| Objective This study aimed to investigate the mechanism of the Qibai Pingfei capsule in reducing the apoptosis of alveolar epithelial cells of COPD based on the CYP1B1/ HIF1α/VEGFA axis.MethodsPart one: the key target gene prediction of QBPF capsule in the prevention and treatment of COPDBioinformatics analysis methods were conducted to analyze the lung tissue microarray data of normal controls and COPD individuals to obtain differentially expressed genes(DEGs),and the main molecule and target genes of QBPF capsules were searched in the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).The DEGs were mapped to targeted genes of the QBPF capsule to construct the “QBPF capsule-target genes” network of the QBPF capsule in the prevention and treatment of COPD,and the protein-protein interaction(PPI)network,hub genes,and enrichment pathways analysis of target genes in the network were conducted to obtain the key targets and pathways of QBPF capsule.Furthermore,the gene set enrichment analysis(GSEA)was further conducted to obtain significant enrichment pathways of COPD,which aimed to obtain the key pathway of the QBPF capsule in COPD.Part two: In vivo study of QBPF capsule intervening COPD model rat The COPD model rats were built by the administration of cigarette smoke(CS)combined with lipopolysaccharides(LPS)tracheal instillation.Hematoxylin-eosin(HE)staining was conducted to assess the pathological changes in lung tissue to evaluate the effects of QBPF capsules in the treatment of pulmonary inflammation and emphysema of the rat model.Enzyme-linked immunosorbent assay(ELISA)was administrated to detect the changes of serum cytokines including IL-1β,TNF-α,NF-κB,IL-4,and IL-10 to evaluate the improvement effects of QBPF capsules on the systemic inflammatory of the rat model.RT-PCR was conducted to detect the expression of HIF1α,CYP1B1,and VEGFA in the lung tissue of rats to assess the effect of QBPF capsules on the expression of these QBPF capsules targeted DEGs.The TUNEL method was applied to evaluate the improvement effect of QBPF capsules on the apoptosis of alveolar epithelial cells.The tissue localization and expression of key proteins,including HIF1α and ARNT of the HIF-1signaling pathway,in the lung were detected by immunofluorescence.Part three: In vitro study of medicated serum of QBPF in intervening cigarette smoke extract(CSE)induced alveolar epithelial cellsCSE-induced alveolar epithelial cells A549 was conducted to construct the in vitro experimental model of COPD.The effect of concentrations of CSE on the viability of A549 cells was detected by the cell counting kit 8(CCK8)method,and the expression of HIF1α mRNA of concentrations of CSE-induced A549 cells was detected using RT-PCR,which aimed to screen the optimal stimulation time and concentration of CSE in inducing A549 cells.The effect of concentrations of medicated serum of QBPF capsule was also detected by the CCK8 method,and the optimal stimulation time and concentration of medicated serum of QBPF capsules were selected.DCFH-DA probes were used to detect the level of intracellular reactive oxygen species(ROS)to evaluate the effect of medicated serum of QBPF capsule on the intracellular ROS of CSE-induced A549 cells.The cell apoptosis was detected by TUNEL,which aimed to evaluate the effect of medicated serum of QBPF capsules on the apoptosis of A549 induced by CSE.The expression and cellular localization of key proteins including HIF1α and ARNT of the HIF-1 signaling pathway were detected by immunofluorescence.The HIF1α knockdown A549 cells were prepared by HIF1α knockdown lentivirus infection.The western blot method was conducted to detect the expressions of HIF1α,VEGFA,ARNT,CYP1B1,and β-actin to evaluate the effect of medicated serum of QBPF capsules on the expression of key proteins of the HIF-1 signaling pathway.ResultsPart one: QBPF capsules mainly targeted the HIF-1 signaling pathway1 This study included the raw data of 96 normal control and 204 COPD individuals,and the DEGs analysis identified 2375 up-regulated DEGs(adjust pvalue<0.05)and 3878down-regulated DEGs(adjust pvalue<0.05).2 QBPF capsules had a total of 242 active ingredients and 244 target genes,of which 157 active ingredients targeted 91 DEGs of COPD,including HIF1α,VEGFA,and CYP1B1,and these DEGs significantly enriched in 30 pathways such as the HIF-1 signal pathway and PI3K-Akt signal pathway.3 The HIF-1 pathway was QBPF capsule mainly targeted signaling