Mechanisms Of Cyr61 In Ventilation-induced Lung Injury

Part 1:The effect of mechanical stretch of A549 cells on cell activity and inflammatory factor IL-8 expressionSummary Objective To investigate the effect of cyclic mechanical stretch of typeⅡalveolar epithelial cells(A549 cells) on the cell apoptosis and the secretion of IL-8. Methods A549 cells were stretched for different periods (Omin,15min,30min,60min, 120min)at strain 15%,0.5Hz. The cell apoptosis was observed in the different periods. mRNA and protein of interleukin IL-8 were assessed by RT-PCR and Enzyme linked immunosorbent assay(ELISA). Results At strain 15%,0.5Hz, the cell apoptosis of A549 cells increased with the stretching time. The mRNA and Protein of interleukin IL-8 increased in a time and dose-dependent manner. Conclusion Mechanical stretch can induce cell apoptosis and secretion of IL-8 in human Alveolar typeⅡA549, which is depend on stretching time.Part 2:The construction of siRNA recombinant expression vector targeting Cyr61 geneSummary Objective To construct SiRNA recombinant expression vector targeting Cyr61 gene, and to screen the stably transfected cell clone. Methods Three SiRNAs sequences based on the sequence of Cyr61 mRNA in the GenBank were designed and synthesized, and a scrambled SiRNA sequences served as negative control. The cDNA was synthesized and inserted into plasmid pGenesil1.1. The plasmids was sequenced to confirm the inserted sequence. RT-PCR and Western blot analysis were used to assess the levels of Cyr61 mRNA and proteins after the constructed plasmids had been transfected into A549 cells. Apoptotic ratio was detected by flow cytometry. Results It was confirmed by restriction endonuclease and sequence analysis that SiRNA recombinant expression vector targeting Cyr61 gene was constructed successfully. Inhibition ratio of Cyr61 SiRNA at mRNA and protein levels were 60.54% and 52.97%. Conlusion The SiRNA recombinant expression vector targeting Cyr61 gene has been constructed successfully and can inhibite the expression of Cyr61 gene in A549 cells significantly.Part 3:The effect of interference on Cyr61 expression and biological activity in A549 cellsSummary Objective To study the effect of mechanical stretch on Cyr61 expression and biological activity in A549 cells. Methods:①A549 cells were given different strain(0%,5%,15%,30%) at frequency 0.5Hz for 2 hours. And then A549 cells were stretched for different periods(0min,15min,30min,60min,120min)at strain 15%,0.5Hz. mRNA and protein of Cyr61 were detected by PCR and western blot.②The A549 cells cultured in vitro were divided into 3 groups, i.e., normal control group, negative plasmid-transfected and stretched group(transfected with blank plasmid 48h before the stretch), active plasmid-transfected and stretched group(transfected with chemosynthetic Cyr61 specific SiRNA 48h before the stretch). Then the cells of stretched group were stretched at strain 15%,0.5Hz for 2h. Apoptosis of the cells following treatments were routinely monitored. Results The mRNA and Protein of Cyr61 increased in a time and dose-dependent manner with the stretch. Conclusion The production and secretion of Cyr61 increased in a time and dose-dependent manner with the cyclic stretch in A549 cells. Stretch can induce apoptosis of A549 cells through up-regulation of Cyr61 which may play a role in ventilator-induced lung injury. Part 4:Study of the Cyr61 transduction mechanism in human alveolar typeⅡepithelial cells by mechanical stretchSummary Objective To investigate the effect of Cyr61 secretion on activity of NF-κB and expression of the downstream inflammatory factors. Methods①To observe the activation of NF-κB, A549 cells were exposed to cyclic strech for Omin,15 min,30 min, 60 min and 120min. Rel A and IκBαwas assessed by western blot.②The A549 cells cultured in vitro were divided into 3 groups, i.e., normal control group, negative plasmid-transfected and stretched group(transfected with blank plasmid 48h before the stretch), active plasmid-transfected and stretched group(transfected with chemosynthetic Cyr61 specific SiRNA 48h before the stretch). Then the cells of stretched group were stretched at strain 15%,0.5Hz for 15min,30min,60min and 120min. Rel A and IkB-αwere assessed by western blot.③With the same intervention as above, mRNA and protein of interleukin(IL)-8 were assessed using RT-PCR and ELISA in different stretch periods. Results Western blot shows that RelA was detected as soon as 15min after the initiation of cyclic stretch, peaked at 30min, and then returned to basal level. IkB-αshowed an opposite change. In active plasmid-transfected and stretched group, the translocation of Rel A and the degradation of I IkB-awere obviously inhibited, and the secretion of IL-8 both in mRNA and protein decreased significantly. Conclusion Mechanical stretch can induce the activation of NF-κB path through activating the secretion of Cyr61, and can lead the lung injury through up-regulating the expression of IL-8.