Fig 1: In vivo mouse studies confirm the role of CCL3 and miR-4456 in COPD (A) H&E staining confirms the macroscopic appearance of pulmonary tissue in the four groups: 1) Control (Ctrl), 2) CSE, 3) CCL3, and 4) CSE + DAPTA. Quantification of the alveolar space is in the lower panels. n = 8,*p < 0.05, vs. the control group, #p < 0.05, vs. the CSE group (B) Airway resistance, Elasticity, Compliance, and total BALF proteins were detected in the four groups same as Figure 5A n = 8,*p < 0.05, vs. the control group, #p < 0.05, vs. the CSE group (C) Inflammatory and oxidative stress markers in the four groups same as Figure 5A n = 8,*p < 0.05, vs. the control group, #p < 0.05, vs. the CSE group (D) Tight junction proteins ZO-1 merge (red and blue), and occludin merge (green and blue) were stained using immunofluorescence in the four groups same as Figure 5A. Data are representative of three independent experiments. Scale bar = 10 µm n = 8, *p < 0.05, vs. the control group, #p < 0.05, vs. the CSE group (E) Real-time PCR assays were performed in the blood and lung to detect ZO-1 and occludin mRNA before and after CSE treatment in the four groups same as Figure 5A. Data are represented as the mean ± standard deviation. n = 8, *p < 0.05, vs. the control group. #p < 0.05, vs. the CSE group. The expression levels were normalized to the expression of GAPDH, n = 3 (F) Western blot for ZO-1 and occludin protein expression in the four groups same as Figure 5A n = 8, *p < 0.05, vs. the control group, **p < 0.01, vs. the control group, #p < 0.05, vs. the CSE group. Protein expression was normalized to ß-actin (G) Real-time PCR assays were performed in the whole blood and lung to detect CCL3 mRNA expression in four groups same as Figure 5A. Data are represented as the mean ± standard deviation. n = 8, *p < 0.05, vs. the control group, #p < 0.05, vs. the CSE group. The expression levels were normalized to the expression of GAPDH (H) Real-time PCR assays were performed in the whole blood and lung to detect CCR5 mRNA expression in four groups same as Figure 5A. Data are represented as the mean ± standard deviation. n = 8, *p < 0.05, vs. the control group, **p < 0.01, vs. the control group, #p < 0.05, vs. the CSE group. The expression levels were normalized to the expression of GAPDH (I) Real-time PCR assays were performed in the whole blood and lung to detect miR-4456 expression in four groups same as Figure 5A. Data are represented as the mean ± standard deviation. n = 8, *p < 0.05, vs. the control group, **p < 0.01, vs. the control group. The expression levels were normalized to the expression of 5S RNA and/or U6 (J) Western blot for ZO-1 and occludin protein expression in the four groups: 1) Control (Ctrl), 2) CSE, 3) CCL3, and 4) CSE + miR-4456. n = 8, *p < 0.05, vs. the control group, #p < 0.05, vs. the CSE group. Protein expression was normalized to ß-actin (K) Tight junction proteins ZO-1 merge (red and blue), and occludin merge (green and blue) were stained using immunofluorescence in the four groups same as Figure 5J. Data are representative of three independent experiments. Scale bar = 10 µm n = 8, *p < 0.05, vs. the control group, #p < 0.05, vs. the CSE group.
Fig 2: Western blot analysis gray value comparison of HLA-A, Fas, CCR5, FasL and HLA-E. ß-actin was the internal reference. Results were represented as the mean ± standard deviation. Paired t test was used for statistical analysis. *P<0.05; **P<0.01. HLA-A, human leukocyte antigen-A; Fas, apoptosis antigen 1; CCR5, C-C chemokine receptor type 5; FasL, apoptosis antigen 1 ligand; HLA-E, human leukocyte antigen class I histocompatibility antigen, alpha chain E.
