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Hispidin attenuates bleomycin-induced idiopathic pulmonary fibrosis via an anti-oxidative effect in A549 cells

Hispidin attenuates bleomycin-induced idiopathic pulmonary fibrosis via an anti-oxidative effect... Idiopathic pulmonary fibrosis (IPF) is a serious and irreversible chronic lung disease. Bleomycin (BLM) is an anticancer drug, which can cause severe lung toxicity. The main target of oxidative stress‑induced lung injury is alveolar epithelial cells, which lead to interstitial fibrosis. The present study investigated whether hispidin (HP), which has excellent anti‑ oxidant activity, attenuates bleomycin‑induced pulmonary fibrosis via anti‑ oxidative effects in A549 cells. We found that hispidin reduced bleomycin‑induced fibrosis of A549 cells by reducing reactive oxygen species (ROS) levels and inhibiting epithelial‑mesenchymal transition. Taken together, our data suggest that hispidin has therapeutic potential in preventing bleomycin‑induced pulmonary fibrosis. Keywords: Bleomycin, Epithelial‑mesenchymal transition, Hispidin, Idiopathic pulmonary fibrosis, Reactive oxygen species Introduction metalloproteinase expression [4]. Oxidative stress is Idiopathic pulmonary fibrosis (IPF) is a serious and irre - reportedly involved in the development of alveolar injury, versible chronic lung disease [1]. IPF has the following inflammation, and fibrosis [5]. In recent years, the inci - characteristics: Excessive accumulation of fibroblasts, dence rate of IPF has increased. Among the environmen- extensive deposition of the extracellular matrix, alveolar tal factors, lung cell damage caused by inhalable particles structure damage, and a gradual decline of lung func- and drug-induced interstitial lung disease have attracted tion [2]. Smoking, sawdust, sand, and silica exposure may extensive attention. For example, bleomycin-induced pul- lead to repeated injury of alveolar epithelial cells. Many monary fibrosis has become a serious clinical problem studies have shown that injury and apoptosis of alveo- [6]. Nintedanib and pirfenidone are the only two antifi - lar epithelial type II cells are important early features of brotic drugs approved for the treatment of this disease IPF [3]. IPF is accompanied by epithelial cell apoptosis, worldwide, and both drugs can actively interfere with cel- epithelial-mesenchymal transition (EMT) and matrix lular redox state and oxidative stress [7, 8]. However, all types of treatments failed to change the natural course of the disease, except for lung transplantation; therefore, a new and effective way of treatment is urgently required. *Correspondence: cuiyudong@126.com; kwon@kribb.re.kr; sunhunan76@163.com Bleomycin (BLM) is a complex glycopeptide with Chen‑ Xi Ren and Xin Jin contributed equally to this work antitumor properties. It is often used as an antibiotic to College of Life Science & Technology, Heilongjiang Bayi Agricultural treat various cancers in the clinic. Compared with other University, Xinyanglu, 163319 Daqing, People’s Republic of China Primate Resources Center, Korea Research Institute of Bioscience tissues, lungs lack the bleomycin-hydrolyzing enzyme. and Biotechnology (KRIBB), 351‑33 Neongme‑gil, Ibam‑myeon, After bleomycin reaches the lung tissue, it leads to the Jeongeup‑si, Jeonbuk 56216, Republic of Korea production of reactive oxygen species (ROS) under the Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Ren et al. Appl Biol Chem (2021) 64:74 Page 2 of 9 action of iron and oxygen, which increases oxidative In light of the above-mentioned gaps in the field, this stress, leading to severe pulmonary toxicity [9]. Oxida- study aimed to explore the preventive and therapeutic tive stress is an important molecular mechanism, lead- effects of hispidin on pulmonary fibrosis.First, bleomycin ing to fibrosis in many organs [10]. The main target of was used to induce pulmonary fibrosis, and then A549 oxidative stress-induced lung injury is alveolar epithelial cells were pretreated with HP. MTT assay and would cells, which can lead to fibrosis [11]. The toxicity of BLM healing test were used to detect cell activity and cell is directly caused by oxidative damage, and the produc- migration, respectively, and western blotting was used to tion of intracellular ROS leads to inflammation and fibro - detect the expression of pulmonary fibrosis- and EMT- sis [12]. The existing research shows that BLM causes the related proteins. Flow cytometry and fluorescent micros - increase of ROS levels in A549 cells, which leads to oxi- copy were used to detect intracellular and mitochondrial dative stress in cells. In addition, BLM, through TGF-β/ ROS levels, as well as mitochondrial membrane potential. Smad signaling pathway, induces EMT of A549 cells [13]. Our results will reveal the preventive and therapeutic Many studies have shown that various plant extracts and effects of HP on fibrotic lung injury through anti-oxida - active components have significant effects on inhibit - tion and anti-fibrosis activities. ing BLM-induced pulmonary fibrosis. Emodin alleviates BLM-induced pulmonary fibrosis in rats through anti- Material and methods inflammatory and antioxidant effects [14]. Submicron Reagents and antibodies infusion of cinnamaldehyde improves BLM-induced A549 cell line was purchased from Daqing Hongtu Bio- IPF by inhibiting inflammation, oxidative stress and technology Co., Ltd. (Heilongjiang, China). PBS and EMT [15]. Studies have shown that mitochondria play DMEM high glucose medium were purchased from an important role in IPF [16]. Some studies suggest that Hyclone; TE/EDTA, penicillin/streptomycin, (P/S), and ROS can promote EMT in certain types of cells [17] and fetal bovine serum (FBS) from Solarbio; Hispidin (HP) mitochondrial ROS is the key