Effects Of Mast Cells In Pulmonary Artery Pressure Increases During The Development Of High Altitude Pulmonary Edema

Background The hypoxic environment at high altitude is the key factor causing High altitude pulmonary edema(HAPE),and its pathogenesis is complicated,closely related to the abnormal increase of Pulmonary artery pressure(PAP).In recent years,many literatures have reported that Mast Cells(MCs)MCs is involved in the formation of chronic pulmonary hypertension,but there are a few reports on the role and mechanismof MCs in the occurrence of acute hypoxic PAP increase and HAPE.MCs are one of the important immune cells,and play an important role as "sentinels" in the pulmonary immune supervision.MCs activation can synthesize of a large number of particles(particles are various bioactive medium),particle release of intracellular active medium,the medium effects on different target cells,produce different biological effects,such as Tryptase and Chymase,can be used as the specificity of the MCs activation and degranulation activation markers,MCs can be divided into two types: one type of MC_T(rich in Tryptase)and the other type of MC_TC(rich in Tryptase and Chymase).Tryptase can interact with IL-4,IL-6 and other mediators to trigger cascades of MCs activation.Chymase can promote local Ang II production independent of the angiotensin-converting enzyme(ACE).5-hydroxytryptamine(5-HT)and Histamine(His)can promote vasoconstriction and change the permeability of blood vessels.Based on literature research,this study investigated the role and mechanism of lung MCs activation degranulation in HAPE related PAP increase under acute hypoxic stress.Scientific problems How does lung MCs(number,activity,type,distribution)change under acute hypoxic stress?? Does the change of MCs participate in the process of HAPE related PAP increase?? Possible mechanism of pulmonary MCs involvement in HAPE related PAP increase?Research Content Design In order to carry out the experimental study,MCs degranulation inhibitor Sodium cromoglycate(SCG)was selected as the tool drug to carry out this study.RBL-2H3 cells(Rat basophilic leukemia cells,which were reported as the model cell line for the study of MCs in literature)were used to substitute for MCs in vitro studies.SCG pretreatment of SD rats and SCG culture of RBL-2H3 cells(representing MCs)were used to conduct this study at the overall body,tissue and cellullar levels.Results 1.Overall level: Study the role of activated pulmonary MCs degranulation in the increase of acute hypoxic PAPThe acute normobaric hypoxic model was established by endotracheal intubation with 15% O₂ ventilation.The changes of PAP and Psa(systemic arterial pressure)in SD rats were monitored dynamically and in real time by Power Lab System.The results showed that SCG pretreatment could reduce the elevation of PAP in SD rats under acute normobaric hypoxic stimulation,but had no significant improvement in the reduction of Psa,suggesting that the activation of pulmonary MCs played a role in or even promoted the elevation of acute hypoxic PAP in SD rats.2.Lung tissue level: study on the mechanism of pulmonary MCs activation in HAPE related PAP increaseBy HE,TB(Toluidine Blue)staining and IHC(Immunohistochemistry)experiment,to study the incidence of pulmonary edema and the changing trend of pulmonary MCs in SD rats under different acute hypobaric and hypoxia conditions(different altitudes(5000 m,7000 m)of the low-pressure chamber and different hypoxia stimulation time(12 h,24 h,48 h,72 h).The results showed:(1)Under acute hypobaric hypoxia stress,the number of MCs increased,and activated in the lung tissues of SD rats.(2)MC_TC type MCs is mainly distributed near pulmonary microvessels,near small bronchus,submucosal interstitium of trachea,and pulmonary capsule.(3)The reproduction effect of acute hypoxic pulmonary edema in hypobaric chamber simulated at altitude of 7000 m is better than 5000 m,and the number and activity of MCs are the highest at 7000 m-12 h.By TB staining,TEM(Transmission Electron Microscopy),WB(Western Blot),IF(Immunofluorescence),ELISA(Enzyme linked immunosorbent Assay)results showed that:(1)Pretreatment with SCG(100 mg/ kg,i.p.)in SD rats,continuous stimulation in animal hypobaric chamber(altitude 7000 m)for 12 h were established as SCG pretreatment and acute hypobaric hypoxia animal model conditions;(2)Under acute hypobaric and hypoxia stimulation,both MC_T and MC_TC types of MCs increased,and MC_TC type of MCs near pulmonary microvessels increased;(3)Increased release of inflammatory mediator IL-6 from MCs activation and Tryptase can initiate the "waterfall effect" of MCs activation.Directly and indirectly release of His,5-HT,and Ang II participate in or even promote the occurrence of acute hypoxic PAP increase.3.Cellullar(MCs)level: Metabolomics study of supernatant of RBL-2H3 cells(MCs)cultured in hypoxia.RBL-2H3 cells(MCs)in each group were treated with SCG for WB test and cell supernatant ELISA test.The results showed:(1)In hypoxic environment,the number and activaty of MCs increased,and the release of His(vasoconstrictor)increased.(2)RBL-2H3 cells were cultured at 37? and 1% O₂ + 5% CO₂ for 48 h as hypoxic conditions,and SCG 10-6 g/ m L as hypoxic drug concentration for 48 h.RBL-2H3 cells were cultured by SCG treatment with hypoxia(1%O₂ + 5%CO₂),and the cell supernatants of each group were sequenced by non-target metabolomics.The results showed:(1)Hypoxic stimulation induced significant changes in amino acid,lipid and glucose metabolism pathways in RBL-2H3 cells.(2)SCG treatment can alleviate the adverse effects of amino acids,especially histidine related metabolites,and regulate lipid metabolism and glucose metabolism.(3)SCG can regulate the metabolic pathways of ammonia,histidine and glutamate.(4)It was speculated that hypoxic stimulation affected histidine metabolism in RBL-2H3 cells(MCs).Through histidine increase,the production of His increased,Hypoxic pulmonary vasoconstrietion(HPV)occurrence and PAP increase were promoted.Conclusion Under acute hypoxic stress,pulmonary MCs were rapidly recruited,increased in number(both MC_T and MC_TC type),enhanced in activity,and increased near pulmonary microvessels.The release of IL-6 increase,and Tryptase participate in the "waterfall effect" of lung MCS activation.Directly or indirectly release increased His,5-HT and Ang II,which participate in or even promote the process of acute hypoxic PAP increase.Under acute hypoxic stress,the activation of MCs under hypoxia stress can increase the production of His through histidine metabolism pathway,leading to constriction of pulmonary small vessels and increase of PAP,further promoting the occurrence of HAPE.