Expressions and effects of autophagy-related genes in bleomycin-induced skin fibrosis of mice

Objective To investigate the expressions and effects of autophagy-related genes in bleomycin-induced skin fibrosis of mice. Methods (1) Totally 72 male BALB/c mice aged 6 weeks were divided into blank control group, simple phosphate buffer solution (PBS) group, and bleomycin group according to the random number table, with 24 mice in each group. Mice in blank control group received no treatment, and 100 μL of PBS and bleomycin (1 mg/mL) were respectively injected subcutaneously in the back skin of mice in simple PBS and bleomycin group, once a day for 28 days. On injection day (ID) 7, 14, 21, and 28, 6 mice in each group were collected to observe the skin change on the back of mice with naked eyes. After the observation, the mice were sacrificed and skin tissue on the back was taken. Skin tissue of mice on ID 28 was collected to measure the thickness of skin tissue by routine hematoxylin-eosin staining and observe skin tissue morphology by Masson staining. Skin tissue on ID 7, 14, 21, and 28 was taken to detect content of hydroxyproline by enzyme linked immunosorbent assay, and mRNA and protein expressions of p62, microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ) and Beclin-1 were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. (2) Skin tissue of mice in blank control group in experiment (1) was taken to culture fibroblasts (Fbs) in 3rd-6th passages. The cells were divided into blank control group, simple PBS group, and bleomycin group according to the random number table, with 6 wells in each group. Cells in blank control group were not stimulated, and cells in simple PBS group and bleomycin group were stimulated with 20 μL of PBS and bleomycin (1 mg/mL) for 72 h, respectively. Cellular immunofluorescence staining was used to observe the expression of LC3 Ⅱ. Data were statistically analyzed with analysis of variance of factorial design, one-way analysis of variance, t test, and Bonferroni correction. Results (1) Skin on the back of mice in blank control group and simple PBS group was thin and ruddy, and the veins were clear on ID 7, 14, 21, and 28. Several raised ridges were visible on the puncture site of mice in simple PBS group from ID 14. Skin on the back of mice was ruddy, with several raised ridges visible on the puncture site of mice in bleomycin group on ID 7, the skin turned slightly white on ID 14, the skin turned white obviously with unclear surrounding blood vessels on ID 21, and the skin turned white and the surrounding blood vessels could not be recognized on ID 28. (2) On ID 28, the skin thicknesses of mice in blank control group and simple PBS group were similar (t=0.79, P>0.05). Compared with that in blank control group and simple PBS group, the skin thickness of mice in bleomycin group was significantly increased (t=0.50, 0.50, P<0.01). (3) On ID 28, the skin tissue structure of mice in blank control group and simple PBS group was similar, with a small amount of orderly arranged collagen and evenly distributed hair follicle; the number of collagen of skin in mice of bleomycin group was increased obviously and arranged disorderly, and the number of hair follicle was decreased significantly. (4) On ID 7, 14, 21, and 28, the content of hydroxyproline in the skin tissue of mice in bleomycin group was significantly higher than that in blank control group and simple PBS group (t=0.99, 0.98, 0.50, 0.51, 0.50, 0.50, 0.52, 0.51, P<0.05 or P<0.01). (5) On ID 7, p62 mRNA expression in the skin tissue of mice in bleomycin group was significantly lower than that in simple PBS group (t=0.93, P<0.05). On ID 14 and 21, the mRNA expressions of p62, LC3 Ⅱ, and Beclin-1 in the skin tissue of mice in bleomycin group were significantly higher than those in blank control group (t=0.74, 0.70, 0.58, 0.49, 0.51, 0.74, P<0.05) and simple PBS group (t=0.94, 0.65, 0.65, 0.77, 0.49, 0.51, P<0.05). On ID 28, the mRNA expressions of p62 and Beclin-1 in the skin tissue of mice in bleomycin group were significantly lower than those in blank control group (t=0.50, 0.44, P<0.05) and simple PBS group (t=0.97, 0.55, P<0.05), and that of LC3 Ⅱ was significantly higher than that in blank control group and simple PBS group, respectively (t=0.51, 0.98, P <0.01). (6) On ID 7, 14, 21, and 28, the protein expressions of LC3 Ⅱ in blank control group, simple PBS group, and bleomycin group were 0.167±0.042, 0.122±0.016, 0.553±0.078, 0.118±0.035, 0.120±0.023, 0.117±0.061, 0.581±0.039, 0.159±0.065, 0.233±0.027, 0.304±0.031, 1.020±0.010, 0.089±0.045. On ID 14, the protein expressions of p62 and Beclin-1 in the skin tissue of mice in bleomycin group were significantly higher than those in blank control group (t=0.86, 0.89, P<0.05) and simple PBS group (t=0.42, 0.89, P<0.05). On ID 21, the protein expressions of p62, LC3 Ⅱ, and Beclin-1 in the skin tissue of mice in bleomycin group were significantly higher than those in blank control group and simple PBS group (t=0.82, 0.45, 0.50, 0.79, 0.51, 0.50, P<0.01). On ID 28, the protein expressions of p62, LC3 Ⅱ, and Beclin-1 in the skin tissue of mice in bleomycin group were significantly lower than those in blank control group and simple PBS group (t=0.77, 0.54, 0.52, 0.50, 0.51, 0.50, P<0.05). (7) After culture for 72 h, the expression of LC3 Ⅱ in Fbs of bleomycin group was significantly lower than that of blank control group and simple PBS group, respectively. Conclusions In the process of bleomycin stimulating skin fibrosis, autophagy-related genes increase firstly and then decrease. When the autophagy process is activated, it is expected to reverse the process of skin fibrosis.