factor of hypoxia-induced from Shanghai Macklin Biochemical Co., Ltd. (Product EMT in alveolar epithelial cells [18]. Here, we showed No: H861853); dimethyl sulfoxide (DMSO) from Sigma; that HP significantly inhibited BLM-induced A549 fibro - BLM from Gibco; 4,5-dimethylthiazol-2-yl)-2,5-diphe- sis by reducing ROS levels, EMT, and mitochondrial nyltetrazolium bromide (MTT reagent from Amreso. All membrane damage. antibodies (anti-β-actin, anti-N-cadherin, anti-E-cad- Hispidin (HP, 6-(3, 4-dihydroxystyryl)-4-hydroxy- herin, anti-vimentin, anti-fibronectin, and anti-slug) were 2-pyrone) and its derivatives are widely distributed in purchased from Santa Cruz (CA, USA). Dihydroethid- edible mushrooms, such as Lentinus edodes [19–21]. HP ium (DHE), Mito-SOX, JC-1, and Hochest stains/probes is extracted from Lentinus edodes, which is widely used were purchased from Solarbio. to treat various diseases [16]. Most of these diseases are caused by the excessive production of free radicals, espe- Cell culture cially ROS. Studies have shown that HP can neutralize A549 lung cancer cells were cultured in DMEM high glu- free radicals and is known for its strong anti-cancer, anti- cose medium containing 10% inactivated FBS and 1% P/S oxidant, and DNA damage preventing activities [22]. It in a humidified incubator with 5% CO . BLM (200  μg/ can also inhibit the growth and migration of tumors [23]. mL) and hispidin (10, 20, or 30 μg/mL) (Additional file  1; HP protects H9c2 cardiomyocytes from hydrogen perox- Fig. S1) were collectively administrated to the A549 cell ide-induced apoptosis by reducing ROS production and line for 48 h. activation of Akt/GSK-3β and ERK1/2 signaling pathways [24, 25]. At the same time, high concentration of HP can Morphological observation lead to high cytotoxicity and induce apoptosis [26]. HP Cells were seeded onto 24-well plates at a density of can effectively protect min6n β Cells from ROS damage 6 × 10 cells/well. Serum-free DMEM was added and [27], C C myotubes from palmitate-induced oxida- exposed to treatments accordingly. Images were acquired 2 12 tive stress [28], Caco-2 cells from acrylamide-induced using an inverted microscope after 48 h. oxidative stress [29], and ARPE-19 cells from hydrogen peroxide-induced damage by activating Nrf2 signaling MTT assay and upregulating downstream targets, including phase II Cell morphology was observed under a light micro- enzyme [30]. HP can prevent the damage caused by oxi- scope. Cells were seeded onto 96-well plates at a density dative stress by scavenging ROS. Due to different effects of 6 × 10 cells/well. Serum-free DMEM was added and of HP under different conditions, studying it further may treatments were performed accordingly. MTT solution help to treat cancer and prevent side effects caused by (final concentration of 0.5  mg/ml in media) was then chemotherapy. added to each well, and the plate was incubated at 37˚C R en et al. Appl Biol Chem (2021) 64:74 Page 3 of 9 for an additional 4 h. After removing all medium in each anti-fibronectin, anti-slug, and anti-β-actin primary well, 100  μl of DMSO was added to each well and the antibodies (1:1000) at 4  °C overnight. Membranes were optical density was measured at 490 nm. washed five times with Tris buffered saline containing Tween (TBST, 150 mM NaCl, 10 mM Tris HCl (pH 7.5) Wound healing assay and 0.2% Tween-20) and were subsequently incubated Cells were seeded onto 24-well plates at a density of with horseradish peroxidase-conjugated goat anti-mouse 2 × 10 cells/well. A scratch was established after the cells IgG or anti-rabbit IgG for 1 h at room temperature. After adhered to the plate. Then, the medium was removed removing excess antibodies by washing with TBST, spe- and cells were washed with PBS three times. Serum- cific conjugates were detected using a chemilumines - free DMEM was added and treatments were performed cence detection system according to the manufacturer’s accordingly. Images were acquired using an inverted protocol. microscope after 0 and 48  h. Image J software was used to calculate the scratch area as follows: Area healing Statistical analysis (%) = [(average gap at 0 h—average gap at 48 h) / average Repeated measures analysis of variance (ANOVA) was gap at 0 h] × 100%. used to analyze changes in time and differences between groups for each experiment. Significance was measured Fluorescence microscopy using independent-samples t-test. All differences were The superoxide anion in mitochondria and mitochon - considered statistically significant if the p-value was less drial membrane potential were measured using the flu - than 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001). All experi- orescent probe MitoSOX/JC-1 and Hoechst dye. Cells ments were performed in triplicates. were seeded onto 24-well plates at a density of 6 × 10 cells/well. Serum-free DMEM was added and treatments Results were performed accordingly. Followed by washing twice BLM inhibits proliferation and promotes fibrosis in A549 with PBS, the cells were incubated with the fluorescent cells probe MitoSOX/JC-1 and Hoechst dye for 15  min at We constructed a cell model of alveolar epithelial cell 37  °C. After the reaction, cells were washed twice with fibrosis in vitro and used BLM was to induce the fibrosis PBS and the superoxide anion in mitochondria, as well of A549 human alveolar epithelial cells. A549 cells were as the mitochondrial membrane potential, were detected treated with BLM (100 or 200  μg/ml) for 48  h. Micro- using an inverted fluorescence microscope. scopic observation showed that with the increase in drug concentration, cell fibrosis increased, and the MTT assay Flow cytometry showed that cell viability decreased (Fig.  1a, Additional The intracellular ROS were measured by DHE probes in file  1; Fig. S2). Moreover, to