The Effect Of FOXO1 On TNF-α-induced Type Ⅱ Alveolar Epithelial Cells Apoptosis And Its Mechanism

1 BackgroundClinically, acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a common severe disease, the mortality rate of which is as high as 50% to 70%in the country, mainly due to addition to mechanical ventilation outside there is still yet not find effective treatment.Various non-cardiac diseases such as severe burns, infection, shock, etc., can secondary systemic inflammatory response syndrome (systemic inflammatory response syndrome, SIRS)with a rapid progression.If the treatment is not timely, it can be developed into a comprehensive multi-organ dysfunction Zheng (multiple system organ dysfunction, MODS).ALI is the performance of SIRS in the lung. The essence of its pathophysiological process is the excessive amplificated inflammation and overactivated oxidative stress. Imbalanced inflammatory induced a large number of reactive oxygen species(ROS), which in turn increasing tumor necrosis factor -α (Tumor Necrosis Factor -α, TNF-α) and other inflammatory factors, resulting diffuse interstitial pulmonary and alveolar epithelial cells edema which leading to serious hypoxemia and respiratory failure, the serve stage of which is called ARDS TNF-α is one of the major inflammatory mediators in ALI/ARDS pathophysiology, it not can mediates amplification of inflammatory response, but also breaks the oxidation-antioxidant system balance by induces endothelial cells and neutrophils producding large amounts of reactive oxygen species, leading to lung tissue apoptosis and necrosis which has a seriously effect on the severity and prognosis of patient.Studies have show that over-amplificatied inflammatory response and oxidative stress induced wide alveolar epithelial apoptosis is an important basis for pathogenesis of ALI. Excessive apoptosis causes not only decrease of structure cells, while aggravating the pathological changes in the lung tissue.Addition clearing damaged structural cells may cause inflammation and aggravate exsisted lung injury.Apoptosis is programmed cell death under certain physiological or pathological conditions, it has great significance for the stability and growth of the body. Apoptotic pathways include extrinsic apoptosis pathway and intrinsic apoptosis pathway. The Bcl2 families play a very important role in the regulation of apoptosis. According to the function they are divided into two categories, one is the anti-apoptotic proteins, including Bcl2, Bcl-xl, Bcl-w, etc., while the other is the pro-apoptotic proteins, including Bax, Bad, Bim, etc.FOXO is one of FOX family, it contains highly conserved DNA domains, namely Forkhead domains, including helix-turn-helix motif, three a helix and a wing-like structure constructed by two large rings. FOXO includes four subtypes in mammals, FOXO1, FOXO3a, FOXO4 and FOXO6. FOXO6 shares the same highly conservative with the other three FOXO proteins, but lacks of PKB phosphorylation sites in the C terminus. FOXOs play an important role in cell differentiation, metabolism, tumorigenesis, skeletal muscle development and other processes. FOXOs’distribution is tissue-specific, whereas FOXO1 is higher expressed in adipose tissue, skeletal muscle and lung tissue Currently, many studies have shown FOXOs can regulate apoptosis process. FOXO3a involves in the process of ROS-induced mouse undifferentiated adipocytes apoptosis by regulating the expression of apoptosis-related genes Bim. FOXOs play an important role in oxidative stress and apoptosis directly as a transcription factor, or co-regulating with other transcription factors involved in gene transcription. Studies have shown that high expression of FOXOs related to the inhibition of cell growth, FOXOs will promote apoptosis when translocating from the cytoplasm to the nucleus.FOXOs as an important regulator of apoptosis is still unclear whether involved the in ALI process. FOXO1 regulated Osteoblasts apoptosis induced by excessive inflammation. Another study showed that TNF-a mediating adipocytes apoptosis can increase expression of proinflammatory cytokines IL-6、MCP-1, while also increase the activity of Akt-dependent FOXO1 Therefore, we hypothesized that FOXO1 may play an important role in apoptosis of alveolar epithelium of ALI. In view of this, we construct ALI cell inflammation model and use recombinant DNA technology and RNAi interference technology to study the fuction of FOXO1 in alveolar epithelial cell apoptosis and its mechanism, expecting to provide new solutions for the treatment and treatment in accordance with ALI/ARDS.2 Objectives(1) By using genetic recombination technology, to explore and verificate the effects of enhanced FOXO1’s expression on TNF-a-induced alveolar epithelial cells apoptosis and expressions of apoptotic genes of bcl2/Bax and Bim.