Effect And Mechanism Of Dexmedetomidine On Mitochondrial Dynamic Equilibrium During Endotoxin-Induced Acute Lung Injury

Objective Sepsis-induced acute lung injury is a complex life-threatening disease in clinic,which is a difficult problem in the field of critical care medicine.Mitochondrial dysfunction is emerging as a key process in the pathophysiology of septic lung injury.Dexmedetomidine(DEX),as a highly selective and potent α2 adrenergic agonist,is commonly used in the intensive care unit and operating room for its sedation and analgesia.Increasing lines of evidence in recent years have demonstrated that DEX conferred anti-inflammatory,anti-apoptotic and antioxidant properties to attenuate the lung injury following ischemia/reperfusion,lipopolysaccharide,and ventilation in animal models.But,the underlying molecular mechanism of action is still ill-defined.For this purpose,the current study was designed to illuminate the effects and mechanism of DEX treatment on the dynamic equilibrium of mitochondrial fusion/fission during endotoxin-induced acute lung injury,which provides a theoretical basis for the clinical use of DEX in the prevention of sepsis-caused acute lung injury.Methods To ascertain whether DEX acted via HIF-1a/HO-1 pathway to preserve mitochondrial dynamics to mitigate septic lung injury,the well-accepted septic lung injury model of LPS exposed to mice and LPS-induced oxidative injury of NR8383 cells were used in the study,which were divided into three parts as follows.The first experimental part was to conduct the mice model of endotoxin-induced acute lung injury by caudal vein injection of LPS 15mg/kg diluted in 2ml saline for 12 h.50ug/kg DEX was administered intraperitoneally 30 min prior to LPS treatment.The degrees of lung tissue injury,changes of oxidative stress indexes in serum and lung tissues,alterations of mitochondrial function and morphology,the expressions of mitochondrial fusion/fission related proteins and genes were observed to verify that DEX could protect against septic acute lung injury through regulating mitochondrial dynamics.The second experimental part was to construct in vitro model of endotoxin-attacking rat NR8383 alveolar macrophages with transfected with HO-1 or HIF-1a siRNA.NR8383 cells were incubated with 10μg/ml LPS for 24 h in the presence of 10μmol/L DEX preincubated for 1h.Cell viability,changes of oxidative stress indexes,alterations of mitochondrial function and morphology,the expressions of HO-1,HIF-1a,mitochondrial fusion/fission related proteins and genes were observed to verify that DEX regulated HIF-1a/HO-1 pathway to preserve the balance of mitochondrial dynamics to reduce the oxidative stress damages induced by LPS.The third experimental part was to establish the model of endotoxin-induced acute lung injury in vivo and in vitro by using HO-1fl/fl/CAG-Cre ERT2 mice and HO-1siRNA-transfected NR8383 cells subjected to LPS.Dimethyloxalylglycine(DMOG),as a HIF-1a stabilizer,was pre-treated before DEX prior to LPS challenge.The degrees of lung tissue injury,cell viability,changes of oxidative stress indexes,alterations of mitochondrial function and morphology,the expressions of HO-1,HIF-1a,mitochondrial fusion/fission related proteins and genes were observed to further clarify that HIF-1a/HO-1 pathway indeed was involved in DEX-mediated dynamic equilibrium of mitochondria.Results 1.The C57BL/6J mice model of endotoxin-induced acute lung injury was successfully established by caudal vein injection of LPS 15mg/kg for 12 h.Compared with LPS group,DEX pretreatment alleviated the pathological damages of lung tissues,decreased the lung injury scores,reduced the levels of TOS,OSI,GSSG,H2O2,while elevated the levels of TAS,GSH,and GSH/GSSG ratio.Meanwhile,mitochondrial ROS and mt DNA contents were decreased,accompanied by relieved mitochondrial swollen,vacuolization and nearly normal shape of cristae with enhanced tubular mitochondria,and reduced rod-shaped or punctate mitochondria.Besides,pretreatment with DEX markedly upregulated the expressions of Mfn1,Mfn2,OPA1 proteins and m RNA,yet downregulated the expressions of Drp1,Fis1 proteins and m RNA.Immunofluorescence study revealed that DEX pretreatment obviously increased the fluorescence intensity of HO-1 and HIF-1a in the nucleus,which indicated that DEX could sustain the dynamic balance of mitochondria to alleviate septic lung injury in vivo.2.NR8383 rat alveolar macrophages were used to establish a cell model of LPSattacking alveolar macrophages.Silencing of HO-1 or HIF-1a by siRNA blocked DEX-mediated preservation of mitochondrial dynamics,characterized by reduced cell viability,elevated the levels of serum TOS,OSI while declined TAS levels,increased mitochondrial ROS and mt DNA contents,as well as more deformed mitochondria manifested as swelling and vacuolation,fractured cristae with enhanced rod-shaped or punctate mitochondria,and reduced tubular mitochondria.Besides,loss of HO-1 or HIF-1a significantly downregulated the expressions of HIF-1a,HO-1,Mfn1,Mfn2 and OPA1 m RNA and proteins,while upregulated the expressions of Drp1 and Fis1 m RNA and proteins,which indicated that DEX acted via HIF-1a/HO-1 pathway to preserve mitochondrial dynamics to mitigate LPS-induced oxidative stress injury in vitro.3.Using HO-1 KO mice and HO-1 siRNA NR8383 cells to conduct the model of endotoxin-induced acute lung injury in vivo and in vitro,DMOG,as an activator of HIF-1a/HO-1 signaling pathway,was pre-administrated prior to DEX treatment.Compared with DEX-pretreated group,precondition of DMOG plus DEX obviously alleviated the pathological damages of lung tissues,inhibited LPS-induced oxidative injury,improved mitochondrial morphology and function,promoted mitochondrial fusion while suppressed fission.However,HO-1 knockout or silencing counteracted the above-mentioned protective effects,which confirmed that HIF-1a acted at its downstream target gene HO-1 to exert the endogenous lung protective effects,and further clarified that DEX could preserve mitochondrial dynamic equilibrium through regulating the HIF-1α/HO-1 signaling pathway so as to alleviate endotoxin-induced acute lung injury.Conclusion In summary,DEX sustained mitochondrial dynamic equilibrium through regulating the HIF-1α/HO-1 signaling pathway to alleviate sepsis-related acute lung injury in vivo and in vitro.The findings provided new breakthroughs in the molecular mechanism of HO-1,and also provided a theoretical basis for the clinical novel use of DEX in the prevention and treatment of sepsis-related acute lung injury.