Oxymatrine attenuates pulmonary fibrosis via APE1‑mediated regulation of the PINK1/Parkin pathway

Figures

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  Figure 1.. OMT treatment reverses bleomycin-induced PF. (A) Schematic diagram of the treatment protocol. (B) H&E staining was used to observe and evaluate lung tissue structure and morphology. (C) Masson's trichrome staining was performed to assess PF. (D) TUNEL staining was used to detect apoptosis. IHC staining was conducted to detect the expression levels of (E) α-SMA and (F) COL1a1. Semi-quantitative analysis of (G) H&E, (H) Masson's, (I) TUNEL, and IHC staining of (J) α-SMA and (K) COL1a1. Scale bar, 100 µm. (G) Data are presented as median and IQR, and statistical significance was analyzed using the Kruskal-Wallis test. (H-K) Data are presented as the mean ± SD, and statistical significance was analyzed using one-way ANOVA; n=6. *P<0.05, ***P<0.001. The treatment schematic was designed using Figdraw (https://www.figdraw.com/). PF, pulmonary fibrosis; OMT, oxymatrine; NAC, N-acetylcysteine; α-SMA, α-smooth muscle actin; COL1a1, collagen type I α 1; IHC, immunohistochemical.

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  Figure 2.. OMT mitigates PF by activating the APE1 and PINK1/Parkin pathways, and promoting autophagy and mitochondrial function restoration. (A) RT-qPCR of the relative expression of APE1 mRNA following APE1 silencing in LLC cells. (B) Western blotting of the relative expression of APE1 protein following APE1 silencing in LLC cells. (C) IHC staining of APE1, PINK1 and Parkin expression in the lung tissue of C57BL/6 mice (scale bar, 100 µm). Semi-quantitative analysis of (D) APE1, (E) PINK1 and (F) Parkin IHC staining. Data are presented as the mean ± SD. Statistical significance was analyzed using one-way ANOVA. ***P<0.001. OMT, oxymatrine; NAC, N-acetylcysteine; APE1, apurinic/apyrimidinic endonuclease-1; PINK1, PTEN-induced kinase 1; KD, knockdown; mdivi-1 mitophagy inhibitor mitochondrial division inhibitor-1, RT-qPCR, reverse transcription-quantitative PCR; LC-3, microtubule-associated proteins 1A/1B light chain 3; TOM20, outer membrane translocase 20; NC, negative control; IHC, immunohistochemical.

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  Figure 3.. OMT regulates APE1 and mitophagy-related proteins and promotes LC3/TOM20 colocalization. (A) Western blotting of the relative expression levels of APE1, PINK1 and Parkin proteins in the lung tissue of C57BL/6 mice. Semi-quantitative analysis of (B) APE1, (C) PINK1 and (D) Parkin. (E) Immunofluorescence analysis of LC-3 and TOM20 expression and colocalization in the lung tissue of C57BL/6 mice (scale bar, 100 µm). (F) Percentage of the yellow fluorescent colocalization area of LC-3 and TOM20. Data are presented as the mean ± SD. Statistical significance was analyzed using one-way ANOVA; n=6. **P<0.01, ***P<0.001. OMT, oxymatrine; NAC, N-acetylcysteine; APE1, apurinic/apyrimidinic endonuclease-1; PINK1, PTEN-induced kinase 1; KD, knockdown; mdivi-1 mitophagy inhibitor mitochondrial division inhibitor-1.

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  Figure 4.. Silencing APE1 reverses the antifibrotic effects of OMT on bleomycin-induced PF. (A) Schematic diagram of the treatment protocol incorporating the OMT + KD-APE1 intervention group. (B) H&E staining was used to observe and evaluate lung tissue structure and morphology. (C) Masson's trichrome staining was performed to assess PF. (D) TUNEL staining was used to detect apoptosis. Immunohistochemical staining was conducted to detect the expression levels of (E) α-SMA and (F) COL1a1. Semi-quantitative analysis of (G) H&E, (H) Masson's, (I) TUNEL, and IHC staining of (J) α-SMA and (K) COL1a1. Scale bar, 100 µm. (G) Data are presented as the median and IQR, and statistical significance was analyzed using the Kruskal-Wallis test. (H-K) Data are presented as the mean ± SD, and statistical significance was analyzed using one-way ANOVA; n=6. *P<0.05, ***P<0.001. The treatment schematic was designed using Figdraw (https://www.figdraw.com/). PF, pulmonary fibrosis; OMT, oxymatrine; NAC, N-acetylcysteine; α-SMA, α-smooth muscle actin; COL1a1, collagen type I α 1; KD, knockdown; APE1, apurinic/apyrimidinic endonuclease-1; IHC, immunohistochemical.

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  Figure 5.. Mitochondrial autophagy inhibitors reverse the effects of OMT against bleomycin-induced PF. (A) Schematic diagram of the treatment protocol incorporating the OMT + mdivi-1 intervention group. (B) H&E staining was used to observe and evaluate lung tissue structure and morphology. (C) Masson's trichrome staining was performed to assess PF. (D) TUNEL staining was used to detect apoptosis. IHC staining was conducted to detect the expression levels of (E) α-SMA and (F) COL1a1. Semi-quantitative analysis of (G) H&E, (H) Masson's, (I) TUNEL, and IHC staining of (J) α-SMA and (K) COL1a1. Scale bar, 100 µm. (G) Data are presented as the median and IQR, and statistical significance was analyzed using the Kruskal-Wallis test. (H-K) Data are presented as the mean ± SD, statistical significance was analyzed using one-way ANOVA; n=6. *P<0.05, ***P<0.001. The treatment schematic was designed using Figdraw (https://www.figdraw.com/). PF, pulmonary fibrosis; OMT, oxymatrine; NAC, N-acetylcysteine; α-SMA, α-smooth muscle actin; COL1a1, collagen type I a 1; KD, knockdown; APE1, apurinic/apyrimidinic endonuclease-1; IHC, immunohistochemical.

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  Figure 6.. Mechanism by which oxymatrine alleviates pulmonary fibrosis by upregulating APE1, activating the PINK1/Parkin pathway and promoting mitochondrial autophagy. The mechanism diagram was self-designed by the authors using Figdraw (https://www.figdraw.com/). α-SMA, α-smooth muscle actin; COL1a1, collagen type I α 1; APE1, apurinic/apyrimidinic endonuclease-1; PINK1, PTEN-induced kinase 1.