(2) By using RNAi technology, to explore and verificate the effects of silencing FOXO1’s expression on TNF-a-induced alveolar epithelial cells apoptosis and expressions of apoptotic genes of bcl2/Bax and Bim.3 Methods and materialsType II alveolar epithelial cells (A549) were obtained from experimental department of Guangzhou General Hospital of Guangzhou Military command and routinely grown in F12K supplemented with 10% fetal bovine serum (FBS) in a humidified chamber supplemented with 5% CO2 at 37℃.3.1 The effect of enhanced FOXO1’s expression on type II alveolar epithelial cells apoptosis induced by TNF-a and its mechanism.3.1.1 The effect of enhanced FOXO1’s expression on TNF-a-induced type II alveolar epithelial cells apoptosisThe cells were divided into four groups:Control group (without any intervention factor), TNF-a group (intervent with TNF-α), FOXO1 group (pre-transfection with GV230-FOXO1 plasmid and intervent with TNF-a), Negative control group (pre-transfection with GV230 empty plasmid and intervent with TNF-a).2% serum concentration of culture medium starved cells for eight hours. Continue to culture the cells for twelve hours after six hours of plasmid infection, subsequently stimulating the cells by TNF-a for twenty four hours. A549 cells apoptosis rates of all groups were detected by flow cytometry.3.1.2 The effect of enhanced FOXO1’s expression on expressions of bcl2/Bax and Bim apoptotic genes.The cells were divided into five groups as indicated above.RT-PCR detected levels of bcl2/Bax, Bim mRNAWestern Blot detected levels of bcl2/Bax, Bim protein3.2 The effect of RNAi inhibiting the expression of FOXO1 on type II alveolar epithelial cells apoptosis induced by TNF-a and its mechanism.3.2.1 The effect of silencing the expression of FOXO1 by siRNA on TNF-a-induced type II alveolar epithelial cells apoptosisThe cells were divided into four groups:Control group (without any intervent factor), TNF-a group (intervent with TNF-α), FOXO1 si group (pre-transfection with FOXO1 siRNA and intervent with TNF-a), Negative control group (pre-transfection with negative siRNA and intervent with TNF-a).2% serum concentration of culture medium starved cells for eighteen hours. The RNA interference lasted for 6 hours, then with TNF-a stimulated for 24 hours.A549 cells apoptosis rates of all groups were detected by flow cytometry.3.2.2 The effect of silencing the expression of FOXO1 on expressions of bcl2/Bax and Bim apoptotic genes.The cells were divided into five groups as indicated above.RT-PCR detected levels of bcl2/Bax, Bim mRNAWestern Blot detected levels of bcl2/Bax, Bim protein3.3 Statistical methodsSPSS20.0 was used for statistical analysis. Measurement datas were expressed as means ± SD (X ± S). One-way ANOVA was used for comparison between the different groups. Datas of each group were first tested for homogeneity of variance, When variance equal, multiple comparison were tested by the least significant difference (LSD) test, if not using the Dunnett’s T3 test. P<0.05 means difference was statistically significant.4 Results4.1The effect of enhanced FOXO1 expression on type Ⅱ alveolar epithelial cells apoptosis induced by TNF-a and its mechanism.4.1.1 The effect of enhanced FOXOl’s expression on TNF-a-induced type Ⅱ alveolar epithelial cells apoptosisCells apoptosis rates of TNF-a group, FOXO1 group and Negative control group were obviously higher than control group, the differences were statistically significance (P<0.05);There is no statistically significance between TNF-a group and Negative group (P>0.05); FOXO1 group was obviously higher than TNF-a group and Negative control group, the difference was statistically significance (P<0.05).4.1.2 The effect of enhanced FOXO1’s expression on bcl2/Bax and Bim mRNA’s expressionsbcl2/Bax mRNA’s expressions of TNF-a group, FOXO1 group and Negative