Haematococcus pluvialis alleviates bleomycin-induced pulmonary fibrosis in mice by inhibiting transformation of lung fibroblasts into myofibroblast

doi:10.12122/j.issn.1673-4254.2025.08.12

Abstract

Abstract:

Objective To investigate the effect of Haematococcus pluvialis (HP) on bleomycin (BLM)-induced pulmonary fibrosis in mice and on TGF-β1-induced human fetal lung fibroblasts (HFL1). Methods Thirty male C57BL/6 mice were randomly divided into control group, BLM-induced pulmonary fibrosis model group, low- and high-dose HP treatment groups (3 and 21 mg/kg, respectively), and 300 mg/kg pirfenidone (positive control) group. The effects of drug treatment for 21 days were assessed by examining respiratory function, lung histopathology, and expression of fibrosis markers in the lung tissues of the mouse models. In TGF-β1-induced HFL1 cell cultures, the effects of treatment with 120, 180 and 240 μg/mL HP or 1.85 μg/mL pirfenidone for 48 h on expression levels of fibrosis markers were evaluated. Transcriptome analysis was carried out using the control cells and cells treated with TGF-β1 and 240 μg/mL HP. Results HP obviously alleviated BLM-induced lung function damage and fibrotic changes in mice, evidenced by improved respiratory function, lung tissue morphology and structure, inflammatory infiltration, and collagen deposition and reduced expressions of fibrotic proteins. HP at the high dose produced similar effect to PFD. In TGF-β1-induced HFL1 cells, treatment with 240 μg/mL HP significantly reduced the mRNA and protein expression levels of α-SMA and FN. Transcriptome analysis revealed that multiple key genes and pathways mediated the protective effect of HP against pulmonary fibrosis. Conclusion HP alleviates pulmonary fibrosis in both the mouse model and cell model, possibly as the result of the synergistic effects of its multiple active components.

Key words: Haematococcus pluvialis, pulmonary fibrosis, bleomycin, fibroblast

Xiao ZHANG, Jingzhou MAN, Yong ZHANG, YunJian ZHENG, Heping WANG, Yijun YUAN, Xi XIE. Haematococcus pluvialis alleviates bleomycin-induced pulmonary fibrosis in mice by inhibiting transformation of lung fibroblasts into myofibroblast[J]. Journal of Southern Medical University, 2025, 45(8): 1672-1681.

Figures/Tables 8

Tab.1 Primer sequences for qRT-PCR

Primer	Forward primer sequence (5'-3')	Reverse primer sequence (5'-3')
Mouse Col1	CTTTGCTTCCCAGATGTCCT	CGGTGTCCCTTCATTCCAG
Mouse Col3	GGCAGTGATGGGCAACCT	TCCCTTCGCACCGTTCTT
Mouse α-SMA	GGGAGTAATGGTTGGAATG	CTCAAACATAATCTGGGTCA
Mouse FN1	CATTGTTACCAACTGGGACG	CCAGAGGCATACAGGGAC
Mouse Col5	GGACTCGGCGGAACATTG	GGGAGTTGAGGGAACCAAA
Mouse GAPDH	TGTTTCCTCGTCCCGTAG	CAATCTCCACTTTGCCACT
Human Col1	AAGGTGTTGTGCGATGACG	TGGTCGGTGGGTGACTCTG
Human Col3	GAGCTGGCTACTTCTCGC	TCTATCCGCATAGGACTGAC
Human α-SMA	GGGGTGATGGTGGGAATG	GCAGGGTGGGATGCTCTT
Human FN1	ATGGAGGAAGCCGAGGTT	AGCGGTTTGCGATGGTAC
Human Col5	TGGCAAGTGGCACAGAATT	TCACCCTCAAACACCTCCTC
Human β-actin	GGAAATCGTGCGTGACATT	CAGGCAGCTCGTAGCTCTT

Fig.1 Effect of HP on pulmonary respiratory function in bleomycin-induced mouse pulmonary fibrosis model. Saline: Control group; BLM: Bleomycin-induced model group; BLM+HP (3 mg/kg): Low-dose HP group; BLM+HP (21 mg/kg): High-dose HP group; BLM+PFD: Positive drug pirfenidone control group. #P<0.05, ##P<0.01 vs Saline group; *P<0.05, **P<0.01 vs BLM group.

