| Strenthening marine power is an important part of building Socialism with Chinese Characteristics.Under this background,the implementation of the “Marine Economic Development Plan” and the “navy moving towards the Deep Blue Strategy” have promoted the vigorous development of diving-related professions.The number of participants in military,commercial and recreational diving is increasing rapidly.In order to balance the pressure inside and outside the body under water,divers must breathe high-pressure gases same as the surrounding environmental pressure.In this process,massive physiological inert gases(such as nitrogen,helium,etc.)will dissolve in blood and tissues.With increasingly decompression,these physiological inert gases dissolved in the body are very easy to overflow and form bubbles.This drives body destruction by mechanical force,blockage of blood vessels and biochemical effects,which is caused by contacting to surrounding tissues.This is such a disease which is called Decompression Sickness(DCS).As a core medical problem in diving activities,DCS happens rapidly,severely and it is hard-handled.The key solution of DCS lies in prevention.Studies have shown that vascular endothelial cell(VEC)damage caused by bubbles play a key role in the pathogenesis of DCS.Air bubbles can directly damage VECs by mechanical action,and aggravate VEC damage by a series of biochemical effects,such as autophagy,pyroptosis,endothelial particles damage,calcium overload and so on.Administration of endothelial protective agents such as aescin not only reduced the damage of VECs,but also significantly reduced the morbidity and mortality of DCSs in rats and Bama pigs.Enhancing the function of VECs may be a potential target for effective prevention of DCS.Hyperbaric oxygen(HBO)is a special medical method to treat diseases by breathing pure oxygen in an environment higher than an atmospheric pressure.Because of its advantage of safe,economic and efficient,it has been widely used in many diseases such as CO poisoning,gas embolism,stroke and so on.Previous studies found that HBO can not only effectively treat DCS,but also reduce the morbidity and mortality of DCS in rats by HBO pretreatment.The mechanism is related to that HBO pretreatment can appropriately increase the level of reactive oxygen species(ROS),mobilize the endogenous protective mechanism and up-regulate the expression of protective proteins.In the study of rat and porcine DCS model,we found that HBO pretreatment can significantly improve the function of VECs,suggesting that HBO pretreatment can effectively combat DCS by activating the internal protective mechanism of VECs.In the study of HBO pretreatment against DCS,we found that the activation of transcription factor Nrf2 and the up-regulation of downstream protein expression may play a key role in the prevention of DCS by HBO pretreatment.In rat pulmonary microvascular endothelial primary cells and rat DCS model,this study aims to observe the role and mechanism of Nrf2 and its downstream proteins in HBO pretreatment against VEC DCS injury.This study also provide reference for the practical application of HBO pretreatment.Objective : Observing the protective effect of HBO pretreatment on rat pulmonary microvascular endothelial cells(PMVECs)touched by bubbles,the role of Nrf2,and Nrf2 downstream proteins in HBO protection.Part II aiming to verify whether Nrf2 enhance VECs function and alleviate DCS damage in HBO pretreatment in vivo..Methods : Rat PMVECs were cultured in vitro.Firstly,transcriptome and western blotting techniques were used to observe the activation of PMVEC Nrf2 and the expression of PMVEC Nrf2 downstream proteins after HBO treatment.Then,during the peak of protein expression,the in vitro cell bubble touch technique was used to simulate the PMVEC DCS damage caused by decompression bubbles.Protein specific inhibitors were used to inhibit protein function to observe the protective effect of HBO pretreatment for PMVECs and the role of PMVEC functional proteins.The efficiency and mechanism of Nrf2 and its downstream proteins on PMVECs after HBO pretreatment is discussed.Finally,according to existing literature reports,combined with the mechanism of HBO pretreatment,we identified the potential signal molecules that be involved in the activation of Nrf2 by HBO pretreatment,and we detected their changes of activation by western blotting.We used specific signal molecular inhibitors to observe their effect for HBO treated-Nrf2,to understand the relationship between signal molecules,and to clarify the mechanism of Nrf2 activation by HBO pretreatment.Results :(1)the variation of Nrf2 activation after HBO treatment and its downstream functional proteins expression : After HBO exposed for 1 h,Nrf2 was activated from the cytoplasm to the nucleus,and the peak is at 4 h.The results of transcriptome showed that the m RNA of Trx-1 and NQO-1 increased significantly after HBO exposure.Western blotting results showed that the expression of both proteins increased significantly at 6 h,and the peak is at 12 h after HBO exposure.(2)The protective effect and mechanism of HBO pretreatment on PMVEC bubble contact injury : After HBO exposureof 12 h,PMVECs were damaged by 2.0 mm bubble touch for 3 h.The results showed that bubble touch could significantly reduce the viability of PMVECs(Bubble vs Air,p < 0.0l),and the HBO pretreatment could significantly reduce the air bubble damage for PMVECs(HBO +Bubble vs Bubble,p < 0.01).The treatment of Trx-1 specific inhibitor,PX-12,and NQO1 specific inhibitor,dicoumarol,could significantly antagonize the protective effect of HBO pretreatment,suggesting that both Trx-1 and NQO-1 participating in protect PMVECs from bubble contact injury by HBO pretreatment.(3)The mechanism of Nrf2 activation by ROS,p38 MAPK,PKC and AKT phosphorylation levels of PMVEC cells increased significantly after HBO exposuration.Nrf2 activation can be inhibited by ROS inhibitor,NAC,and P38 MAPK inhibitor,SB203580,while inhibition of PKC and AKT had no significant effect on the activation of Nrf2.In addition,removing of ROS can also inhibit the phosphorylation of p38 MAPK after HBO exposure.(4)The results of Western blotting and immunohistochemistry showed that there was obvious nuclear translocation of Nrf2 in lung tissue cells at 4 h after HBO exposure,that is,Nrf2 was activated,and the expression of Nrf2 downstream proteins,Trx-1 and NQO-1 were significantly increased at 12 h after Nrf2 exposure.Compared with the normal control group,the lung W/D score,the pathological score of lung tissue,the content of inflammatory cytokines IL-1 β and TNF-αin plasma and the related targets of internal injury,MDA,ET-1 and ICAM-1 in DCS model group were significantly higher than those in normal control group.Pretreatment with HBO for 12 h in advance could significantly reduce the changes of these injury issues.These protective effects of HBO could be significantly antagonized by Nrf2 inhibitor ML385.Conclusion : HBO pretreatment can protect PMVECs against bubble contact injury by increasing ROS production,activating p38MAPK/Nrf2 signal pathway and up-regulating the expression of protective proteins Trx-1 and NQO-1.In vivo HBO pretreatment can enhance the function of VECs and protect them against DCS damage by activating Nrf2,up-regulating the expression of Nrf2 downstream protein,Trx-1 or NQO-1. |
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