Mechanisms Of Acute Hypoxia-induced Disturbance Of Water Balance In Rats

IntroductionPlateau is an important strategic direction for our army.However,the plateau environment,seriously affect the combat effectiveness of the army.How to proceed from the need of military struggle,do a good job at high altitude troops to ensure the health,is an important content in the study of high altitude medicine.When people enter the plateau,there will be acute mountain sickness,including high altitude pulmonary edema,high altitude cerebral altitude(HACE)and acute mountain sickness(AMS).High altitude pulmonary edema and high altitude cerebral edema are the results of the disorder of water metabolism.Even if there is no acute high altitude disease,the relative health of people is also a common phenomenon of drinking water loss and weight loss.Therefore,high altitude can cause the body to dehydration or edema of water metabolism disorder manifestation.Animal experiments showed that acute hypobaric hypoxia can significantly reduce the amount of drinking water in rats.As early as 1981,the changes of drinking water volume in rats at simulated altitude of 5400 m were observed.It was found that in the first days of hypoxia,the water intake of rats was only about 30% of the normal value.Although the volume of urine was significantly reduced,the degree of reduction was lighter than that of drinking water,the urine volume was relatively increased,and the body dehydration,weight loss and hematocrit increased.The study of their mechanism suggested that acute hypoxia in rats can not be explained by osmotic pressure receptors or volume receptors.However,we noticed that in preliminary experiment,once the rats were exposed to hypoxia,the drinking behavior was immediately reduced,and the drinking behavior was recovered rapidly.We hypothesized that the decrease of drinking water in rats with acute hypoxia may be related to the central nervous system,and the decrease of drinking water may be due to the inhibition of hypoxia on the neurons and the inhibition of the excitatory neurons.Involved in the regulation of the thirst for a lot of nerve nuclei.studies have shown that the anterior cingulate cortex(ACC)is the senior cortex in the central nervous system,and the circumventricular organs(CVOs)is the initiator of the thirsty signal.Subfornical organ(SFO)is located in the central part of CVOs,surrounded by a large number of blood vessels and choroid,a and is vulnerable to oxygen partial pressure for lacking of blood brain barrier.Therefore,it is speculated that the neurons in the SFO area are likely to directly perceive the changes of oxygen partial pressure,and then affect the drinking behavior of animals.It is found that the transient receptor potential-vanilloids receptors(TRPVs)on the SFO neurons are the key molecules of thirsty regulation,including TRPV1 and TRPV4.By injecting TRPV4 agonist into the brain,it was found that the water consumption of rats was significantly decreased.It is also proved that TRPV4 is very sensitive to hypoxia and is an important factor in hypoxic pulmonary vasoconstriction.We hypothesized that hypoxia may directly activate TRPV4 on the SFO neurons,then lead to the decrease of water consumption.Human and animals on plateau hypoxia acclimatization is completed in the overall regulation under the neurohumor.In addition to the neural mechanisms,humoral mechanisms may play an important role in the reduction of drinking water.The results showed that Adropin is a secreted small molecule protein encoded by Enho gene,which could regulate the metabolism of the body,and the central injection of Adropin could significantly reduce the amount of drinking water.We also observed that the Adropin content in the serum and brain was increased in hypoxic rats.Therefore,Adropin maybe one of the humoral mechanisms of the decrease of the drinking water of the rats.In moderate hypoxia,the body is compensatory,but severe hypoxia can cause damage to the blood brain barrier(BBB)and increase the permeability of BBB.BBB is composed of vascular endothelial cells and its tight junction,basement membrane and the surrounding astrocytes,foot and peripheral cells.It is extremely stable in homeostasis maintenance barrier in the central environment.The causes of BBB damage in severe hypoxia include endothelial cell apoptosis and tight junction protein degradation,but the underlying molecular mechanisms are not fully elucidated.Therefore,exploring the cause and molecular mechanism of BBB damage will be helpful to find HACE and AMS prevention and treatment methods.In summary,this paper based on the previous work,drinking water from hypoxia decrease and BBB damage in two directions,the mechanism of water metabolism in rats of acute hypoxia,which not only helps to improve our understanding of the mechanism of high altitude acclimatization,but also lay a solid theoretical basis for us to do the medical team at high altitude security.Material and Methods1.Feeding animal to establish a rat model of hypoxic chambers by this study.Under the condition of 3700 m,4000m and 6000 m,the occurrence and molecular mechanism of the decrease of drinking water were studied,and the damage of blood brain barrier was studied under the condition of simulated 8000 m hypoxia.The hypoxia model was constructed in vitro by using a hypoxic cell culture incubator and a confocal microscopy of three gas chamber.2.Using rat cranial catheter technique,10 min before hypoxia,intracerebroventricular injection of TRPV1 inhibitor SB-705498,TRPV4 inhibitor Gadolinium and HC-067047,the corresponding time after hypoxia for accurate measurement of animal drinking amount,intracranial temperature change and the change of osmotic pressure.In vitro SFO primary neurons and HEK293 cells,plasmid and siRNA intervention were used to establish TRPV4 overexpression and knockdown model.Besides,Westernblot,Co-IP,IF,ELISA and Fura-3 calcium imaging techniques were used to