Experimental Studies On Hypoxia And Nacn-Induced Lung Injuries And Their Mechanisms

Hydrogen cyanide(HCN),a widely used high toxic industrial chemical,has long been employed as a chemical warfare agent since the First Wold War (WWI). It is generally released in vapor and could be quickly inhaled into the body. Both sodium cyanide and potassium cyanide are also cyanogenic compounds.They can mimic the effect of hydrogen cyanide in body. This kind of chemicals can generate cyanide ion, which could immediately find its target site existed in the respiratory chian on the respiratory chain,combind with the enzyme of cytochrome aa3 and inhibit its activity to transfer electrons.The consequent results includes the decreases of oxidative phosphorylation and ATP generation in cells. Therefore, people with cyanide intoxication will die from respiratory failure and circulatory failure within minutes.Hypobaric hypoxia is the main deleterious environmental factor to health in high-altitude. As altitude raising, the air becomes thinner, which means less oxygen is in the atmosphere. The lungs get less oxygen with each breath and the amount of oxygen in blood is declined. Acute exposure in it causes high-altitude sickness such as acute mountain sickness(AMS), high altitude cerebral edema (HACE) and high altitude pulmonary edema (HAPE) as well.Because of the use of cyanide in war and in terrorism,the possibility of cyanide intoxication for people in high-altitude and other hypoxic condition is also existed. Clinical features of combined injury of hypoxia and cyanide intoxication is unclear.A few result is reported about the mechanism of this combind injuries,especially in lung.Alveolar epithelial type II cells(AEC II)are important structure cells that line the alveolar surface .They act as the stem cell for the alveolar epithelium to proliferate and further differentiate into type I cells, and play a critical role in the repair of lung injuries.The main character of Acute Lung Injury (ALI) is pneumonedema and alveolar collapse, there are many erythrocytic and plasmic-fibrous exudation in alveolar. Pneumonedema, alveolar and dysfunction of gas exchange is closely related to the decrease of pulmonary surfactant (PS). Hypoxia can induce the dysfunction of AEC II, that in turn hampers the synthesis and secretion of PS. The'epithelial hypothesis'linked apoptotic death of alveolar epithelial cells with the pathogenesis of acute lung injury.In order to well understand the lung injury induced by cyanide intoxication and the joint effect of hypoxia and sodium cyanide on the AEC II, we established an animal model with treatments under a hypobaric condition simulating the high altitude.Changes of the histomorphology, pulmonary surfactant and the functions in rats lung were synchronously investigated. Cell intoxication model with cyanide was also established in cultured primary AEC II cells. Alteration of AEC II's function and apoptosis-associated genes were determined.Method:1. in vivo studies: male SD rats were randomly divided into 2 groups: the hypobaric hypoxia group (HH)in 4000m high altitude and the normoxia group (NH) in 308m altitude. The animals for the experiment of hypoxia were processed in a artificial hypobaric chamber to simulating the air pressure of 4000m altitude (61kPa).Sodium cyanide (NaCN)was injected subcutaneously to the rats in both the two groups at the dose of 3.6mg/kg. Histology of lung tissue, SP-A and apoptosis gene of AEC II were observed with HE and immunohistochemistry staining. Changes of vasopermeability, contents in BALF, oxidative stress were detected with biochemistry.2. in vitro studies: Primary cultured alveolar epithelial type II cells were intoxicated with sodium cyanide: Cell morphology and viability were observed. Protein expression of SP-A, HIF-1α, P53 were detected with Western Blot. Medical intervention on the function of AEC II was investigated with LY294002 and pifithrin-α.Results:Part one: in vivo studies1. Pathological injuries of the lungs were observed in the rats treated with NaCN intoxication and/or hypoxia.That includes widespread edema and hemorrhage. Meanwhile,less lamina-body containing in alveoli and endothelium swollen were manifested in TEM.2. Immunohistochemical staining demonstrated increased expressions of HIF-1αand P53. SP-A expressions in the lungs, however,were declined in rats treated with sodium cyanide and/or acute hypobaric hypoxia.3. Compared with the normoxia rats, the relative value of lung vs body and the wet/dry weight ratio of lung were significantly higher in HH(P<0.05).NaCN intoxication and acute hypobaric hypoxia produced a more significant increasing effect(P<0.05).4. Compared with the normoxia animals,the cell count in BALF of the rats was significantly higher in HH(P<0.01).NaCN intoxication and acute hypobaric hypoxia produced a more significant increasing effect(P<0.01).5. Compared with normoxia animals,the total protein level in BALF was significantly higher in the rats of HH group(P<0.01).NaCN intoxication and acute hypobaric hypoxia produced a more significant increasing effect(P<0.01).6. Normoxia and hypobaric hypoxia significantly increased the phospholipids content in rats BALF(P<0.05). Phospholipids content in the lung tissues of the rats, however, was obviously decreased. Acute hypobaric hypoxia and cyanide intoxication produced a more significant increasing effect on the phospholipids content in the rats BALF(P<0.05).7. The activity of LDH, ACP and AKP in BALF of the rats intoxicated with cyanide were reached the highest level at 0.5h, the values in the hypoxia group are significantly higher than those in nomoxia group.8. Decreased SOD, GSH-PX activity and GSH content were determined both in lung tissues and in BALF. These parameters reached their peak value at 0.5h. The content of MDA was increased and presented a the highest level at 0.5h in NaCN intoxicated animals, correspondingly the activity of SOD, GSH-PX, content of GSH in lung tissue and BALF reached the lowest at 0.5h in NaCN intoxicated animals under hypoxia, but they were still lower than that of control and NaCN intoxicated group(P<0.01).Part two: in vitro studies1. The results from our in vitro studies demonstrated the toxicological effect of cyanide on primary AEC cells. Morphology changes of AEC II after treatment with cyanide include the cycloidal cytosome, reduced process and abnormal nuclei. Some cells displayed characters of suspention cells.2. The viability of ACC II significantly decreased by cyanide at the concentration rage of 0.05 mmol/L to 5.00 mmol/L (P<0.01). 3. The expression of SP-A decreased markly in AEC II treated with hypoxia and NaCN intoxication,but HIF-1αand P53 protein expression was increased obviously.4. When AEC II were pretreated with LY294002 or/and PFT-αto inhibit HIF-1α/P53 activity,the expression of SP-A decreased markly(P<0.01).Conclusions:Hypoxia and NaCN intoxication can lead to an acute lung injury in early stage that produced the morphological and functional change of AEC II. HIF-1α/P53 may mediate the apoptosis activation in AEC II under hypoxia/NaCN intoxication.