detect the change in cell cells. Cells were seeded in 24-well plates at a density of migration upon BLM-induced cell fibrosis, we performed 6 × 10 cells per well. Serum free DMEM medium was a wound healing assay. Would healing assay showed that added and exposed to treatments accordingly, followed the migration of A549 cells was enhanced after treatment by PBS wash (twice), as well as DHE and Hoechst incu- with BLM for 48  h (Fig.  1b, c). Western blotting results bation for 15 min at 37 °C. After the reaction, cells were showed that expression levels of EMT-related proteins washed twice with PBS and observed using flow cytom - N-cadherin, vimentin, fibronectin, and slug were upregu - etry to detect intracellular ROS levels. lated, and those of E-cadherin were downregulated. A549 cells developed cellular fibrosis through EMT (Fig.  1d– Western blotting h). These results indicated that BLM inhibited cell prolif - Serum-free DMEM was added to cells and treatments eration and promoted cell migration and fibrosis in A549 were performed accordingly. The cells were recov - cells. ered and harvested. Harvested cells were lysed (20  mM HEPES-OH, pH 7.0; 50 mM NaCl; 10% glycerol and 0.5% BLM increases ROS levels and decreases mitochondrial Triton X 100) and total protein was extracted. Then, pro - membrane potential in A549 cells teins were denatured for 5  min, exposed to 12% sodium After treatment of A549 cells with BLM (100 or 200 μg/ dodecyl sulfate–polyacrylamide gel electrophoresis for mL) for 48  h, fluorescence microscopy showed that the separation, and transferred into nitrocellulose mem- level of intracellular ROS was significantly increased branes. Skim milk (5%) was used to block the membranes (Fig.  2a), and the same effect was also observed using for 30  min at room temperature. They were then incu - flow cytometry (Fig.  2b). Fluorescence microscopy also bated with polyclonal rabbit anti-N-cadherin, as well showed that ROS levels in mitochondria increased as mouse monoclonal anti-E-cadherin, anti-vimentin, (Fig.  2c) and the mitochondrial membrane potential Ren et al. Appl Biol Chem (2021) 64:74 Page 4 of 9 Fig. 1 Bolemycin inhibits the proliferation of A549 cells and promotes fibrosis. a The morphology of cellular fibrosis was observed under the microscope. Cell viability was detected using MTT. b, c Cell migration was detected using would healing assay. d The expression levels of EMT‑related proteins were detected using western blotting. e–h The related protein expression levels are represented as the mean ± standard deviation. Data are presented as the mean ± standard error of the mean of three different samples. *P < 0.05, **P < 0.01, and ***P < 0.001 HP inhibits BLM‑induced fibrosis in A549 cells decreased (Fig.  2d). These results suggested that BLM Subsequently, we treated A549 cells with BLM (200  μg/ increased ROS levels and caused mitochondrial dysfunc- mL) and HP (10, 20, and 30 μg/mL) for 48 h. Microscopic tion in A549 cells. observation showed that HP significantly inhibited BLM- induced cell fibrosis (Fig.  4a). At the same time, MTT HP inhibits the increase of ROS levels in A549 cells assay showed that there was no significant difference in and mitochondria dysfunction induced by BLM the activity of A549 cells treated with BLM alone or com- A549 cells were treated with BLM (200  μg/mL) and HP bined with HP for 48  h (Fig.  4b). Wound healing assay (10, 20, and 30  μg/mL) for 48  h. Fluorescence micros- results showed that HP significantly inhibited cell migra - copy showed that HP significantly inhibited the increase tion induced by BLM (Fig. 4c, d). Western blotting results of ROS levels in cells induced by BLM (Fig.  3a). Flow showed that HP significantly inhibited the changes in the cytometry also showed the same effect (Fig.  3b). At the expression levels of EMT-related proteins induced by same time, HP significantly inhibited the increase in ROS BLM, thus inhibiting EMT and cell fibrosis of A549 cells levels and the decrease of membrane potential induced (Fig.  4e–i). These results indicated that hispidin signifi - by BLM (Fig.  3c, d). These results indicated that HP sig - cantly inhibited the migration and fibrosis of A549 cells nificantly inhibited the increase in ROS levels and mito - induced by BLM. chondrial dysfunction induced by BLM in A549 cells. R en et al. Appl Biol Chem (2021) 64:74 Page 5 of 9 Fig. 2 Bolemycin increased the ROS levels of in A549 cells. a Intracellular ROS levels were detected using flow cytometry, b as well as DHE staining. c ROS levels in mitochondria were detected using Mito‑SOX staining. d The mitochondrial membrane potential was detected using JC‑1 staining. **P < 0.01, and ***P < 0.001 also believe that oxidative stress is closely related to Discussion pulmonary fibrosis [37, 39]. IPF is a chronic, irreversible and usually fatal lung BLM is an antibiotic used to treat various tumors, but disease [31], which is highly prevalent in the elderly. it causes severe pneumonia and pulmonary fibrosis [40]. Patients with IPF suffer from long-term cough and The injury and dysfunction of pulmonary epithelial cells dyspnea, which is difficult to diagnose, and most are considered to be an important initial and central pro- patients are already at the advanced stage when they cess of fibrosis [3], BLM induces the transformation of are diagnosed [32]. Today, the prevalence and inci- lung epithelial cells into mesenchymal cells through EMT dence rate of IPF is increasing [33, 34] and the survival process, gradually transforms the morphology of lung time of patients is about 2–3 years after diagnosis [35]. epithelial cells into mesenchymal phenotype, inhibits the Therefore, there is an urgent need for more effective proliferation of epithelial cells and further promotes the diagnostic and treatment