Fig.2 Effects of HP on lung tissue histology and collagen deposition in mice with bleomycin-induced pulmonary fibrosis. ##P<0.01 vs Saline group; **P<0.01 vs BLM group.

Fig.3 Western blotting (A) and qRT-PCR (B) for assessing the effect of HP on expressions of fibrotic proteins in the lung tissue of mice with bleomycin-induced pulmonary fibrosis. #P<0.05, ##P<0.01 vs saline group; *P<0.05, **P<0.01 vs BLM group.

Fig.4 Effect of different concentrations of HP on expression levels of fibrotic proteins in TGF-β1-induced HFL1 cell model. CCK-8 was used for detecting changes in proliferation of HFL1 cells treated with different concentrations of HP. Western blotting was used to detect the expression levels of fibrotic proteins of the treated cells. Except for the control group, all the other groups were induced with 5 ng/mL TGF-β1. ##P<0.01 vs Control group; *P<0.05, **P<0.01 vs TGF-β1 group.

Fig.5 Immunofluorescence (A) and qRT-PCR (B) for detecting the effects of high-dose HP (240 μg/mL) on expression levels of fibrotic proteins in TGF-β1-induced HFL1 cells. Model: TGF-β1-induced cells; HP: Cells treated with 240 μg/mL HP. #P<0.05, ##P<0.01 vs Control group. *P<0.05, **P<0.01 vs Model group.

Fig.6 Transcriptome analysis of the effects of high-dose HP (240 μg/mL) on pulmonary fibrosis-associated gene expression levels and major pathways in TGF-β1-induced HFL1 cell models. C1, C2, and C3: Three replicates of control cells; M1, M2, and M3: Three replicates of TGF-β1-induced cells; HP1, HP2, and HP3: Three replicates of cells treated with 240 μg/mL HP. A: PCA analysis. B: DEGs analysis. C: Venny diagram. D: Gene expression. E: Enrichment plot.

Tab.2 Differentially expressed genes related to fibrosis

Gene	Full name	Expression in the model	Function
ANKRD1	Ankyrin repeat domain-containing protein 1	Upregulation	Pro-fibrosis^[17]
GPRC5A	G protein-coupled receptor class C group 5 member A	Downregulation	Anti-fibrosis^[18]
HAPLN1	Hyaluronan and proteoglycan link protein 1	Upregulation	Pro-fibrosis ^[19]
POSTN	Periostin	Upregulation	Pro-fibrosis^[20]
CCL2	C-C motif chemokine 2	Downregulation	Anti-fibrosis^[21]
SLIT2	Slit homolog 2 protein	Downregulation	Anti-fibrosis^[22]
VSIR	V-type immunoglobulin domain-containing suppressor of T-cell activation	Downregulation	Anti-fibrosis^[23]
ADAMTS4	A disintegrin and metalloproteinase with thrombospondin motifs	Upregulation	Pro-fibrosis^{[24, 25]}
PLK1	Serine/threonine-protein kinase	Upregulation	Pro-fibrosis ^{[26, 27]}
HHIP	Hedgehog-interacting protein	Upregulation	Pro-fibrosis ^[28]
KCNN4	Intermediate conductance calcium-activated potassium channel protein 4	Upregulation	Pro-fibrosis ^{[29, 30]}
CDK1	Cyclin-dependent kinase 1	Upregulation	Pro-fibrosis ^{[31, 32]}
CMKLR1	Chemokine-like receptor 1	Upregulation	Pro-fibrosis ^{[33, 34]}

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