Effect Of Methylprednisolone On Expression Of Aquaporin-5in Rats With Neurogenic Pulmonary Edema Induced By Adnephrin

Neurogenic Pulmonary Edema (NPE) which also called centralpulmonary edema is one of the most serious complications of the CNSdiseases.It is usually caused by the craniocerebral injury or suddenly increasedintracranial pressure due to other diseases of the central nervous systemdeprive of primary heart, lung and kidney diseases.In recent years,incidence ofthis disease which usually infected by EV71has been a significant rise.Itinduces that abundant interstitial fluid is detained in pulmonary interstitial andalveoli,then forms the interstitial or alveolar pulmonary edema.The clinicalsymptoms of The NPE caused by severe hand-foot-and-mouth is dyspnea,coughing up pink foam sputum, hemoptysis, respiratory failure and so on.Like other severe case, the state of this illness progresses rapidly, dangerously,and treats difficult, following a high mortality rate (90%)[1].Although manyresearches has been done about its pathogenesis, the precise mechanism of theneurogenic pulmonary edema is still not very clear. In its pathological changes,pulmonary permeability is a distinctive performance.AQP5widely distribute on the surface of the alveolar type I epithelialcells and also located in the surface of glandular epithelial cells in thenasopharynx and respiratory, airway epithelial cell cavity membrane,whichalmost express in lung tissue. As rapid liquid transport channel on fluidtransport function,AQP5plays an important role to maintain the normalphysiological function and water transportation in lung. Methylprednisolonesodium succinate which is a synthetic glucocorticoid is commonly used totreat hand-foot-mouth disease.It be classified to medium efficiency. Inaddition to intensive anti-inflammatory effect,it can improve nervoustransmition,relief inflammation and swelling[2],reduce vasoconstrictor andblood clots forming caused by catecholamine[3],reduce capillary permeability and pulmonary edema.At the same time, because of blocking the vicious cycleof cerebral edema pulmonary edema,Methylprednisolone is an effectivetherapy in preventing from cerebral edema.Through observing different doseof methylprednisolone sodium succinate influences on AQP5express-ion,pathomorphological change and wet and dry weight ratio from neurogenicpulmonary edema induced by epinephrine in rat lung tissue,we explore theprotective role of methylprednisolone sodium succinate on neurogenicpulmonary edema.Objective:To observe different dose of methylprednisolone sodium succinateinfluences on AQP5expression,pathomorphological change and wet and dryweight ratio from neurogenic pulmonary edema induced by epinephrine in ratlung tissue,we explore the protective role of methylprednisolone sodiumsuccinate on neurogenic pulmonary edema.Methods:1The preparation of animal modelMale or female Wistar rats,1month,Weight100±8g, epinephrine isinjected to intraperitoneal, the group of neurogenic pulmonary edema isinjected2.7mg/kg epinephrine through intraperitoneal(1mg:1ml).Observationabout20min, Most rats appear rapid breathing frequency after intraperitonealinjection around about5min, respiratory difficulty after15min.If they cansurvive longer than20min, the model is successful.The rats is anesthesiaed by10%chloral hydrate through intraperitoneal injection, then put on theoperation table, gross. From median abdominal open thorax, tracheal ligation,take out the lungs.2Animal groups50Wistar rats is randomly divided into control group (n=10), the groupof neurogenic pulmonary edema (NPE group, n=10),the treatment group usedby methylprednisolone (n=30). three sub-groups is divided at different dose,each group is average10rats. The treatment groups is immediatelyintraperitoneal injected after animal model prepare successful. 3Observation lung MorphologicalAfter the rats is anesthetized,quickly open the chest, expose thepipes,ligate the bottom of tracheal,remove the lung entirely, observe the colorand morphology of lung.4Determination W/DAbsorb water and blood on surface of middle of the right lung. At thesame time, the wet weight of the right lung in electronic balance, then placethe middle of right lung at65℃72h in the oven to Gross weight and weighdry weight, calculate wet/dry weight ratio (W/D) of the right middle lobe oflung.5Observation pathological morphologyRespectively left lung is fixed into the4%formaldehyde until sinked tothe bottom of the bottle at4℃storage,then dehydrate, transparent, embedding,paraffin, and HE dyed, observe pulmonary pathological changes.6Immunohistochemical staining of AQP5with Streptomycin-affinity biotin-peroxidase method (SABC).After slice is waxed, hydrated,removed endogenous oxidase with3%hydrogen peroxide, repaired the antigen with microwave,next step accord toKit instructions. A positive control is known positives result, a negative resultis replaced with PBS. AQP5antibodies is diluted to the concentration of1:400.Positive expressive AQP5cell is brown-yellow cytoplasm appear under thelight microscope. Each slice randomly is selected to examine the absorbancevalue (absorbance,A)in10(200X) View,then we calculate the average of theabsorbance value to reflect the expression of AQP5.7Examination the expression of AQP5mRNA with Real-time quantitativePCRTaked the right upper lobe of the lung tissue，Reference manual totalRNA extraction of pulmonary cells in rats of each group, and then reversetranscription into cDNA, some cDNA as template.with TRIzol reagent(purchased from Invitrogen Corporation)，Well PCR reaction preparationsolution in PCR amplification reaction with RealtimePCR tool. The same reaction conditions. AQP5(synthesis by Invitrogen Corporation), primersequences：GAPDHβ-actin：: upstream primer5’-GGCATGGACTGTGGTCATGA-3’; downstream primer5’-TTCACCACCATGGAGAAGGC-3’;(244bp),AQP5upstream