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. 2020 Nov:78:153298.
doi: 10.1016/j.phymed.2020.153298. Epub 2020 Aug 1.

Biochanin-A ameliorates pulmonary fibrosis by suppressing the TGF-β mediated EMT, myofibroblasts differentiation and collagen deposition in in vitro and in vivo systems

Sai Balaji Andugulapati  1 , Karthik Gourishetti  1 , Satya Krishna Tirunavalli  1 , Taslim Babru Shaikh  1 , Ramakrishna Sistla  2
Affiliations

Biochanin-A ameliorates pulmonary fibrosis by suppressing the TGF-β mediated EMT, myofibroblasts differentiation and collagen deposition in in vitro and in vivo systems

Sai Balaji Andugulapati et al. Phytomedicine. 2020 Nov.

Abstract

Background: Idiopathic Pulmonary Fibrosis (IPF) is a progressive inflammatory disorder driven by a fibrotic cascade of events such as epithelial to mesenchymal transition, extracellular matrix production and collagen formation in the lungs in a sequential manner. IPF incidences were raising rapidly across the world. FDA approved pirfenidone and nintedanib (tyrosine kinase inhibitors) are being used as a first-line treatment drugs for IPF, however, neither the quality of life nor survival rates have been improved because of patient noncompliance due to multiple side effects. Thus, the development of novel therapeutic approaches targeting TGF-β mediated cascade of fibrotic events is urgently needed to improve the survival of the patients suffering from devastating disease.

Purpose: The aim of this study was to investigate and validate the anti-fibrotic properties of Biochanin-A (isoflavone) against TGF-β mediated fibrosis in in vitro, ex vivo, in vivo models and to determine the molecular mechanisms that mediate these anti-fibrotic effects.

Methods: The therapeutic activity of BCA was determined in in vitro/ex vivo models. Cells were pre-treated with BCA and incubated in presence or absence of recombinant-TGF-β to stimulate the fibrotic cascade of events. Pulmonary fibrosis was developed by intratracheal administration of bleomycin in rats. BCA treatment was given for 14 days from post bleomycin instillation and then various investigations (collagen content, fibrosis gene/protein expression and histopathological changes) were performed to assess the anti-fibrotic activity of BCA.

Results: In vitro/ex vivo (Primary normal, IPF cell line and primary IPF cells/ Precision cut mouse lung slices) experiments revealed that, BCA treatment significantly (p < 0.001) reduced the expression of TGF-β modulated fibrotic genes/protein expressions (including their functions) which are involved in the cascade of fibrotic events. BCA treatment significantly (p < 0.01) reduced the bleomycin-induced inflammatory cell-infiltration, inflammatory markers expression, collagen deposition and expression of fibrotic markers in lung tissues equivalent or better than pirfenidone treatment. In addition, BCA treatment significantly (p < 0.001) attenuated the TGF-β1/BLM-mediated increase of TGF-β/Smad2/3 phosphorylation and resulted in the reduction of pathological abnormalities in lung tissues determined by histopathology observations.

Conclusion: Collectively, BCA treatment demonstrated the remarkable therapeutic effects on TGF-β/BLM mediated pulmonary fibrosis using IPF cells and rodent models. This current study may offer a novel treatment approach to halt and may be even rescue the devastating lung scarring of IPF.

Keywords: BALF; Bleomycin; Collagen; Extra-cellular matrix; Inflammation; Myofibroblasts; TGF-β.

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Conflict of interest statement

Declaration of Competing Interest The authors declare no competing or financial interests.

