Effect Of Rapamycin On NLRC4 Inflammasome Activity In Ventilator-induced Lung Injury In Rats

Objective Ventilator-induced lung injury（VILI）model of rats was established through mechanical ventilation at high tidal volumes.The influence of Rapamycin（RAPA）on the activity of NOD-like receptors C4（NLRC4）in the lung tissue of rats was evaluated,and the influence of Rapamycin on VILI and its mechanism were discussed.Methods Thirty-six healthy and clean male SD rats,aged 6-8 weeks,weighing 200-250g,were randomly divided into three groups（n=12 each）:control group（group C）,group VILI and rapamycin intervention group（group RAPA）.Group RAPA received intraperitoneal injection of rapamycin(4 mg·kg^-1·d^-1)once a day for three consecutive days before surgery,while group C and group VILI were given equal volume of saline as control.The rats were anesthetized by intraperitoneal injection of 2%pentobarbital sodium 80 mg·kg^-1,BP and HR of rats were monitored by puncture and catheterization of left femoral artery.The right external jugular vein catheter for intravenous cisatracurium pump to maintain muscle relaxation.After tracheotomy,a 14G tracheal tube was inserted into the trachea.Spontaneous respiration was retained in group C,group VILI and group RAPA were connected to RWD407 small animal ventilator for mechanical ventilation,parameters were set as follows:RR 80 times/min,Fi O₂21%,PEEP 0 cm H₂O（1 cm H₂O=0.098 k Pa）,I∶E 1∶1,V_T20 ml/kg,continuous mechanical ventilation for 4 h.Blood samples of femoral artery were collected for blood gas analysis,Pa O₂was recorded,and the concentrations of interleukin-1β（IL-1β）and interleukin-18（IL-18）in serum were detected according to the instructions of the ELISA kit.Rapid thoracotomy under deep anesthesia to extract lung tissue,ligation of the right lung and lavage of the left lung with PBS precooling at 4℃,then the bronchoalveolar lavage fluid（BALF）of the left lung was collected,the neutrophils count as well as the concentrations of IL-1βand IL-18 were determined.The upper lobe of the right lung was fixed with 4%paraformaldehyde to make paraffin embedded sections,and then lung tissue was stained with HE under light microscope to observe the pathological results,and the lung injury was evaluated.The ratio of wet weight to dry weight（W/D）of the middle lobe of the right lung was measured.The remaining right lung tissue was stored in a refrigerator at-80℃.The expression of mammalian target of rapamycin（m TOR）,NLRC4 and cysteine aspartic protease（caspase-1）was detected by Western blot,the expression of NLRC4 mRNA was detected by RT-PCR.Results The lung tissue structure of rats in group C was still intact,no obvious injury was observed,no obvious edema was observed in the lung interstitium,and the alveolar cavity was clearly visible.Compared with group C,lung tissue structure of rats in group VILI was damaged,lung interstitium was edema,alveolar walls thicken,or even rupture and fusion,and a large amount of exudate could be seen in lung interstitium and alveolar cavity.The pathological changes of lung tissue in group RAPA were less than that in group VILI,the alveolar structure was relatively intact,the lung interstitium and alveolar exudate were relatively reduced,and the degree of lung injury was less than that in group VILI.Meanwhile,lung tissue W/D ratio,lung injury score,neutrophil count in BALF,concentrations of IL-1βand IL-18 in serum and BALF were increased in group VILI and group RAPA,while Pa O₂decreased.The expressions of m TOR,NLRC4,caspase-1 and NLRC4 mRNA in lung tissues were up-regulated（P<0.01）.Compared with group VILI,W/D,lung injury score,neutrophil count in BALF,concentrations of IL-1βand IL-18 in serum and BALF were decreased in group RAPA,while Pa O₂increased.The expressions of m TOR,NLRC4,caspase-1 and NLRC4mRNA in lung tissues were down-regulated（P<0.05）.Conclusion The mechanism by which rapamycin alleviates VILI in rats may be related to inhibiting activation of m TOR signaling pathway and inhibiting the activity of NLRC4 inflammasomes in rats.