Dexmedetomidine Attenuates Lipopolysaccharide-induced Acute Lung Injury By Inhibiting Mitochondria-dependent Apoptosis Via PI3K/AKT

Acute lung injury(ALI)and acute respiratory distress syndrome are well-defined and readily recognized clinical disorders caused by numerous clinical insults to the lung or due to predispositions to lung injury.ALI is a frequent complication following sepsis in critically ill patients and lipopolysaccharide(LPS)is thought to be the most important pathogen that leads to the development of ALI in sepsis.Apoptosis has been reported plays important role in the ALI.Apoptosis is executed by caspases,which can be activated by two main pathways.The nonmitochondrial or "extrinsic" pathway is initiated by binding the extracellular ligand(TNF-a,and TNF-related apoptosis-inducing ligand TRAIL).The second or"intrinsic" pathway is mediated via activation of the mitochondria.Mitochondrial regulation of apoptosis is mediated through the release of cytochrome c,apoptosis-inducing factor,and second mitochondrial-derived activator of caspases(smac),all of which are regulated by members of the Bcl-2 family of proteins.The Bcl family consists of both antiapoptotic(Bcl-2,Bcl-xL)and proapoptotic(BAK,BAX)factors.The antiapoptotic members of this family prevent apoptosis by sequestering proforms of death-driving cysteine proteases or by preventing the release of the aforementioned mitochondrial apoptogenic factors.In contrast,the proapoptotic members of this family,such as BAK,trigger the release of mitochondrial apoptogenic factors into the cytoplasm by acting on the mitochondrial permeability transition(PT)pore,thereby leading to caspase activation.Translocation of cytochrome c from mitochondria to the cytosol through mitochondrial transition pores is an important route of caspase activation.Cytochrome c triggers the release of apoptosome assembly from apoptotic protease activating factor-1,ATP,and procaspase-9,which activates caspase-3 and caspase-7,leading to cellular alterations.Thus alterations in mitochondrial membrane integrity via proapoptotic factors and the subsequent mitochondrial release of cytochrome c have very important roles in the apoptotic signaling cascade.Dexmedetomidine(DEX),a highly selective and potent a2-adrenoreceptor agonist,provides excellent sedation and analgesia with minimal cardiovascular effects.Previous studies have shown that DEX attenuates LPS,ischemia-reperfusion and ventilator-induced lung injury in animal models;however,the mechanism remains unclear.Additionally,the anti-inflammatory and antiapoptotic effects of DEX have been demonstrated in previous studies.DEX has also been reported to have an effect on mitochondrial permeability transition pore(mPTP)in neutrophil and isolated rat hearts following ischemia/reperfusion injury,and to inhibited H202-induced alveolar epithelial cells apoptosis,but these mechanisms are unclear.Furthermore,DEX has been demonstrated the activation of PI3K/AKT signaling viaa2-adrenoreceptor agonist.The present study hypothesized that DEX may provide protective effect against LPS-induced ALI by alleviating oxidative stress and mitochondria-dependent apoptosis via activation of PI3K/AKT.MethodsPart 1 Dexmedetomidine attenuates lipopolysaccharide-induced acute lung injury in rats ALI was induced by intratracheal administration of LPS.Briefly,the animals were intramuscularly anesthetized with an injection of sodium pentobarbital(30 mg/kg).The rats were placed in a supine position on a warming device and the trachea was surgically exposed by a cervical middle line incision in the skin.The rats were subsequently challenged intratracheally with either 0.5 ml sterile normal saline(NS)alone or 0.5 ml NS with LPS(5 mg/kg body weight;Escherichia coli 0111:B4;Sigma-Aldrich,St.Louis,MO,USA)stabbing the trachea with a microsyringe.Rats were divided into 4 groups:(1)Control:the rats were challenged intratracheally with 0.5 ml NS alone;(2)LPS group:the rats were challenged intratracheally with LPS(5 mg/kg);(DEX-L group):the rats were pretreated with DEX(10 ?g/kg)for 30 min,then challenged intratracheally with LPS(5 mg/kg);(DEX-H group):the rats were pretreated with DEX(50 ?g/kg)for 30 min,then challenged intratracheally with LPS(5 mg/kg).The lung histopathological changes were observed by HE-staining;the lung microvascular permeability were measured by Evans blue