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. 2019 Sep 17;9(22):6646–6664. doi: 10.7150/thno.33741

Figure 7.

Figure 7

Transplantation of HUMSCs stimulated macrophage synthesis of MMP-9 in the left lungs of rats with PF. (A) Left lung sections were subjected to immunohistochemistry with an anti-ED1 antibody for macrophage labeling. On Days 7 and 14, the macrophages in the left lung were extensively activated. However, from Day 21 to 49, the number significantly decreased. Transplantation of HUMSCs stimulated the activation of macrophages. Immunohistostaining of anti-iNOS (B) and anti-CD163 (C) were performed to detect M1 and M2 macrophages in the lung respectively. (D) The quantitative results showed that M2 macrophages significantly increased, whereas M1 macrophages statistically decreased in the group of BLM+HUMSCs (HD) on Day 49. Magnified image of the boxed area (arrow) demonstrated that highly active M2 macrophages were present in the alveoli. (E) The contents of MMP-9 and MMP-2 in the rats' left lungs were detected through western blotting. The quantitative results indicated that MMP-9 significantly increased following the transplantation of HUMSCs, whereas no significant alteration was found for MMP-2. (F) The proteolytic activities of total MMPs in the left lung tissue lysates were measured by the gelatin fluorometric assay. (G) Representative image of gelatin zymography showing MMPs activity in left lung tissue lysate. (H and I) Gelatin zymography results showed that MMP-9 activity was significantly increased, whereas MMP-14 or other MMPs was significantly decreased in the group of BLM+HUMSCs (HD) at Day 49. (J) Quantitate real-time PCR analysis of relative expression of rat MMP-9 mRNA in lungs (n = 3/group). To further explore whether increases in MMP-9 came from HUMSCs or macrophages, the left lung tissue sections from the BLM+HUMSCs (HD) group on Day 49 were subjected to double-staining with either (K) anti-MMP-9 (green) and anti-ED1 (red) antibodies, (L) anti-MMP-9 (green) and anti-human nuclei (red) antibodies, or (M) anti-ED1 (green) and anti-human nuclei (red) antibodies. The results indicated that MMP-9 was predominantly synthesized and secreted by macrophages. Additionally, transplanted HUMSCs did not differentiate into macrophages or synthesize MMP-9. Dotted circles indicate alveoli. n= 4 animals per group. ✱ vs the Normal group, p < 0.05. # vs the BLM group, p < 0.05. ♠ vs the BLM+HUMSCs (LD) group, p < 0.05. ⧫ vs the BLM+HUMSCs (HD) group, p < 0.05.