The present study aimed to determine whether isorhamnetin (Isor), a natural antioxidant polyphenol, has antifibrotic effects in a murine model of bleomycin-induced pulmonary fibrosis. A C57 mouse model of pulmonary fibrosis was established by intraperitoneal injection of a single dose of bleomycin (3.5 U/kg), and then Isor (10 and 30 mg/kg) was administered intragastrically. The level of fibrosis was assessed by hematoxylin and eosin and Sirius red staining. α-smooth muscle actin and type I collagen levels in lung tissues were determined by western blotting and immunohistochemistry (IHC). Epithelial-mesenchymal transition (EMT), endoplasmic reticulum stress (ERS) and related signaling pathways were examined by western blotting and IHC.
Pulmonary fibrosis is a chronic and progressive disease characterized by alveolar epithelial injury and abnormal collagen production (
Pulmonary fibrosis is caused by abnormal proliferation of myofibroblasts and fibroblasts, which secrete excessive extracellular matrix (ECM) proteins (
The endoplasmic reticulum (ER), an important organelle, serves as a key role in biological synthesis, including correct protein folding, secretion and membrane protein post-translational modifications (
Isorhamnetin (Isor; molecular formula, C16H12O7), a flavonol aglycone isolated from the plant
In the present study, the effect of Isor on bleomycin (BLM)-induced pulmonary fibrosis was investigated. The results demonstrated that Isor mitigated pulmonary fibrosis induced by BLM. Mechanistically, the results revealed that Isor-mediated ERS prevention was partially dependent on the regulation of EMT progression. Based on the present findings, Isor might serve as a potential therapeutic strategy for the treatment of pulmonary fibrosis.
Recombinant human TGFβ1 was purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). Isor was purchased from Baomanbio (Shanghai, China). BLM was purchased from Hisun Company (Zhejiang, China). Antibodies targeting collagen I (dilution, 1:6,000; cat. no. ab138492), α-smooth muscle actin (α-SMA; dilution, 1:300; ab32575) and 78 kDa glucose-regulating protein (GRP78)/binding immunoglobulin protein (BiP; dilution, 1:1,200; cat. no. BM0134) were obtained from Abcam (Cambridge, UK). Antibodies targeting TGFβ1 (dilution, 1:1,000; cat. no. AM4195), protein kinase R-like endoplasmic reticulum kinase (PERK; dilution, 1:1,200; cat. no. BM0524) and E-cadherin (dilution, 1:1,200; cat no. BM0537) were obtained from Abzoom Biolabs, Inc. (Dallas, TX, USA). Antibodies targeting vimentin (dilution, 1:1,200; YT4880), phosphorylated (p)-PERK (dilution, 1:2,000; cat. no. YP1055), DNA damage-inducible transcript 3 (DDIT3; also known as CHOP; dilution, 1:1,200; cat. no. YT0911), eukaryotic translation initiation factor 2 subunit α (eIF2α; dilution, 1:1,200; cat. no. YT1507) and p-eIF2α (dilution, 1:1,000; cat. no. YP0093) were obtained from ImmunoWay Biotechnology, Plano, TX, USA. Horseradish peroxidase (HRP)-coupled sheep anti-rat (dilution, 1:15,000; cat. no. SA001) or sheep anti-rabbit (dilution, 1:15,000; cat. no. SA009) secondary antibodies were obtained from Auragene Technology, Co., Inc. (Changsha, China).
A total of 15 male 4-week old C57 mice (20-25 g in weight; SLRC Laboratory Animal Company, Changsha, China) were housed in rooms with a 12-h light/dark cycle at 25°C and 40-70% humidity for 1 week prior to the experiment. Mice were fasted for 12 h and had
Human A549 cells and human bronchial epithelial cells (HBECs) were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C with 5% CO2. These cells were divided into five groups: The control group, the TGFβ1 group, and three TGFβ1 + Isor groups. Cells in the TGFβ1 group were stimulated with TGFβ1 alone (5 ng/ml) for 48 h. Cells in the TGFβ1 + Isor groups were treated with 5 ng/ml TGFβ1 and 25, 50 or 100
The lung specimens were fixed in 4% paraformaldehyde at room temperature for 48 h, dehydrated in a graded alcohol series and embedded in paraffin blocks. Sections were blocked with 3% hydrogen peroxide for 15 min at room temperature. Five-micron-thick sections were then stained with H&E for 10 min for routine examination, or with Sirius red for 8 min to visualize collagen deposition at room temperature. For immunohistochemistry staining, sections were stained with the aforementioned antibodies, developed with 3,3′-diaminobenzidine, and counterstained with hematoxylin. Samples were viewed using a light microscope (magnification for H&E staining, ×100 and ×400; magnification for immunohistochemistry staining, ×200 and ×400).
