Novel strategies targeting hepatic stellate cells to reverse liver fibrosis
Shajari, Shiva
DOI:
10.33612/diss.144700577
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
2020
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Shajari, S. (2020). Novel strategies targeting hepatic stellate cells to reverse liver fibrosis. University of
Groningen. https://doi.org/10.33612/diss.144700577
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
1
INTRODUCTION AND
AIM OF THE THESIS
Shiva Koets-Shajari, Han Moshage, Klaas Nico Faber
6
Discussion
Liver fibrosis is a significant health problem worldwide with an unmet need for effective drug-based treatment. Current therapeutic options are eliminating the disease-causing conditions, and, in advanced cases, liver transplantation. In the past decades, accumulating evidence has identified Hepatic Stellate Cells (HSC) as the major contributors to fibrogenesis in the liver following their transdifferentiation from quiescent into activated myofibroblast-like cells. Although the underlying mechanisms of HSC activation have been studied in detail, no effective drugs are available to halt or reverse hepatic stellate cell activation. Two natural molecules, melatonin and esculetin, have been demonstrated to harbor therapeutic properties in variety of liver diseases, mostly through anti-oxidant effects. (1–8). In this thesis, we established direct anti-fibrotic effects of melatonin and esculetin on HSC activation. Both molecules inhibit 5-lipoxygenase (5-LO) activity, which is an essential enzyme for the production of leukotrienes and lipoxins (9), factors that promote HSC activation. Thus, we propose that melatonin and esculetin may be used for treating liver fibrosis and should be subject of further clinical investigations. One of the hallmarks of HSC activation is their transition to become highly proliferative. In theory, an antiproliferative agent could therefore halt or even reverse liver fibrosis by suppressing HSC proliferation, but should not prevent recovery of functional liver tissue that was lost due to liver disease. We investigated the therapeutic effect of hydroxyurea (HU), an FDA-approved anti-cancer medicine, on liver fibrosis and our data shows that a relatively small dose of HU strongly reduces HSC proliferation and collagen deposition without suppressing hepatocyte proliferation that is essential for liver regeneration. The combination of a safe dose of HU with a 5-LO inhibitor agent, such as melatonin or esculetin, may therefore be a promising drug cocktail that targets various key aspects of HSC activation and leads to regression of liver fibrosis.
In addition, we discovered in the work presented in this thesis that Hormone-sensitive lipase (HSL), an enzyme with retinyl ester hydrolase (REH) activity, is expressed in HSCs and has a role in vitamin A metabolism.
The highlights of our findings are summarized and discussed in more detail below.
In Chapter 2, we discovered that HSL is present in quiescent HSC (qHSC) and partly co-localizes
with cytoplasmic lipid droplets that are characteristic for qHSC. Pharmacological super-activation of HSL using isoproterenol accelerated retinyl ester loss during super-activation, supporting a role for HSL in hydrolyzing retinyl esters in qHSC as a 3rd REH besides adipose triglyceride lipase
(ATGL) and patatin-like phospholipase domain-containing protein 3 (PNPLA3). Retinyl content in liver and plasma remained unchanged in ATGL-null mice and HSL-null mice, while retinyl
6
esters significantly accumulated in adipose tissue of HSL-null mice (10). This suggest that the 3 REHs can compensate for each other’s absence in HSC to maintain efficient release of retinyl from the liver to the circulation. In contrast, retinyl esterification for vitamin A storage in the liver depends for up to 90% on lecithin retinyl acyltransferase (LRAT), as LRAT-null mice have almost no hepatic retinoid stores (11–14). A triple (Atgl-Lipe-Pnpla3)-knock-out mouse should be generated to analyze whether we now have all REHs identified in HSC.
Individuals with genetic HSL deficiency are prone to develop hepatic steatosis, systemic insulin resistance and diabetes (15). Xia et al. demonstrated that HSL-null mice (total knock out or adipose tissue-specific knock out) suffer from age-dependent hepatic steatosis. Liver-specific HSL-null mice, on the other hand, do not accumulate triglycerides (TGs) in the liver and do not show any sign of hepatic inflammation or fibrosis. The authors concluded that adipose tissue is a target for therapeutic strategies to prevent and treat hepatic steatosis(16). This may include identification of HSL activators for improving patient’s health based on their HSL expression status (16,17). According to our data, targeting HSL must be evaluated cautiously since HSL has a role in vitamin A homeostasis in HSC. Super-activation of HSL and subsequent release of retinyl may trigger quiescent HSC to undergo activation. Also boosting HSL activity may disturb the retinyl/retinyl esters balance. Therefore, monitoring of serum retinyl levels and/or hepatic vitamin A metabolism is necessary.
