| PART ? The Effects and Mechanism of Midkine in Regulation of ACE-Ang ? Mediated Pulmonary Vascular Endothelial Cell InjuryOBJECTIVE We aimed to elucidate the effects and molecular mechanism of midkine in regulation of ACE-Ang ? mediated pulmonary vascular endothelial cell injury.METHODS 1.Stimulate murine pulmonary microvascular using midkine,and detect m RNA and protein,evaluate ACE hydrolytic activity by FAPGG;detect Ang ? level catalyzed by ACE by enzyme-linked immunosorbent assay(ELISA)after adding Ang I to the medium;2.Screen candidate receptors participating midkine signalling pathway;block the receptors by small molecular inhibitors,and detect the protein expression and hydrolytic activity of ACE,to clarify the key receptor in the regulation of midkine;3.Add Ang I to the cell medium and continue culturing the cells,and detect ROS activity by DCFH-DA and the viability of endothelial cells by CCK-8;4.Detect the expression of intercellular junction molecules including VE-Cadherin and ZO-1 by immunofluorescence,to elucidate the permeability of vascular endothelium;5.Detect the expression of cellular inflammatory adhesive molecule VCAM-1,to clarify the inflammation of endothelial cells.RESULTS 1.The expression of m RNA and protein of ACE in pulmonary microvascular endothelial cell was significantly increased by midkine stimulation;2.The hydrolytic activity and protein expression of ACE was significantly decreased,and the production of Ang ? catalyzed by ACE reduced by adding DAPT inhibiting the activity of Notch 2 receptor;3.The ROS release of vascular endothelial cells was significantly increased and the activity reduced after midkine stimulation.Inhibition of Notch 2 could significantly reduce ROS release by vascular endothelial cells and improve cell viability;4.Immunofluorescence revealed that the expression inter-cellular injunction protein VE-Cadherin and ZO-1 was significantly decreased,and Western blots indicated the decrease of VE-Cadherin protein expression.Blocking Notch 2 could significantly improve intercellular protein expression and intercellular permeability;5.Immunofluorescence and Western blots revealed that the expression of surface inflammatory adhesive molecule VCAM-1 was significantly increased after midkine stimulation and addition of Ang I to vascular endothelial cell.Blocking Notch 2 activity could significantly decrease the expression of VCAM-1 and alleviate cell inflammatory response.CONCLUSION Vascular endothelial cell injury was regulated by midkine of ACE-Ang ? via Notch 2 receptor.PART ? The Effects and Mechanism of Midkine in Regulation of ACE-Ang ? Mediated Sepsis Induced Acute Lung InjuryOBJECTIVE We aimed to validate the effects mechanism of midkine in regulation of ACE-Ang ? mediated lung injury in sepsis.METHODS 1.Adeno-associated virus(AAV)carrying vector interfering midkine expression was established.Mouse lung tissue was infected by AAV via transtracheal infusion,and the efficiency of infection was tested by immunofluorescence,and the interfering efficiency evaluated by RT-q PCR and Western blot respectively;2.Mouse sepsis model was established by cecal ligation and puncture(CLP),and the peripheral blood,bronchial alveolar lavage fluid(BALF)and lung tissue sample were obtained 24 hours after model establishment;3.Midkine,ACE and Ang ? levels in plasma was tested via ELISA in CLP,AAV interfering CLP,and sham group;midkine expression in lung tissue was detected via Western blots and immunohistochemistry;ACE expression in lung tissue was detected via Western blots;4.H&E staining and lung histopathological score was used to evaluate the severity of lung injury;5.Cell counts was used to evaluate inflammatory cell infiltration in BALF,ELISA was used to detect TNF-? level in BALF;and MPO activity was used to evaluate neutrophil infiltration in lung tissue;6.Protein concentration in BALF was measured by BCA method,and lung edema and pulmonary vascular permeability was evaluated via mouse lung wet/dry ratio;7.Mouse lung ACE protein expression was detected by Western blots,ACE hydrolytic activity was tested by FAPGG and Ang ? was detected by ELISA after Ang I was added to lung tissue homogenate.RESULTS 1.Immunofluorescence revealed effective infection of midkine to mouse lung tissue and sufficient interfering midkine protein expression in mouse lung tissue(more than 70%);2.Plasma midkine was significantly elevated in CLP mouse compared with sham [ng/L,747.3(703.6-811.1)vs.87(64.5-199.5),P = 0.0095];and lung midkine expression was also increased(P < 0.05).AAV transtracheal injection could significantly reduce midkine expression in lung tissue in CLP mouse;3.Significant pathological lung injury was induced by mouse CLP model.Midkine interfering in lung tissue could alleviate mouse lung pathological injury and reduce lung histopathological injury score(11.3 ± 1.6 vs.7.0 ± 1.1,P = 0.038);4.Cell counts and TNF-? level was significantly elevated in CLP mouse compared with sham and MPO activity in lung tissue was also elevated(P < 0.05).Midkine interference could reduce inflammatory counts(P < 0.05)and TNF-?