The Mechanism Of Apoptosis On Rat Alveolar Epithelial Cells Induced By Acute Oxidative Stress

Oxidants play an important role in the pathogenesis of various airway diseases,and ithas been shown to induce two distinct types of cell death: programmed cell death(apoptosis) and necrosis. Genes controlls programmed cell death in two sorts, one isinhibiting cell death, the other is promoting cell death. The two sorts of genes interact witheach other in order to control cell normal death. ROS is increased in oxidative stress,which results in injury widely. However, whether and how hyperoxia induce apoptosis inalveolartype cells when respired high concentration oxygen in short-term remains unclear.To address these questions, we make use of respiring high concentration oxygen to inducealveolar epithelial Cell oxidative stress and test the matter of cytosolic acidification, andthen to observe the mechanisms of oxidative stress in cell apoptosis.Methods: WISTER-mice, weighing150-200g, were studied as follows:theexperimental mice were devided into four groups randomly and exposed to85-100％oxygen in a self-made chamber and the control animals were kept in normal room air.(1)The MDA,SOD,GSH,CAT,T-AOC,NO,NOS were detected in plasmaand lung homogenates in experimental4h,8h,12h,16h. Alveolar type cells apoptosis wasdetected by TUNEL technique.(2)To detect the effects of eNOS and iNOS in early stage of Alveolar epithelial cellsapoptosis induced by oxydative stress, RT-PCR and Western blot analysis were used.(3)To investigate the mechanisms of apoptosis in Alveolar epithelial cells inducedby oxydative stress, RT-PCR and Western blot test to detect Bax/Bcl-2mRNA,Bax/Bcl-2and AKT/mTOR protein changing were used.(4)To observe the role of intracellular acidification in alveolar epithelial Cellsinjured by oxydative stress, BCECF-AM Flowcytometry analysis was used. Results:(1)Compared with the control group, the levels of MDA,NO,NOS in plasm andlung tissue increased markedly in all hyperoxia groups (P＜0.01), and at the same timeSOD,GSH,CAT,T-AOC fell down accordingly(P＜O.O1). Correspondly, the number ofapoptosis cell increased to9.13±3.2％,17.47±3.5％,19.22±4.5%and11.03±2.8％in4hgroup,8h.group,12h.group and16h group respectively compared with the control group.(2)eNOSmRNA/protein expression was clear in control group, increased in4hgroup, increased obviously in8h.group,12h.group and16h group. iNOSmRNA/proteinexpression was far lower than that of eNOS in the control group, slightly increase in16hgroup. Index of plasma and lung tissue and the number of cell apoptosis had a significantpositive correlation.(3)During the ROS-induced Alveolar epithelial cells injuries,the apoptosis rate wasappeared correspond with the increased level of ROS. The ratio of Bax/Bcl-2mRNA andprotein was increased severely. The Akt/phospho-Akt at Ser-473andmTOR/phospho-mTOR at Ser-2481protein expressions decreased obviously.(4)Compared with control group, the pH-sensitive fluorescent probe BCECF-AMappeared cytoplasmic acidification in hyperoxia groups. FL1/FL2ratio decreased and CellProliferation Index increased significantly.Conclusions:1. By observing the apoptosis cellular nuclear morphology induced by oxydativestress,we considered that just for short-term respiring of high concentrate oxygen caninduce apoptosis in alveolar epithelial Cells and result in acute lung injury,and with thetime going,the injure aggravate.2. Our test results suggest that NO/NOS may play an very important role in apoptosisof rat alveolar epithelial Cells when exposed to hyperoxia. Especially at the early stage,eNOS is of prime inportance.3. In our test, BaxmRNA and Bax protein level was increased, Bcl-2mRNA and Bcl-2protein level was decreased, it suggested that ROS was related to apoptosis through Mitochondria injuring.By observing that the Akt/phospho-Akt at Ser-473protein,mTOR/phospho-mTOR at Ser-2481protein expressions obviously decreased, it indicatesthat Akt/mTOR signal pathway may be involved in apoptosis induced by Oxidative Stres.All of these suggested that Mitochondrial pathway may be the fundamental deathmechanism induced by oxydative stress at the early stage.4. By observing the changing of bcl-2/Bax and Akt/mTOR, it suggesteed that with themitochondria be injured,the lysosomal appeareed instability, and mitochondrial deathpathway maybe the main mechanism in ROS-induced alveolar epithelial Cells death. Onthe other hand, the lysosomal instability may aggravate the injury which happened onmitochondria in the early stage cauded by ROS.The balance between mitochondria andlysosome is important to cell survival.5.Lately,study on apoptosis suggested that there was intracelluar abnormal signalingtransduction. Intracellular acidification in these abnormal signaling, is a character ofapoptosis. Intracellular pH adjust maybe tightly related to apoptosis. FL1/FL2ratio andintracellular pH were all decreased, and cell number with acidic intracellular pH increasedinduced by Oxidative Stres. It suggests that oxydative stress induces alveolar epithelialCells death may be related to cytosolic acidification. Cytosolic acidification maybe alongwith apoptosis, moreover also result in mitochondrial and lysosomal injury, aggravatemitochondrial-lysosome cell death pathway,which mechanism remain be furtherinvestigated.