methods. The specific patho - process of fibrosis [3, 13, 36, 38]. In this study, bleomy- genesis of the disease is not clear, but more studies have cin was used to construct the pulmonary fibrosis model. shown that alveolar epithelial type II cells can be used A549 cells were treated with different concentrations as a driver of idiopathic pulmonary fibrosis [36] and the of bleomycin for 48  h. The results showed that the cells occurrence of IPF is related to alveolar epithelial cell showed obvious fibrosis at the concentration of 200  μg/ injury [37] and EMT [38]. Moreover, some researchers Ren et al. Appl Biol Chem (2021) 64:74 Page 6 of 9 Fig. 3 Hispidin inhibits bolemycin‑induced ROS. a Intracellular ROS levels were detected using flow cytometry, b as well as DHE staining. c ROS levels in mitochondria was detected using Mito‑SOX staining. d The mitochondrial membrane potential was detected using JC‑1 staining. **P < 0.01, and ***P < 0.001 mL. MTT assay showed that the proliferation of cells was cell morphology and EMT related proteins were signifi - inhibited at 200  μg/mL. The EMT related proteins also cantly inhibited, it can be seen from the above that HP changed significantly after BLM treatment. Some studies has a significant inhibitory effect on the fibrosis of A549 have shown that bleomycin can induce ROS, which may alveolar epithelial cells. The specific mechanism has not be an important reason for lung epithelial cell injury and been explored, but we know that the target signal path- EMT transformation [41]. At the same time, mitochon- way of HP is TGF- β/ Smad signal pathway. At the same dria in cells are the most important position to produce time, studies have shown that anlotinib attenuates BLM ROS in cells [42], which is closely related to pulmonary induced pulmonary fibrosis through TGF-β1 signal - fibrosis [43]. Therefore, DHE fluorescent dye detection ing pathway [45], BLM through TGF-β/ Smad signaling showed that intracellular ROS increased significantly pathway induces EMT of cultured A549 cells [13], pae- after BLM treatment, Mito-SOX and JC-1 fluorescent oniflorin inhibits TGF-β mediated EMT of pulmonary dye detection showed that mitochondrial ROS increased fibrosis through Smad dependent pathway, etc. These and mitochondria were damaged. Then we focused on reports suggest that TGF-β/ Smad signaling pathway is HP, which can neutralize free radicals [22]. By further closely related to the process of pulmonary fibrosis. searching the data, we found that HP is a protein kinase Based on the above information, we speculate that C β Inhibitors, and protein kinase C β overexpression HP may attenuates BLM induced pulmonary fibrosis by can induce fibrosis in human proximal tubular epithe - TGF- β/ Smad signaling pathway, which will also become lial cells [44]. We speculate that HP may inhibit cellular the focus of our next research. This study will also pro - fibrosis. After treating fibrotic cells with HP, we found vide new ideas for the treatment of IPF. that the level of ROS decreased significantly, fibrotic R en et al. Appl Biol Chem (2021) 64:74 Page 7 of 9 Fig. 4 Eec ff t of Hispidin on bolemycin‑induced EMT. a The morphology of cellular fibrosis was observed under the microscope. b Cell viability was detected using MTT. c, d Cell migration was detected using wound healing assay. e Expression levels of EMT‑related proteins were detected using western blotting. f–i The related protein expression levels were represented as the mean ± standard deviation. Data are presented as the mean ± standard error of the mean of three different samples. *P < 0.05, **P < 0.01, and ***P < 0.001 Authors’ contributions Supplementary Information CXR, XJ, DXP, HNS and TK performed the experiments and wrote the article. The online version contains supplementary material available at https:// doi. CXR, XJ, DXP, XYG, LYY, YDC, HNS and TK performed the data analysis. YDC, org/ 10. 1186/ s13765‑ 021‑ 00646‑x. HNS and TK reviewed and edited the manuscript. TK acquired the funding. All authors read and approved the final manuscript. Additional file 1: Fig. S1. Cell viability was measured by MTT assay. Cells Funding were treated with different concentrations of hispidin (HP) (10, 20, 30 This research was supported by the Basic Science Research Program through μg/mL) incubated for 48 h. Data are presented as the mean ± standard the National Research Foundation of Korea funded by the Ministry of Educa‑ error of the mean of three different samples. *P < 0.05, **P < 0.01, and tion (2020R1I1A2052417), The Korean Research Institute of Bioscience and ***P < 0.001. Fig. S2. a The morphology of cellular fibrosis was observed Biotechnology Research Information System (RBM0112112). under the microscope. b Intracellular ROS levels were detected using flow cytometry. c, d Cells were treated with different concentrations of Availability of data and materials Bolemycin (BLM) and hispidin (HP) incubated for 48 h. Data are presented The datasets used and analyzed in this study are available from the corre‑ as the mean ± standard error of the mean of three different samples. *P < sponding author upon reasonable request. 0.05, **P < 0.01, and ***P < 0.001. Declarations Acknowledgements This research was supported by the project of the scientific research team Competing interests support plan of Heilongjiang Bayi Agricultural University ( TDJH201904), China. The authors declare that they have no competing interests. Ren et al. Appl Biol Chem (2021) 64:74 Page 8 of 9 Author details 20. 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Hispidin attenuates bleomycin-induced idiopathic pulmonary fibrosis via an anti-oxidative effect in A549 cells

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Springer Journals
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Copyright © The Author(s) 2021