Fig 3: CCL3 promotes epithelial tight junction injury by binding with CCR5 (A) Tight junction proteins ZO-1 (red), occludin (green), and merged (blue + orange) were stained in 16HBE cells (left panel) and A549 cells (right panel) by immunofluorescence. Presented as means ± standard error. n = 3, *p < 0.05, vs. the control group. Data are representative of three independent experiments. Scale bar = 10 µm (B) Transepithelial electrical resistance (TER) after CCL3 treatment (10 mg/ml), as a cell function test 7 days after plating the airway epithelial cells on coated permeable filters. Presented as means ± standard error. n = 3, *p < 0.05, vs. the control group (C) Western blot analysis to detect protein expression of ZO-1, claudin and occludin in 16HBE cells and A549 cells using different concentrations of CCL3. n = 3, *p < 0.05, vs. the control group, **p < 0.01, vs. the control group (CCL3: 0 ng/ml). The expression levels were normalized to the expression of ß-actin (left panel). n = 3 (D) Real-time PCR assays of ZO-1 (upper) and occludin (lower) mRNA expression in 16HBE cells and A549 cells using different concentrations of CCL3. n = 3, *p < 0.05, vs. the Ctrl group, **p < 0.01, vs. the control group. Protein expression was normalized to GAPDH (E) Tight junction proteins ZO-1 (red), occludin (green) and merged (blue and orange) were stained by immunofluorescence in the four groups: 1) Ctrl (PBS:10 mg ml-1), 2) CSE (10 mg ml-1), 3) CCL3 (CCL3 10 mg ml-1), and 4) CSE (10 mg ml-1)+DAPTA (0.1 mg ml-1) in 16HBE cells (left panel) and A549 cells (right panel). Presented as means ± standard error. n = 3, *p < 0.05, vs. the control group. #p < 0.05, vs. the CSE group. Data are representative of three independent experiments. Scale bar = 10 µm (F) TER in the four groups same as Fig3E in 16HBE cells (left panel) and A549 cells (right panel). The results are shown as a function test on 7 days after plating the airway epithelial cells on coated permeable filters. Presented as means ± standard error. n = 3,*p < 0.05, vs. the control group, **p < 0.01, vs. the control group, #p < 0.05, vs. the CSE group (G) Real-time PCR assays to detect ZO-1 mRNA (upper panel) and occludin mRNA (lower panel) expression in 16HBE cells and A549 cells in four groups same as Fig3E. n = 3,*p < 0.05, vs. the control group, #p < 0.05, vs. the CSE group. The expression levels were normalized to the expression of GAPDH (left panel) (H) Western blot analysis to detect ZO-1protein, occludin protein, CCL3 protein and CCR5 protein expression in 16HBE cells and A549 cells in four group same as Fig3E. Protein expression levels were normalized to ß-actin expression. n = 3, *p < 0.05, vs. the control group, #p < 0.05, vs. the CCL3 group. Protein expression was normalized to ß-actin.
Fig 4: Immunohistochemical staining of CCR5 among the four groups at a magnification of ×400. (A) Negative expression of CCR5 in normal colorectal mucosa. (B) Weak positive expression of CCR5 in colorectal adenoma. (C) Weak positive expression of CCR5 in early colorectal cancer. (D) Strong positive expression of CCR5 in advanced colorectal cancer. CCR5, C-C chemokine receptor type 5.
Fig 5: Higher expression of CCL3 in the supernatants of PBMCs cells (A) Human Cytokine Array for the parallel determination of relative levels of cytokines and chemokines in the supernatants of PBMCs cells. Downregulated proteins are shown in blue, and upregulated proteins are shown in red, as the mean of all specimens included (n = 48). non-smokers: Ctrl-1 to Ctrl-8, GOLD 1:G1-1 to G1-9, GOLD2: G2-1 to G2-9, GOLD3:G3-1 to G3-10, GOLD4:G4-1 to G4-12 (B) Expression of CCL3 in no-COPD and COPD patients in the human GEO database (n = 53) (C) Real-time PCR assays were performed in 16HBE, A549, BEpic, and PAEC cells to detect CCL3 mRNA expression before and after CSE treatment. n = 3, *p < 0.05, vs. the control group (left panel). The expression levels were normalized to the expression of GAPDH. ELISA assays were performed in 16HBE, A549, BEpic, and PAEC cells to detect CCL3 protein expression before and after CSE treatment. n = 3, *p < 0.05, vs. the control group (right panel) (D) Correlation between FEV1 (%predicted) and CCL3 expression in patients with COPD. r = 0.7175, p < 0.001, n = 40 (E) Real-time PCR assays were performed in 16HBE, A549, BEpic, and PAEC cells to detect CCR5 mRNA expression before and after CSE treatment. *p < 0.05, vs. the control group (F) Western blot assays were performed in 16HBE, A549, BEpic, and PAEC cells to detect CCR5 protein expression before and after CSE treatment. n = 3, *p < 0.05, vs. the control group. The expression levels were normalized to the expression of ß-actin.
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