Total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific, Inc.). A total of 2
Protein was extracted from lung tissues and cells with RIPA lysis buffer (Auragene Technology, Co., Inc., Shenzen, China). Protein concentrations were determined with bicinchoninic acid protein assay reagents (Auragene Technology, Co., Inc.) according to the manufacturer’s instructions. Equal amounts of protein (10
Cells were fixed in 4% paraformaldehyde for 30 min at room temperature. After three 5-min washes with PBS, the cells were blocked in PBS containing 3% goat serum (OriGene Technologies, Inc., Beijing, China) and 0.5% Triton X-100 for 1 h at room temperature. Afterwards, the cells were incubated with the indicated primary antibodies at 4°C overnight and then incubated with Cy3-conjugated goat anti-rabbit IgG(H+L) secondary antibody (1:500) for 1 h at room temperature (catalog no. SA012; Auragene Technology, Co., Inc.). The coverslips were counter-stained with DAPI and imaged with a confocal laser scanning microscope (AE31; Motic Group, Co., Ltd., Xiamen, China).
Data from three independent experiments were expressed as the mean ± standard deviation and processed using SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). Differences between groups were evaluated by one-way analysis of variance followed by Tukey’s test. P<0.05 was considered to indicate a statistically significant difference.
In accordance with previous studies (
Recent studies have demonstrated that pulmonary ERS is involved in the pathogenesis of BLM-induced pulmonary fibrosis (
ERS-induced EMT contributes to fibrotic remodeling in the lungs (
To investigate the possible pathways by which Isor inhibits ERS, the expression levels of total and phosphorylated PERK were determined by western blotting. PERK phosphorylation levels significantly increased in the BLM-induced group compared with the control group, while a dramatic decrease in p-PERK was observed in the Isor-treated BLM groups (
To further validate the antifibrotic effect of Isor,
Exposure to TGFβ1 caused A549 cells and HBECs to undergo EMT, during which the morphology of A549 cells changed from polygonal or cobblestone-like to a spindle-like shape, and that of HBECs changed from a short fusiform shape to a long fusiform shape (
The present study demonstrated for the first time that Isor could inhibit pulmonary fibrosis
Increasing evidence has shown that EMT is involved in the formation of myofibroblasts and serves an important role in the pathogenesis of pulmonary fibrosis. In addition, inhibition of EMT could prevent epithelial cells from transforming into myofibroblasts, alleviating pulmonary fibrosis. For example, NLR family pyrin domain containing 3 (NLRP3) modulates EMT through the TGFβ1 signaling pathway, inhibiting pulmonary fibrosis (
Multiple studies have confirmed that ERS and UPR signaling activation are involved in the pathogenesis of idiopathic pulmonary fibrosis, which includes elevated expression of GRP78, activating transcription factor 6 (ATF6), PERK, and inositol-requiring enzyme-1 (IRE-1) (
According to recent reports, ERS can induce EMT and the progression of pulmonary fibrosis. Inhibition of ERS could alleviate pulmonary fibrosis (
In summary, the present study revealed that Isor inhibited EMT and ERS in BLM-induced pulmonary fibrosis via the PERK pathway. These findings may provide novel insights into the pathogenesis of pulmonary fibrosis. Additional investigations are necessary to elucidate the full antifibrotic potential of Isor as a concomitant therapy for patients with lung fibrosis, including that produced during BLM treatment, especially with regard to timing of its administration.
Not applicable.
The present study was supported by the Science and Technology Innovation Program of Hunan Province (grant no. 2017SK50515).
The analyzed datasets generated during the study are available from the corresponding author on reasonable request.
YY designed the study and obtained funding. QZ performed the animal experiments and tests. QZ, MT, BO, CL and CH performed the cell function assay. MT and YY provided major contributions to the writing of the manuscript. All authors read and approved the final manuscript.
All experiments involving animals were approved by the Ethics Committee of Hunan Normal University Medical College (Changsha, China).
Not applicable.
The authors declare that they have no competing interests.