detect protein expression of TRPV4 and calcium channel opening.3.Using rat cranial catheter technology in rats 7d before hypoxia,AVV-shRNA target specificity to knockdown TRPV4 expression of CVOs,through the central injection of recombinant Adropin protein,TRPV4 inhibitor HC-067047,CamKK inhibitor STO-609,peripheral injection of Adropin antibody method,animal drinking amount,IF detection and Westernblot detection of neuronal activity TRPV4-CamKK-AMPK pathway measuring changes in corresponding time after hypoxia;4.Using rat cranial catheter technique,rat 48 h before hypoxia,reducing SIRT-3 to knockdown siRNA target by intraperitoneal injection of 1h before hypoxia,different concentrations of Minocycline,blood brain barrier function was detected by colloidal gold electron microscopy and Evans blue method;Using Westernblot,qRT-PCR,IF,luciferase reporter method for detection of HIF-1? /SIRT-3/PHD-2 signaling pathway and the expression of tight junction protein changes in HBMECs cells.Results1.Compared with normoxia group,hypoxia rats in 4000 m for 6h will not cause the change of osmotic pressure and intracranial blood temperature changes,but the amount of water decreased by 75%;compared with the hypoxia group,third intraventricular injection of TRPV4 inhibitor Gadolinium and HC-067047 can significantly increase the amount of water,while the TRPV1 inhibitor SB-705498 can cause increase in drinking water;in addition,compared with the wild type TRPV4-/-mice,the amount of water under normoxic conditions did not change significantly,but water consumption increased significantly in TRPV4-/-mice under hypoxic conditions.Whether it is the overexpression TRPV4 of HEK293 cells or cultured SFO neurons,hypoxia or TRPV4 agonist 4?-PDD will make the intracellular calcium increase rapidly,causes the calcium ion pulse.however,knockdown of TRPV4 in HEK293 cells or SFO neurons,hypoxia could hardly produce calcium ion pulse;knockdown of HO-2 expression in TRPV4 HEK293 cells and primary cultured SFO neurons after hypoxia significantly weakened pulse calcium ions,but TRPV4 agonist 4?-PDD or inhibitor Gadolinium could still change the regulation of intracellular calcium ion;the presence of TRPV4 and HO-2 in SFO neurons to form two dimers.2.Compared with normoxia group,Adropin protein in brain tissue hypoxia and serum 3700 m CVOs increased,while CVOs decreased the number of c-Fos positive cells;compared with the hypoxia group,intracerebroventricular injection of Adropin neutralizing antibody after drinking water decreased 35%,the number of c-Fos positive cells increased.Normoxic condition,rat central injection of recombinant Adropin protein after drinking water reduced the amount of CVOs the number of c-Fos positive cells;compared with the control group,TRPV4 on recovery of water intake and the number of c-Fos positive cells after injection of recombinant Adropin protein.Compared with the control group,the recombinant Adropin protein increased expression of CVOs region p-CamKK and p-AMPK,reduced the number of c-Fos positive cells in rats,while reducing the amount of drinking water,while the TRPV4 inhibitor HC-067047 or CamKK inhibitor STO-609,could reduced the expression of p-CamKK and p-AMPK in CVOs,increased the number of c-Fos positive cells,partial recovery of rats drinking water.3.Compared with normoxia group,8000 m hypoxia increased Evans blue and gold colloid blood brain barrier permeability of vascular endothelial cells.hypoxia decreased TEER,and increased the protein and mRNA expression of HIF-1?,MMP9,MMP2,VEGF,ZO-1,Occludin and Claudin-5,HIF-1? transcriptional activity of VEGF also increased.Compared with the control group,Minocycline dose dependently reduced Evans blue and gold colloid permeability,increased endothelial cell TEER,MMP9,MMP2 and down regulate the expression of VEGF protein and mRNA,but only Minocycline can increase HIF-1? protein expression but not mRNA,but at the same time,the upregulation of ZO-1 protein and mRNA expression of Occludin,Claudin-5,HIF-1?,adding ? at the same time in the treatment of Minocycline inhibitor YC-1 or adding HIF-1? agonist DMOG,could regulate the above changes.Compared with normoxia group,hypoxia induced upregulation of HIF-1? and inhibit the expression of SIRT-3 and PHD-2 protein,but compared with the hypoxia group,Minocycline also increased SIRT-3 and PHD-2 protein expression,SIRT-3 knockdown after Minocycline inhibition of HIF-1? and SIRT-3 decreased,PHD-2 up-regulated.Conclusion1.Acute hypoxia caused the decrease of drinking water,and the severe acute hypoxia could damage the blood-brain barrier.2.The decrease of drinking water in rats with acute hypoxia was closely related to the opening of TRPV4 neurons in midbrain SFO area.TRPV4 and HO-2 exist in the cell membrane of SFO region in the form of complex.In hypoxia,the decrease of drinking water in rats might be caused by the changes of [Ca2+]i in the SFO area,which was caused by the changes of oxygen partial pressure and TRPV4,and the inhibitory effect of HO-2.3.The mechanism of acute hypoxia induced the increase of small molecular protein Adropin expression in rats.Adropin acts on the TRPV4 calcium channel in the central region of CVOs,resulting in the phosphorylation of its downstream calcium dependent Camkk-AMPK signaling pathway,activation of TRPV4 inhibitory neurons,and the decrease of water consumption in rats.4.Severe hypoxic injury of blood brain barrier,Minocycline through activation of SIRT-3/PHD-2 signaling pathway,promoting the degradation of HIF-1?.Minocycline decreased the expression of VEGF and MMP-2,MMP-9,up-reuglated endothelial tight junction protein expression,protected blood brain barrier.The experimental results indicate that the decrease of drinking water is the result of active inhibition of central nervous system.For people staying in serious high altitude hypoxia,Minocycline is expected to become a potential drug for prevention and treatment of high altitude cerebral edema.