HSL is a multifunctional enzyme with a broad range of substrates, including TGs, diacylglycerols (DGs), monoacylglycerols, retinyl esters, cholesterol esters (CEs) and other lipids(18). As previously described, in liver homogenate of HSL-null mice, cholesteryl ester hydrolyzing (CEH) activity is significantly reduced when fed a normal or high-cholesterol diet, while TG and DG hepatic-lipase activities remain intact. Isolated hepatocytes from HSL-null mice contain higher levels of CE, suggesting an important role for HSL as CEH in hepatocytes (19). We found that not only qHSC, but also cholangiocytes and Kupffer cells in the rat liver highly express Lipe, the encoding gene for HSL. Cholangiocytes maintain the cholesterol equilibrium in bile and liver and express proteins involved in cholesterol synthesis (20). Therefore, HSL might be particularly important in controlling cholesterol homeostasis in cholangiocytes. HSL also contributes to hydrolyzing CE in mouse and human macrophages (21,22). In line with this, the primary function of HSL in Kupffer cells, the liver-resident macrophages, may also be as a CEH, although there is no direct evidence for it yet. Altogether, HSL harbors several lipase activities and is present in various hepatic cell types, but its cell type-specific activity will be largely determined by the availability of the specific substrates. Recent single-cell sequencing techniques (23,24) allow us to investigate the cell-type specific function of HSL in more detail. This, together with cell
type--specific knock out models will further illuminate the detailed functions of HSL in the liver. Additionally, we observed that the expression of Lipe and Pnpla3 drops during HSC activation, while Atgl mRNA levels remain unchanged. This raises the question what is the function of ATGL in fully-activated HSC (aHSC) and, if ATGL hydrolyzes a specific substrate in aHSC, how essential this substrate is for the cells. Further research on the involvement of such metabolic enzymes in aHSC will provide us a better insight in HSC transdifferentiation mechanisms that may lead to new therapeutic strategies.
Melatonin has demonstrated protective effects in various liver disease models induced by chemicals (e.g. benzene), mycotoxins (e.g. ochratoxin A) and drugs, such as adriamycin, cyclosporine and acetaminophen (25–29). Moreover, melatonin ameliorates the harmful effects of radiation (30) and excessive alcohol consumption (7). Various hepatic pathologies are improved by melatonin treatment, including hepatic steatosis (31,32), fibrosis (8), cirrhosis (33) and hepatocarcinoma (34). Protective actions of melatonin include prevention of oxidative damage (33), improving mitochondrial function (35), inhibiting hepatic neutrophil infiltration (36), suppressing necrosis (37) and apoptosis (5), and ameliorating liver fibrosis (8). Very recently, melatonin has also been used as a safe and effective drug to alleviate Covid-19-associated symptoms (38–49). Melatonin supplements reduced the levels of pro-inflammatory cytokines in Covid-19 patients (38,39) and ameliorated pulmonary lesions associated with inflammation and oxidative stress (41). Moreover, melatonin decreases the likelihood of a positive Covid-19 laboratory test result (43,50). Despite such an extensive body of evidence on systemic and hepatoprotective effects of melatonin and its strong safety profile, there are no data available on the clinical use of melatonin for treating liver injuries. In Chapter 3, we demonstrated that
melatonin directly suppresses HSC activation by modulating the transcription of RAR-related orphan receptor alpha (RORα) leading to downregulation of Alox-5, the encoding gene for 5-LO. Melatonin is available over-the-counter as supplement for regulating the sleep-wake cycle through initiation of sleep (51), which was also used in Covid-19 patients to reduce sleep disturbances and delirium (46). The influence of melatonin on the sleep pattern can be a limiting factor for using it in the treatment of liver fibrosis. However, by identifying the mechanism via which RORα acts we may be able to activate RORα using other ligands that do not affect the sleep-awake cycle. On the other hand, it would be interesting to determine whether people with low endogenous levels of melatonin are more prone to liver fibrosis development, or alternatively, whether people who are using melatonin for a long time show protection against the development of liver fibrosis. Such studies will require large population cohorts, such as Lifelines (52, https://www.lifelines.nl/researcher), and/or in populations with
6
high use of melatonin, such as in the United Kingdom (53).