control group were obviously lower than control group, the differences were statistically significance (P<0.05);There is no statistically significance between TNF-a group and Negative control group (P>0.05); FOXO1 group was obviously lower than TNF-a group and Negative control group, the difference was statistically significance (P<0.05).Bim mRNA’s expressions of TNF-a group, FOXO1 group and Negative control group were obviously higher than control group, the differences were statistically significance (P<0.05);There is no statistically significance between TNF-a group and Negative control group (P>0.05); FOXO1 group was obviously higher than TNF-a group and Negative control group, the difference was statistically significance (P<0.05).4.1.3 The effect of enhanced FOXO1’s expression on bcl2/Bax and Bim proteins’ expressionsBcl2/Bax protein’s ration of TNF-a group, FOXO1 group and Negative control group were obviously lower than control group, the differences were statistically significance (P<0.05);There is no statistically significance between TNF-a group and Negative control group (P>0.05); FOXO1 group was obviously lower than TNF-a group and Negative control group, the difference was statistically significance (P<0.05).Bim protein’s expressions of TNF-a group, FOXO1 group and Negative control group were obviously higher than control group, the differences were statistically significance (P<0.05);There is no statistically significance between TNF-a group and Negative control group (P>0.05); FOXO1 group was obviously higher than TNF-a group and Negative control group, the difference was statistically significance (P<0.05). 4.2The effect of inhibiting the expression of FOXO1 by RNAi on type II alveolar epithelial cells apoptosis induced by TNF-a and its mechanism. 4.2.1The effect of silencing expression of FOXO1 by siRNA on TNF-a-induced type II alveolar epithelial cells apoptosisCells apoptosis rates of TNF-a group and Negative control group were obviously higher than control group, the differences were statistically significance (P<0.05);There is no statistically significance between TNF-a group and Negative control group (P>0.05); cells apoptosis rate of FOXO1si group was obviously lower than TNF-a group and Negative control group, the difference was statistically significance (P<0.05).4.2.2 The effect of silencing the expression of FOXO1 on bcl2/Bax and Bim mRNA’s expressionsbcl2/Bax mRNA’s expression of TNF-a group, FOXO1si group, and Negative control group were obviously lower than control group, the differences were statistically significance (P<0.05);There is no statistically significance between TNF-a group and Negative control group (P>0.05); FOXO1si group was obviously higher than TNF-α group and Negative control group, the difference was statistically significance (P<0.05).Bim mRNA’s levels of TNF-a group and Negative control group were obviously higher than control group, the differences were statistically significance (P<0.05);There is no statistically significance between TNF-a group and Negative control group (P>0.05); FOXO1si group was obviously lower than TNF-a group and Negative control group, the difference was statistically significance (P<0.05).4.2.3 The effect of silencing the expression of FOXO1 on bcl2/Bax and Bim proteins’ expressionsbcl2/Bax proteins’ expression of TNF-a group, FOXO1si group, and Negative control group were obviously lower than control group, the differences were statistically significance (P<0.05);There is no statistically significance between TNF-a group and Negative control group (P>0.05); FOXO1si group was obviously higher than TNF-a group and Negative control group, the difference was statistically significance (P<0.05).Bim protein’s expression of TNF-a group and Negative control group were obviously higher than control group, the differences were statistically significance (P<0.05);There is no statistically significance between TNF-a group and Negative control group (P>0.05); FOXO1 si group was obviously lower than TNF-a group and Negative control group, the difference was statistically significance (P<0.05).5ConclusionFOXO1 play a favoring role in TNF-a-induced type II alveolar epithelial cells apoptosis. This may result from up regulating the expression of pro-apoptotic gene Bim, and down regulating the ratio of apoptotic gene bcl2/Bax.