Figures

Image, graphical abstract
Graphical abstract
Fig 1
Fig. 1
Fibrotic gene/markers were over-expressed in IPF cells and BCA showed selective inhibition towards IPF cells NHLF, DHLF and LL29 cells were cultured and subjected to RT-qPCR analysis for the specified transcripts A-B) Gene expression analysis. Parallel dishes were subjected to immunofluorescence for α-SMA, Fibronectin and COL1A1. C-F) Immunofluorescence analysis in NHLF, DHLF and LL29 cells, respectively. Cells (5 × 105) were cultured and treated with BCA or vehicle for 72 h then subjected to RT-qPCR. Gene expression analysis in G) PCLMS, H) NHLF, I) LL29 cells and J) DHLF was performed using specified primers. Results were shown as Mean ± SEM, n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
Fig 2
Fig. 2
BCA treatment attenuated the TGF-β-induced expression of EMT genes/markers: Cells were pre-treated with BCA for 2 h, thereafter rTGF-β added to the cells and cultured for 72 h. Further, cells were subjected to RT-qPCR for the specified transcripts in A-D) NHLF, E-H) DHLF, I-K) LL29. Parallel dishes were subjected to immunofluorescence assay for FN-1 protein. L-N) Immunofluorescence analysis in NHLF, DHLF and LL29 cells, respectively. Results are shown as Mean ± SEM, n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
Fig 3
Fig. 3
BCA treatment ameliorated the TGF-β-induced expression of ECM markers: Cells were pre-treated with BCA for 2 h, thereafter rTGF-β added to the cells and cultured for 72 h. Further, cells were subjected to RT-qPCR for the specified transcripts in A-D) NHLF, E-H) DHLF, I-L) LL29.  Parallel dishes were subjected to immunofluorescence analysis for α-SMA protein. M-O) Immunofluorescence analysis in NHLF, DHLF and LL29 cells, respectively. Results were shown as Mean ± SEM, n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
Fig 4
Fig. 4
BCA treatment attenuated the TGF-β/smad3 axis induced expression of collagen depositing markers: Cells were pre-treated with BCA for 2 h, thereafter rTGF-β added to the cells and cultured for 72 h. Further, cells were subjected to RT-qPCR for the specified transcripts in A-D) NHLF, E-H) DHLF, I-L) LL29.  M-O) Smad7 mRNA expression in NHLF, DHLF and LL29 cells, respectively. Results were shown as Mean ± SEM, n = 3. ∗ p < 0.05, ∗∗ p < 0.01,∗∗∗ p < 0.001.
Fig 5
Fig. 5
Role of BCA on TGF-β-induced fibrotic genes/markers expression in PCMLS: PCMLS were pre-treated with BCA for 2 h, thereafter rTGF-β (mouse) added to the cells and cultured for 72 h. Further, PCMLS were subjected to RT-qPCR for the specified transcripts. A-G) Gene expression analysis.  Graphs in panels represent fold change in gene expression normalized to GAPDH. PCMLS derived cells were subjected to immunofluorescence analysis for H) FN-1, I) COL1A1 and J) E-cad. Results were shown as Mean ± SEM, n = 3. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.
Fig 6
Fig. 6
BCA treatment attenuated the expression and function of pro-fibrotic markers through TGF-β/smadsignaling cascade: LL29 cells or PCMLS were pre-treated with BCA for 2 h, thereafter rTGF-β added to the cells and cultured for 72 h. Further, cells were subjected to immunoblot analysis A and E) Immunoblot for specified antibodies. The graphs represent densitometric quantification for the specified proteins in B-D) LL29 and F-I)PCMLS. J-M) After attaining 100% confluency of NHLF, PCMLS derived cells, LL29 and DHLF cells, cells were pre-treated with BCA for 2 h and then scratch was made, thereafter cells were cultured for 72h in presence or absence of rTGF-β1. The graphs represent time kinetics of wound confluence percentage, calculated by image J software Results were shown as Mean ± SEM, n = 3. ∗ p < 0.05,∗∗ p < 0.01,∗∗∗ p < 0.001.
Fig 7
Fig. 7
Protective role of BCA on BLM-induced pulmonary fibrosis: A) Schematic representation of BLM Induced pulmonary fibrosis. Post bleomycin (BLM) instillation, rats were orally treated with BCA (5 mg/kg), BCA (10 mg/kg), pirfenidone (50 mg/kg) for a period of two weeks. BLM group and sham control animals received vehicle suspension and saline respectively. After the treatment period, rats were anesthetized and BALF fluid and lung tissue was collected. B) Alkaline phosphatase C) and lactate dehydrogenase (LDH) levels were measured in BALF D) neutrophil count and E) WBC count was estimated in BALF. F) Lung index G) Hydroxyproline content. Data was shown as Mean ± S.E.M (n = 4). * p < 0.05, ** p < 0.01 vs. BLM group.
Fig 8
Fig. 8
BCA treatment attenuated the fibrotic marker's expression by modulating the TGF-β/smad pathway in in vivo: After treatment, lung tissues were collected and part of tissues was subjected for RT-qPCR or immunoblot analysis for the specified transcripts or antibodies respectively. A-H) Gene expression analysis I) Representative images of western blot, J-L) Graph represents densitometric quantification of the specified proteins; Results were shown as Mean ± SEM; n = 4. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. the BLM alone group.
Fig 9
Fig. 9
Protective effect of BCA on bleomycin-induced pathological changes After treatment with BCA (5 mg/kg) or (10 mg/kg) or pirfinidone (50 mg/kg) vehicle, lungs were isolated and subjected for histopathology. A and B) Representative pictures (10 ×) of A) H&E-stained, B) Masson's trichrome-stained sections. The Black arrow points at typical areas of fibrosis, manifesting as group of cells with protruding cytoplasm (myofibroblasts). C, D and E) Immunohistochemistry for FN-I, COL1A1 and E-cad in lung sections. The black arrows at typical areas represent the increased expression of protein. Images were taken under original magnification 20 ×. Graphs in panels represent the score of F) Fibrosis and G) Ashcroft score, score numbers of 0–8, were evaluated by experienced pathologists in a blinded fashion. H) Table represents the semi-quantitative evaluation of protein expression (FN-I, COL1A1 and E-cad) in specified treatment groups. Data was expressed as Mean ± S.E.M. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. the BLM alone group.
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