RORα is involved in several key physiological processes, such as metabolism and cell proliferation (54,55). In endometrial cancer, stimulation of RORα suppresses proliferation of tumor cells and metastasis by down-regulating beta-catenin (56). RORα is highly expressed in hepatocytes (57). Thus, one can argue that melatonin-induced activation of RORα may decrease cell proliferation of hepatocytes and thereby liver regeneration. However, it has been demonstrated that melatonin actually promotes liver regeneration by reducing hepatocyte apoptosis (7). The selective regulatory function of RORα is not completely understood. According to our data, RORα inhibition also suppresses HSC proliferation. Whether this inhibition is a direct regulatory effect of RORα is an interesting question to explore.
Esculetin is a natural compound present in various herbs and plants, which harbors several pharmacological properties, including inflammatory, diabetic, cancer and anti-fibrotic actions (58,59), in part due to its inhibitory effect on lipoxygenases (60). In Chapter 4,
we examined the direct effect of esculetin on human HSC activation in vitro, followed by an in
vivo experiment where mice were exposed for 4 weeks to liver fibrosis-inducing CCl4 and treated with esculetin in the final 2 weeks. Esculetin significantly reversed human HSC proliferation and activation in vitro and suppressed liver fibrosis in the CCl4-treated mice. In the animal model, esculetin suppressed Collagen1a1 and αSma RNA and protein levels. Moreover, it strongly suppressed expression and serum levels of tissue inhibitor of MMP-1 (TIMP1), which allows collagen degradation by matrix metalloproteinases (MMPs). In addition, esculetin enhanced the hepatic GSH/GSSG ratio in CCl4-treated mice, indicating that it improved the anti-oxidant status of the liver.
HSC are the major producers of the extracellular matrix (ECM) deposited in liver fibrosis. Turnover of deposited ECM is subsequently regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). In healthy liver, the production and degradation of ECM are tightly balanced. In injured liver, however, activated HSC overexpress TIMPs that leads to inhibition of MMPs and subsequently impairs ECM degradation (61,62). One way to reverse fibrosis is to increase the degradation of excessive ECM by enhancing MMPs activity, which can be achieved by reducing TIMPs expression (63). We found that esculetin significantly suppressed CCl4-induced TIMP-1, while MMP9 levels were slightly reduced and MMP2 and MMP13 levels remained unchanged. Thus, the esculetin-induced reduction in collagen deposition in CCl4
degradation.
Lipoxygenases (LOXs) are lipid peroxidizing enzymes, involved in the production of leukotrienes that initiate and maintain inflammatory responses. Three main LOXs of pharmacological importance are 5-LO, 12-LO and 15-LO (64). Kupffer cells express all these LOXs (65,66), while hepatocytes have been shown to express epidermal-type lipoxygenase, eLOX3 (encoded by
Aloxe3), particularly under enhanced insulin sensitivity condition (67). According to our data, Alox-5 and Alox12/15 are also expressed by rat hepatocytes, however, the protein levels and
activities of these enzymes remain to be established. Esculetin is non-competitive inhibitor of 5-LO and 12/15-lipoxygenase (12/15-LO) activity (60). Still, esculetin treatment also significantly suppressed the CCl4-induced Alox12/15 mRNA levels in mouse livers, while it did not affect
Alox5 expression. The effect of esculetin on Alox12/15 mRNA levels may be secondary to the
suppression of liver injury and fibrosis but may further contribute to improvement of liver health due to esculetin treatment.
Leukotriene B4 (LTB4), a product of the lipoxygenase pathway, is a chemoattractant of neutrophils that promote hepatic inflammation (68). A recent study showed that esculetin reduced LTB4 plasma levels in a rat arthritis model (69). A decrease in LTB4 production might be also be a mechanism of esculetin in suppressing inflammation and fibrosis in livers of CCl4 -exposed mice. It would therefore be interesting to investigate whether HSC express LTB4 receptors, and if so, whether the migratory capacity of HSC would be impaired by esculetin-mediated reduction of LTB4 production. The leukotriene B4 receptor type 1 (BLT1) and type 2 (BLT2) are expressed by keratinocytes, but not by dermal fibroblasts(70). So far, there are no reports about the expression of these two LTB4 receptors in HSC.
