The Study On The Dexmedetomidine Hydrochloride Pretreatment Effect On Rat Hyperoxic Acute Lung Injury

Objective:1.To establish rat model of hyperoxic acute lung injury and to evaluate the effect.2.To observe whether the effect of dexmedetomidine hydrochloride pretreatment on hyperoxic acute lung injury in rats is dose-dependent.3.The effect of PI3K/Akt signaling pathway in the treatment of hyperoxic acute lung injury in rats with dexmedetomidine pretreatment.Method:1.To establish rat model of hyperoxic acute lung injury and to evaluate its effect.80 SD rats were randomly divided into Oxygen Group and Hyperoxia Group.The rats in Hyperoxia Group were then randomly divided into 24 h Subgroup,48 h Subgroup and 72 h Subgroup based on the oxygen inhaling time.80 Rats are in 4 groups with20 rats in each group.The rats in Oxygen Group were fed in oxygen environment,while the rats in Hyperoxia Group were fed in a hyperoxic chamber with temperature at 25 ?,humidity at 50%,the oxygen concentration kept at 90% and CO2 concentration less than 0.5%;the sodium lime,anhydrous calcium chloride,thermometer and hygrometer were placed in the chamber;the oxygen supply was continuously uninterrupted every day and the mental status,feeding,and skin mucous membrane of rats were observed in different time point.the rat arterial blood in Oxygen Group and Hyperoxia Group were drawn and centrifuged for spare and the blood gas were analyzed.The lung tissue was opened in the thoracic cavity,and the whole lung tissue was observed by the naked eye.The left lung tissue was immersed in 10% formalin specimen and stained with HE.The pathological changes of lung tissue were observed under light microscope and pulmonary pathology score was made.The right lung tissue was taken as a whole and the blood and moisture on the surface of it was wiped by napkin and?W/D?mass ratio was measured on the electronic balance scale.Plasma TNF-? and IL-1?,IL-6 concentration were measured at the concentration of plasma and water in the lungs.2.To observe whether the effect of dexmedetomidine hydrochloride on hyperoxic acute lung injury in rats is dose-dependent or not.The acute lung injury model was established.The arterial blood gas analysis,lung histopathological examination and early apoptosis rate of lung cells were taken as the indexes to detect whether the effect of different concentrations of dexmedetomidine hydrochloride on hyperoxia acute lung injury is a dose-dependent or not.80 healthy SD rats were randomly divided into four groups: Hyperoxia Group?H?,Dexmedetomidine Hydrochloride2.5?g/?kg·h?Group?DL?,Dexmedetomidine Hydrochloride 5?g/?kg?Group?DM?,Dexmedetomidine Hydrochloride 10?g/?kg·h?Group?DH?.Rats in 4 groups were placed in the hyperoxic chamber respectively for 48 h feeding and specimens were taken for test.The rats in DL Group were given dexmedetomidine 2.5?g/?kg·h?,added to 20 ml 5% glucose,0.5ml/h,caudal vein continued pumping and immediately into hyperoxic chamber;The rats in DM Group were given dexmedetomidine 5?g/?kg·h?,added to 20 ml 5% glucose,0.5ml/h,caudal vein continued pumping and immediately into hyperoxic chamber;The rats in DH Group were given dexmedetomidine 10?g/?kg.h?,added to 20 ml 5% glucose,0.5ml/h,tail vein continued pumping and immediately into hyperoxic chamber;The rats in H group were given the same amount of sugar,the caudal vein continued pumping and immediately intohyperoxic chamber.The rats in each group were taken out from the chambers and narcotized respectively after 48 h modeling.The rat oxygen index?OI?and respiratory index?RI?were calculated based on neck arterial blood gas anaylysis;The left low lung tissue was immersed in 10% formalin specimen and stained with HE.The pathological changes of lung tissue were observed under light microscope and pulmonary pathology score was made.The right lung tissue was taken out to test the early apoptosis rate of lung tissue e by flow cytometry.3.The effect of PI3K/Akt signaling pathway in the treatment of hyperoxic acute lung injury in rats with