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2468-0834
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10.1186/s13765-021-00646-x
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Abstract

Idiopathic pulmonary fibrosis (IPF) is a serious and irreversible chronic lung disease. Bleomycin (BLM) is an anticancer drug, which can cause severe lung toxicity. The main target of oxidative stress‑induced lung injury is alveolar epithelial cells, which lead to interstitial fibrosis. The present study investigated whether hispidin (HP), which has excellent anti‑ oxidant activity, attenuates bleomycin‑induced pulmonary fibrosis via anti‑ oxidative effects in A549 cells. We found that hispidin reduced bleomycin‑induced fibrosis of A549 cells by reducing reactive oxygen species (ROS) levels and inhibiting epithelial‑mesenchymal transition. Taken together, our data suggest that hispidin has therapeutic potential in preventing bleomycin‑induced pulmonary fibrosis. Keywords: Bleomycin, Epithelial‑mesenchymal transition, Hispidin, Idiopathic pulmonary fibrosis, Reactive oxygen species Introduction metalloproteinase expression [4]. Oxidative stress is Idiopathic pulmonary fibrosis (IPF) is a serious and irre - reportedly involved in the development of alveolar injury, versible chronic lung disease [1]. IPF has the following inflammation, and fibrosis [5]. In recent years, the inci - characteristics: Excessive accumulation of fibroblasts, dence rate of IPF has increased. Among the environmen- extensive deposition of the extracellular matrix, alveolar tal factors, lung cell damage caused by inhalable particles structure damage, and a gradual decline of lung func- and drug-induced interstitial lung disease have attracted tion [2]. Smoking, sawdust, sand, and silica exposure may extensive attention. For example, bleomycin-induced pul- lead to repeated injury of alveolar epithelial cells. Many monary fibrosis has become a serious clinical problem studies have shown that injury and apoptosis of alveo- [6]. Nintedanib and pirfenidone are the only two antifi - lar epithelial type II cells are important early features of brotic drugs approved for the treatment of this disease IPF [3]. IPF is accompanied by epithelial cell apoptosis, worldwide, and both drugs can actively interfere with cel- epithelial-mesenchymal transition (EMT) and matrix lular redox state and oxidative stress [7, 8]. However, all types of treatments failed to change the natural course of the disease, except for lung transplantation; therefore, a new and effective way of treatment is urgently required. *Correspondence: cuiyudong@126.com; kwon@kribb.re.kr; sunhunan76@163.com Bleomycin (BLM) is a complex glycopeptide with Chen‑ Xi Ren and Xin Jin contributed equally to this work antitumor properties. It is often used as an antibiotic to College of Life Science & Technology, Heilongjiang Bayi Agricultural treat various cancers in the clinic. Compared with other University, Xinyanglu, 163319 Daqing, People’s Republic of China Primate Resources Center, Korea Research Institute of Bioscience tissues, lungs lack the bleomycin-hydrolyzing enzyme. and Biotechnology (KRIBB), 351‑33 Neongme‑gil, Ibam‑myeon, After bleomycin reaches the lung tissue, it leads to the Jeongeup‑si, Jeonbuk 56216, Republic of Korea production of reactive oxygen species (ROS) under the Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Ren et al. Appl Biol Chem (2021) 64:74 Page 2 of 9 action of iron and oxygen, which increases oxidative In light of the above-mentioned gaps in the field, this stress, leading to severe pulmonary toxicity [9]. Oxida- study aimed to explore the preventive and therapeutic tive stress is an important molecular mechanism, lead- effects of hispidin on pulmonary fibrosis.First, bleomycin ing to fibrosis in many organs [10]. The main target of was used to induce pulmonary fibrosis, and then A549 oxidative stress-induced lung injury is alveolar epithelial cells were pretreated with HP. MTT assay and would cells, which can lead to fibrosis [11]. The toxicity of BLM healing test were used to detect cell activity and cell is directly caused by oxidative damage, and the produc- migration, respectively, and western blotting was used to tion of intracellular ROS leads to inflammation and fibro - detect the expression of pulmonary fibrosis- and EMT- sis [12]. The existing research shows that BLM causes the related proteins. Flow cytometry and fluorescent micros - increase of ROS levels in A549 cells, which leads to oxi- copy were used to detect intracellular and mitochondrial dative stress in cells. In addition, BLM, through TGF-β/ ROS levels, as well as mitochondrial membrane potential. Smad signaling pathway, induces EMT of A549 cells [13]. Our results will reveal the preventive and therapeutic Many studies have shown that various plant extracts and effects of HP on fibrotic lung injury through anti-oxida - active components have significant effects on inhibit - tion and anti-fibrosis activities. ing BLM-induced pulmonary fibrosis. Emodin alleviates BLM-induced pulmonary fibrosis in rats through anti- Material and methods inflammatory and antioxidant effects [14]. Submicron Reagents and antibodies infusion of cinnamaldehyde improves BLM-induced A549 cell line was purchased from Daqing Hongtu Bio- IPF by inhibiting inflammation, oxidative stress and technology Co., Ltd. (Heilongjiang, China). PBS and EMT [15]. Studies have shown that mitochondria play DMEM high glucose medium were purchased from an important role in IPF [16]. Some studies suggest that Hyclone; TE/EDTA, penicillin/streptomycin, (P/S), and ROS can promote EMT in certain types of cells [17] and fetal bovine serum (FBS) from Solarbio; Hispidin (HP) mitochondrial ROS is the key factor of hypoxia-induced