Activated HSC are highly proliferative and suppression thereof is an index for the regression of hepatic fibrosis (71). Inhibition of HSC proliferation can be achieved through targeting two fibrotic factors: platelet-derived growth factors (PDGFs) and transforming growth factor-beta 1 (TGF-β1) (72–74). However, TGF-β1-based therapeutics have not yet been established, mainly because of the complexity of this pathway that makes cell-type targeting of TGF-β1 very challenging (74). Such complications can be associated to PDGF-targeting therapeutic approaches as well, since no medication has been developed yet to inhibit PDGF signaling pathways. Hydroxyurea is an anti-proliferative drug used for the treatment of variety of diseases, including chronic myelogenous leukemia, psoriasis, melanoma, ovarian cancer, polycythemia vera, sickle cell anemia and HIV (75). It is generally well-tolerated with very few side effects. Hydroxyurea prevents proliferation of metastatic cells by suppressing DNA replication through
6
inhibition of the enzyme ribonucleotide reductase (RNR) that converts ribonucleotides into deoxyribonucleotides (76). In Chapter 5, we assessed the effect of hydroxyurea on cultured
HSC and subjected mice to CCl4-induced fibrosis, where animals were given a daily dose of hydroxyurea during 4 weeks of CCl4 treatment. Hydroxyurea significantly and dose-dependently inhibited HSC proliferation in vivo and in vitro. Hydroxyurea did, however, not change the expression of Collagen1a1 and αSma in fully-activated HSC in vitro nor in CCl4-induced fibrosis in mice. Remarkably, hydroxyurea still attenuated collagen deposition in CCl4-treated mice, suggesting that the antifibrogenic effect of hydroxyurea is primarily due to its anti-proliferative actions. An effective anti-fibrotic agent should not block liver regeneration, which is essential for the restoration of functional liver tissue following liver injury. We found that, although hydroxyurea strongly suppresses HSC proliferation, it has no significant impact on regenerative hepatocyte proliferation. This cell type-specific effect of hydroxyurea might be due to a difference in uptake and/or metabolism of hydroxyurea between HSC and hepatocyte. Further investigation is required to determine the proper dose of HU and its potential impact on liver parenchymal cells. However, this data suggests the potential efficacy of HU therapy in patients with liver fibrosis.
Given the current view that effective therapies for liver fibrosis may require cocktails of drugs with complementing activities (77,78), the work described in my thesis indicates that the combination of hydroxyurea, with its capacity to suppress HSC proliferation, and melatonin or esculetin, which also suppress HSC activation, could be a promising formula for the treatment of liver fibrosis. All of these compounds, hydroxyurea, melatonin and esculetin, are readily available and low-priced. Although this is beneficiary for patients, especially in low income regions, it is not easy to find sponsors to initiate placebo-controlled double-blinded randomized clinical trials for such compounds. Unfortunately, such inexpensive drugs without patent position do not make strong business models for pharmaceutical companies to pursue such expensive clinical trials. Thus, great opportunities for treating common diseases by natural and affordable compounds are unfortunately lost in this way. Such agents may depend on circumstantial evidence (as from of population cohorts) to get support for their therapeutic action. In an ideal situation, non-profit organizations such as WHO, universities and research centers would invest on such therapeutic potentials based on adequate research results and recommendations. Still, I wonder whether hydroxyurea, melatonin and/or esculetin will ever end up in modern medicine treatments for liver fibrosis.
REFERENCES
1. Huang SX, Mou JF, Luo Q, Mo QH, Zhou XL, Huang X, et al. Anti-Hepatitis B Virus Activity of Esculetin from Microsorium fortunei in Vitro and in Vivo. Molecules. 2019;24(19):1–15.
2. Tien YC, Liao JC, Chiu CS, Huang TH, Huang CY, Chang W Te, et al. Esculetin ameliorates carbon tetrachloride-mediated hepatic apoptosis in rats. Int J Mol Sci. 2011;12(6):4053–67.
3. Are D, Axis MS, Stacchiotti A, Grossi I, Garc R, Petro G De, et al. Melatonin E ff ects on Non-Alcoholic Fatty Liver.
4. Hu C, Zhao L, Tao J, Li L. Protective role of melatonin in early-stage and end-stage liver cirrhosis. J Cell Mol Med. 2019;23(11):7151–62.
