Induction of HO-1 by 5, 8-Dihydroxy-4′,7-Dimethoxyflavone via Activation of ROS/p38 MAPK/Nrf2 Attenuates Thrombin-Induced Connective Tissue Growth Factor Expression in Human Cardiac Fibroblasts

Abstract

Heme oxygenase-1 (HO-1) has been shown to exert as an antioxidant and anti-inflammatory enzyme in cardiovascular inflammatory diseases. Flavonoids have been demonstrated to display anti-inflammatory and antioxidant effects through the induction of HO-1. 5,8-Dihydroxy-4${}^{\prime }$,7-dimethoxyflavone (DDF), one of the flavonoid compounds, is isolated from Reevesia formosana. Whether DDF induced HO-1 expression on human cardiac fibroblasts (HCFs) remained unknown. Here, we found that DDF time- and concentration-dependently induced HO-1 protein and mRNA expression, which was attenuated by pretreatment with reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC) in HCFs. DDF-enhanced ROS generation was attenuated by NAC, but not by either diphenyleneiodonium chloride (DPI, Nox inhibitor) or MitoTempol (mitochondrial ROS scavenger). Interestingly, pretreatment with glutathione (GSH) inhibited DDF-induced HO-1 expression. The ratio of GSH/GSSG was time-dependently decreased in DDF-treated HCFs. DDF-induced HO-1 expression was attenuated by an inhibitor of p38 MAPK (p38i VIII) or siRNA, but not by MEK1/2 (PD98059) or JNK1/2 (SP600125). DDF-stimulated p38 MAPK phosphorylation was inhibited by GSH or p38i VIII. Moreover, DDF-induced HO-1 expression was mediated through Nrf2 phosphorylation and translocation into the nucleus which was attenuated by NAC or p38 siRNA. DDF also stimulated antioxidant response element (ARE) promoter activity which was inhibited by NAC, GSH, or p38i VIII. Interaction between Nrf2 and the ARE-binding sites on the HO-1 promoter was revealed by chromatin immunoprecipitation assay, which was attenuated by NAC, GSH, or p38i VIII. We further evaluated the functional effect of HO-1 expression on the thrombin-induced fibrotic responses. Our result indicated that the induction of HO-1 by DDF can attenuate the thrombin-induced connective tissue growth factor expression. These results suggested that DDF-induced HO-1 expression is, at least, mediated through the activation of the ROS-dependent p38 MAPK/Nrf2 signaling pathway in HCFs. Thus, the upregulation of HO-1 by DDF could be a candidate for the treatment of heart fibrosis.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Growing evidence has indicated that oxidative stress participates in several cardiovascular diseases. In the cardiovascular system, fibroblasts are the most abundant cell population and play an important role in cardiac development, cardiac repair and remodeling, and cardiac fibrosis in response to mechanical force, chemical signals, or various insults. The activation of fibroblasts expresses profibrotic cytokines and positively amplifies the effects via autocrine/paracrine mechanisms. For example, cardiac fibrosis is characterized by excessive deposition of fibrotic extracellular matrix (ECM) and synthesis and deposition of collagen which directly cause left ventricular (LV) dysfunction, alter electrical conduction, and increase coronary resistance [1]. The changes in these functions might be related to the generation of reactive oxygen species (ROS) through the induction of inflammatory mediators via the activation of several signaling components in the cardiac fibroblasts [2, 3]. Accumulating evidence shows that an imbalance between ROS formation and antioxidant defense mechanisms associated with increased inflammatory responses triggers the initiation and progression of cardiovascular diseases. Under pathological conditions, elevated ROS production can result in oxidative damage to DNA, proteins, and lipids and changes in cellular functions leading to cell death. Indeed, several studies have indicated that dysregulated ROS production has a crucial role in a host of cardiac diseases, including heart failure (HF), cardiac hypertrophy, cardiac ischemia-reperfusion injury (I/RI), and ischemic cardiomyopathy [3–6]. Therefore, the development of an effective antioxidant strategy to target these components could provide a beneficial intervention for the management of cardiovascular diseases.

Connective tissue growth factor (CTGF), a cysteine-rich secreted protein, belongs to a set of structurally related proteins of the CCN family. CTGF is involved in angiogenesis and cellular differentiation under normal circumstances. In addition, CTGF also modulates wound healing and fibrosis in pathological conditions [7, 8]. In tissue response to injury and fibrotic reaction, CTGF could stimulate the proliferation of fibroblasts, their differentiation towards myofibroblasts, and enhancement of extracellular matrix (ECM) production. In granulation tissue and various fibrotic disorders, the higher levels of CTGF were detected in various organs including the heart [9]. Therefore, CTGF might play a key role in the development of various cardiac diseases such as myocardial fibrosis [10]. The expression of CTGF, an immediate early gene, is regulated by a variety of cell types and stimuli, including thrombin [11–13]. Altieri et al. found that thrombin triggers primary human atrial fibroblasts to differentiate to myofibroblasts enriched for α-smooth muscle actin (αSMA), fibronectin, and type I collagen [14]. Dabigatran, a thrombin inhibitor, can attenuate cardiac fibrosis induced by high-pressure overload and improve global cardiac function [14, 15]. Therefore, the expression of CTGF induced by thrombin might be a key player in the pathogenesis of cardiac fibrosis.