Effects of Isor on BLM-induced pulmonary fibrosis. (A) Representative images from hematoxylin and eosin staining showing lung pathologic abnormalities (magnification, ×100 and ×400). (B) Representative images from Sirius red staining showing collagen deposition (magnification, ×200 and ×400). (C) The protein expression levels of α-SMA and collagen I were assessed by western blot analysis, and the densitometry values were normalized to β-actin. (D) Immunohistochemistry staining of α-SMA in lung tissue sections (magnification, ×200 and ×400). Data are presented as the mean ± standard deviation (n=3). *P<0.05 and ***P<0.001, with comparisons indicated by lines. Isor, isorhamnetin; BLM, bleomycin; α-SMA, α-smooth muscle actin.
Effects of Isor on BLM-induced pulmonary endoplasmic reticulum stress markers. (A) The protein expression levels of CHOP and GRP78 were assessed by western blot analysis, and the densitometry values were normalized to β-actin. (B) The mRNA expression levels of CHOP and GRP78 were determined by reverse transcription-quantitative polymerase chain reaction. (C) Immunohistochemistry staining of CHOP in lung tissue sections (magnification, ×200 and ×400). Data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01 and ***P<0.001, with comparisons indicated by lines. Isor, isorhamnetin; BLM, bleomycin; CHOP, DNA damage-inducible transcript 3; GRP78, 78 kDa glucose-regulating protein.
Effects of Isor on endoplasmic reticulum stress-mediated epithelial-mesenchymal transition. (A) The protein expression levels of E-cadherin and Vimentin, were evaluated by western blot analysis, and the densitometry values were normalized to β-actin. (B) Quantitative statistical analysis of E-cadherin. (C) Quantitative statistical analysis of Vimentin. (D) The protein expression levels of TGFβ1 in lung tissue sections was detected by western blot analysis. (E) Quantitative statistical analysis of TGFβ1. Data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01 and ***P<0.001, with comparisons indicated by lines. Isor, isorhamnetin; TGFβ1, transforming growth factor β1.
Effects of Isor on the expression of PERK and eIF2α. (A) The protein expression levels of total and phosphorylated PERK and eIF2α were examined by western blot analysis, and the densitometry values were normalized to β-actin. (B) Immunohistochemical staining of p-PERK in lung tissue sections (magnification, ×200 and ×400). Data are presented as the mean ± standard deviation (n=3). ***P<0.001, with comparisons indicated by lines. Isor, isorhamnetin; PERK, protein kinase R-like endoplasmic reticulum kinase; eIF2α, eukaryotic translation initiation factor 2 subunit α; p-, phosphorylated.
Effects of Isor on the expression of fibrosis-related factors in TGFβ1-induced A549 cells and HBECs. The protein expression levels of α-SMA and collagen I were assessed by western blot analysis in A549 cells and in HBECs, and the densitometry values were normalized to β-actin. Data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01 and ***P<0.001, with comparisons indicated by lines. Isor, isorhamnetin; TGFβ1, transforming growth factor β1; HBECs, human bronchial epithelial cells; α-SMA, α-smooth muscle actin.
Effects of Isor on the expression of endoplasmic reticulum stress markers in TGFβ1-induced A549 cells and HBECs. The protein expression levels of CHOP and GRP78 were assessed by western blot analysis in A549 cells and in HBECs, and the densitometry values were normalized to β-actin. Data are presented as the mean ± standard deviation (n=3). **P<0.01 and ***P<0.001, with comparisons indicated by lines. Isor, isorhamnetin; TGFβ1, transforming growth factor β1; HBECs, human bronchial epithelial cells; CHOP, DNA damage-inducible transcript 3; GRP78, 78 kDa glucose-regulating protein.
Effects of Isor on TGFβ1-induced epithelial-mesenchymal transition in A549 cells and HBECs. (A) Morphological changes in Isor-treated A549 cells and HBECs in the presence of TGFβ1 were observed by phase contrast microscopy (magnification, ×200). (B) The protein expression levels of E-cadherin and Vimentin were assessed by western blot analysis, and the densitometry values were normalized to β-actin. (C) Immunofluorescent staining for E-cadherin and Vimentin in Isor-treated A549 cells and HBECs in the presence of TGFβ1 (magnification, ×400). Data are presented as the mean ± standard deviation (n=3). *P<0.05, **P<0.01 and ***P<0.001, with comparisons indicated by lines. Isor, isorhamnetin; TGFβ1, transforming growth factor β1; HBECs, human bronchial epithelial cells.