The effective-compound compatibility of JHF inhibits fibroblast activation in pulmonary fibrosis by enhancing PINK1/PARK2-mediated mitophagy

Abstract

This work aimed to elucidate the anti-PF mechanism of ECC-JHF.The effects of ECC-JHF on lung fibrosis and fibroblast activation were investigated by establishing a BLM-induced PF rat model and a transforming growth factor-beta (TGF-β)-induced fibroblast activation model. Furthermore, the effects of ECC-JHF on Nrf2 signaling and mitophagy were explored both in vivo and in vitro. In the PF model rats, ECC-JHF mitigated pathological damage, reduced collagen deposition, decreased levels of malondialdehyde (MDA) and P62, and increased levels of total superoxide dismutase (T-SOD) as well as the expression of Nrf2, HO-1, PINK1, PARK2, and LC3B in lung tissues. These results suggest that the anti-PF mechanism of ECC-JHF may be associated with the inhibition of oxidative stress and the enhancement of mitophagy. The medium dose of ECC-JHF and pirfenidone were similar in improving pulmonary fibrosis in rats. In the TGF-β-induced lung fibroblast activation, ECC-JHF inhibited fibroblast activation by downregulating the levels of fibronectin, alpha-smooth muscle actin (α-SMA), and collagen I. Additionally, ECC-JHF upregulated the level of Nrf2 and its target proteins, including HO-1 and NQO1, as well as mitophagy-related proteins PINK1, PARK2, and LC3B. This led to an increase in the co-localization of TOM20 and LC3, thereby enhancing mitochondrial autophagy. The application of Nrf2 siRNA and Nrf2 inhibitors significantly diminished the effects of ECC-JHF on Nrf2 signaling, PINK1/PARK2-mediated mitophagy, and fibroblast activation. ECC-JHF exerts a protective effect against PF by suppressing fibroblast activation through the upregulation of Nrf2 and PINK1/PARK2-mediated mitophagy, it provides a new target and strategy for the treatment of pulmonary fibrosis.

Keywords: Effective-compound combination; Fibroblasts activation; Mitophagy; Nrf2; Pulmonary fibrosis.

Fig. 1

The therapeutic effect of ECC-JHF on BLM-induced PF in rats. (A) The general process of animal experiments. (B) The compounds of ECC-JHF and their chemical structure. (C) HE and Masson staining (magnification, × 200). (D) The Szapiel and Ashcroft scores of lung tissue (Alveolitis and fibrosis scores). (E-F) Immunohistochemical analysis of α-SMA, Col III and IHS score assessment (magnification, × 200). All data were expressed as mean ± SE (n = 6)., ^**p < 0.01, vs. control group. ^#p < 0.05, ^##p < 0.01, vs. BLM group.

Fig. 2

ECC-JHF suppresses oxidative stress and enhances mitochondrial autophagy in lung tissue. (A) The activity of T-SOD. (B) The content of MDA. (C) Immunohistochemical analysis of Nrf2, HO-1, and IHS score assessment (magnification, × 200). (D) Transmission electron microscopy: the red arrows indicate autophagosomes and the blue indicate mitochondria (magnification, × 30000). (E) Immunohistochemical analysis of PINK1, Park2, p62 and LC3B and IHS score assessment (magnification, × 200). All data were expressed as mean ± SE (n = 6). ^*p < 0.05, ^**p < 0.01, vs. control group. ^#p < 0.05, ^##p < 0.01, vs. BLM group.

Fig. 3

Inhibitory effect of ECC-JHF on fibroblast activation. (A) Relative mRNA levels of ACTA2, FN1 and COL1A1. (B) The protein expression levels of α-SMA, FN and COL1 were detected by western blotting. (C) Immunofluorescent staining of the protein expression levels of α-SMA and FN (magnification, × 200). All data were expressed as mean ± SE. ^*p < 0.05, ^**p < 0.01, vs. control group. ^#p < 0.05, ^##p < 0.01, vs. model group.

Fig. 4

Effect of ECC-JHF on Nrf2 signaling and mitophagy in TGFβ1-induced HFL1 cells. (A) The protein expression levels of Nrf2, HO-1 and NQO1. (B) The protein expression levels of PINK1, PARK2 and LC3B. (C) Immunofluorescent staining of the co-localization of TOM20 and LC3 (magnification, × 400). All data were expressed as mean ± SE. ^*p < 0.05, ^**p < 0.01, vs. control group. ^#p < 0.05, ^##p < 0.01, vs. model group.

Fig. 5

ECC-JHF inhibit TGF-β1-induced fibroblast activation by activting Nrf2 singaling, strengthening mitophagy. Effect of Nrf2 in ECC -treated HFL1 cells. After transfection with siNrf2 for 24 h, HFL1 cells were treated with ECC-JHF (30.63 and 61.25 µg/ml) and TGF-β1 (5 ng/ml) for 24 h. Then, cells were collected for western blotting. (A-G)The protein expression levels of α-SMA, FN, Nrf2, HO-1, and PINK1, PARK2. All data were expressed as mean ± SE. ^*p < 0.05, ^**p < 0.01, vs. control group in normal cells. ^#p < 0.05, ^##p < 0.01, vs. model group in normal cells. ^△p < 0.05, ^△△p < 0.01, vs. control group in Nrf2 siRNA cells.

Fig. 6

Nrf2-dependence for the anti-PF effect of ECC-JHF. (A) HE and Masson staining (magnification, × 200). (B-C) The Szapiel and Ashcroft scores of lung tissue (Alveolitis and fibrosis scores). (D-F) Immunohistochemical analysis of α-SMA, Col III and IHS score assessment (magnification, × 200). All data were expressed as mean ± SE (n = 6). ^*p < 0.05, ^**p < 0.01, vs. control group. ^#p < 0.05, ^##p < 0.01, vs.BLM group. ^△p < 0.05, ^△△p < 0.01, vs. ECC-JHF group.

Fig. 7

ECC-JHF improves BLM-induced PF in rats by activating Nrf2, enhancing mitophagy. (A-G)The protein expression levels of Nrf2, HO-1, NQO1, PINK1, PARK2 and LC3B. All data were expressed as mean ± SE (n = 3). ^*p < 0.05, ^**p < 0.01, versus the control group. ^#p < 0.05, ^##p < 0.01, vs. BLM group. ^△p < 0.05, ^△△p < 0.01, vs. ECC-JHF group.