Study On The Protective Effect Of Nicorandil On Acute Lung Injury In Mice And Its Mechanism

PartⅠProtective Effects and Mechanism of Nicorandil on Acute Lung Injury in MiceAcute lung injury（ALI）/acute respiratory distress syndrome（ARDS）is one of the most common clinical critical illnesses.ALI/ARDS is caused by various pulmonary endogenous and exogenous pathogenic factors,leading to uncontrolled inflammation and alveolar edema.ALI is featured by the infiltration of massive inflammatory cells,release of inflammatory factors,excessive oxidative stress,and impaired pulmonary vascular endothelium barrier,contributing to acute pulmonary edema and alveolar collapse.The pathological alternations result in severe aortic/blood flow imbalance and intractable hypoxemia.ARDS has posed threats to public heath and aggravated the social and economic burden due to its high incidence,serious clinical presentation and poor prognosi.Despite advances in mechanical ventilation strategies in ARDS treatment,the prognosis of ARDS patients remains poor,and the mortality rate still reaches to 40%.Therefore,there is an urgent need to explore the molecular targets related to the pathological development of ALI/ARDS,and find out the effective prevention and treatment for ALI/ARDS.ATP-sensitive potassium channel(K_ATPTP channel)is a special type of inward rectifier potassium channel,providing a unique connection between cellular metabolic status and membrane excitability.K_ATPTP channels participate in numerous cellular processes including cell apoptosis,migration,and angiogenesis.Previous studies showed that the opening of K_ATPchannels can alleviate vascular endothelial cells injury and regulate the activation of inflammatory cells.Mounting evidence revealed that K_ATPTP channels are related with the development of respiratory diseases.The opening of K_ATPTP channels can effectively improve hypoxia-induced pulmonary vascular remodeling and inhibit airway remodeling during chronic airway diseases.In addition,K_ATPTP channels are involved in the regulation of various inflammatory responses.The mortality of K_ATP-knockout mice is significantly increased in sepsis;K_ATPTP channel gene knockout aggravates the inflammatory response in the acute liver injury.Although K_ATPTP channels play an important role in inflammatory responses,the effect of KATP channels opening in ALI remains unclear.Therefore,this study investigated the protective effects of K_ATPTP channel opener nicorandil on ALI and the related mechanisms in vivo and in vitro.1.The protective effects of Nicorandil on acute lung injury in miceObjective:To observe the effects of K_ATPTP channel opener nicorandil on lipopolysaccharide（LPS）-induced ALI,we detected the pathological damage,neutrophil infiltration,release of inflammatory factors and pulmonary vascular endothelial cells injury in mice.Methods:LPS 5 mg/kg airway instillation was used to induce ALI in mice.Nicorandil（12.5,25,50 mg/kg）was given by intragastric administration 3 days before LPS injection,dexamethasone treatment group as positive control.The mice were sacrificed 24 hr after LPS treatment.The lesions were observed by haematoxylin and eosin staining（H&E）.Immunohistochemistry（IHC）was used to detect neutrophil infiltration in lung tissue.Enzyme-linked immunosorbent assay（ELISA）was used to detect imflammatory factors in lung tissue.Microplate reader was used to analysis the myeloperoxidase（MPO）activity in bronchoalveolar lavage fluid（BALF）.BCA concentration was used to determine the protein level in BALF.Western blotting was used to detect endothelial specific protein.Results:LPS administration significantly induced alveolar septum thickening,intra-alveolar hemorrhage,fibrin deposition and inflammatory cell infiltration;pretreatment with nicorandil reduced the pathological damage of lung tissue in a concentration-dependent manner.The infiltration of neutrophils in lung tissue and the protein content in BALF was significantly increased in LPS group,and nicorandil significantly inhibited these alternations.Nicorandil can significantly reduce LPS-induced tumor necrosis factor-α（TNF-α）and interleukin-1β（IL-1β）levels in lung tissue,and ameliorate the up-regulation of inducible nitric oxide synthase（iNOS）,increase LPS-induced down-regulation of endothelial nitric oxide synthase（eNOS）and vascular endothelial cadherin（VE-cadherin）expression.Conclusion:The results showed that opening of K_ATPTP channel alleviatd LPS-induced ALI in mice,which may be related to the inhibition of inflammatory responses and protecting pulmonary vascular endothelial barrier function.2.The regulation of Nicorandil on LPS-induced endothelial dysfunctionObjective:Human pulmonary artery endothelial cells（HPAECs）stimulated by LPS were used for detecting the K_ATPTP channel opener nicorandil on cell apoptosis,inflammatory response,oxidative stress and the underlying mechanism.Methods:HPAECs were divided into six groups:control;LPS group:LPS 1μg/ml;LPS+nicorandil group:LPS 1μg/ml+nicorandil 100μM（or 1,10,100μM）;LPS+nicorandil+glibenclamide group:LPS 1μg/ml+nicorandil 100μM+glibenclamide10μM;nicorandil group:nicorandil 100μM;glibenclamide group:glibenclamide 10μM.CCK8（cell counting kit）was used to detect the cytotoxicity of nicorandil on HPAECs.Hoechst33342 staining was used to detect the effects of nicorandil and glibenclamide on cell apoptosis.Quantitative real-time PCR（qRT-PCR）was used to detect the levels of inflammatory mediators.U937-endothelial cell co-culture system was established to detect the adhesion of HPAECs to leukocytes.Immunofluorescence