from Shanghai Macklin Biochemical Co., Ltd. (Product EMT in alveolar epithelial cells [18]. Here, we showed No: H861853); dimethyl sulfoxide (DMSO) from Sigma; that HP significantly inhibited BLM-induced A549 fibro - BLM from Gibco; 4,5-dimethylthiazol-2-yl)-2,5-diphe- sis by reducing ROS levels, EMT, and mitochondrial nyltetrazolium bromide (MTT reagent from Amreso. All membrane damage. antibodies (anti-β-actin, anti-N-cadherin, anti-E-cad- Hispidin (HP, 6-(3, 4-dihydroxystyryl)-4-hydroxy- herin, anti-vimentin, anti-fibronectin, and anti-slug) were 2-pyrone) and its derivatives are widely distributed in purchased from Santa Cruz (CA, USA). Dihydroethid- edible mushrooms, such as Lentinus edodes [19–21]. HP ium (DHE), Mito-SOX, JC-1, and Hochest stains/probes is extracted from Lentinus edodes, which is widely used were purchased from Solarbio. to treat various diseases [16]. Most of these diseases are caused by the excessive production of free radicals, espe- Cell culture cially ROS. Studies have shown that HP can neutralize A549 lung cancer cells were cultured in DMEM high glu- free radicals and is known for its strong anti-cancer, anti- cose medium containing 10% inactivated FBS and 1% P/S oxidant, and DNA damage preventing activities [22]. It in a humidified incubator with 5% CO . BLM (200  μg/ can also inhibit the growth and migration of tumors [23]. mL) and hispidin (10, 20, or 30 μg/mL) (Additional file  1; HP protects H9c2 cardiomyocytes from hydrogen perox- Fig. S1) were collectively administrated to the A549 cell ide-induced apoptosis by reducing ROS production and line for 48 h. activation of Akt/GSK-3β and ERK1/2 signaling pathways [24, 25]. At the same time, high concentration of HP can Morphological observation lead to high cytotoxicity and induce apoptosis [26]. HP Cells were seeded onto 24-well plates at a density of can effectively protect min6n β Cells from ROS damage 6 × 10 cells/well. Serum-free DMEM was added and [27], C C myotubes from palmitate-induced oxida- exposed to treatments accordingly. Images were acquired 2 12 tive stress [28], Caco-2 cells from acrylamide-induced using an inverted microscope after 48 h. oxidative stress [29], and ARPE-19 cells from hydrogen peroxide-induced damage by activating Nrf2 signaling MTT assay and upregulating downstream targets, including phase II Cell morphology was observed under a light micro- enzyme [30]. HP can prevent the damage caused by oxi- scope. Cells were seeded onto 96-well plates at a density dative stress by scavenging ROS. Due to different effects of 6 × 10 cells/well. Serum-free DMEM was added and of HP under different conditions, studying it further may treatments were performed accordingly. MTT solution help to treat cancer and prevent side effects caused by (final concentration of 0.5  mg/ml in media) was then chemotherapy. added to each well, and the plate was incubated at 37˚C R en et al. Appl Biol Chem (2021) 64:74 Page 3 of 9 for an additional 4 h. After removing all medium in each anti-fibronectin, anti-slug, and anti-β-actin primary well, 100  μl of DMSO was added to each well and the antibodies (1:1000) at 4  °C overnight. Membranes were optical density was measured at 490 nm. washed five times with Tris buffered saline containing Tween (TBST, 150 mM NaCl, 10 mM Tris HCl (pH 7.5) Wound healing assay and 0.2% Tween-20) and were subsequently incubated Cells were seeded onto 24-well plates at a density of with horseradish peroxidase-conjugated goat anti-mouse 2 × 10 cells/well. A scratch was established after the cells IgG or anti-rabbit IgG for 1 h at room temperature. After adhered to the plate. Then, the medium was removed removing excess antibodies by washing with TBST, spe- and cells were washed with PBS three times. Serum- cific conjugates were detected using a chemilumines - free DMEM was added and treatments were performed cence detection system according to the manufacturer’s accordingly. Images were acquired using an inverted protocol. microscope after 0 and 48  h. Image J software was used to calculate the scratch area as follows: Area healing Statistical analysis (%) = [(average gap at 0 h—average gap at 48 h) / average Repeated measures analysis of variance (ANOVA) was gap at 0 h] × 100%. used to analyze changes in time and differences between groups for each experiment. Significance was measured Fluorescence microscopy using independent-samples t-test. All differences were The superoxide anion in mitochondria and mitochon - considered statistically significant if the p-value was less drial membrane potential were measured using the flu - than 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001). All experi- orescent probe MitoSOX/JC-1 and Hoechst dye. Cells ments were performed in triplicates. were seeded onto 24-well plates at a density of 6 × 10 cells/well. Serum-free DMEM was added and treatments Results were performed accordingly. Followed by washing twice BLM inhibits proliferation and promotes fibrosis in A549 with PBS, the cells were incubated with the fluorescent cells probe MitoSOX/JC-1 and Hoechst dye for 15  min at We constructed a cell model of alveolar epithelial cell 37  °C. After the reaction, cells were washed twice with fibrosis in vitro and used BLM was to induce the fibrosis PBS and the superoxide anion in mitochondria, as well of A549 human alveolar epithelial cells. A549 cells were as the mitochondrial membrane potential, were detected treated with BLM (100 or 200  μg/ml) for 48  h. Micro- using an inverted fluorescence microscope. scopic observation showed that with the increase in drug concentration, cell fibrosis increased, and the MTT assay Flow cytometry showed that cell viability decreased (Fig.  1a, Additional The intracellular ROS were measured by DHE probes in file  1; Fig. S2). Moreover, to detect the change in