Heme oxygenase- (HO-) 1 is considered to be a potential therapeutic target for human diseases, including cardiovascular inflammatory diseases. HO-1 has been shown to protect against inflammatory responses and to be induced by various stimuli and oxidative stresses [16, 17]. HO-1 can suppress cardiomyocyte senescence and improve heart function in myocardial infarction and aged mice [18]. Emerging evidence has revealed that the NF-E2-related factor (Nrf2)/HO-1 signaling pathway is a critical regulator of cardiovascular homeostasis via the suppression of oxidative stress, which can prevent oxidative stress-associated cardiac remodeling and heart failure [19, 20]. Chinese herbal medicines have attracted attention as the treatment of inflammatory diseases. In particular, the Sterculiaceae family has been discovered to exhibit several biological activities, such as anti-inflammatory and antioxidant activities [21, 22]. Several components extracted from Chinese herbal medicines contain flavonoids that possess anti-inflammatory and antioxidant effects on pathophysiological conditions including cardiovascular diseases. 5,8-Dihydroxy-4${}^{\prime }$,7-dimethoxyflavone (DDF) is one of the flavonoids isolated from Reevesia formosana. These studies prompted us to explore whether DDF can attenuate the effects of thrombin on CTGF expression in HCFs.

The upregulation of HO-1 by various stimuli is tightly modulated through different signaling pathways in various types of cells [23]. For instance, EGF-induced HO-1 expression is mediated through EGFR, NADPH oxidase (Nox), and ROS production in HT-29 cells [24]. Mitogen-activated protein kinases (MAPKs) have also been demonstrated to upregulate HO-1 expression in response to diverse stimuli [25, 26]. Sodium arsenite upregulates HO-1 expression via the activation of JNK1/2 in rat hepatocytes [27] and via ERK1/2 and p38 MAPK pathways through the depletion of glutathione (GSH) and the increase in oxidative stress in chicken hepatoma cells [28]. Moreover, there are several transcriptional regulations involved in HO-1 gene expression through the activation of Sp1, activating-protein 1 (AP-1), or Nrf2 modulated by various signaling pathways [29, 30]. Thus, the objective of this study is aimed at dissecting whether DDF can induce HO-1 expression and attenuate the thrombin-induced CTGF expression, shared with similar mechanisms such as Nox/ROS, MAPKs, and Nrf2 in HCFs. The present results revealed that DDF-induced HO-1 expression is, at least, mediated through the activation of the ROS-dependent p38 MAPK/Nrf2 signaling pathway which inhibited the thrombin-induced CTGF expression in HCFs.

2. Material and Methods

2.1. Materials

Dulbecco’s modified Eagle’s medium (DMEM)/F-12 and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). 5, 8-Dihydroxy-4${}^{\prime }$,7-dimethoxyflavone (DDF, Figure 1) was kindly provided by the Natural Products Laboratory of Dr. Yann-Lii Leu (Chang Gung University, Taiwan). Hybond C membrane and enhanced chemiluminescence (ECL) Western blotting detection system were purchased from GE Healthcare Biosciences (Buckinghamshire, England, UK). Antibodies against phospho-p38 and phospho-Nrf2 were purchased from Cell Signaling (Danvers, MA). Actinomycin D (Act.D), cycloheximide (CHI), N-acetyl cysteine, SP600125, GSH, trolox, diphenyleneiodonium chloride (DPI), PD98059, and p38 inhibitor VIII (p38i VIII) were purchased from Biomol (Plymouth Meeting, PA). Anti-β-actin antibody was purchased from Biogenesis (Boumemouth, UK). Anti-GAPDH (#MCA-1D4) was obtained from EnCor Biotechnology (Gainesville, FL). Anti-HO-1 pAb and Glutathione (GSSG/GSH) detection kits were purchased from Enzo Life Sciences (Farmingdale, NY). CTGF antibody (sc-25440) was purchased from Santa Cruz (Santa Cruz, CA, USA). Mitotempol, dihydroethidium (DHE), and CM-H₂DCFDA were purchased from Molecular Probes (Eugene, OR, USA). SDS-PAGE reagents were purchased from MDBio Inc. (Taipei, Taiwan). Thrombin and other chemicals were purchased from Sigma (St. Louis, MO, USA).

[figure omitted; refer to PDF]

In this study, we found that the ho-1 gene is activated by DDF in HCFs. DDF time- and concentration-dependently induces HO-1 protein expression, which had no significant effect on cell viability. The upregulation of HO-1 protein by DDF is mediated through a new HO-1 mRNA synthesis, which is attenuated by Act D. Real-time PCR analysis further revealed that DDF induces HO-1 mRNA expression, which is attenuated by Act.D but not cycloheximide in HCFs. These results suggested that DDF-induced ho-1 gene expression is primarily regulated at the transcriptional level.