method;the lung edema were evaluated by wet-to-dry(W/D)ratio.To investigate the underlying mechanisms of DEX treatment in LPS-induced ALI,terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)staining,serum lipid peroxidation,cytochrome c release and the expression levels of Bax,Bcl-2 and cleaved caspase 3 were investigated by western blot.Part 2 Dexmedetomidine attenuates lipopolysaccharide-induced activation of mitochondria-dependent apoptosis in alveolar epithelial cellsA549 cells were obtained from ScienCell(San Diego,CA,USA)and grown at 37? in 5%CO2 in Dulbecco's modified Eagle medium(DMEM,Sigma-Aldrich;Merck Millipore),containing low glucose,penicillin(100 U/ml,Sigma-Aldrich;Merck Millipore),streptomycin(100 units,Sigma-Aldrich;Merck Millipore)and 10%bovine serum(Sigma-Aldrich;Merck Millipore).The cells were stimulated with 5?g/ml LPS for 12 h.A549 cells were divided into 4 groups:(1)Control:the cells were stimulated with PBS;(2)LPS group:the cells were stimulated with LPS(5 ?g/ml);(DEX-L group):the cells were pretreated with DEX(10 ?g/l)for 30 min,then challenged intratracheally with LPS(5 ?g/1);(DEX-H group):the cells were pretreated with DEX(50 ?g/l)for 30 min,then challenged intratracheally with LPS(5 mg/kg).The reactive oxygen species(ROS)production were measured by DCFH-DA,the mitochondrial membrane potential(MMP)were measured by JC-1;the cellular ATP were measured by a luciferase-based assay;the cell apoptosis were measured by annexin V-PI method;the cytochrome c release and the expression levels of Bax,Bcl-2 and cleaved caspase 3 were investigated by western blot.Part 3 Dexmedetomidine attenuates lipopolysaccharide-induced activation of mitochondria-dependent apoptosis via PI3K/AKT in alveolar epithelial cellsA549 cells were obtained from ScienCell(San Diego,CA,USA)and grown at 37? in 5%CO2 in Dulbecco's modified Eagle medium(DMEM,Sigma-Aldrich;Merck Millipore),containing low glucose,penicillin(100 U/ml,Sigma-Aldrich;Merck Millipore),streptomycin(100 units,Sigma-Aldrich;Merck Millipore)and 10%bovine serum(Sigma-Aldrich;Merck Millipore).The cells were stimulated with 5?g/ml LPS for 12 h.A549 cells were divided into 4 groups:(1)Control:the cells were stimulated with PBS;(2)LPS group:the cells were stimulated with LPS(5 ?g/ml);(DEX group):the cells were pretreated with DEX(50 ?g/1)for 30 min,then challenged intratracheally with LPS(5 ?g/l);(LY294002 group):the cells were pretreated with LY294002(20?M)for 30 min,then challenged intratracheally with LPS(5 mg/kg).The reactive oxygen species(ROS)production were measured by DCFH-DA,the mitochondrial membrane potential(MMP)were measured by JC-1;the cellular ATP were measured by a luciferase-based assay;the cell apoptosis were measured by annexin V-PI method;the cytochrome c release and the expression levels of Bax,Bcl-2 and cleaved caspase 3 were investigated by western blot.ResultsPart 1 Dexmedetomidine attenuates lipopolysaccharide-induced acute lung injury in rats1.Dexmedetomidine attenuates lipopolysaccharide-induced lung injuryLung tissue of rats in the LPS group showed accumulation of a large number of neutrophils in the intra-and interalveolar space,a thickened alveolar wall,less alveolar space,interstitial congestion and edema.Lung tissue of rats in the DEX-L group showed accumulation of a large number of neutrophils in the intra-and interalveolar space,a thickened alveolar wall,less alveolar space,interstitial congestion and edema.DEX(50 mg/kg)treatment was found to markedly attenuate these signs of LPS-induced lung injury.The W/D in control group is 4.01 ± 0.35.The W/D in LPS group was significantly increased to 5.63 ± 0.51(P<0.05 compared to control group).The W/D in DEX-L group was 5.35 ± 0.49(P>0.05 compared to LPS group).The W/D in DEX-H group was 4.57± 0.42(P<0.05 compared to LPS group).The EB concentration in the lung in control group is 12.58 ± 2.15?g/100 mg dry tissue.The EB concentration in the lung in LPS group was significantly increased to 27.89 ± 5.38?g/100 mg dry tissue(P<0.05 compared to control group).The EB concentration in the lung in DEX-L group was 25.85± 5.08?g/100 mg dry tissue(P>0.05 compared to LPS group).The EB concentration in the lung in DEX-H group was 17.60±4.13?ig/100 dry tissue(P<0.05 compared to LPS group).2.Dexmedetomidine attenuates lipopolysaccharide-induced apoptosisThe expression of Bax and Bcl-2 in the lung in LPS group