The comparison of arterial oxygen saturation (SpO2)
The SpO2 was assessed using a pulse oximeter to evaluate the lung function of gas exchange. The results indicated that, during the 49 days of the experiment, the SpO2 remained at 98.8 ± 0.4% in the Normal group. The SpO2 of the BLM group markedly decreased on Day 7 and remained around 81.6 ± 1.4% until Day 49, which was significantly lower than that in the Normal group. For the BLM + Nintedanib, BLM + Pirfenidone, and BLM + HUMSCs groups, the SpO2 significantly decreased during Day 7 to Day 21, which was similar to that in the BLM group. From Day 21 to Day 49, no increment in SpO2 was observed for the BLM + Nintedanib and BLM + Pirfenidone groups. A significant increase in SpO2 was observed in the BLM + HUMSCs group since Day 28, and the level continuously increased to 92 ± 2.3% on Day 49, which was significantly improved as compared with other treatment groups (Figs. 2A-2B).
The comparison of respiratory rate
The results of the respiratory function revealed that the breathing frequency remained stable in the Normal group, with 4-5 breaths recorded in two seconds. The respiratory rate marked increased during Day 7 to Day 49 in the BLM group. For the BLM+Nintedanib, BLM+Pirfenidone, and BLM+HUMSCs groups, the trends of increased respiratory rate were similar to the BLM group during Day 7 to Day 21. With HUMSCs transplanted on Day 21, the BLM+HUMSCs exhibited a reduced respiratory rate since Day 28 as compared with other treatment groups. The trend remained until Day 49 (Figures 2C-2D).
The comparison of body weight
The results of body weight showed that the weight in the Normal group gradually increased over time. The BLM group displayed a flatter slope in weight gain since Day 7 after BLM injection; although the weight still increased over time, a significant reduction was observed as compared to those in the Normal group, and this trend continued until Day 49. For the BLM+Nintedanib, BLM+Pirfenidone, and BLM+HUMSCs groups, the curves of the body weight were similar to those in the BLM group after Day 7 as the weight increased in a slower slope and were significantly lower than those in the Normal group. Although the rats in the BLM+Nintedanib and BLM+Pirfenidone groups exhibited an increase in weight from Day 7 to Day 49, no significant difference was found when comparing to the BLM group, and a significant reduction was seen as compared to the Normal group. The weight of the BLM+HUMSCs group showed a significant increase since Day 28 when comparing to those in the BLM group. The weight trend remained until Day 39, indicating that transplantation of HUMSCs was superior to other treatments in recovering weight in rats with PF (Figure 2E).
The comparison of alveolar space of left lung
The changes in left-lung alveolar volume were quantified utilizing an MRI scan. With the existence of alveoli in both the left and right lungs, black signals were predominantly seen in the image of the Normal group (Figure 3A and Supplemental Figure 2). Due to inflammation and white blood cell infiltration, white signals were seen in the left lung of the BLM group on Day 7. The alveolar space almost disappeared in the left lung and was mostly occupied by consolidated tissues since Day 14; the condition persisted up to Day 49 (Figures 3A-3B and Supplemental Figure 3). For the BLM+Nintedanib, BLM+Pirfenidone, and BLM+HUMSCs groups, white signals appeared in the left lung during Day 7 to Day 21, indicating the loss of alveolar space and replacement with the consolidated tissues. During Day 28 and Day 49, alveolar space in the left lung was still occupied by the white consolidated tissues (Figures 3A-3B and Supplemental Figures 4-6). However, black alveolar space could be seen since Day 28 in the left lung of the BLM+HUMSCs group (Figures 3A-3B).
The comparison of alveolar structure in left lung
On Day 49 after BLM injection, the left lung tissues were obtained and subjected to H&E stain. From low to high magnifications, the central areas of the images showed that the alveolar structure was intact in the Normal group, with the connective tissues primarily surrounded the bronchus and rarely appeared among alveoli. In the BLM group, intact alveoli only appeared in the outer regions of the left lung, and the central areas were mostly cell-infiltrated. In the BLM+Nintedanib and BLM+Pirfenidone groups, healthy alveoli were also observed in the outer regions of the left lung, and the central areas were infiltrated by a substantial amount of cells. On the contrary, the area with cell infiltration significantly decreased, and the alveolar space markedly increased in the central area of the BLM+HUMSCs group (Figures 4A-4B). By summing data from all left lung sections stained with H&E, the statistical analyses showed that the left lung volume significantly shrank in the BLM group, in which the volume of alveolar structure decreased, whereas 70% of the left lung was occupied by the consolidated tissues (Figures 4D-4F).
The left lung volume, the air space, and the cell infiltration area in the BLM+Nintedanib and BLM+Pirfenidone groups were similar to the BLM group as no statistical differences were seen (Figures 4D-4F). The left lung volume and the air space in the BLM+HUMSCs group were both comparable with those in the Normal group. In addition, the volume of connective tissues and cell infiltration significantly decreased as compared to those of the BLM group (Figures 4D-4F).