Nox family is the major intracellular source of ROS. Nox-dependent ROS generation has been shown to induce HO-1 expression by various stimuli in both in vitro and in vivo studies [35, 39–42]. In HCFs, we found that DDF could induce ROS generation which was attenuated by pretreatment with NAC (ROS scavenger), but not with DPI (Nox inhibitor) and MitoTempol (mitochondrial ROS scavenger). Further, NAC is able to inhibit the DDF-mediated ROS generation and HO-1 expression. However, the structure of NAC has a thiol (-SH) group which is different from Trolox. Thiol groups are also the major structure of GSH, which act as reducing agents. On the other hand, NAC is also known as a precursor of the synthesis of GSH. The difference between GSH and Trolox on the DDF-induced HO-1 expression may be due to their different chemical properties and reaction with ROS in HCFs. Especially, Trolox has been reported to stimulate Nrf2-mediated HO-1 expression protecting human and murine primary alveolar type II cells from injury by cigarette smoke [43]. In a recent study, flavonoids induced GSH synthesis and HO-1 expression which protected against oxidative stress [44]. In this study, we found that DDF treatment decreased intracellular GSH/GSSG ratio and increased ROS levels leading to HO-1 expression in HCFs. These results demonstrated that DDF-induced HO-1 expression may be due to the depletion of GSH in HCFs, consistent with the report demonstrated by Oguro et al. (1996) [45]. These results were further supported by the data that pretreatment with GSH also attenuated the DDF-induced HO-1 expression in HCFs.

MAPKs consist of three subfamilies, including ERK1/2, JNK1/2, and p38 MAPK which modulate physiological and pathological processes. Several studies have demonstrated that MAPKs relay the signaling from the cell surface into the nucleus [46] involved in the initiation of gene expression such as HO-1 induced by oxidative stress in various types of cells [47–49]. These three MAPKs have been shown to be involved in the expression of HO-1 induced by dihydroquercetin in macrophages and Kupffer cells [26]. The induction of HO-1 by andrographolide in astrocytes [50] and by nitric oxide in HeLa cells [48] partly mediated by p38 MAPK and ERK1/2 signaling. In addition, both JNK1/2 and p38 MAPK have been shown to be involved in lonchocarpine-induced HO-1 expression in brain glial cells [37]. In this study, we found that DDF-induced HO-1 expression was attenuated by the inhibitor of p38 MAPK, but not of MEK1/2 or JNK1/2 in HCFs, suggesting the involvement of p38 MAPK in these responses. These results are consistent with that IL-10 [34], butein [38], and cadmium [49] induced HO-1 expression mediated through p38 MAPK in various types of models. Moreover, we also found that ROS generation can stimulate p38 phosphorylation which was inhibited by pretreatment with NAC and p38 inhibitor, implying that p38 MAPK is a downstream component of ROS-mediated response in HCFs.

The transcription factor Nrf2 is considered to modulate and activate antioxidant response element (ARE) in promoter regions, which regulates the expression of antioxidant and detoxifying genes such as HO-1. The Keap1-Nrf2 pathway is the major regulator of cytoprotective responses to endogenous and exogenous stresses caused by ROS and electrophiles [51]. In normal conditions, Nrf2 is sequestered in the cytoplasm by Keap1 and promotes its degradation by the ubiquitin proteasome. Under the stress conditions, the modification of –SH group on Keap1 or phosphorylation of Nrf2 promotes dissociation of the Nrf2-Keap1 complex. Nrf2 translocates into the nucleus and binds to ARE sequences then increases transcription of Nrf2-regulated phase II antioxidant enzyme, attenuating ROS generation [52]. Oxidative stress refers to elevated intracellular levels of ROS that cause damage to protein, lipid, and DNA. However, recent studies have indicated that a slight increase of ROS is necessary to regulate biological and physiological processes and also benefits cell signaling processes. In HCFs, we found that Nrf2 was involved in the DDF-induced HO-1 expression through the accumulation of phosphorylated Nrf2 in the nucleus of HCFs. Thus, we have observed that pretreatment with NAC, GSH, and p38 inhibitor significantly decreased Nrf2 expression, determined by immunofluorescence staining. Moreover, we further revealed that NAC, GSH, and p38 inhibitor blocked Nrf2 binding to the ARE binding site in the HO-1 promoter. Thus, we suggested that Nrf2 may bind to the ARE sequence in the HO-1 promoter and finally induces HO-1 expression in HCFs.