were respectively 279.5%± 32.5%and 50.4%± 5.9%(P<0.05 compared to control group).The expression of Bax and Bcl-2 in the lung in DEX-L group were respectively 259.9%±27.2%and 54.6%± 67.3%(P>0.05 compared to LPS group).The expression of Bax and Bcl-2 in the lung in DEX-H group were respectively 175.1%± 19.5%and 92.2%±8.5%(P<0.05 compared to LPS group).The expression of cleaved caspase-3 in LPS group was 294.1%± 38.7%(P<0.05 compared to control group).The expression of cleaved caspase-3 in the lung in DEX-L group was 310.8%± 41.5%(P>0.05 compared to LPS group).The expression of cleaved caspase-3 in the lung in DEX-H group was 168.3%± 19.6%(P<0.05 compared to LPS group).The TUNEL-positive cell in control group is 1.2 ± 0.5/field.The TUNEL-positive cell in LPS group is significantly increased to 47.4 ± 5.3/field(P<0.05 compared to control group).The TUNEL-positive cell in DEX-L group is 41.6±6.1/field(P>0.05 compared to LPS group).The TUNEL-positive cell in DEX-H group is 24.9 ± 4.9/field(P<0.05 compared to LPS group).3.Dexmedetomidine attenuates lipopolysaccharide-induced oxidative stressThe serum lipid peroxides in control group.is 0.38± 0.05?mol/l.The serum lipid peroxides in LPS group is significantly increased to 0.67 ± 0.09 ?mol/1(P<0.05 compared to control group).The serum lipid peroxides in DEX-L group is 0.64 ± 0.08(P>0,05 compared to LPS group).The serum lipid peroxides in DEX-H group is 0.45± 0.07 ?mol/l(P<0.05 compared to LPS group).Part 2 Dexmedetomidine attenuates lipopolysaccharide-induced activation of mitochondria-dependent apoptosis in alveolar epithelial cells1.Dexmedetomidine attenuates lipopolysaccharide-induced mitochondrial injuryJC-1,the potential-sensitive fluorescent dye,forms aggregates in normally polarized mitochondria and monomers in damaged and depolarized mitochondria.The color of this dual-emission probe changed from red-orange to green as the mitochondrial membrane turned depolarized.The control cells were clearly red.LPS exposure rapidly caused MMP dissipation,as shown by the increase in green fluorescence and the concomitant disappearance of red fluorescence.Pretreatment with DEX(50 ?g/l)significantly attenuated the changes in MMP,as indicated by the repression of green fluorescence and restoration of red fluorescence.Intracellular ATP was determined using a luciferase-based assay.The intracellular ATP in LPS group was decreased to 57.4%± 3.8%of that in control group(P<0.05 compared to control group).The intracellular ATP in DEX-L group was 60.1%± 3.5%of that in control group(P>0.05 compared to LPS group).The intracellular ATP in DEX-H group was 87.5%± 5.2%of that in control group(P<0.05 compared to LPS group).2.Dexmedetomidine attenuates lipopolysaccharide-induced mitochondria-dependent apoptosisThe expression of Bax and Bcl-2 in LPS group were respectively 269.3%±30.2%and 49.3%± 6.3%(P<0.05 compared to control group).The expression of Bax and Bcl-2 in DEX-L group were respectively 253.5%± 26.4%and 53.0%±6.9%(P>0.05 compared to LPS group).The expression of Bax and Bcl-2 in DEX-H group were respectively 169.3%± 17.5%and 91.7%± 7.3%(P<0.05 compared to LPS group).The expression of cleaved caspase-3 and cytosolic cytochrome c in LPS group were respectively 284.5%± 32.8%and 438.8%± 35.2%(P<0.05 compared to control group).The expression of cleaved caspase-3 and cytosolic cytochrome c in DEX-L group were respectively 267.0%± 25.9%and 411.5%± 29.3%(P>0.05 compared to LPS group).The expression of cleaved caspase-3 and cytosolic cytochrome c in DEX-H group were respectively 133.9%± 13.5%and 186.8%±20.5%(P<0.05 compared to LPS group).The cell apoptosis ratio in control group is 2.9%± 0.7%.The cell apoptosis ratio in LPS group is significantly increased to 21.5%± 2.5%(P<0.05 compared to control group).The cell apoptosis ratio in DEX-L group is 20.7%± 3.1%(P>0.05 compared to LPS group).The cell apoptosis ratio in DEX-H group is 11.4%± 2.1%(P<0.05 compared to LPS group).3.Dexmedetomidine attenuates lipopolysaccharide-induced oxidative stressA fluorescent probe,DCFH-DA,was used as a specific marker for quantitative mitochondrial ROS accumulation.The intensity of DCF fluorescence in LPS group was increased to 266.4%± 25.1%of that in control group(P<0.05 compared to control group).The intensity of DCF fluorescence in DEX-L group was 250.6%±27.4%of that in control group(P>0.05 compared to LPS group).The intensity of DCF fluorescence in DEX-H group was 175.0%? 