primer5’-GTGACAGACAAGCCAATGGATAA-3’;downs-trea m primers5’-GCCCTCTTAATAGGAAACCAGAT-3’(287bp).8AQP5Western blotInferior lobe of right lung tissue500mg and1ml Extracts Homogenateextraction with glass Homogenizer，4℃,12000r/min centrifugal5min, takethe supernatant by adding volume2χ on the SDS sample buffer,100℃boilin5min. Polyacrylamide gel electrophoresis, equal amounts of total proteinon each hole. Using water bath by Western blotting protein transferred toPVDF membranes. Room temperature oscillations,5%skim milk powderclosed membrane2H, adding1:250the first antibody, slowly shake2H atroom temperature, then add1:6500of HRP labeled second antibody, Shake2Hat room temperature, ECL color rendering.We use Gel-pro gel analysissoftware, determination of the mean optical density values (OD value) toAQP5optical density values of protein expression. AQP5measuring proteincontent and references in β-actin expression comparison AQP5relativeexpression levels of the protein.Results:1Observation on general situation of ratThe rat of NPE group breath significantly faster than normal group,Moist rales can be heard at the bottom of Lungs，the different doses ofmethylprednisolone group of rats is not found significantly acceleratedbreathing agitation,however manifest increase survival time.2Observation on morphology of lung tissueControl group appearance of the lung is white, not edematous.the NPEgroup of lungs is significantly larger, edema, extravasated blood, distributelarge dark red bleeding on surface,bubble-like liquid is full of trachea. Lungtissue of the methylprednisolone group is larger than NPE group,edema rarelyand distribute dark red dots or small flake hemorrhage. focireduced in size, edema reduction, and the lung appear dark red dots or small Flake hemorrhagefoci.As increased the dose of drug, lung hemorrhage, edema was significantlyreduced.3Lung W/D ratioW/D ratio(6.6080±0.4319) of the NPE group significantly higher than thecontrol group (4.0870±0.5279)(P<0.05), W/D ratio of the treatment groupssignificantly lower than the NPE Group (P<0.05).Compared with the threegroups (5.8279±0.8050)、(5.2430±0.3359)、(4.5900±0.5390), the group oftreated with MP30mg/kg is minimum.4The changes of pathologyUnder the light microscope, there is not congestion,hemorrhage inalveolar cavity.The structure of alveoli can be clearly seen in the controlgroup of lung. were free of, alveolar clearer. In the NPE group,we findabnormal structure of alveoli, the widened alveolar wall, overextended alveoliand a lot of bleeding and Inflammatory cells is full of alveolarcavities.Compared to the NPE group,the changes of pathology is significantlyrelieve.5AQP5immunohistochemical staining in Lung tissueIOD represents relative AQP5expression in immunohistochemicalstaining results.In control group, AQP5is significantly expressed on top ofthe type I alveolar epithelial cell.Compared to control group (0.5572±0.0834),AQP5expression is less in the NPE Group (0.0784±0.0095).AQP5expression of treated group A(0.2385±0.0676),B(0.3441±0.0673)C(0.4432±0.0653) is a noticeable increase than the NPE group. In three groups,groupA, group B group C(0.2385±0.0676)、(0.3441±0.0673)、(0.4432±0.0653),Group C is highest density of AQP5expression in statistically significant(P<0.05).6AQP5mRNA expressionThe expression of AQP5mRNA can be seen at287bp. Compared withthe control group, AQP5expression of The NPE group significantlydecreased(P<0.05). The expression AQP5mRNA of methylprednisolone treated group significantly enhanced expression than the NPE group(P<0.05).In three groups(group A, group B, group C), group C expresshighestAQP5mRNA, statistically significant (P<0.05).7The expression level of AQP5proteinThe expression of AQP5protein in the NPE Group (0.2165±0.0202)significantly reduced compared to the control group (0.6470±0.1518)(P<0.05), The group of methylprednisolone A (0.3641±0.0202), B(0.3788±0.1347), C (0.5243±0.0937) AQP5protein express more than the NPE Group(0.2165±0.0202) in lung tissue and AQP5expression substantially improved(P<0.05); In group A, group B, group C(0.3641±0.0202)(0.3788±0.1347)(0.5243±0.0937) compared to three groups, group C (0.5243±0.0937)expression markedly enhanced, statistically significant (P<0.05).Conclusion:1Intraperitoneal injection of epinephrine injection in rats (1mg:1ml)，Wecan find the lungs of the NPE group is significantly more larger, oedematous,edema, distribute large dark red bleeding on the surface, foamy liquid in thetrachea.Under the light microscope,there is not hyperemia,and hemorrhage inalveolar cavity,the structure of alveolar is normal in the control group,whilethe lungs of the NPE group lost normal structure of alveolar,widen alveolarwall,excessive expanded alveolus,alveolar cavity is filled with a lot ofbleeding and inflammatory cells.In addition,wet/dry weight ratio of lungs isincreased significantly under microscope in HE staining.. To sum up, thepreparation of model is successful.2The lung tissue of rats is significantly reduced the expression of AQP5compared with normal control group.The phenomenon Confirm that AQP5play a protective role in neurogenic pulmonary edema induced byepinephrine. Thus we presume water clearance capacity is decline due to theexpression of AQP5reduced And then contribute to the formation ofneurogenic pulmonary edema.3The wet/dry ratio (W/D) is reduced after use methylprednisolonesodium succinate to Intervene neurogenic pulmonary edema of rats model.This phenomenon illustrate that methylprednisolone sodium succinatecan raise AQP5.So we presume that methylprednisolone sodium succinate canTreat neurogenic pulmonary edema by raise AQP5and enhance alveolarwater clearance capacity.As the dose of methylprednisolone increases,there isdose-dependent of AQP5, enhance alveolar water clearance capacity, reliefpneumonedema.Those Mechanism may be effective ways to treat pulmonaryedema.