Thrombin has been shown to play a crucial role in heart hypertrophy and postinjury remodeling processes [14, 53]. Moreover, CTGF is an important component in several pathogeneses of heart diseases [10], which is induced by thrombin [12, 13]. The present results demonstrated that in HCFs, thrombin significantly induced CTGF expression which was attenuated by DDF through the upregulation of HO-1. These findings are consistent with previous studies indicating that inhibition of thrombin response could attenuate cardiac fibrosis and improve cardiac function [14, 15]. Thus, DDF could be beneficial for the treatment of heart failure and cardiac fibrosis. Based on our data, DDF could block CTGF expression induced by thrombin, because pretreatment of DDF with 6 h can inhibit CTGF induction. Huang et al. (2020) found that HO-1 and CTGF present an inverse correlation in a diabetic retinopathy rat model [54]. Riboflavin treatment has beneficial effects on diabetic cardiomyopathy, which could result from raising myocardial HO-1 and decreasing myocardial CTGF levels at the same time [55]. Our data also demonstrated that overexpression of HO-1 can attenuate CTGF expression induced by thrombin in HCFs, this finding verified that the inhibitory effects of DDF on CTGF expression triggered by thrombin, at least partially, come from HO-1 induction. Moreover, the detailed mechanisms by which DDF inhibits thrombin-stimulated CTGF induction is an important issue for further investigation.

5. Conclusions

In conclusion, we demonstrated that DDF-induced HO-1 expression is, at least, mediated through the activation of the ROS-dependent p38 MAPK/Nrf2 signaling pathway and attenuates the thrombin-stimulated CTGF induction (Figure 9). Thus, DDF treatment may be a potential therapeutic intervention for the management of heart diseases. However, the limitations of this study were that there was no evidence to clarify the HO-1 expression induced by DDF which protected against the heart diseases in vivo. Therefore, it is important to further translate the results of cell culture into an animal study. The results obtained from in vivo study could provide the possibility of therapeutic application of DDF in the management of heart diseases.

Authors’ Contributions

CCY, LDH, HHL, HCT, JHS, YLL, and CMY designed and conducted the study. CCY, LDH, HHL, JHS, and HCT performed and collected the data. CCY, LDH, HHL, HCT, JHS, and CMY analyzed and interpreted the data. CCY and CMY prepared the manuscript. CCY, LDH, HHL, HCT, JHS, YLL, and CMY reviewed the manuscript. CCY, LDH, HHL, HCT, JHS, YLL, and CMY approved the final manuscript.

Acknowledgments

We appreciated Dr. Chen-yu Wang for his suggestions and construction of plasmids applied in this study. This work was supported by the Ministry of Science and Technology, Taiwan (Grant numbers: MOST107-2320-B-039-071-MY2, MOST108-2320-B-039-061, MOST109-2320-B-039-061, MOST108-2320-B-182-014, and MOST109-2811-B-039-525); China Medical University, Taiwan (Grant numbers: CMU109-MF-09); Chang Gung Medical Research Foundation, Taiwan (Grant numbers: CMRPG5F0203, CMRPG5J0142, and CMRPG5J0143).

Glossary

Abbreviations

Act. D:Actinomycin D

ARE:Antioxidant response element

BSA:Bovine serum albumin

CHI:Cycloheximide

ChIP:Chromatin immunoprecipitation

DCF-DA:2${}^{\prime }$, 7${}^{\prime }$-dichlorofluorescein diacetate

CTGF:Connective tissue growth factor

DHE:Dihydroethidium

DMEM/F-12:Dulbecco’s modified Eagle’s medium/Ham’s F-12

DPI:Diphenyleneiodonium chloride

ECL:Enhanced chemiluminescence

ECM:Extracellular matrix

EGFR:Epidermal growth factor receptor

ERK:Extracellular regulated protein kinase

FBS:Fetal bovine serum

FITC:Fluorescein isothiocyanate

GAPDH:Glyceraldehyde-3-phosphate dehydrogenase

GST:Glutathione S-transferase

HO-1:Heme oxygenase-1

HCF:Human cardiac fibroblast

JNK:c-Jun-NH2-terminal kinase

Keap1:Kelch ECH associating protein 1

LV:Left ventricle

MAPK:Mitogen-activated protein kinase

NAC:N-acetyl-L-cysteine

NADPH:Nicotinamide adenine dinucleotide phosphate

Nrf2:NF-E2-related factor 2

PBS:Phosphate-buffered saline

PKC:Protein kinase C

PMSF:Phenylmethylsulfonyl fluoride

ROS:Reactive oxygen species

RT-PCR:Reverse transcription-polymerase chain reaction

SDS-PAGE:Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SiRNA:Small interfering RNA

Sp1:Specificity protein 1

TNF-α:Tumor necrosis factor alpha

Tris-HCl:Tris [hydroxymethyl] aminomethane hydrochloride

TTBS:Tween-Tris-buffered saline

VCAM-1:Vascular cell adhesion molecule-1.

References

[1] B. López, A. González, S. Ravassa, J. Beaumont, M. U. Moreno, G. San José, R. Querejeta, J. Díez, "Circulating biomarkers of myocardial fibrosis," Journal of the American College of Cardiology, vol. 65 no. 22, pp. 2449-2456, DOI: 10.1016/j.jacc.2015.04.026, 2015.