19.4%of that in control group(P<0.05 compared to LPS group).Part 3 Dexmedetomidine attenuates lipopolysaccharide-induced activation of mitochondria-dependent apoptosis via PI3K/AKT in alveolar epithelial cells1.Dexmedetomidine activates the PI3K/AKT signalingThe expression of p-AKT(Thr308)and p-AKT(Ser473)in LPS group were respectively 96.1%± 11.1%and 112.5%± 9.6%of that in control group(P>0.05 compared to control group).The expression of p-AKT(Thr308)and p-AKT(Ser473)in DEX group were respectively increased to 387.1%± 28.5%and 341.9%± 26.4%of that in control group(P<0.05 compared to LPS group).The expression of p-AKT(Thr308)and p-AKT(Ser473)in LY924002 group were respectively 168.5%±17.7%and 159.3%? 15.8%of that in control group(P<0.05 compared to DEX group).2.Dexmedetomidine attenuates lipopolysaccharide-induced mitochondrial injury via PI3K/AKTJC-1,the potential-sensitive fluorescent dye,forms aggregates in normally polarized mitochondria and monomers in damaged and depolarized mitochondria.The color of this dual-emission probe changed from red-orange to green as the mitochondrial membrane turned depolarized.The control cells were clearly red.LPS exposure rapidly caused MMP dissipation,as shown by the increase in green fluorescence and the concomitant disappearance of red fluorescence.Pretreatment with DEX significantly attenuated the changes in MMP,as indicated by the repression of green fluorescence and restoration of red fluorescence.However,LY924002 reversed the protective effects of DEX,as shown by the increase in green fluorescence and the concomitant disappearance of red fluorescence.Intracellular ATP was determined using a luciferase-based assay.The intracellular ATP in LPS group was decreased to 57.4%? 3.8%of that in control group(P<0.05 compared to control group).The intracellular ATP in DEX group was improved to 87.5%± 5.2%of that in control group(P<0.05 compared to LPS group).The intracellular ATP in LY924002 group was 64.7%? 4.5%of that in control group(P<0.05 compared to DEX group).3.Dexmedetomidine attenuates lipopolysaccharide-induced mitochondria-dependent apoptosis via PI3K/AKTThe expression of Bax and Bcl-2 in LPS group were respectively 269.3%±30.2%and 49.3%± 6.3%(P<0.05 compared to control group).The expression of Bax and Bcl-2 in DEX group were respectively 169.3%± 17.5%and 91.7%± 7.3%(P<0.05 compared to LPS group).The expression of Bax and Bcl-2 in LY924002 group were respectively 233.6%? 22.5%and 51.3%± 7.7%(P<0.05 compared to DEX group).The expression of cleaved caspase-3 and cytosolic cytochrome c in LPS group were respectively 284.5%± 32.8%and 438.8%± 35.2%of that in control group(P<0.05 compared to control group).The expression of cleaved caspase-3 and cytosolic cytochrome c in DEX group were respectively 133.9%± 13.5%and 186.8%± 20.5%of that in control group(P<0.05 compared to LPS group).The expression of cleaved caspase-3 and cytosolic cytochrome c in LY924002 group were respectively 271.8%± 29.5%and 435.2%± 33 7%of that in control group(P<0.05 compared to DEX group).The cell apoptosis ratio in control group is 2.9%± 0.7%.The cell apoptosis ratio in LPS group is significantly increased to 21.5%± 2.5%(P<0.05 compared to control group).The cell apoptosis ratio in DEX group is 11.4%? 2.1%(P<0.05 compared to LPS group).The cell apoptosis ratio in LY924002 group is 19.8%?3.3%(P<0.05 compared to DEX group).4.Dexmedetomidine attenuates lipopolysaccharide-induced oxidative stress via PI3K/AKTA fluorescent probe,DCFH-DA,was used as a specific marker for quantitative mitochondrial ROS accumulation.The intensity of DCF fluorescence in LPS group was increased to 266.4%± 25.1%of that in control group(P<0.05 compared to control group).The intensity of DCF fluorescence in DEX group was 175.0%±19.4%of that in control group(P<0.05 compared to LPS group).The intensity of DCF fluorescence in LY924002 group was 250.8%± 21.4%of that in control group(P<0.05 compared to DEX group).Conclusions1.Dexmedetomidine attenuates LPS-induced oxidative stress and apoptosis,improves acute lung injury in rats.2.Dexmedetomidine inhibits LPS-induced activation of oxidative stress and mitochondria-dependent apoptosis in alveolar epithelial cells.3.Dexmedetomidine inhibits LPS-induced activation of mitochondria-dependent apoptosis via PI3K/AKT in alveolar epithelial cells...