Pathological changes in skin tissues
HE stain and Masson stain results of skin tissues indicated that the dermis of mice in the control group had proper thickness, clear structure, and a large amount of underlying adipose tissue (Fig. 1a, e). Skin tissues of mice in the model group at different time points exhibited significant thickening of the dermis and disordered structure with increasing model establishment timethere was inflammatory cell filtration and collagen deposition (Fig. 1b–d, f–h). Of these features, the inflammatory cell infiltration was the most pronounced in the 7-day group (Fig. 1b), and the collagen deposition and skin fibrosis were the more pronounced in the 28- and 42-day group (Fig. 1g, h). After NaHS intervention, the skin tissues of mice in the treatment group exhibited a clear structure, the dermis became thinner, the lipid layer thickened, inflammatory cell filtration and collagen deposition significantly decreased compared to the model group on 7-day (Fig. 2c, d) and on 42 day (Fig. 2h, i; m, n). Compared to that in the low dose treatment group, the improvement in the high dose group was even more clearly detectable (Fig. 2e, j, o).
Pathological changes in lung tissues
HE stain and Masson stain results of lung tissues indicated that the alveoli of mice in the control group were transparent and bright, the structure was intact (Fig. 1i, m). Compared to the control group, the model group at different time points all exhibited obvious inflammatory cell infiltration, alveolar collapse, disordered structure, increased collagen deposition, and thickening of alveolar septa (Fig. 1j–l; m, o, p). Thereinto, the inflammatory cell infiltration was more evident in the 7-day group (Fig. 1j); whereas the alveolar collapse, patchy fusion, collagen deposition, and pulmonary parenchymal fibrosis were the more evident in the 28- and 42-day group (Fig. 1o, p). After NaHS intervention, lung tissue pathological changes in mice from the treatment group were significantly relieved, inflammatory cell filtration and collagen deposition decreased, alveolar structure improved, the alveoli were more transparent and bright, and the degree of inflammation and fibrosis decreased on 7-day (Fig. 3c, d) and on 42 day (Fig. 3h, i; m, n).
Changes in the plasma H2S concentrations
Compared to the control group, the plasma H2S concentrations for mice in the model group at different time points all decreased. The mean plasma H2S concentration for mice in the 7-day control group was 24.96 ± 4.12 μM. The concentration in the 7-day model group was 22.79 ± 1.54 μM; the concentration decreased compared to that in the control group, but the difference was not statistically significant. The concentrations in the 28- and the 42-day control groups were 24.66 ± 3.52 and 24.41 ± 2.57 μM, The concentrations in the 28-day model group and the 42-day model groups were 18.59 ± 3.32 and 16.49 ± 2.46 μM, respectively compared to that in the control group, the differences were each statistically significant (Fig. 4).
H2S reduces the expression level of HYP in the lung tissues of mice induced by BLM on the 42th day
Compared to that in the control group, the HYP levels in the lung tissues of mice in the model group at different time points all significantly increased. For example in the 42-day group, after NaHS intervention, the HYP concentration in the treatment group significantly decreased. The decreasing trend in the high dose treatment group was more evident than that in the low dose treatment group, but the difference was not statistically significant (Fig. 5).
H2S inhibits the expression of α-SMA, Col-I, Col-III and FN in lung tissues on the 42th day
Compared to that in the control group, the mRNA expression levels of α-SMA, Col-I, and Col-III in the lung tissues of mice in the model group at different time points were significantly upregulated. For example in the 42-day group, after NaHS intervention, the expression levels of the above indicators for mice in the treatment group were significantly downregulated, and the differences were statistically significant. The trend of downregulation in the high dose treatment group was more evident than that in the low dose treatment group, but the difference was not statistically significant (Fig. 6).
Compared to that in the control group, the protein expression levels of α-SMA and FN in the lung tissues of mice in the model group were significantly upregulated, and in the 42-day group after NaHS intervention, the expression levels of the above indicators for mice in the treatment group were significantly downregulated, and the differences were statistically significant. Compared to that in the low dose treatment group, the trend of downregulation of the expression levels of the above proteins in the high dose treatment group was more evident, but the difference was not statistically significant (Fig. 7).
H2S reduces the number of ED-1-positive cells and MCP-1-positive cells in lung tissues on the 7th day
Alveolitis was the most clearly observed in the 7-day model group. Therefore, the 7-day group was used for routine ED1 and MCP-1 immunohistochemistry to investigate the effects of H2S on inflammatory cytokines. The results of immunohistochemistry indicated that compared to the control group, the 7-day model group exhibited significant inflammatory cell infiltration and an increased number of ED1 and MCP-1 positive expression cells (Figs. 8b, 9b). After NaHS intervention, the expression levels of the above positive cells were significant downregulated (Figs. 8c, d, 9c, d). However, the difference between the low dose treatment group and the high dose treatment group was not statistically significant.
H2S inhibits the expression of TGF-β1 and p-Smad2/3 in lung tissues on the 28th day and the 42th day
Compared to the control group, the protein expression levels of TGF-β1 in lung tissues of mice in the model group at different time points were significantly upregulated, particularly the 28- and 42-day groups. After NaHS intervention, the protein expression level of TGF-β1 of mice in the treatment group was significantly downregulated, and the difference was statistically significant; however, the difference between the low dose treatment group and the high dose treatment group was not statistically significant. Meanwhile, compared to the control group, the protein expression levels of p-Smad2/3 in the 42 days model group lung tissues were significantly upregulated. After NaHS administration, the protein expression level of p-Smad2/3 in the treatment group was significantly downregulated, and the difference was statistically significant (Fig. 10).