pathway,and the HIF1α and VEGFA in this pathway were in the top three hub genes of QBPF capsules targeted genes.Part two: QBPF capsule affected the expression of key molecules of CYP1B1/HIF1α/VEGFA axis and reduced the apoptosis of alveolar epithelial cells in COPD rats1 The results of HE indicated that the lung of the COPD model rat showed structure destruction and increased alveolar mean linear intercept(AMLI)(P<0.05).The results of TUNEL indicated that a large number of apoptotic cells exist in the alveoli of COPD model rat.The results of ELISA indicated that,in the serum of COPD rats,the expression of the pro-inflammatory cytokine,like IL-1β(P<0.01),TNF-α(P<0.01),NF-κB(P<0.01)were up-regulated,and the expression of anti-inflammatory cytokines like IL-4(P<0.01)and IL-10(P<0.01)were down-regulated.2 QBPF capsules could reduce the inflammation of lung tissue,the destruction of alveolar structure,the alveolar epithelial cells apoptosis,the AMLI(P<0.01),the expression of serum pro-inflammatory cytokine including IL-1β(P<0.01),TNF-α(P<0.01),and NF-κB(P<0.01),and increased the expression anti-inflammatory cytokines like IL-4(P<0.01)and IL-10(P<0.01),of COPD rats.3 In the COPD model rat versus control rat,the expression of CYP1B1 mRNA and HIF1αmRNA in lung tissue and the HIF1α protein in alveolar epithelial cells were up-regulated,and the expression of VEGFA mRNA in lung tissue and the ARNT protein in alveolar epithelial cells were down-regulated.QBPF capsule could reduce the expression of CYP1B1 mRNA and HIF1α mRNA in lung tissue and HIF1α protein in alveolar epithelial cells of COPD model rat,and increase the expression of VEGFA mRNA in lung tissue and ARNT protein in alveolar epithelial cells of COPD model rat.Part three: QBPF capsule could affect the expression of key molecules in the CYP1B1/HIF1α/VEGFA axis and reduce CSE-induced apoptosis of alveolar epithelial cells A5491 The medicated serum of QBPF capsules could improve the cell viability of alveolar epithelial cells A549,and the cell viability began to decline after 12 hours.CSE could reduce the viability of A549 cells,and the 60% CSE treated A549 cells showed the highest expression of HIF1α mRNA(P<0.05)at 24 hours.2 The 60% CSE increased the level of ROS in alveolar epithelial cells A549 cells and reduced the apoptosis of alveolar epithelial cells A549.The medicated serum of QBPF capsules reduced the increased ROS level and the apoptosis of alveolar epithelial cells A549 cells induced by 60% CSE.3 The results of immunofluorescence indicated that 60% CSE could increase the expression of HIF1α and decrease the expression of ARNT protein in A549 cells.The medicated serum of QBPF capsules reduced the expression of HIF1α and increased ARNT protein expression of CSE-induced A549 cells.4 The results of western blot showed that the medicated serum of QBPF capsules could reduce the expression of HIF1α and increase the expression of ARNT and VEGFA(P<0.05)in CSE-induced A549 cells.After knocking down the expression of HIF1α in A549 cells,the expression of HIF1α was significantly decreased,and the medicated serum of QBPF capsules can reduce the expression of HIF1α and CYP1B1 protein and increase the expression of VEGFA and ARNT protein in CSE induced HIF1α knockdown A549 cells.In addition,the medicated serum of QBPF capsules could reduce the expression of CYP1B1(P<0.05)which was upregulated by 60% CSE,the HIF1αknockdown A549 cells showed the same CYP1B1 expression trend as A549 cells when induced by 60% CSE or treated by QBPF(P<0.05).Conclusions1 There were many dysregulated genes in the lung tissue of COPD,and the DEGs,targeted by QBPF capsules,of COPD might be involved in the mechanism of QBPF in the prevention and treatment of COPD,of which HIF1α,VEGFA,and CYP1B1 might be the main targeted genes and the HIF-1 signaling pathways might be the key pathway of QBPF capsules.2 QBPF capsules could reduce the increased apoptosis of alveolar epithelial cells in COPD model rat and alveolar epithelial cells A549 induced by CSE,and the intracellular ROS level in CSE inducing A549.3 The apoptosis of alveolar epithelial cells in COPD might be related to the increased expression of CYP1B1 and HIF1α and the decreased expression of ARNT and VEGFA.The mechanism of QBPF capsules in reducing apoptosis of alveolar epithelial cells might be related to the decreased expression of HIF1α and CYP1B1,and the increased expression of ARNT and VEGFA. |
PDF Full Text Request