| Objective:To investigate whether platelet-derived growth factor-BB(PDGF-BB)can promote the proliferation of PASMC in neonatal rats with HPH by regulating the expression of HIF-1α,thereby promoting pulmonary vascular remodeling and increasing pulmonary artery pressure,by targeting the key factors of PDGF-BB→HIF-1α signaling pathway and to study the feasibility of reversing the pathological process of pulmonary vascular remodeling in neonatal rats with HPH,so as to block the disease from the upstream regulatory pathway of HIF-1α.Methods:Part I: Neonatal rat pulmonary vascular smooth muscle cells(PASMC)were isolated and cultured by tissue adherence method and identified by immunofluorescence.Pasmcs from neonatal rats were transfected with recombinant adenovirus with or without PDGF-BB,PDGF-BB transcription inhibitor(STI571),and cultured under hypoxic or normoxic conditions to establish cell models.The observation time points were 24 h,48h and 72 h of hypoxia.Transmission electron microscope observation and CCK8 cell proliferation experiment were used to detect the morphological changes and proliferation rate of the cells in each group.RT-PCR and Western blot were used to detect the m RNA and protein expression levels of PDGF-BB,HIF-1α,Ki67 and p27kip1 in cells.Part II:Neonatal rats were transfected with adenovirus carrying or not carrying PDGF-BB through tail vein,and given PDGF-BB transcriptional inhibitor by gavage(STI571)intervention.After hypoxia,HPH model of newborn rats was established,and the control group was established.Neonatal rats were killed at 3d,7d,14 d and 21 d after modeling,lung tissues were collected,immunofluorescence was used to determine the transfection effect of PDGF-BB in each group,and the expression site and protein expression level of PDGF-BB,HIF-1 α and proliferation-related proteins Ki67 and p27kip1 in pulmonary vessels of each group were determined by immune double-labeling method.The m RNA and protein levels of PDGF-BB,HIF-1 α,Ki67 and p27kip1 were detected by RT-PCR and Western blot.Part III: Neonatal rats were treated by tail vein transfection with PDGF-BB adenovirus with or without PDGF-BB transcription inhibitor(STI571)by intragastric administration.The HPH model was established by hypoxia.The pulmonary artery pressure was measured on day 3,7,14 and 21 after modeling.The lung and heart tissues were extracted,the ultrastructure of lung tissue was observed by transmission electron microscope,and the indexes of pulmonary vascular remodeling(MA%and MT%)were measured,and the right ventricular hypertrophy index(RVHI)was calculated.Results:Part I: The proliferation rate of PASMC in hypoxic group was significantly higher than that in normoxic group,and the proliferation rate of CCK-8 cells and Ki67 were significantly higher in hypoxic group than that in normoxic group,while the expression of p27kip1 was decreased.At 24 h,48h and 72 h,the expression of PDGF-BB m RNA in hypoxic group was significantly higher than that in normoxic group.The overexpression of PDGF-BB enhanced the proliferation rate of PASMC and the expression of Ki67 m RNA and protein.The expression of PDGF-BB was inhibited in the PDGF-BB inhibition group,and the proliferation rate of PASMC and the expression of Ki67 m RNA and protein were significantly lower than those in the hypoxia group.Part II: The RVSP of neonatal rats on days 3,7,14,and 21 of hypoxia was higher than that of the corresponding normoxic control group,suggesting that the neonatal rat model of HPH was successfully established.The results of immunofluorescence showed that PDGF-BB was positively expressed in the pulmonary vascular smooth muscle cells of neonatal rats in the HPH group and the hypoxia +PDGF-BB overexpression group.The adenovirus carrying PDGF-BB was successfully transfected into the lung tissue of neonatal rats.The expression levels of the PDGF-BB,HIF-1α and Ki67 in HPH group and hypoxia+PDGF-BB overexpression group were significantly higher than those in normoxia group,while the expression of p27kip1 in HPH group and hypoxia +PDGF-BB overexpression group was lower than that in normoxia group.RT-PCR and Western blot showed that the expression of PDGF-BB,HIF-1α and Ki67 in PASMC of neonatal rats in HPH group and HPH+PDGF-BB overexpression group was increased,while the expression of p27kip1 was decreased on days 3,7,14 and 21 of hypoxia.Adenovirus-targeted PDGF-BB overexpression in the tail vein increased the expression of exogenous PDGF-BB in the lung tissue of HPH newborn rats,and intragastric administration of PDGF-BB inhibitor(STI571)decreased the expression of PDGF-BB and HIF-1α in the lung tissue.Overexpression of PDGF-BB increased the expression of HIF-1α,promoted the expression of Ki67,a pro-proliferation factor,and decreased the expression of p27kip1,an anti-proliferation factor,and proliferation of PASMC.Inhibition of PDGF-BB overexpression by STI571 produced the opposite effect.Part III: RVSP of neonatal rats on days 3,7,14,and 21 of hypoxia was higher than that of the corresponding normoxic control group,suggesting that the neonatal rat model of HPH was successfully established.HPH neonatal rats had pulmonary vascular remodeling on day 7 of hypoxia,HPH+PDGF-BB overexpression group had pulmonary vascular remodeling on day 3 of hypoxia,and the degree was higher than that of HPH neonatal rats,while PDGF-BB inhibition group had pulmonary vascular remodeling on day 7 of hypoxia and the degree was significantly milder.Overexpression of PDGF-BB increased and inhibition of PDGF-BB reduced pulmonary artery pressure in neonatal rats with HPH at each time point of hypoxia.RVHI in HPH neonatal rats overexpressing PDGF-BB was higher than that in HPH group and normoxia control group,and increased pulmonary artery pressure.Conclusion:PDGF-BB may participate in the proliferation of PASMC by activating the expression of HIF-1α,thus promoting pulmonary vascular remodeling and increasing pulmonary artery pressure in neonatal rats with HPH.PDGF-BB→HIF-1α signaling pathway plays an important role in the pathogenesis of neonatal HPH. |
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