cell cells. Cells were seeded in 24-well plates at a density of migration upon BLM-induced cell fibrosis, we performed 6 × 10 cells per well. Serum free DMEM medium was a wound healing assay. Would healing assay showed that added and exposed to treatments accordingly, followed the migration of A549 cells was enhanced after treatment by PBS wash (twice), as well as DHE and Hoechst incu- with BLM for 48  h (Fig.  1b, c). Western blotting results bation for 15 min at 37 °C. After the reaction, cells were showed that expression levels of EMT-related proteins washed twice with PBS and observed using flow cytom - N-cadherin, vimentin, fibronectin, and slug were upregu - etry to detect intracellular ROS levels. lated, and those of E-cadherin were downregulated. A549 cells developed cellular fibrosis through EMT (Fig.  1d– Western blotting h). These results indicated that BLM inhibited cell prolif - Serum-free DMEM was added to cells and treatments eration and promoted cell migration and fibrosis in A549 were performed accordingly. The cells were recov - cells. ered and harvested. Harvested cells were lysed (20  mM HEPES-OH, pH 7.0; 50 mM NaCl; 10% glycerol and 0.5% BLM increases ROS levels and decreases mitochondrial Triton X 100) and total protein was extracted. Then, pro - membrane potential in A549 cells teins were denatured for 5  min, exposed to 12% sodium After treatment of A549 cells with BLM (100 or 200 μg/ dodecyl sulfate–polyacrylamide gel electrophoresis for mL) for 48  h, fluorescence microscopy showed that the separation, and transferred into nitrocellulose mem- level of intracellular ROS was significantly increased branes. Skim milk (5%) was used to block the membranes (Fig.  2a), and the same effect was also observed using for 30  min at room temperature. They were then incu - flow cytometry (Fig.  2b). Fluorescence microscopy also bated with polyclonal rabbit anti-N-cadherin, as well showed that ROS levels in mitochondria increased as mouse monoclonal anti-E-cadherin, anti-vimentin, (Fig.  2c) and the mitochondrial membrane potential Ren et al. Appl Biol Chem (2021) 64:74 Page 4 of 9 Fig. 1 Bolemycin inhibits the proliferation of A549 cells and promotes fibrosis. a The morphology of cellular fibrosis was observed under the microscope. Cell viability was detected using MTT. b, c Cell migration was detected using would healing assay. d The expression levels of EMT‑related proteins were detected using western blotting. e–h The related protein expression levels are represented as the mean ± standard deviation. Data are presented as the mean ± standard error of the mean of three different samples. *P < 0.05, **P < 0.01, and ***P < 0.001 HP inhibits BLM‑induced fibrosis in A549 cells decreased (Fig.  2d). These results suggested that BLM Subsequently, we treated A549 cells with BLM (200  μg/ increased ROS levels and caused mitochondrial dysfunc- mL) and HP (10, 20, and 30 μg/mL) for 48 h. Microscopic tion in A549 cells. observation showed that HP significantly inhibited BLM- induced cell fibrosis (Fig.  4a). At the same time, MTT HP inhibits the increase of ROS levels in A549 cells assay showed that there was no significant difference in and mitochondria dysfunction induced by BLM the activity of A549 cells treated with BLM alone or com- A549 cells were treated with BLM (200  μg/mL) and HP bined with HP for 48  h (Fig.  4b). Wound healing assay (10, 20, and 30  μg/mL) for 48  h. Fluorescence micros- results showed that HP significantly inhibited cell migra - copy showed that HP significantly inhibited the increase tion induced by BLM (Fig. 4c, d). Western blotting results of ROS levels in cells induced by BLM (Fig.  3a). Flow showed that HP significantly inhibited the changes in the cytometry also showed the same effect (Fig.  3b). At the expression levels of EMT-related proteins induced by same time, HP significantly inhibited the increase in ROS BLM, thus inhibiting EMT and cell fibrosis of A549 cells levels and the decrease of membrane potential induced (Fig.  4e–i). These results indicated that hispidin signifi - by BLM (Fig.  3c, d). These results indicated that HP sig - cantly inhibited the migration and fibrosis of A549 cells nificantly inhibited the increase in ROS levels and mito - induced by BLM. chondrial dysfunction induced by BLM in A549 cells. R en et al. Appl Biol Chem (2021) 64:74 Page 5 of 9 Fig. 2 Bolemycin increased the ROS levels of in A549 cells. a Intracellular ROS levels were detected using flow cytometry, b as well as DHE staining. c ROS levels in mitochondria were detected using Mito‑SOX staining. d The mitochondrial membrane potential was detected using JC‑1 staining. **P < 0.01, and ***P < 0.001 also believe that oxidative stress is closely related to Discussion pulmonary fibrosis [37, 39]. IPF is a chronic, irreversible and usually fatal lung BLM is an antibiotic used to treat various tumors, but disease [31], which is highly prevalent in the elderly. it causes severe pneumonia and pulmonary fibrosis [40]. Patients with IPF suffer from long-term cough and The injury and dysfunction of pulmonary epithelial cells dyspnea, which is difficult to diagnose, and most are considered to be an important initial and central pro- patients are already at the advanced stage when they cess of fibrosis [3], BLM induces the transformation of are diagnosed [32]. Today, the prevalence and inci- lung epithelial cells into mesenchymal cells through EMT dence rate of IPF is increasing [33, 34] and the survival process, gradually transforms the morphology of lung time of patients is about 2–3 years after diagnosis [35]. epithelial cells into mesenchymal phenotype, inhibits the Therefore, there is an urgent need for more effective proliferation of epithelial cells and further promotes the diagnostic and treatment methods. The specific