[2] A. Nabeebaccus, M. Zhang, A. M. Shah, "NADPH oxidases and cardiac remodelling," Heart Failure Reviews, vol. 16 no. 1,DOI: 10.1007/s10741-010-9186-2, 2011.

[3] H. Tsutsui, S. Kinugawa, S. Matsushima, "Oxidative stress and heart failure," American Journal of Physiology. Heart and Circulatory Physiology, vol. 301 no. 6, pp. H2181-H2190, DOI: 10.1152/ajpheart.00554.2011, 2011.

[4] S. Cadenas, "ROS and redox signaling in myocardial ischemia-reperfusion injury and cardioprotection," Free Radical Biology & Medicine, vol. 117, pp. 76-89, DOI: 10.1016/j.freeradbiomed.2018.01.024, 2018.

[5] C. Di Filippo, S. Cuzzocrea, F. Rossi, R. Marfella, M. D'Amico, "Oxidative Stress as the Leading Cause of Acute Myocardial Infarction in Diabetics," Cardiovascular Drug Reviews, vol. 24 no. 2, pp. 77-87, DOI: 10.1111/j.1527-3466.2006.00077.x, 2006.

[6] M. Seddon, Y. H. Looi, A. M. Shah, "Oxidative stress and redox signalling in cardiac hypertrophy and heart failure," Heart, vol. 93 no. 8, pp. 903-907, DOI: 10.1136/hrt.2005.068270, 2007.

[7] H. Ihn, "Pathogenesis of fibrosis: role of TGF- β and CTGF," Current Opinion in Rheumatology, vol. 14 no. 6, pp. 681-685, DOI: 10.1097/00002281-200211000-00009, 2002.

[8] D. R. Brigstock, "The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family," Endocrine Reviews, vol. 20 no. 2, pp. 189-206, DOI: 10.1210/edrv.20.2.0360, 1999.

[9] A. Daniels, M. van Bilsen, R. Goldschmeding, G. J. van der Vusse, F. A. van Nieuwenhoven, "Connective tissue growth factor and cardiac fibrosis," Acta Physiologica (Oxford, England), vol. 195 no. 3, pp. 321-338, DOI: 10.1111/j.1748-1716.2008.01936.x, 2009.

[10] Y. Ramazani, N. Knops, M. A. Elmonem, T. Q. Nguyen, F. O. Arcolino, L. van den Heuvel, E. Levtchenko, D. Kuypers, R. Goldschmeding, "Connective tissue growth factor (CTGF) from basics to clinics," Matrix Biology, vol. 68-69, pp. 44-66, DOI: 10.1016/j.matbio.2018.03.007, 2018.

[11] H. Y. Chen, C. H. Lin, B. C. Chen, "ADAM17/EGFR-dependent ERK activation mediates thrombin-induced CTGF expression in human lung fibroblasts," Experimental Cell Research, vol. 370 no. 1, pp. 39-45, DOI: 10.1016/j.yexcr.2018.06.008, 2018.

[12] W. C. Ko, B. C. Chen, M. J. Hsu, C. T. Tsai, C. Y. Hong, C. H. Lin, "Thrombin induced connective tissue growth factor expression in rat vascular smooth muscle cells via the PAR-1/JNK/AP-1 pathway," Acta Pharmacologica Sinica, vol. 33 no. 1, pp. 49-56, DOI: 10.1038/aps.2011.178, 2012.

[13] R. C. Chambers, P. Leoni, O. P. Blanc-Brude, D. E. Wembridge, G. J. Laurent, "Thrombin is a potent inducer of connective tissue growth factor production via proteolytic activation of protease-activated receptor-1," The Journal of Biological Chemistry, vol. 275 no. 45, pp. 35584-35591, DOI: 10.1074/jbc.M003188200, 2000.

[14] P. Altieri, M. Bertolotto, P. Fabbi, E. Sportelli, M. Balbi, F. Santini, C. Brunelli, M. Canepa, F. Montecucco, P. Ameri, "Thrombin induces protease-activated receptor 1 signaling and activation of human atrial fibroblasts and dabigatran prevents these effects," International Journal of Cardiology, vol. 271, pp. 219-227, DOI: 10.1016/j.ijcard.2018.05.033, 2018.

[15] A. Dong, P. Mueller, F. Yang, L. Yang, A. Morris, S. S. Smyth, "Direct thrombin inhibition with dabigatran attenuates pressure overload-induced cardiac fibrosis and dysfunction in mice," Thrombosis Research, vol. 159, pp. 58-64, DOI: 10.1016/j.thromres.2017.09.016, 2017.

[16] S. A. Rushworth, X.-L. Chen, N. Mackman, R. M. Ogborne, M. A. O’Connell, "Lipopolysaccharide-induced heme oxygenase-1 expression in human monocytic cells is mediated via Nrf2 and protein kinase C," Journal of Immunology, vol. 175 no. 7, pp. 4408-4415, DOI: 10.4049/jimmunol.175.7.4408, 2005.

