Fig 2: Hes activated autophagy in BLM-induced fibrosis via EI24. (A) The relative protein expressions of α-SMA, COLLAGEN I and FIBRONECTIN in lung tissues were determined by western blot. Data were expressed with the normalization with β-ACTIN. (B) The relative protein expressions of EI24, BECLIN1, LC3II/LC3I and P62 in lung tissues were examined by western blot. Data were expressed with the normalization with β-ACTIN. **P < 0.01 vs. sham; ##P < 0.01 vs. BLM; &P < 0.05 vs. BLM + 100 mg/kg Hes. Note: Hes: hesperidin; EI24: etoposide-induced protein 2.4; BLM: bleomycin; α-SMA: alpha smooth muscle Actin.

Fig 3: Hes inhibited fibrosis by regulating EI24-mediated autophagy in TGF-β1-induced RLE-6TN cells. RLE-6TN cells were transfected with si-EI24. After 48 h of transfection, cells were collected to treat with 10 ng/mL TGF-β1 for 48 h, as well as 100 µM Hes and 1 mM 3-MA for 24 h. (A) The relative protein expression of EI24 was examined by western blot. Data were expressed with the normalization with β-ACTIN. **P < 0.01 vs. control (con). (B) The relative protein expressions of EI24, BECLIN1, LC3II/LC3I and P62 were examined by western blot. Data were expressed with the normalization with β-ACTIN. **P < 0.01 vs. control (con); ##P < 0.01 vs. TGF-β1; &P < 0.05 vs. TGF-β1 + 100 µM Hes. (C) The expression level of LC3B was detected by IF. Scale bar = 25 µm. **P < 0.01 vs. control (con); ##P < 0.01 vs. TGF-β1; &P < 0.05 vs. TGF-β1 + 100 µM Hes. (D) The relative protein expressions of α-SMA, COLLAGEN I and FIBRONECTIN were determined by western blot in RLE-6TN cells. Data were expressed with the normalization with β-ACTIN. **P < 0.01 vs. control (con); ##P < 0.01 vs. TGF-β1; &P < 0.05 vs. TGF-β1 + 100 µM Hes. Note: Hes: hesperidin; TGF-β1: transforming growth factor β1; EI24: etoposide-induced protein 2.4; IF: immunofluorescence; α-SMA: alpha smooth muscle Actin.

Fig 4: Hes activated autophagy in TGF-β1-induced RLE-6TN cells. RLE-6TN cells were treated with 10 ng/mL TGF-β1 for 48 h, as well as 25, 50 and 100 µM Hes for 24 h. (A) The relative protein expressions of EI24, BECLIN1, LC3II/LC3I and P62 were examined by western blot. Data were expressed with the normalization with β-ACTIN. (B) The expression level of LC3B was detected by IF. Scale bar = 25 µm. **P < 0.01 vs. control (con); #P < 0.05 and ##P < 0.01 vs. TGF-β1. Note: Hes: hesperidin; TGF-β1: transforming growth factor β1; EI24: etoposide-induced protein 2.4; IF: immunofluorescence.

Fig 5: Hes regulated EI24 to mitigate BLM-induced fibrosis of rat lung tissue. Rats were intratracheally treated with 6 IU/kg BLM on day 7, intragastrically administered with 100 mg/kg Hes from day 1 to day 28, and treated with 30 μL of ad-sh-EI24 (5 × 10 transducing unit) from day 1 to day 28. (A) Rats body weight on day 28. (B) Measurement of the lung tissues wet/dry ration. (C) Detection of lung resistance. (D) Examination of the hydroxyproline concentration. (E) Pathological examination by H&E staining. (F) The fibrosis was determined by masson trichome staining. **P < 0.01 vs. sham; ##P < 0.01 vs. BLM; &P < 0.05 vs. BLM + 100 mg/kg Hes. Note: Hes: hesperidin; EI24: etoposide-induced protein 2.4; BLM: bleomycin H&E: hematoxylin and eosin.