5. Mohamed Y, Basyony MA, El-Desouki NI, Abdo WS, El-Magd MA. The potential therapeutic effect for melatonin and mesenchymal stem cells on hepatocellular carcinoma. BioMedicine. 2019;9(4):24. 6. Hong RT, Xu JM, Mei Q. Melatonin ameliorates experimental hepatic fibrosis induced by carbon
tetrachloride in rats. World J Gastroenterol. 2009;15(12):1452–8.
7. Zhang JJ, Meng X, Li Y, Zhou Y, Xu DP, Li S, et al. Effects of melatonin on liver injuries and diseases. Int J Mol Sci. 2017;18(4):1–27.
8. Bona S, Rodrigues G, Moreira AJ, Di Naso FC, Dias AS, Da Silveira TR, et al. Antifibrogenic effect of melatonin in rats with experimental liver cirrhosis induced by carbon tetrachloride. JGH Open. 2018;2(4):117–23.
9. Mashima R, Okuyama T. The role of lipoxygenases in pathophysiology; new insights and future perspectives. Redox Biol [Internet]. 2015;6:297–310. Available from: http://dx.doi.org/10.1016/j. redox.2015.08.006
10. Taschler U, Schreiber R, Chitraju C, Grabner GF, Romauch M, Wolinski H, et al. Adipose triglyceride lipase is involved in the mobilization of triglyceride and retinoid stores of hepatic stellate cells. Vol. 1851, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. 2015. p. 937–45. 11. Mhatre V. Ho, Ji-Ann Lee and KCM. 基因的改变NIH Public Access. Bone [Internet]. 2012;23(1):1–7.
Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624763/pdf/nihms412728.pdf 12. Liu L, Gudas LJ. Disruption of the lecithin:retinyl acyltransferase gene makes mice more susceptible to
vitamin A deficiency. J Biol Chem. 2005;280(48):40226–34.
13. Shirakami Y, Gottesman ME, Blaner WS. Diethylnitrosamine-induced hepatocarcinogenesis is suppressed in lecithin: Retinyl acyltransferase-deficient mice primarily through retinoid actions immediately after carcinogen administration. Carcinogenesis. 2012;33(2):268–74.
14. D’Ambrosio DN, Clugston RD, Blaner WS. Vitamin A metabolism: An update. Nutrients. 2011;3(1):63– 103.
15. Albert JS, Yerges-Armstrong LM, Horenstein RB, Pollin TI, Sreenivasan UT, Chai S, et al. Null mutation in hormone-sensitive lipase gene and risk of type 2 diabetes. N Engl J Med. 2014;370(24):2307–15. 16. Xia B, Cai GH, Yang H, Wang SP, Mitchell GA, Wu JW. Adipose tissue deficiency of hormone-sensitive
lipase causes fatty liver in mice. PLoS Genet. 2017;13(12):1–17.
17. Lan YL, Lou JC, Lyu W, Zhang B. Update on the synergistic effect of HSL and insulin in the treatment of metabolic disorders. Ther Adv Endocrinol Metab. 2019;10:1–10.
18. Kraemer FB, Shen WJ. Hormone-sensitive lipase: Control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis. J Lipid Res. 2002;43(10):1585–94.
19. Sekiya M, Osuga JI, Yahagi N, Okazaki H, Tamura Y, Igarashi M, et al. Hormone-sensitive lipase is involved in hepatic cholesteryl ester hydrolysis. J Lipid Res. 2008;49(8):1829–38.
6
20. Yoo KS, Lim WT, Choi HS. Biology of cholangiocytes: From bench to bedside. Gut Liver. 2016;10(5):687– 98.
21. Jepson CA, Harrison JA, Kraemer FB, Yeaman SJ. Down-regulation of hormone-sensitive lipase in sterol ester-laden J774.2 macrophages. Biochem J. 1996;318(1):173–7.
22. Belkner J, Stender H, Holzhutter HG, Holm C, Kuhn H. Macrophage cholesteryl ester hydrolases and hormone-sensitive lipase prefer specifically oxidized cholesteryl esters as substrates over their non-oxidized counterparts. Biochem J. 2000;352(1):125–33.
23. Krenkel O, Hundertmark J, Ritz T, Weiskirchen R, Tacke F. Single Cell RNA Sequencing Identifies Subsets of Hepatic Stellate Cells and Myofibroblasts in Liver Fibrosis. Cells. 2019;8(5):503.