Furthermore, the number and the circumference of alveoli in the unit area in the outer region of left lung where alveoli remained were quantified to evaluate the effectiveness of gas exchange by alveoli. From low to high magnifications of the H&E stained sections, the alveolar size in the outer region of left lung was smaller in the Normal group; therefore, the number of alveoli in the unit area was higher, and the total alveolar circumference for gas exchange was longer. The alveolar size in the outer region in the BLM group from Day 7 to Day 49 was significantly larger than that in the Normal group, resulting in a decrease in the total number and alveolar circumference of alveoli in the unit area. The number of alveoli and the total alveolar circumference in the unit area of the BLM+Nintedanib and BLM+Pirfenidone groups did not significantly differ from those in the BLM group. For the BLM+HUMSCs group, the number of alveoli and the alveolar circumference in the unit area significantly were increased as compared with those in the BLM group, indicating that transplantation of HUMSCs improves the efficiency of gas exchange (Figures 4A, 4C, 4G-4H).
The comparison of overall appearance of left lung
On Day 49, the overall appearances of the right and left lungs showed that white alveolar structures with intact and smooth alveoli could be seen in the Normal group. The left lung markedly shrunk in the BLM group, with healthy alveoli only presented in the outer region of the left lung and no alveoli but pathological tissues appeared in its central area. Moreover, the pathological tissues in the central area of the left lung displayed dry and flat features. For the BLM+Nintedanib and BLM+Pirfenidone groups, no apparent improvements in the overall appearance of the left and right lungs were observed. However, the volume and the white alveolar regions of the left lung were both markedly preserved in the BLM+HUMSCs group on Day 49 (Figure 5A).
The comparison of collagen deposition
The left lung sections were stained with Sirius red to label collagen red (Figures 5B-5C). Collage predominantly surrounded the bronchus and blood vessels in the Normal group. The area of collagen stained in red significantly increased in the BLM group. The level of collagen deposition in the BLM+Nintedanib and BLM+Pirfenidone groups did not decrease when comparing to that of the BLM group. The level of collagen deposition in the BLM+HUMSCs was not only lower than those in the BLM, BLM+Nintedanib and BLM+Pirfenidone groups but also comparable with that in the Normal group (Figures 5B-5D).
The comparison of myofibroblast activation
Anti-α-SMA antibody was used to identify myofibroblasts (Figures 6A-6C). The images of the Normal group showed that only a very limited number of α-SMA-positive cells existed, primarily surrounded the bronchus and blood vessels. A substantial amount of α-SMA appeared in the connective tissues in the BLM group. The expression level of α-SMA was assessed by Western blotting, which showed that α-SMA significantly increased in the BLM group in contrast to that in the Normal group. The expression level of α-SMA in the BLM+Nintedanib and BLM+Pirfenidone groups showed a decreasing trend, but not a significant amelioration as compared with that in the BLM group (Figures 6A-6C). The expression of α-SMA significantly decreased in the BLM+HUMSCs group as compared with that in the BLM group (Figures 6A-6C).
The comparison of cell number in bronchoalveolar lavage
Cell number measured in BALF was used to estimate the amplitude of pulmonary inflammation. The results showed that the inflammatory cell counts significantly increased in the BLM group (Figure 6D). The inflammatory cell counts in the BLM+Nintedanib and BLM+Pirfenidone groups were lower than that in the BLM group but were higher than that in the Normal group. The inflammatory cell count in the BLM+HUMSCs were significantly lower than those in the BLM, BLM+Nintedanib, and BLM+Pirfenidone groups, and were comparable with that in the Normal group (Figure 6D).
The comparison of macrophage activation and MMP-9 synthesis
Anti-ED1 antibody was used to identify macrophage (Figures 7A-7B). The results showed that only a limited number of macrophages existed in the Normal group. The number of macrophages increased, with a smaller size in the BLM group. In the BLM+Nintedanib and BLM+Pirfenidone groups, a large number of macrophages with a smaller size substantially appeared in the connective tissues of the left lung. In the BLM+HUMSCs group, macrophages with a larger size were observed in the connective tissues and among the alveolar space of left lung (Figure 7A-7B). Anti-CD86 antibody was further applied to mark M1 macrophages. The results showed that the number of M1 macrophages was higher in the BLM, BLM+Nintedanib, and BLM+Pirfenidone groups. Transplantation of HUMSCs significantly reduced the number of M1 macrophages (Figures 7C and 7E). When using anti-CD163 antibody to label M2 macrophages, it showed that the number of M2 macrophages was scarce in the BLM, BLM+Nintedanib, and BLM+Pirfenidone groups, whereas the number of M2 macrophages was further increased in the BLM+HUMSCs group (Figures 7D and 7F).
Western blotting was applied to quantify the expression of matrix metallopeptidase 9 (MMP-9). The results showed that no statistical differences were identified among the Normal, BLM, BLM+Nintedanib, and BLM+Pirfenidone groups. The MMP-9 level markedly elevated in the BLM+HUMSCs group, suggesting transplantation of HUMSCs increased MMP-9 expression, which could help collagen degradation in the fibrotic region of lung (Figure 7G).
The comparison of TLR-4 expression
The protein level of toll-like receptor-4 (TLR-4) was quantified using Western blotting. The results showed that no statistical significance in TLR-4 expression was observed between the Normal, BLM, BLM+Nintedanib, and BLM+Pirfenidone groups. The TLR-4 content in the BLM+HUMSCs group significantly increased, suggesting that transplantation of HUMSCs elevated TLR-4 expression, which could help the regeneration of type II alveolar epithelial cells in the fibrotic areas of lung (Figure 7H).