[17] J. Huang, X. D. Shen, S. Yue, J. Zhu, F. Gao, Y. Zhai, R. W. Busuttil, B. Ke, J. W. Kupiec-Weglinski, "Adoptive transfer of heme oxygenase-1 (HO-1)-modified macrophages rescues the nuclear factor erythroid 2-related factor (Nrf2) antiinflammatory phenotype in liver ischemia/reperfusion injury," Molecular Medicine, vol. 20 no. 1, pp. 448-455, DOI: 10.2119/molmed.2014.00103, 2014.

[18] H. Shan, T. Li, L. Zhang, R. Yang, Y. Li, M. Zhang, Y. Dong, Y. Zhou, C. Xu, B. Yang, H. Liang, X. Gao, H. Shan, "Heme oxygenase-1 prevents heart against myocardial infarction by attenuating ischemic injury-induced cardiomyocytes senescence," eBioMedicine, vol. 39, pp. 59-68, DOI: 10.1016/j.ebiom.2018.11.056, 2019.

[19] S. Zhou, W. Sun, Z. Zhang, Y. Zheng, "The role of Nrf2-mediated pathway in cardiac remodeling and heart failure," Oxidative Medicine and Cellular Longevity, vol. 2014,DOI: 10.1155/2014/260429, 2014.

[20] X. Zhang, Y. Yu, H. Lei, Y. Cai, J. Shen, P. Zhu, Q. He, M. Zhao, "The Nrf-2/HO-1 signaling axis: a ray of hope in cardiovascular diseases," Cardiology Research and Practice, vol. 2020,DOI: 10.1155/2020/5695723, 2020.

[21] L. M. R. Al Muqarrabun, N. Ahmat, "Medicinal uses, phytochemistry and pharmacology of family Sterculiaceae: a review," European Journal of Medicinal Chemistry, vol. 92, pp. 514-530, DOI: 10.1016/j.ejmech.2015.01.026, 2015.

[22] H. S. Chang, M. Y. Chiang, H. Y. Hsu, C. W. Yang, C. H. Lin, S. J. Lee, I. S. Chen, "Cytotoxic cardenolide glycosides from the root of Reevesia formosana," Phytochemistry, vol. 87, pp. 86-95, DOI: 10.1016/j.phytochem.2012.11.024, 2013.

[23] R. Motterlini, R. Foresti, "Heme Oxygenase-1 As a Target for Drug Discovery," Antioxidants & Redox Signaling, vol. 20 no. 11, pp. 1810-1826, DOI: 10.1089/ars.2013.5658, 2014.

[24] G. S. Lien, M. S. Wu, M. Y. Bien, C. H. Chen, C. H. Lin, B. C. Chen, "Epidermal growth factor stimulates nuclear factor- κ B activation and heme oxygenase-1 expression via c-Src, NADPH oxidase, PI3K, and Akt in human colon cancer cells," PLoS One, vol. 9 no. 8,DOI: 10.1371/journal.pone.0104891, 2014.

[25] D. Martin, A. I. Rojo, M. Salinas, R. Diaz, G. Gallardo, J. Alam, C. M. Ruiz de Galarreta, A. Cuadrado, "Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol," The Journal of Biological Chemistry, vol. 279 no. 10, pp. 8919-8929, DOI: 10.1074/jbc.m309660200, 2004.

[26] M. Zhao, J. Chen, P. Zhu, M. Fujino, T. Takahara, S. Toyama, A. Tomita, L. Zhao, Z. Yang, M. Hei, L. Zhong, J. Zhuang, S. Kimura, X. K. Li, "Dihydroquercetin (DHQ) ameliorated concanavalin A-induced mouse experimental fulminant hepatitis and enhanced HO-1 expression through MAPK/Nrf2 antioxidant pathway in RAW cells," International Immunopharmacology, vol. 28 no. 2, pp. 938-944, DOI: 10.1016/j.intimp.2015.04.032, 2015.

[27] T. Kietzmann, A. Samoylenko, S. Immenschuh, "Transcriptional regulation of heme oxygenase-1 gene expression by MAP kinases of the JNK and p38 pathways in primary cultures of rat hepatocytes," The Journal of Biological Chemistry, vol. 278 no. 20, pp. 17927-17936, DOI: 10.1074/jbc.M203929200, 2003.

[28] Y. Shan, J. Pepe, T. H. Lu, K. K. Elbirt, R. W. Lambrecht, H. L. Bonkovsky, "Induction of the heme oxygenase-1 gene by metalloporphyrins," Archives of Biochemistry and Biophysics, vol. 380 no. 2, pp. 219-227, DOI: 10.1006/abbi.2000.1921, 2000.

[29] M. Exner, E. Minar, O. Wagner, M. Schillinger, "The role of heme oxygenase-1 promoter polymorphisms in human disease," Free Radical Biology & Medicine, vol. 37 no. 8, pp. 1097-1104, DOI: 10.1016/j.freeradbiomed.2004.07.008, 2004.