dexmedetomidine.80 healthy SD rats were randomly divided into four groups: H?hyperoxia?Group,DM?dexmedetomidine group?,WD?wortmannin dexmedetomidine group?W?wortmannin?group;4 groups of rats were placed in hyperoxia chamber for feeding.The rats in DM Group were given dexmedetomidine5?g/?kg·h?,added to 20 ml 5% glucose,0.5ml/h,caudal vein continued pumping and immediately into hyperoxic chamber;The rats in WD Group were given wortmannin dexmedetomidine,wortmannin 30?g/kg slowly injected into caudal vein,dexmedetomidine 5?g/?kg·h?added to 20 ml 5% glucose,0.5ml/h,caudal vein continued pumping and immediately into hyperoxic chamber.The rats in W Group were slowly injected 30?g/kg of wortmannin via caudal vein first,and given the same amount of 5% glucose,0.5ml/h,caudal vein continued pumping,immediately into the hyperoxic chamber.The rats in H group were given the same amount of 5%glucose,caudal vein continued pumping,immediately into the hyperoxic chamber.48 h after the end of modeling,The rats of each group were taken out from the chambers and anesthetized to death after 48 h modeling.The rat chest were opned and the left and right lung tissue were taken out.The left lung tissue was immersed in 10% formalin specimen and stained with HE.The pathological changes of lung tissue were observed under light microscope and pulmonary pathology score was made.The right lung sub-parts were quickly placed in liquid nitrogen.The expression of PI3 K,AKT,P-Akt,GSK-3?,P-GSK-3? and protein were detected by Western blotting.Result:1.?1?Lung Tissue Observation Results: The lung tissue in Oxygen Group was in uniform color,pink color,soft and flexible touched by hand.No bleeding,tissue edema and necrosis was seen.In the Hyperoxia Group 24 h,the lung tissue was pale red,and a very small amount of scattered and different sizes of bleeding point were on the surface;In the Hyperoxia Group 48 h,the lung tissue was in slightly dark red,more obvious congestion,slightly edema,accompanied by a small amount of points or small flaky bleeding.In the Hyperoxia Group 72 h,the lung tissue was in severe injury,dark red,obvious lung tissue edema.A number of patchy bleeding can be seen and chest visible translucent or pale red pleural effusion,and even nose and mouth appear bloody secretions were seen in some severe injury.?2?Lung Tissue HE Staining Results and Lung Pathological Score: The alveolar structure in Oxygen Group was clear and complete with uniform alveolar size,smooth alveolar wall.No white blood cells and exudate in alveolar cavity was seen and no widening alveolar space,and no inflammatory cell infiltration;In the Hyperoxia Group 24 h,the alveolar structure was clear,alveolar cavity with a small amount of exudate associated with a small amount of red blood cells leakage,a very small number of telangiectasia,alveolar septum slightly thickened;In the Hyperoxia Group 48 h,the alveolar lacunae exudate was increased,with some red blood cell leakage,obvious angiotelectasis,enlarged alveolar cavity,thickening alveolar septum,and the majority of alveolar septal fracture associated with a large number of WBC exudation;In the Hyperoxia Group 72 h,the lung tissue was diffuse and the lung tissue structure was unclear.The alveolar wall rupture was fused into thepulmonary bullous.The alveolar cavity and the interstitial lung were filled with WBC,accompanied by edema;severe injury was with formation of hyaline membrane?P<0.05?.In the lung pathological score comparison,the score in DM Group and the DL Group?P<0.05?were decreased compared with the score in H group.There was significant decreasing score between the score in DM Group and the DL Group?P<0.05?.The difference was not statistically significant between the score in DM Group and H Group?P>0.05?.?3?Blood Gas Analysis: The oxygen index?OI?in Oxygen Group was no obvious difference?P>0.05?.In the Hyperoxia Group,the OI was in decreasing with the hyperoxia exposure time compared with Oxygen Group?P<0.05?;There was no significant change in Oxygen Group?P>0.05?.The Hyperoxia