobility group protein B1（HMGB1）,which can provide further basis for the mechanism of VILI.Methods There were forty SPF-grade healthy male C57BL/6 mice,aged 6-8 weeks,weighing 20-25 g.The mice were adaptively fed a standard rodent diet and freely with water（autoclaving）in a standard animal laboratory at a temperature of 22-24℃,air humidity of 30-70%,12:12 hour light–dark cycle for one week.Before anesthesia,they were fasted for 12 h and drank freely.Then,the mice were randomly divided into 4 groups（n=10 each）by the random number table method:control group（group C）,mechanical ventilation group（group M）,negative control group（group NC）and HSF1 siRNA pretreatment group（group siRNA）.The 5 nmol siRNA was diluted to 50μl with sterile phosphate buffer solution（PBS）.The mixture was swirled and rewarmed for 15 min at room temperature.Sodium pentobarbital（60 mg/kg）were used to anesthetize by intraperitoneal injection,and the NC-siRNA and HSF1 siRNA transfection complexes were injected into the airway of the NC group and the siRNA group,respectively,with50μl（5 nmol siRNA/50μl）.The group C and group M were intratracheal injected the same amount of PBS.All mice were anesthetized after 48 h.The longitudinal incision was cut at 1 cm in the suprasternal can cave in mice,and an endotracheal tube was inserted after tracheotomy.Group C kept spontaneous breathing after tracheotomy for 4 h,and the remaining of groups were mechanically ventilated（VT 35 ml/kg,RR 75 breaths/min,I/E1:2,FiO₂ 21%,PEEP 0 cm H₂0）for 4 h.The mice were closely observed during the experiment.Pentobarbital sodium were added intermittently to maintain anesthesia according to the state of mice.Mice were kept warm by heating blanket to prevent low temperature.The blood of right femoral artery was collected after endotracheal intubation and mechanical ventilation for arterial blood gas analysis.,The sternum was cut to expose the complete lung after the mice were sacrificed by aorta bloodletting under deep anesthesia.The superior lobe of right lung was weighed as wet weight（W）after drying surface moisture by using a clean filter paper,and then placed in a drying oven at 70℃ for 48 h to a constant weighing as dry weight（D）,then the wet/dry weight（W/D）ratio of the lung tissue was calculated and recorded.The middle lobe of the right lung was stained by 10%formalin with HE.The light microscope（x100）was used to observe pathological changes of pulmonary tissue,and the pulmonary tissue damage was scored by score standard.The remaining right lung tissue was placed in liquid nitrogen rapid freezing and then placed in an ultra-low temperature refrigerator at-80℃ for standby.The right main bronchial tube was clamped in a low temperature environment.Sterile PBS was injected by tracheal tube to lavage the left lung,repeating three times.The bronchoalveolar lavage fluid（BALF）was collected.Supernatant was extracted after centrifugation.The concentrations of TNF-α,IL-1βand HMGB1 in BALF were tested by enzyme-linked immunosorbent assay（ELISA）.Expression of HSF1 and HMGB1mRNA in lung tissue（by quantitative real-time polymerase chain reaction）and expression of HSF1 and HMGB1 protein in lung tissue（by Western Blot）were determined.Results Compared with group C,PaO₂ was significantly decreased after the end of ventilation,the lung injury score,W/D ration and concentration of TNF-α、IL-1β、HMGB1 in BALF and the expression of HMGB1 mRNA and protein in lung tissue were significantly increased（P<0.05）in group M,group NC and group siRNA（P<0.05）.Compared with group NC and group M,there were a significant decrease in PaO₂after the end of ventilation and HSF1 mRNA and protein,and a significant increase in lung injury score,W/D ration,concentration of TNF-α、IL-1β、HMGB1 in BALF and the expression of HMGB1 mRNA and protein in lung tissue in group siRNA（P<0.05）.Conclusion HSF1 is involved in the endogenous protective mechanism underlying VILI in mice,which may be related to the down-regulation of HMGB1 expression and attenuation of inflammatory response in lung tissues.