[30] C. A. Piantadosi, M. S. Carraway, A. Babiker, H. B. Suliman, "Heme oxygenase-1 regulates cardiac mitochondrial biogenesis via Nrf2-mediated transcriptional control of nuclear respiratory factor-1," Circulation Research, vol. 103 no. 11, pp. 1232-1240, DOI: 10.1161/01.RES.0000338597.71702.ad, 2008.

[31] S. Papaiahgari, Q. Zhang, S. R. Kleeberger, H. Y. Cho, S. P. Reddy, "Hyperoxia stimulates an Nrf2-ARE transcriptional response via ROS-EGFR-PI3K-Akt/ERK MAP kinase signaling in pulmonary epithelial cells," Antioxidants & Redox Signaling, vol. 8 no. 1-2, pp. 43-52, DOI: 10.1089/ars.2006.8.43, 2006.

[32] Z. Han, S. Varadharaj, R. J. Giedt, J. L. Zweier, H. H. Szeto, B. R. Alevriadou, "Mitochondria-derived reactive oxygen species mediate heme oxygenase-1 expression in sheared endothelial cells," The Journal of Pharmacology and Experimental Therapeutics, vol. 329 no. 1, pp. 94-101, DOI: 10.1124/jpet.108.145557, 2009.

[33] M. Yu, D. Wang, M. Xu, Y. Liu, X. Wang, J. Liu, X. Yang, P. Yao, H. Yan, L. Liu, "Quinocetone-induced Nrf2/HO-1 pathway suppression aggravates hepatocyte damage of Sprague-Dawley rats," Food and Chemical Toxicology, vol. 69, pp. 210-219, DOI: 10.1016/j.fct.2014.04.026, 2014.

[34] T. S. Lee, L. Y. Chau, "Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice," Nature Medicine, vol. 8 no. 3, pp. 240-246, DOI: 10.1038/nm0302-240, 2002.

[35] C. C. Lin, W. N. Lin, R. L. Cho, C. C. Yang, Y. C. Yeh, L. D. Hsiao, H. C. Tseng, C. M. Yang, "Induction of HO-1 by mevastatin mediated via a Nox/ROS-dependent c-Src/PDGFR α /PI3K/Akt/Nrf2/ARE cascade suppresses TNF- α -induced lung inflammation," Journal of Clinical Medicine, vol. 9 no. 1,DOI: 10.3390/jcm9010226, 2020.

[36] G. Wang, T. Hamid, R. J. Keith, G. Zhou, C. R. Partridge, X. Xiang, J. R. Kingery, R. K. Lewis, Q. Li, D. G. Rokosh, R. Ford, F. G. Spinale, D. W. Riggs, S. Srivastava, A. Bhatnagar, R. Bolli, S. D. Prabhu, "Cardioprotective and antiapoptotic effects of heme oxygenase-1 in the failing heart," Circulation, vol. 121 no. 17, pp. 1912-1925, DOI: 10.1161/circulationaha.109.905471, 2010.

[37] Y.-H. Jeong, J.-S. Park, D.-H. Kim, H.-S. Kim, "Lonchocarpine Increases Nrf2/ARE-Mediated Antioxidant Enzyme Expression by Modulating AMPK and MAPK Signaling in Brain Astrocytes," Biomolecules & Therapeutics, vol. 24 no. 6, pp. 581-588, DOI: 10.4062/biomolther.2016.141, 2016.

[38] Z. Wang, S. O. Ka, Y. Lee, B. H. Park, E. J. Bae, "Butein induction of HO-1 by p38 MAPK/Nrf2 pathway in adipocytes attenuates high-fat diet induced adipose hypertrophy in mice," European Journal of Pharmacology, vol. 799, pp. 201-210, DOI: 10.1016/j.ejphar.2017.02.021, 2017.

[39] P. L. Chi, C. C. Lin, Y. W. Chen, L. D. Hsiao, C. M. Yang, "CO induces Nrf2-dependent heme oxygenase-1 transcription by cooperating with Sp1 and c-Jun in rat brain astrocytes," Molecular Neurobiology, vol. 52 no. 1, pp. 277-292, DOI: 10.1007/s12035-014-8869-4, 2015.

[40] R. L. Cho, C. C. Yang, H. C. Tseng, L. D. Hsiao, C. C. Lin, C. M. Yang, "Haem oxygenase-1 up-regulation by rosiglitazoneviaROS-dependent Nrf2-antioxidant response elements axis or PPAR γ attenuates LPS-mediated lung inflammation," British Journal of Pharmacology, vol. 175 no. 20, pp. 3928-3946, DOI: 10.1111/bph.14465, 2018.

[41] S. H. Latham Birt, R. Purcell, K. M. Botham, C. P. D. Wheeler-Jones, "Endothelial HO-1 induction by model TG-rich lipoproteins is regulated through a NOX4-Nrf2 pathway," Journal of Lipid Research, vol. 57 no. 7, pp. 1204-1218, DOI: 10.1194/jlr.m067108, 2016.