patho - process of fibrosis [3, 13, 36, 38]. In this study, bleomy- genesis of the disease is not clear, but more studies have cin was used to construct the pulmonary fibrosis model. shown that alveolar epithelial type II cells can be used A549 cells were treated with different concentrations as a driver of idiopathic pulmonary fibrosis [36] and the of bleomycin for 48  h. The results showed that the cells occurrence of IPF is related to alveolar epithelial cell showed obvious fibrosis at the concentration of 200  μg/ injury [37] and EMT [38]. Moreover, some researchers Ren et al. Appl Biol Chem (2021) 64:74 Page 6 of 9 Fig. 3 Hispidin inhibits bolemycin‑induced ROS. a Intracellular ROS levels were detected using flow cytometry, b as well as DHE staining. c ROS levels in mitochondria was detected using Mito‑SOX staining. d The mitochondrial membrane potential was detected using JC‑1 staining. **P < 0.01, and ***P < 0.001 mL. MTT assay showed that the proliferation of cells was cell morphology and EMT related proteins were signifi - inhibited at 200  μg/mL. The EMT related proteins also cantly inhibited, it can be seen from the above that HP changed significantly after BLM treatment. Some studies has a significant inhibitory effect on the fibrosis of A549 have shown that bleomycin can induce ROS, which may alveolar epithelial cells. The specific mechanism has not be an important reason for lung epithelial cell injury and been explored, but we know that the target signal path- EMT transformation [41]. At the same time, mitochon- way of HP is TGF- β/ Smad signal pathway. At the same dria in cells are the most important position to produce time, studies have shown that anlotinib attenuates BLM ROS in cells [42], which is closely related to pulmonary induced pulmonary fibrosis through TGF-β1 signal - fibrosis [43]. Therefore, DHE fluorescent dye detection ing pathway [45], BLM through TGF-β/ Smad signaling showed that intracellular ROS increased significantly pathway induces EMT of cultured A549 cells [13], pae- after BLM treatment, Mito-SOX and JC-1 fluorescent oniflorin inhibits TGF-β mediated EMT of pulmonary dye detection showed that mitochondrial ROS increased fibrosis through Smad dependent pathway, etc. These and mitochondria were damaged. Then we focused on reports suggest that TGF-β/ Smad signaling pathway is HP, which can neutralize free radicals [22]. By further closely related to the process of pulmonary fibrosis. searching the data, we found that HP is a protein kinase Based on the above information, we speculate that C β Inhibitors, and protein kinase C β overexpression HP may attenuates BLM induced pulmonary fibrosis by can induce fibrosis in human proximal tubular epithe - TGF- β/ Smad signaling pathway, which will also become lial cells [44]. We speculate that HP may inhibit cellular the focus of our next research. This study will also pro - fibrosis. After treating fibrotic cells with HP, we found vide new ideas for the treatment of IPF. that the level of ROS decreased significantly, fibrotic R en et al. Appl Biol Chem (2021) 64:74 Page 7 of 9 Fig. 4 Eec ff t of Hispidin on bolemycin‑induced EMT. a The morphology of cellular fibrosis was observed under the microscope. b Cell viability was detected using MTT. c, d Cell migration was detected using wound healing assay. e Expression levels of EMT‑related proteins were detected using western blotting. f–i The related protein expression levels were represented as the mean ± standard deviation. Data are presented as the mean ± standard error of the mean of three different samples. *P < 0.05, **P < 0.01, and ***P < 0.001 Authors’ contributions Supplementary Information CXR, XJ, DXP, HNS and TK performed the experiments and wrote the article. The online version contains supplementary material available at https:// doi. CXR, XJ, DXP, XYG, LYY, YDC, HNS and TK performed the data analysis. YDC, org/ 10. 1186/ s13765‑ 021‑ 00646‑x. HNS and TK reviewed and edited the manuscript. TK acquired the funding. All authors read and approved the final manuscript. Additional file 1: Fig. S1. Cell viability was measured by MTT assay. Cells Funding were treated with different concentrations of hispidin (HP) (10, 20, 30 This research was supported by the Basic Science Research Program through μg/mL) incubated for 48 h. Data are presented as the mean ± standard the National Research Foundation of Korea funded by the Ministry of Educa‑ error of the mean of three different samples. *P < 0.05, **P < 0.01, and tion (2020R1I1A2052417), The Korean Research Institute of Bioscience and ***P < 0.001. Fig. S2. a The morphology of cellular fibrosis was observed Biotechnology Research Information System (RBM0112112). under the microscope. b Intracellular ROS levels were detected using flow cytometry. c, d Cells were treated with different concentrations of Availability of data and materials Bolemycin (BLM) and hispidin (HP) incubated for 48 h. Data are presented The datasets used and analyzed in this study are available from the corre‑ as the mean ± standard error of the mean of three different samples. *P < sponding author upon reasonable request. 0.05, **P < 0.01, and ***P < 0.001. Declarations Acknowledgements This research was supported by the project of the scientific research team Competing interests support plan of Heilongjiang Bayi Agricultural University ( TDJH201904), China. The authors declare that they have no competing interests. Ren et al. Appl Biol Chem (2021) 64:74 Page 8 of 9 Author details 20. 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Journal

Applied Biological ChemistrySpringer Journals

Published: Dec 1, 2021

Keywords: Bleomycin; Epithelial-mesenchymal transition; Hispidin; Idiopathic pulmonary fibrosis; Reactive oxygen species

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