Group increased gradually with the hyperoxia exposure time?P<0.05?.?4?No obvious change of levels of IL-1?,IL-6 and TNF-? in plasma in Oxygen Group as time went by.The levels of IL-1?,IL-6 and TNF-? in plasma increased as time went by in Hyperoxia Group?P<0.05?.?5?Lung Tissue W/D Ratio: no obvious W/D Ratio change in Oxygen Group?P>0.05?;The W/D ratio of lung tissue was gradually increased as time went by in Hyperoxia Group?P<0.05?.2.?1?Lung Tissue Cell Early Apoptosis Rate: The early apoptotic rate of the lung tissue in DL Group and DM Group was lower than that of H Group?P<0.05?,but there was significant difference between the DM group and the DL group?P<0.05?.There was no significant difference between DH Group and H Group.The apoptotic rate of lung tissue cells was significantly different between DM group and DH group?P<0.05?.Removal of super safety dose DH group showed a linear trend.?2?Lung Histopathological Changes and Lung Pathology Score: H Group alveolar structure was in disorder,with thicken alveolar wall,individual alveolar wall fracture;part of the fusion into the lung bullae,obvious edema and widening alveolar septum,and a large number of inflammatory cell exudation can be seen;DH Group performance similar to H Group,which lung tissue structure was slightly disturbed,part of the alveolar wall fracture,and fusion into the lung bullae,and alveolar septum obvious inflammatory cell infiltration;DM Group alveolar structure was clear,with a slight congestion and edema of the lung tissue,dark red,the lung surface with a small amount of punctate bleeding,which alveolar septum has a very small number of inflammatory cell infiltration.The histopathological scores and pathologic changes of lung tissue were consistent.?P<0.05?.There was significant difference between DH group and H group?P>0.05?.There was significant difference between DM group and DH group?P<0.05?.Removal of super safety dose DH group showed a linear trend.?3?Blood Gas Analysis: comparison between DL Group & DM Group and H Group& RI Group?P>0.05?;comparison between DL Group & DM Group and H Group& O Group?P<0.05?;Removal of super safety dose DH group showed a linear trend.3.?1?Lung Tissue Lung Pathological Changes and Lung Pathological Score: H Group alveolar tissue damage area was extensive,and individual alveolar structure showed unclear,alveolar cavity and pulmonary interstitial filled with a large number of white blood cells,with hyaline membrane formation.DM Group expansion of pulmonary capillary congestion,accompanied by leukocyte adhesion,alveolar cavity exudate and a lot of red blood cell leakage.Part of the alveolar septum thickening associated with alveolar septal fracture,surrounded by a large number of leukocyte aggregation was exudative changes.WD group pathological changes with H Group.The pulmonary pathological score and lung pathology were consistent.There was a significant difference between the group with dexmedetomidine and the group without dexmedetomidine?P<0.05?,and there was also significant difference between the group with wortmannin and the group without wortmannin?P<0.05?,and wortmannin and dexmedetomidine has interaction,so the wortmannin has an effect on hydrochloride?P<0.05?.?2?Western Blot Result: The dexmedetomidine could affect the expression of PI3K_P110,p-AKT and p-GSK-3?,and the effect of dexmedetomidine was significantly affected by wortmannin blocking the pathway.Both dextromethorphan and wortmannin did not affect the expression of P13K_P85,AKT and GSK-3?.Conclusion:1.48 h in halation of high concentration of oxygen more than 90% can successfully copy the typical HALI model,lung injury at 48⁷2h peak.2.5?g/?kg·h?dexmedetomidine hydrochloride can improve the acute lung injury caused by hyperoxia and improve the rat oxygenation index,and can significantly reduce the early apoptotic rate of alveoli,which role in the safe dose range was dose-dependent.3.PI3K/AKT signal transduction pathway may be involved in the effect of dexmedetomidine hydrochloride on hyperoxic acute lung injury.