Effect Of RAGE On The Expression Of MMPs/TIMPs In Lung Epithelial A549 Cells Induced By TNF-?

Objective: To determine the effects of tumor necrosis factor-?(TNF-?)on the expressions of matrix metalloproteinases(MMPs)and tissue inhibitors of metalloproteinases(TIMPs)in lung epithelial A549 cells,and investigate the role and mechanism of receptor advanced glycation end products(RAGE)in the metabolism of extracellular matrix(ECM)after inflammatory injury of lung epithelial cells,providing an experimental basis for targeted regulation of ECM remodeling in the course of acute respiratory distress syndrome(ARDS).Methods:(1)MTT assay was used to detect the effects of 1ng/mL,10ng/mL and 50ng/mL TNF-? on the proliferation of A549 cells.(2)The expression of interleukin-8(IL-8) gene was detected by quantitative real-time polymerase chain reaction(qRT-PCR)in vitro model of ARDS which built by using 10ng/mL TNF-? to induce A549 cells.(3)QRT-PCR was used to test the effects of TNF-? on the expressions of RAGE gene and the family genes of MMPs and TIMPs in A549 cells.(4)Western blot and ELISA were used to detect the effects of TNF-? on the protein expressions of RAGE and the family factors of MMPs and TIMPs,and activation of NF-?B signaling pathway in A549 cells.(5)Western blot was used to detecte the protein expressions of MMP-2,MMP-3 and MMP-9 and the phosphorylation of NF-?B signaling pathway inA549 cells after the gene expression of RAGE knocked down by RNA interference.Results:(1)50ng/mL TNF-? significantly inhibited the activity of lung epithelial A549 cells(P<0.05),while 1ng/mL and 10ng/mL TNF-? had no significant effect on the viability of A549 cells(P>0.05).(2)10ng/mL TNF-? increased the mRNA expressions of RAGE and IL-8 mRNA levels in cells(P<0.01).(3)Compared with the control group,the mRNA expressions of MMP-1,MMP-2,MMP-3,MMP-7,MMP-9,MMP-10,MMP-12,MMP-13,MMP-14,MMP-15,MMP-16,MMP-19,MMP-25 and MMP-27 were significantly increased(P<0.05 or P<0.01),and the mRNA expressions of MMP-17 and MMP-28 were down-regulated significantly(P<0.05 or P<0.01)in the family of MMPs after the cells were pretreated with TNF-?.In the family of TIMPs,the mRNA expressions of TIMP-1 and TIMP-2 were significantly increased(P<0.01),and the mRNA expressions of TIMP-3 and TIMP-4 were down-regulated(P<0.01)after the cells were pretreated with TNF-?.At the same time,TNF-? can significantly increase the protein expressions levels of RAGE,MMP-2,MMP-3 and TIMP-2(P<0.05 or P<0.01),and promote the expressions and secretions of MMP-9 and TIMP-1(P<0.05 or P<0.01),but reduce the protein expressions levels of TIMP-3 and TIMP-4 in A549 cells.(4)TNF-? activated the NF-?B p65 signaling pathway in lung epithelial A549 cells,and this effect can be blocked by the pathway inhibitor BMS-345541.Knocking down the gene expression of RAGE can also affect the phosphorylation of NF-?B p65 and inhibit the protein expression of MMP-2,MMP-3 and MMP-9.Conclusion: TNF-? can induce inflammatory reaction on lung epithelial A549 cells and cause changes in the expression profile of MMPs/TIMPs which mediate ECM metabolism.RAGE can participate the process of ECM metabolism during the inflammatory reaction of lung epithelial A549 cells by regulating MMPs/TIMPs,and its mechanism may be related to the activation of NF-?B p65 signaling pathway.