Synergetic Effect Of α-lipoic Acid With Keratinocyte Growth Factor On Protecting Alveolar Epithelial TypeⅡ Cells Of Rat Fetus From Hyperoxia-induced Injury

Part Ⅰ Prolonged exposure to hyperoxia inhibits the protective effect of KGF on ATIICs of rat fetus[Objective] To explore the effects of keratinocyte growth factor (KGF) on alveolar epithelial type Ⅱ cells (ATIICs) exposed to hyperoxia.[Methods] Primary culture of AEC II from the Sprague-Dawley rat fetuses was studied under room air and95%of O2for0h、0.5h、1h、4h、8h and12h. Various concentrations of KGF (0、15、25、50、75、100ng/ml) were added into the cell culture. The levels of intracellular reactive oxygen species (ROS), cleaved caspase-3, LDH and proliferation of AEC Ⅱ were measured using flow cytometer, western blot, ELISA and the MTT assays, respectively.[Results] Exposure to hyperoxia resulted in significant increase of intracellular ROS production in AEC Ⅱ cells compared to the room air group. Expression of the cleaved caspase-3was up-regulated after AEC Ⅱ cells were exposed to hyperoxia for4and8hours. At the same time, addition of25ng/ml and75ng/ml KGF led to decreased expression of cleaved caspase-3compared to the control. Proliferation of AEC Ⅱ was decreased and production of LDH was increased under hyperoxia in a time-dependent manner. The lower doses of KGF (<50ng/ml) resulted in further proliferation of AEC Ⅱ and lower production of LDH after being exposed to hyperoxia for0.5and1hour. After exposure to hyperoxia for8hours,50ng/ml and75ng/ml of KGF were required to increase proliferation of AEC Ⅱ in vitro. After12-hour culture of AEC Ⅱ under hyperoxia, up to100ng/ml KGF did not result in higher proliferation than the control.[Conclusions] KGF promoted proliferation and inhibited apoptosis of rat fetal ACE Ⅱ in room air or under temporary exposure to hyperoxia in vitro. However, prolonged exposure to hyperoxia may decrease the sensitivity of AEC Ⅱ to KGF and limit its protective effects on lung injury. Part II Mechanism of decreased sensitivity of ATIICSs to KGF after prolonged exposure to hyperoxia[Objective] The aim of this study was to explore the mechanism of decreased sensitivity of ATIICSs to KGF induced by prolonged exposure to hyperoxia[Methods] Primary culture of ATIICs from the Sprague-Dawley rat fetuses was examined under room air and95%of O2. Various KGF concentrations (0to100ng/mL) were added into the cell culture. Levels of intracellular reactive oxygen species, necrosis, and proliferation of ATIICs were measured using flow cytometry, ELISA, and MTT assays, respectively. RT-PCR was performed to detect KGFR mRNA expression. Western blot was employed to detect the expression of KGFR, phospho-p53, HDAC1, and acetylated H3and H4.[Results] Obvious reduction of KGFR mRNA occurred after4h exposure to hyperoxia. Moreover, exposure for8h to12h induced much lower KGFR mRNA expression than that of the room air group. KGFR protein expression in the ATIICs was decreased remarkably with prolonged exposure, and lower KFGR expression was observed from24h to36h under hyperoxic condition. Phospho-p53(ser392) expression of ATIICs in the hyperoxic group was significantly higher than that of the room air group at4,8, and12h after hyperoxic exposure. HDAC-1was markedly upregulated after4and8h hyperoxic exposure and reached a plateau level at4h exposure compared with that of the room air condition group, and the total p53levels remained unchanged. Acetylation level of H4decreased significantly after4and8h exposure to hyperoxia in a time-dependent manner.[Conclusions] Oxidative stress suppressed the KGFR gene and protein expression after prolonged exposure to hyperoxia. Further investigation demonstrated that down-regulation of KGF receptor via activation of p53and subsequent recruitment of HDAC1induced by oxidative stress contributed to KGF resistance. HDAC promoted H4deacetylation leading to transcriptional inhibition of the KGFR gene. Part III Synergetic effect of a-lipoic acid with KGF on protecting ATIICs of rat fetus from hyperoxia-induced injury[Objective] To explore the potential mechanism of the synergetic effect of a-lipoic acid with keratinocyte growth factor (KGF) on protecting alveolar epithelial type II cells (ATIICs) from hyperoxia-induced injury.[Methods] Primary culture of ATIICs from the Sprague-Dawley rat fetuses was examined under room air and95%of O2.25ng/mL of KGF and0.5mM of a-lipoic were added into the cell culture. Levels of intracellular reactive oxygen species, necrosis, and proliferation of ATIICs were measured using flow cytometry, ELISA, and MTT assays, respectively. Western blot was employed to detect the expression of KGFR, phospho-p53.[Results] The level of intracellular ROS in a-LA group was significantly lower than that of the hyperoxic group, and the difference became more remarkable with prolonged exposure. The phospho-p53(ser392) expression was reduced significantly in a-LA group than that in the control after4and8h hyperoxic exposure. The KGFR expression of ATIICs in the a-LA group were2.47-and1.58-fold higher than those in the control under hyperoxic condition for12and24h, respectively. Markedly higher viability and lower necrosis of ATIICs were detected both in KGF and a-LA-KGF groups under room air and after4h hyperoxic exposure, compared with that in the blank. After prolonged hyperoxic exposure for8and12h, significant protective effect was observed only in the a-LA-KGF group compared with the other groups. Furthermore, no significant difference was found between the blank and KGF groups.[Conclusions] Application of KGF combined with a-lipoic acid could inhibit KGF resistance to provide maximum protection to ATIICs from hyperoxic injury.