staining and western blotting were used to detect oxidative stress and the expression of VE-cadherin.Western blotting was used to analyse apoptosis-related proteins,adhesion molecules,NF-κB and MAPK pathways.Fluorescence microscopy was used to detect the levels of reactive oxygen species（ROS）induced by LPS.Immunofluorescence was used for analysis NADPH oxidase4（Nox4）,manganese superoxide dismutase（MnSOD）,VE-cadherin and nuclear translocation of p65.Western blotting was used to detect the apoptosis-related proteins,inflammatory mediators andexpression levels of Nox4,MnSOD,and VE-cadherin,as well as NF-κB and MAPK pathways.Results:Nicorandil inhibited LPS-induced apoptosis of HPAECs in a concentration dependent manner,and down-regulated the expression of cleared caspase3,caspase9,and CHOP.Nicorandil suppressed the release of inflammatory factors,such as TNF-α,cyclooxygenase 2（COX2）,iNOS,intercellular adhesion molecule-1（ICAM-1）,vascular cell adhesion molecule-1-1,VCAM-1),and up-regulated the expression of eNOS and VE-cadherin.Nicorandil reduced the adhesion of leukocytes to the activated HPAECs.In addition,nicorandil significantly inhibited LPS-induced release of reactive oxygen species（ROS）,down-regulated NADPH oxidase 4（Nox4）and upregulated manganese superoxide dismutase（MnSOD）.Moreover,nicorandil can inhibit LPS-induced activation of NF-κB and MAPK pathways.Glibenclamide can block the above effects of nicorandil.Conclusion:The opening of K_ATPTP channel can reduce LPS-induced apoptosis of HPAECs,inhibit LPS-induced inflammation,regulate LPS-induced oxidative stress,and exert endothelial protective function.3.The effects of Nicorandil on LPS-induced endothelial progenitor cell dysfunctionObjective:Endothelial progenitor cells（EPCs）were isolated and cultured to investigate the role of K_ATPTP channel in regulating the migration,angiogenesis,and adhesion molecule expression.Methods:Peripheral blood EPCs were isolated and cultured by density gradient centrifugation.EPCs were identified by morphological observation and immunostaining.EPCs were divided as follows:control;LPS group:LPS 1μg/ml;LPS+nicorandil group:LPS 1μg/ml+nicorandil（1,10,and 100μM）.Nicorandil was given 1 hr prior to LPS administration.Transwell assay was used to detect migration;matrigel assay was used to detect angiogenesis;western blotting was used to detect the expression of adhesion molecules,and NF-κB,p38 MAPK pathways.Results:EPCs were presented with“cobblestone”morphology,and expressed endothelial specific molecules platelet endothelial cell adhesion molecule-1（PECAM-1/CD31）and VE-cadherin.EPCs showed the ability to phagocytosis Dil-ac-LDL and bind to FITC-UEA-I.Nicorandil improved LPS-induced EPCs migration and angiogenesis in a concentration dependent way,and downregulated the expression of ICAM-1 and VCAM-1.Nicorandil inhibited LPS-induced the phosphorylation of NF-κB p65 and p38 MAPK signaling pathways.Conclusion:K_ATPTP channels are involved in the regulation of EPCs function,and improving LPS-induced EPCs angiogenesis and migration.The protective effects of K_ATPTP channels may be related to NF-κB and p38/MAPK signaling pathways.PartⅡ The antitumor effect of self-assembled Paclitaxel nanofibers for non-small cell lung cancerLung cancer is one of the most common malignancies,which results in the highest mortality rate in cancer-caused death.Although operation is the main strategy to cure lung cancer,most patients are diagnosed in advanced stage losing the opportunity for surgery.Chemotherapy plays an important role in lung cancer management.Paclitaxel（Ptx）is a vital drug in the history of chemotherapy,however the efficacy of Ptx alone or in combination is less than 50%,and the severe side effects caused by Ptx limits its clinical use.Therefore,it is important to find a safe and effective drug delivery system for Ptx with few side effects.Objective: Based on the concept of "carrier free",paclitaxel-loaded nanofibers（P-NFs,Ptx-SA）were prepared.By comparing with Ptx,the anti-tumor effects of P-NFs were detected in vitro as well as the underlying mechanisms.Methods: P-NFs were synthesized using Ptx and succinic acid（SA）.Human non-small cell lung cancer（NSCLC）cell lines A549 and H460 were used for analyzing the anti-tumor effects.CCK-8（Cell Counting Kit-8）method was used to detect the cytoxitity of different concentrations of P-NFs and Ptx.Hoechst staining and flow cytometry were used to detect cell apoptosis.Scratch test was used to detect the cell migration.Western blotting was used to detect the expression of endoplasmic reticulum stress and apoptosis-related protein in A549 cells and H460 cells.Results: Both P-NFs and Ptx inhibited the viability of A549 and H460 cells in a concentration-dependent manner（10,20,40,80,160,320 n M）,and the effects of P-NFs was significantly stronger than that of Ptx.At the same dose,P-NFs significantly induced A549 and H460 apoptosis and inhibited cell migration.Western blotting showed that P-NFs significantly up-regulated protein kinase RNA-like endoplasmic reticulum kinase（PERK）,inositol-dependent enzyme 1α（IRE1α）,and the phosphorylation of JNK;P-NFs significantly induced the expression of CHOP and bax/bcl-2 compared with Ptx.Conclusion: This study showed that P-NFs,as a novel nano-drug delivery system synthetizesd based on the concept of "carrier freed",showed stronger anti-tumor effects in vitro compared with Ptx,which may be related to the regulation of endoplasmic reticulum stress-related protein expression.P-NFs as novel delivery system for treating non-small cell lung cancer deserve further study.