24. Chang N, Tian L, Ji X, Zhou X, Hou L, Zhao X, et al. Single-Cell Transcriptomes Reveal Characteristic Features of Mouse Hepatocytes with Liver Cholestatic Injury. Cells. 2019;8(9):1–17.
25. Sharma S, Rana SVS. Melatonin improves liver function in benzene-treated rats. Arh Hig Rada Toksikol. 2013;64(2):219–27.
26. Malir F, Ostry V, Pfohl-Leszkowicz A, Malir J, Toman J. Ochratoxin A: 50 years of research. Toxins (Basel). 2016;8(7):12–5.
27. Karakilçik AZ, Bitiren M, Zerin M, Çelik H, Aksoy N. Melatonin increased vitamin C and antioxidant enzyme values in the plasma, heart, liver, and kidney of Adriamycin-treated rats. Turkish J Biol. 2015;39(6):325–931.
28. Shokrzadeh M, Ahmadi A, Naghshvar F, Chabra A, Jafarinejhad M. Prophylactic efficacy of melatonin on cyclophosphamide-induced liver toxicity in mice. Biomed Res Int. 2014;2014.
29. Liang YL, Zhang ZH, Liu XJ, Liu XQ, Tao L, Zhang YF, et al. Melatonin Protects against Apoptosis-Inducing Factor (AIF)-Dependent Cell Death during Acetaminophen-Induced Acute Liver Failure. PLoS One. 2012;7(12).
30. Shirazi A, Mihandoost E, Ghobadi G, Mohseni M, Ghazi-Khansari M. Evaluation of radio-protective effect of melatonin on whole body irradiation induced liver tissue damage. Cell J. 2013;14(4):292–7. 31. Tiao MM, Huang LT, Chen CJ, Sheen JM, Tain YL, Chen CC, et al. Melatonin in the regulation of liver
steatosis following prenatal glucocorticoid exposure. Biomed Res Int. 2014;2014.
32. Sun H, Huang FF, Qu S. Melatonin: A potential intervention for hepatic steatosis. Lipids Health Dis [Internet]. 2015;14(1):1–6. Available from: http://dx.doi.org/10.1186/s12944-015-0081-7
33. Colares JR, Schemitt EG, Hartmann RM, Licks F, Do Couto Soares M, Dal Bosco A, et al. Antioxidant and anti-inflammatory action of melatonin in an experimental model of secondary biliary cirrhosis induced by bile duct ligation. World J Gastroenterol. 2016;22(40):8918–28.
34. Wang TH, Hsueh C, Chen CC, Li WS, Yeh CT, Lian JH, et al. Melatonin inhibits the progression of hepatocellular carcinoma through microRNA let7i-3p mediated RAF1 reduction. Int J Mol Sci. 2018;19(9):1–15.
35. Karnewar S, Vasamsetti SB, Gopoju R, Kanugula AK, Ganji SK, Prabhakar S, et al. Mitochondria-targeted esculetin alleviates mitochondrial dysfunction by AMPK-mediated nitric oxide and SIRT3 regulation in endothelial cells: Potential implications in atherosclerosis. Sci Rep [Internet]. 2016;6(April):1–18. Available from: http://dx.doi.org/10.1038/srep24108
36. Xu L, Zhang W, Kwak M, Zhang LJ, Lee PCW, Jin JO. Protective effect of melatonin against polymicrobial sepsis is mediated by the anti-bacterial effect of neutrophils. Front Immunol. 2019;10(JUN):1–11. 37. Lin YW, Chen TY, Hung CY, Tai SH, Huang SY, Chang CC, et al. Melatonin protects brain against ischemia/
38. Zhang R, Wang X, Ni L, Di X, Ma B, Niu S, et al. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID- 19 . The COVID-19 resource centre is hosted on Elsevier Connect , the company ’ s public news and information . 2020;(January).
39. Anderson G, Reiter RJ. Melatonin: Roles in influenza, Covid-19, and other viral infections. Rev Med Virol. 2020;30(3):1–10.
40. Hooper PL. COVID-19 and heme oxygenase: novel insight into the disease and potential therapies. Cell Stress Chaperones. 2020;
41. Margarita V, Giménez M, Inserra F, Tajer CD, Mariani J. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID- 19 . The COVID-19 resource centre is hosted on Elsevier Connect , the company ’ s public news and information . 2020;(January).