[42] M. He, H. Pan, C. Xiao, M. Pu, "Roles for redox signaling by NADPH oxidase in hyperglycemia-induced heme oxygenase-1 expression in the diabetic retina," Investigative Ophthalmology & Visual Science, vol. 54 no. 6, pp. 4092-4101, DOI: 10.1167/iovs.13-12004, 2013.

[43] E. M. Messier, K. Bahmed, R. M. Tuder, H. W. Chu, R. P. Bowler, B. Kosmider, "Trolox contributes to Nrf2-mediated protection of human and murine primary alveolar type II cells from injury by cigarette smoke," Cell Death & Disease, vol. 4 no. 4,DOI: 10.1038/cddis.2013.96, 2013.

[44] Y. C. Yang, C. K. Lii, A. H. Lin, Y. W. Yeh, H. T. Yao, C. C. Li, K. L. Liu, H. W. Chen, "Induction of glutathione synthesis and heme oxygenase 1 by the flavonoids butein and phloretin is mediated through the ERK/Nrf2 pathway and protects against oxidative stress," Free Radical Biology & Medicine, vol. 51 no. 11, pp. 2073-2081, DOI: 10.1016/j.freeradbiomed.2011.09.007, 2011.

[45] T. Oguro, M. Hayashi, S. Numazawa, K. Asakawa, T. Yoshida, "Heme oxygenase-1 gene expression by a glutathione depletor, phorone, mediated through AP-1 activation in rats," Biochemical and Biophysical Research Communications, vol. 221 no. 2, pp. 259-265, DOI: 10.1006/bbrc.1996.0583, 1996.

[46] L. Chang, M. Karin, "Mammalian MAP kinase signalling cascades," Nature, vol. 410 no. 6824, pp. 37-40, DOI: 10.1038/35065000, 2001.

[47] R. Kacimi, J. Chentoufi, N. Honbo, C. S. Long, J. S. Karliner, "Hypoxia differentially regulates stress proteins in cultured cardiomyocytes: role of the p38 stress-activated kinase signaling cascade, and relation to cytoprotection," Cardiovascular Research, vol. 46 no. 1, pp. 139-150, DOI: 10.1016/s0008-6363(00)00007-9, 2000.

[48] K. Chen, M. D. Maines, "Nitric oxide induces heme oxygenase-1 via mitogen-activated protein kinases ERK and p38," Cellular and Molecular Biology (Noisy-le-Grand, France), vol. 46, pp. 609-617, 2000.

[49] J. Alam, C. Wicks, D. Stewart, P. Gong, C. Touchard, S. Otterbein, A. M. Choi, M. E. Burow, J. Tou, "Mechanism of heme oxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial cells. Role of OF p38 kinase and Nrf2 transcription factor," The Journal of Biological Chemistry, vol. 275, pp. 27694-27702, DOI: 10.1074/jbc.M004729200, 2000.

[50] S. Y. Wong, M. G. K. Tan, P. T. H. Wong, D. R. Herr, M. K. P. Lai, "Andrographolide induces Nrf2 and heme oxygenase 1 in astrocytes by activating p38 MAPK and ERK," Journal of Neuroinflammation, vol. 13 no. 1,DOI: 10.1186/s12974-016-0723-3, 2016.

[51] E. Kansanen, H. K. Jyrkkänen, A. L. Levonen, "Activation of stress signaling pathways by electrophilic oxidized and nitrated lipids," Free Radical Biology & Medicine, vol. 52 no. 6, pp. 973-982, DOI: 10.1016/j.freeradbiomed.2011.11.038, 2012.

[52] A. Loboda, M. Damulewicz, E. Pyza, A. Jozkowicz, J. Dulak, "Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism," Cellular and Molecular Life Sciences, vol. 73 no. 17, pp. 3221-3247, DOI: 10.1007/s00018-016-2223-0, 2016.

[53] R. Pawlinski, M. Tencati, C. R. Hampton, T. Shishido, T. A. Bullard, L. M. Casey, P. Andrade-Gordon, M. Kotzsch, D. Spring, T. Luther, J. I. Abe, T. H. Pohlman, E. D. Verrier, B. C. Blaxall, N. Mackman, "Protease-activated receptor-1 contributes to cardiac remodeling and hypertrophy," Circulation, vol. 116 no. 20, pp. 2298-2306, DOI: 10.1161/circulationaha.107.692764, 2007.

[54] Y. Huang, C. Qian, J. Zhou, J. Xue, "Investigation of expression and influence of CTGF and HO-1 in rats with diabetic retinopathy," Experimental and Therapeutic Medicine, vol. 19, pp. 2291-2295, DOI: 10.3892/etm.2019.8395, 2019.

[55] G. Wang, W. Li, X. Lu, X. Zhao, "Riboflavin Alleviates Cardiac Failure in Type I Diabetic Cardiomyopathy," Heart International, vol. 6 no. 2,DOI: 10.4081/hi.2011.e21, 2018.