Effects of PBEF on HPMEC and A549 cells and the pathogenesis of ARDS

Abstract:

Objective：To investigate the role of pre-B-cell colony enhancing factor（PBEF） in the pathogenesis of acute respiratory distress syndrome（ARDS）. Methods：Human pulmonary microvascular endothelial cell line（HPMEC） and human type Ⅱ alveolar epithelial cell line（A549） were constructed by increasing human recombinant PBEF（rPBEF） concentrations（0，50，100，500，1 000，2 000 ng/mL） in a stepwise manner. The cell viabilities to rP－BEF were tested by MTT assay. And then the cells were stimu－lated with 100 and 1 000 ng/mL rPBEF while the blank control group was set up. The cell cycle and cell apoptosis were analyzed using flow cytometry（FCM）. Morphological changes of apoptosis were observed by transmission electron microscopy. The expres－sions of caspase-3，B-cell lymphoma/leukemia-2 gene were detected by Western blot. The expressions of aquaporin 1，interleukin-8 and interleukin-1β were detected using RT-PCR and Western blot. Results：The cell viabilities were significantly inhibited in the rPBEF group with 100，500，1 000，2 000 ng/mL rPBEF compared with those in blank control group（P<0.05）. FCM showed that the percentage of S phase in 100 and 1 000 ng/mL rPBEF groups[HPMEC：（43.847±1.272）%，（63.300±2.102）%；A549：（36.247±2.045）%，（46.400±1.346）%）] were significantly higher than those in the blank control group[HPMEC：（19.347±2.052）%；A549：（17.297±0.800）%]（all P=0.000） and the apoptic rate in 100 and 1 000 ng/mL rPBEF groups[HPMEC：（11.317±0.533）%，（15.227±0.637）%；A549：（6.120±0.439）%，（8.633±0.497）%] were significantly higher than those in the control group[HPMEC：（8.433±0.600）%；A549：（3.877±0.666）%）]（PHPMEC=0.001，PHPMEC=0.000；PA549=0.002，PA549=0.000）. Transmission electron microscopy showed typical apoptosis，such as heterochromatin concentrated which was set in the nuclear membrane and visible apoptotic bodies in 1 000 ng/mL rPBEF group. The expressions of Bcl-2 protein in 100 and 1 000 ng/mL rPBEF groups[HPMEC：（0.418±0.043），（0.190±0.012）；A549：（1.276±0.212），（0.601±0.164）] were significantly decreased（all P=0.000） and the ex－pressions of caspase-3 protein were significantly increased in 100 and 1 000 ng/mL rPBEF groups[HPMEC：（0.763±0.030），（1.170±0.056）；A549：（0.217±0.010），（0.375±0.032）]，respectively，compared with those of blank control group（PHPMEC=0.000，PHPMEC=0.000；PA549=0.001，PA549=0.000）. The expressions of AQP1 gene and protein in 100 and 1 000 ng/mL rPBEF groups were statisti－cally lower than blank control group[mRNA：HPMEC（0.543±0.113），（0.287±0.093） vs. （1.050±0.155）（P=0.002，P=0.000），A549（0.823±0.104），（0.463±0.184） vs. （1.317±0.215）（P=0.013，P=0.001）；protein：HPMEC（0.494±0.038），（0.233±0.030） vs. （0.824±0.067）（all P=0.000），A549（0.850±0.157），（0.484±0.118） vs. （1.344±0.136）（P=0.005，P=0.000）] while the expressions of IL-8 and IL-1βgene and protein were statistically higher than blank control group in 100 and 1 000 ng/mL rPBEF groups（P<0.05）. Conclusion：PBEF may induce the apoptosis of HPMEC and A549 by down-regulating the expression of Bcl-2 and up-regulating the expression of caspase-3 and PBEF could induce low expression of AQP1 which suggests that PBEF may play an critical role in the development of ARDS.