Fig 1: ERY has a protective role in the disturbance of MMP and anti-MMP activity in CS-induced emphysema in rats. (A) The protein expression of MMP-2, MMP-9 and TIMP-1 as measured by immunohistochemical staining (original magnification, ×400). (B) Protein expression levels of MMP-2, MMP-9 and TIMP-1 as measured by western blotting, with quantification relative to β-actin. For TIMP-1, the upper band was considered to be non-specific binding. Data are presented normalized to the control and are the mean ± standard deviation. Control rats were exposed to normal air and received daily saline injections. CS rats were exposed to CS for 12 weeks. CS + ERY rats were exposed to CS for 12 weeks and received daily injections of 100 mg/kg ERY. *P<0.05 vs. control group and #P<0.05 vs. CS group; n=12 per group. CS, cigarette smoke; ERY, erythromycin; MMP, metalloprotease; TIMP-1, tissue inhibitor of metalloproteinases.
Fig 2: Validation of DEPs identified from the comparisons of the N, L and LP groups. (A) Western blot analysis of proteins including THBS4, THBS1, IL1RL1, GLUL and TIMP1. With the exception of the IL1RL1 band, all bands on the Western blot were consistent with the bioinformatic analysis results. RT-qPCR analysis of the following genes: THBS4 (B), THBS1 (C), IL1RL1 (D), GLUL (E) and TIMP1 (F). The mRNA levels of Thbs4, Thbs1, IL1RL1, and Glul were consistent with the trends observed in the bioinformatic analyses. The data are shown as the mean ± SD values; *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig 3: TIMP-1 triggers the chemotactic migration of primary human monocytes via CD74. (a,b) Recombinant TIMP-1 induces phosphorylation of AKT (a) and ERK1/2 (b) in primary human monocytes in a dose-dependent manner. Cell lysates from isolated human monocytes stimulated with different concentrations of TIMP-1 were analyzed by Western blot using anti-pAKT, anti-AKT, anti-pERK1/2, and anti-ERK1/2 antibodies, as indicated. (c) Dose-dependent Chemotaxis of human primary human monocytes in response to TIMP-1 as determined by the Transwell migration assay (n = 4 biological replicates). (d) The Transwell assay showed the involvement of CD74, but not CXCR4, in the TIMP-1-mediated Chemotaxis of monocytes. The blocking anti-CD74 antibody (LN2) and the CXCR4 antagonist (AMD3100) were used to test the causal role of both receptors in TIMP-1-triggered Chemotaxis (n = 4 biological replicates). (e) TIMP-1 triggers the 3D Chemotaxis of human monocytes in a dose-dependent manner (red traces). The unstimulated control (gray traces) reflects random motility processes. A representative experiment is shown. Migration trajectories of 30 randomly selected cells per treatment group were recorded. Heat-inactivated (HI) TIMP-1 and CCL2 were used as negative and positive controls, respectively. (f) Quantification of (e) using the forward migration index (FMI) as the Chemotaxis activation parameter. Shown are the migration trajectories of one experiment out of n = 3 independent experiments performed. (g) Involvement of CD74, but not CXCR4, in TIMP-1-triggered migration, as determined by 3D real-time Chemotaxis of human monocytes. (h) Quantification of (g) using the FMI as the Chemotaxis activation parameter. Shown are the migration trajectories of one experiment out of n = 3 independent experiments performed. Migration trajectories of 30 randomly selected cells per treatment group were recorded. Data are means ± SD.
Fig 4: TIMP-1 promotes the activation of serine/threonine kinases in THP-1 monocytic cells. Cells were incubated under serum-free conditions in the absence (untreated or 0) and presence of recombinant TIMP-1 (100 ng/mL) for different time intervals, as indicated. (a) Total-protein cell lysates were obtained and analyzed using a membrane-based phosphokinase array, detecting 46 different kinase phosphorylation sites (seven of the phosphorylated substrates are indicated; the respective phosphorylated amino acids in brackets). (b) Densitometric quantification of the spots in (a) and representation as a heatmap. The quantification is based on mean values from duplicate determinations in percent to untreated control cells (100%). The heatmap shown provides an overview of the changes in phosphorylation of all 46 measured kinase substrates/sites at all three determined time points. (c) Selection of the heatmap in (b) with a focus on those substrates/sites with the most-substantial alterations. (d) Representation of the densitometric quantification according to the selection of substrates/sites depicted in (c) as a bar diagram. The dotted red line indicates the baseline value of the control. (e) Western blot analysis of cell lysates using antibodies against phosphorylated AKT (p-AKT), total Akt (AKT), and β-actin as the loading control.
Fig 5: UA intervention effects on the mRNA and protein expression of the NOX4/ROS and RhoA/ROCK1 signaling pathways. (A,B) RT-qPCR analysis of Nox4, Rhoa, Rock1, Alphasma, Col1a1, mmp1, and Timp1 mRNA expression levels in each group. (C–E) WB analysis of the protein expression levels in each group. (F) ROS contents in each group detected by the DCFH-DA assay. The data are presented as the means ± SEMs of three replicates. ∗p < 0.05, versus the CON group; #p < 0.05, versus the NOX4GV 358Lv group.
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