42. Reiter RJ, Abreu-Gonzalez P, Marik PE, Dominguez-Rodriguez A. Therapeutic Algorithm for Use of Melatonin in Patients With COVID-19. Front Med. 2020;7(May):1–7.
43. Jehi L, Ji X, Milinovich A, Erzurum S, Rubin B, Gordon S, et al. Individualizing risk prediction for positive COVID-19 testing: results from 11,672 patients. Chest. 2020;(January).
44. El-missiry MA, El-missiry ZMA, Othman AI. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID- 19 . The COVID-19 resource centre is hosted on Elsevier Connect , the company ’ s public news and information . 2020;(January).
45. Maruta H, He H. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID- 19 . The COVID-19 resource centre is hosted on Elsevier Connect , the company ’ s public news and information . 2020;(January). 46. Wiwanitkit V. Delirium, sleep, COVID-19 and melatonin. Sleep Med. 2020;(January).
47. García IG, García IG, Rodriguez-Rubio M, Rodriguez-Rubio M, Mariblanca AR, Mariblanca AR, et al. A randomized multicenter clinical trial to evaluate the efficacy of melatonin in the prophylaxis of SARS-CoV-2 infection in high-risk contacts (MeCOVID Trial): A structured summary of a study protocol for a randomised controlled trial. Trials. 2020;21(1):1–4.
48. Banerjee A, Czinn SJ, Reiter RJ, Blanchard TG. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID- 19 . The COVID-19 resource centre is hosted on Elsevier Connect , the company ’ s public news and information . 2020;(January).
49. Kloc M, Ghobrial RM, Kubiak JZ. The Role of Genetic Sex and Mitochondria in Response to COVID-19 Infection. Int Arch Allergy Immunol. 2020;77030:1–6.
50. Zhou Y, Hou Y, Shen J, Kallianpur A, Zein J, Culver DA. Zh ou et af t Zh ou al af t. 2020. 1–29 p. 51. Fu Z, Jiao Y, Wang J, Zhang Y, Shen M, Reiter RJ, et al. Cardioprotective Role of Melatonin in Acute
Myocardial Infarction. Front Physiol. 2020;11(April):1–15. 52. https://www.lifelines.nl/researcher
53. Frisher M, Gibbons N, Bashford J, Chapman S, Weich S. Melatonin, hypnotics and their association with fracture: a matched cohort study. Age Ageing. 2016;45(6):801–6.
54. Jetten AM. Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism. Nucl Recept Signal. 2009;7.
6
55. Ding K, Yu ZH, Yu C, Jia YY, He L, Liao CS, et al. Effect of gga-miR-155 on cell proliferation, apoptosis and invasion of Marek’s disease virus (MDV) transformed cell line MSB1 by targeting RORA. BMC Vet Res. 2020;16(1):1–9.
56. Sun X, Dongol S, Qiu C, Xu Y, Sun C, Zhang Z, et al. MiR-652 promotes tumor proliferation and metastasis by targeting RORA in endometrial cancer. Mol Cancer Res. 2018;16(12):1927–39. 57. Molinaro A, Caesar R, L’homme L, Koh A, Ståhlman M, Staels B, et al. Liver-specific RORα deletion
does not affect the metabolic susceptibility to western style diet feeding. Mol Metab [Internet]. 2019;23(March):82–7. Available from: https://doi.org/10.1016/j.molmet.2019.02.010
58. Hsia CW, Lin KC, Lee TY, Hsia CH, Chou DS, Jayakumar T, et al. Esculetin, A coumarin derivative, prevents thrombosis: Inhibitory signaling on PLCγ2-PKC-AKT activation in human platelets. Int J Mol Sci. 2019;20(11).
59. Li L, Greene I, Readhead B, Menon MC, Kidd BA, Uzilov A V., et al. Novel therapeutics identification for fibrosis in renal allograft using integrative informatics approach. Sci Rep. 2017;7(November 2016):1– 14.
60. Keizo S, Hiromichi O, Shigeru A. Selective inhibition of platelet lipoxygenase by esculetin. Biochim Biophys Acta (BBA)/Lipids Lipid Metab. 1982;713(1):68–72.