(P < 0.05)in BALF in CLP mouse,and reduce MPO activity(P < 0.05),alleviating mouse lung inflammatory response;5.Protein in BALF and lung wet/dry ratio was significantly elevated in CLP,and interfering midkine could effectively decrease protein concentration and reduce lung wet/dry ratio,and ameliorate pulmonary edema in CLP model;6.ACE protein expression was significantly increased in lung tissue in CLP model and its hydrolytic activity was also increased;Ang ? was elevated after adding Ang I.Interfering midkine in mouse lung tissue reduce ACE expression and hydrolytic activity and decrease Ang ? level after adding Ang I(P < 0.05).CONCLUSION Midkine could mediate acute lung injury induced by sepsis via ACE-Ang ? pathwayPart ? The Correlation between Midkine-ACE –Angiotensin ? and the Severity and Prognostics in Septic Patients.OBJECTIVE We aimed to investigate the correlations between midkine-ACE-angiotensin ? and prognostics,severity and organ dysfunction in sepsis.METHODS The septic patients diagnosed according to sepsis-3 admitted to the Department of Critical Care Medicine,Zhongda Hospital from September,2017 to March,2018 were enrolled in this study.1.The demographic and baseline information at admission was recorded,including gender,age and co-morbidities;2.The clinical features were recorded including diagnosis,the site of infection,sequential organ failure assessment(SOFA)score,existence of shock,complications of acute respiratory distress syndrome(ARDS)and acute kidney injury(AKI);3.The therapeutic managements were recorded,including vasopressors,mechanical ventilation and continuous renal replacement therapy(CRRT);4.The laboratory results were recorded,including blood cell counts,biochemistry and arterial blood gas analysis;5.As for those patients with invasive arterial blood pressure monitor(Pi CCO),variables including extravascular lung water index(EVLWI),pulmonary vascular permeability index(PVPI)and systemic vascular resistance index(SVRI)were recorded;6.All patients were followed-up to day-28,and the survival and death status were recorded,also the death date for those deceased;7.The peripheral blood samples were obtained at admission and plasma was separated and extracted.The plasma midkine,ACE and Ang ? were tested by ELISA.RESULTS A total of 26 septic patients were included in this study,and the results revealed that,1.The average age of included patients was 63.1±19 years-old,and 21 patients were male(80.9%).Lung was the most common infection site(15 patients,57%).The 28-day mortality rate was 28.6%;2.Patients in nonsurvivor group had significantly higher SOFA score compared with those in survivor group(11.9 ± 3.2 vs.7.2 ± 3.1,P = 0.002),and higher proportion of patients with CRRT(6/8 vs.1/18,P = 0.001),shortened ventilation-free days [day,0(0-5)vs.10(5-15.3),P = 0.001] and CRRT-free days [day,0(0-2.5)vs.7.5(4.8-18.3),P = 0.002].Twenty-two patients were complicated with ARDS(8 mild,8 moderate and 6 severe)and 13 patients with AKI;3.Plasma midkine was correlated with outcomes in sepsis.Plasma midkine was greatly elevated in non-survivors compared with survivors [ng/L,763.6(404.7-1305)vs.268.5(147.8-511.4),P = 0.0387].The ROC analysis yielded the area under curve of 0.773(95% CI 0.549-0.997,P = 0.039)of midkine in 28-day mortality prediction.The cut-off of 760ng/L was decided by J statistics,with sensitivity of 0.57 and specificity of 0.94.The mortality was significantly different grouping the patients according to the cut-off(Log-rank P = 0.0024),yielding the hazard ratio of 6.16(95% CI 0.83-45.52);4.Plasma midkine was correlated with severity of sepsis.Septic patients complicated with moderate/severe ARDS has significantly higher plasma midkine compared with those with non/mild ARDS [ng/L,522.3(336.6-960.1)vs.243.8(110.3-478.9),P = 0.0135].Patients with AKI had higher plasma midkine compared with those without [ng/L,489.8(259.2-1058)vs.427.9(129.6-510.3),P = 0.0973];5.Pi CCO was performed on 11 patients,and plasma midkine was linear correlated with PVPI(P = 0.049),but not relevant with EVLWI(P = 0.063)or SVRI(P = 0.299);6.Plasma midkine has a trend of linear correlation to ACE(P = 0.0676)and Ang ?(P = 0.070).ConclusionCONCLUSION Plasma midkine was correlated with 28-day mortality and organ dysfunction in sepsis. |
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