| ObjectiveThe apoptosis of alveolar epithelial cells plays a key role in acute lung injury (ALI) by inflammatory mediators such as TNF-α. Apoptosis-related molecules, NF-κB and Bcl-2 family are important apoptosis regulators in alveolar epithelial cells. Clarity the apoptosis mechanism of alveolar epithelial cells in ALI is conducive to the treatment of ALI.In this study, we used recombinant human TNF-αto stimulate alveolar typeⅡcells A549. The rate of apoptosis was investigated by flow cytometry, the expression of Bcl-2 and Bax was tested by RT-PCR, immunohistochemisty and Western blotting at different times in order to understand the effect of apoptosis and the expression of Bcl-2/Bax in alveolar typeⅡepithelial cells by TNF-α.We employed RNA interference (RNAi) to inhibit the expression of apoptotic gene Bax to investigate the effect of TNF-αon the apoptosis in alveolar typeⅡepithelial cells. Then we also use RNAi to inhibit the expression of NF-κB/p65 to clarify the function and mechanism of NF-KB/p65 in TNF-αinduced apoptosis of alveolar typeⅡepithelial cells.Methods Part 1:Alveolar typeⅡepithelial cells A549 were stimulated by TNF-α(10ng/ml) in vitro when the cell density at 80%-90%. The rate of apoptosis was tested by flow cytometry, the level of the Bcl-2 and Bax mRNA were tested by RT-PCR, the expression of Bcl-2 and Bax protein were tested by Western blotting and immunohistochemisty.Part 2:A549 cells were divided into four groups according to RNAi treatment: control group, TNF-a treated group, Bax siRNA group and control siRNA group. Chemically synthesized small interfering RNA (siRNA) directed against human Bax gene was transfected into A549 cells by cationic liposome. RNAi effect was investigated by RT-PCR, Western blotting and immunohistochemisty, and the rate of apoptosis was investigated by flow cytometry.Part 3:A549 cells were divided into four groups according to RNAi treatment: control group, TNF-a treated group, NF-κB/p65 siRNA group and control siRNA group. Chemically synthesized siRNA directed against human NF-κB/p65 gene was transfected into A549 cells by cationic liposome. RNAi effect was investigated by RT-PCR and immunohistochemisty, and the rate of apoptosis was investigated by flow cytometry. Furthermore, the expression of Bcl-2, Bax was investigated by RT-PCR and Western blotting.ResultsPart 1:The analysis of flow cytometry revealed that the rate of apoptosis of A549 cells was gradually increased in a time-dependent manner (P<0.05). The mRNA and protein expression of Bcl-2 mRNA and protein were significantly decreased compared with TNF-a Oh group (P<0.05). In contrast, the level of mRNA and protein of Bax were significantly increased (P<0.05).Part 2:Bax gene was knocked down significantly in Bax RNAi group(P<0.05). At the same time, apoptosis was induced by TNF-αtreated group and control siRNA group. However, it was abolished in Bax siRNA group by the down-regulation of Bax gene compared to TNF-αtreated group and control siRNA group(P<0.05).Part 3:Compared with normal control group, TNF-αsignificantly increased the level of NF-κB/p65 mRNA and protein in A549 cells (P<0.05). Chemically synthesized siRNA significantly inhibited the expression of NF-κB/p65mRNA and protein in A549 cells (P<0.05). The rate of apoptosis in NF-KB/p65 siRNA group was significantly lower than other groups (P<0.05). The level of Bcl-2 protein decreased significantly, oppositely the level of Bax protein increased.Conclusion1. Low concentration of TNF-αin 10ng/ml induces apoptosis in A549 and promotes the expression of Bax and inhibits the expression of Bcl-2;2. Bax gene silencing significantly reduced TNF-αinduced apoptosis in A549 cells;3. Silencing NF-κB/p65 gene inhibits TNF-α-mediated apoptosis in alveolarⅡepithelial cells via regulating the expression of Bcl-2 and Bax;4. NF-κB/p65 plays an important role on apoptosis of alveolar epithelial cells. |
PDF Full Text Request