61. Roeb E. Matrix metalloproteinases and liver fibrosis ( translational aspects ). Matrix Biol [Internet]. 2018;68–69:463–73. Available from: https://doi.org/10.1016/j.matbio.2017.12.012
62. Geervliet E, Bansal R. Matrix Metalloproteinases as Potential Biomarkers and Therapeutic Targets in Liver Diseases. Cells. 2020;9(5).
63. Roehlen N, Crouchet E, Baumen TE. Liver Fibrosis : Mechanistic Concepts and. Cells. 2020. 1–43 p. 64. Of PI, Human IW. Pseudoperoxidase Investigations of. 2014;21(13):3894–9.
65. Li L, Liu YR, Gao S, Li JF, Li SS, Zhang DD, et al. Inhibition of 5-lipoxygenase pathway attenuates acute liver failure by inhibiting macrophage activation. J Immunol Res. 2014;2014.
66. Lazic M, Inzaugarat ME, Povero D, Zhao IC, Chen M, Nalbandian M, et al. Reduced dietary omega-6 to omega-3 fatty acid ratio and 12/15-lipoxygenase deficiency are protective against chronic high fat diet-induced steatohepatitis. PLoS One. 2014;9(9):1–11.
67. Higgins CB, Zhang Y, Mayer AL, Fujiwara H, Stothard AI, Graham MJ, et al. Hepatocyte ALOXE3 is induced during adaptive fasting and enhances insulin sensitivity by activating hepatic PPARγ. JCI insight. 2018;3(16):1–18.
68. Takeda T, Komiya Y, Koga T, Ishida T, Ishii Y, Kikuta Y, et al. Dioxin-induced increase in leukotriene B4 biosynthesis through the aryl hydrocarbon receptor and its relevance to hepatotoxicity owing to neutrophil infiltration. J Biol Chem. 2017;292(25):10586–99.
69. Rzodkiewicz P, Gąsińska E, Gajewski M, Bujalska-Zadrozny M, Szukiewicz D, Maśliński S. Esculetin reduces leukotriene B4 level in plasma of rats with adjuvant-induced arthritis. Reumatologia. 2016;54(4):161–4.
70. Luo L, Tanaka R, Kanazawa S, Lu F, Hayashi A, Yokomizo T, et al. A synthetic leukotriene B4 receptor type 2 agonist accelerates the cutaneous wound healing process in diabetic rats by indirect stimulation of fibroblasts and direct stimulation of keratinocytes. J Diabetes Complications [Internet]. 2017;31(1):13–20. Available from: http://dx.doi.org/10.1016/j.jdiacomp.2016.09.002
71. Ezhilarasan D, Sokal E, Najimi M. Hepatic fibrosis: It is time to go with hepatic stellate cell-specific therapeutic targets. Hepatobiliary Pancreat Dis Int [Internet]. 2018;17(3):192–7. Available from: https://doi.org/10.1016/j.hbpd.2018.04.003
72. Kinnman N, Goria O, Wendum D, Gendron MC, Rey C, Poupon R, et al. Hepatic stellate cell proliferation is an early platelet-derived growth factor-mediated cellular event in rat cholestatic liver injury. Lab Investig. 2001;81(12):1709–16.
73. Ying HZ, Chen Q, Zhang WY, Zhang HH, Ma Y, Zhang SZ, et al. PDGF signaling pathway in hepatic fibrosis pathogenesis and therapeutics (Review). Mol Med Rep. 2017;16(6):7879–89.
74. Dewidar B, Soukupova J, Fabregat I, Dooley S. TGF-β in Hepatic Stellate Cell Activation and Liver Fibrogenesis: Updated. Curr Pathobiol Rep. 2015;3(4):291–305.
75. Mcgann PT, Ware RE, Children C. Hydroxyurea therapy for sickle cell anemia. Expert Opin Drug Saf. 2018;14(11):1749–58.
76. Elford HL. Effect of hydroxyurea on ribonucleotide reductase. Biochem Biophys Res Commun. 1968;33(1):129–35.
77. Juan PT, Paul M, Andres FC. Novel targeted therapies for the management of liver fibrosis. Expert Opin Emerg Drugs. 2020 Mar;25(1):59-70.doi: 10.1080/14728214.2020.1735350. Epub 2020 Mar 4
78. Christian T, Scott LF, Detlef S, Massimo P. Hepatic fibrosis: Concept to treatment. J Hepatol. 2015 Apr;62(1 Suppl):S15-24. doi: 10.1016/j.jhep.2015.02.039.
