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
The liver has a unique regenerative response towards injuries. After removal of the harmful source, the mass, architecture and function of liver tissue can be entirely restored. However, in chronic conditions when the injury is long-lasting and persistent, the healing response shifts to fibrogenesis where scar-forming connective tissue gradually disrupts the liver function leading to cirrhosis and increasing the risk for liver cancer. Cirrhosis, as the terminal stage of progressive liver fibrosis, is the 12th most common cause of death worldwide. Over a million patients die from cirrhosis every year, which is 2.2% of all deaths annually [1,2]. Liver fibrosis develops in response to a variety of triggers, such as viral hepatitis, excess alcohol consumption, non-alcoholic fatty liver disease (NAFLD), autoimmune hepatitis, hemochromatosis, as well as hereditary diseases, including progressive familial intrahepatic cholestasis (PFIC), Wilson’s disease, haemochromatosis and α1-anti-trypsin deficiency. In the final stages of cirrhosis, the liver may fail with systemic complications, such as portal hypertension-induced variceal bleeding, ascites, hepatic encephalopathy, bacterial infection and often hepatocellular carcinoma (HCC). Although there is no medicine to directly target (advanced) fibrosis, accumulating clinical and experimental evidence show that liver fibrosis can be halted and even reversed using combination therapies [3–6]. The common ground for all therapeutic approaches consists of attenuating pathological mechanisms and simultaneously promoting liver regeneration. Liver fibrosis begins with deposition of connective tissue (components of the extracellular matrix (ECM), particularly collagen I, collagen III and fibronectin) in and around inflamed or damaged areas [7]. The source of the abnormal ECM are myofibroblasts. Exposure to a variety of triggers, including inflammatory molecules and oxidative stress, myofibroblasts, in particular Hepatic Stellate Cells (HSC), undergo cell-intrinsic changes to become ‘activated’. Activated myofibroblasts are migratory, contractile and proliferative and mostly derive from two mesenchymal origins, Hepatic Stellate Cells and portal myofibroblasts. Extracellular signals from other cells, such as macrophages, hepatocytes, liver sinusoidal endothelial cells, natural killer (T) cells, platelets and B cells, promote HSC activation. The external stimuli from other cells are mainly paracrine factors, such as platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ) and other growth factors together with cytokines and chemokines [8]. The function and morphology of HSC-derived myofibroblasts are very similar to those that originate from portal myofibroblast. Thus, the therapeutic strategies that aim to block the trans-differentiation from the mesenchymal phenotype to the fibroblast phenotype are similar and will target both types. However, HSCs appear to be the primary target for direct treatment of liver fibrosis, as they are the major source of myofibroblasts (over 80%) in rodent models of liver fibrosis, including chronic CCl4 exposure, bile duct ligation, the 3,5-diethoxycarbonyl-1,4- dihydrocollidin diet and Mdr2-knockout mice [9].
1
Quiescent Hepatic Stellate Cells (qHSCs)
In healthy conditions, HSCs are in a ‘quiescent’ state populating about 15% of all cells in the liver. qHSCs are located in the subendothelial space of Disse between hepatocytes and sinusoids that carry nutrient- and oxygen-rich blood to the hepatocytes [10,11]. HSCs are relatively small in size compared to hepatocytes and characterized by large cytoplasmic lipid droplets. The HSC lipid droplets are specialized organelles for storage and metabolism of retinoids (e.g. vitamin A) and store about 80-90% of total retinoids of the liver and about 50-80% of total retinoids in the body. HSCs are responsible for maintaining stable levels of circulating retinyl in blood at around 2 µmol/L in humans and 1-1.5 µmol/L in rodents[1]. Most retinoids in the cytoplasmic droplets are stored in the form of retinyl esters, predominantly as retinyl palmitate. The composition and size of these lipid droplets are dependent on dietary intake. Besides retinoids, other lipids, such as triglycerides, phospholipids, cholesterol and free fatty acids, are abundantly deposited in HSC lipid droplets [11,12].
The uptake, transport and storage of dietary vitamin A is a complex process. In the intestinal epithelium, dietary carotenes are converted to retinyl esters and transported to the liver via circulation within chylomicrons. Hepatocytes take up the chylomicrons and hydrolyze retinyl esters to retinyl, which is distributed from the hepatocyte to the circulation bound to the retinyl binding protein 4 (RBP4). HSCs absorb retinyls from the blood and convert it back to retinyl esters and store it in lipid droplets [13–16]. As HSC activation is trigged, cells rapidly lose their lipid droplets, including the vitamin A content, while transdifferentiating into myofibroblasts. As a consequence, vitamin A deficiency is associated with advanced liver fibrosis [13].
Vitamin A homeostasis in HSC is achieved by enzymes that esterify and hydrolyse retinyl and retinyl esters, respectively. Lecithin retinyl acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) are responsible for retinyl esterification [12,17]. Two esterases, adipose triglyceride lipase/patatin-like phospholipase domain containing 2 (ATGL/PNPLA2) and adiponutrin (ADPN/PNPLA3), have been identified as retinyl ester hydrolases (REH) in HSC [18–20]. LRAT appears to be essential for hepatic retinyl esterification, as LRAT-null mice are prone to develop vitamin A deficiency (VAD) [21,22]. For retinyl ester hydrolysis, PNPLA3 seems to play a rate-limiting role, as specific PNPLA3 gene variants in humans are associated with hepatic retinyl ester accumulation and serum retinyl levels(20). In contrast, ATGL-null mice have normal hepatic vitamin A levels implying that ATGL, while being an established REH, is not essential for maintaining hepatic retinoid metabolism [18]. The expression of vitamin A-associated enzymes, such as LRAT or PNPLA3, rapidly drops during HSC activation, along with
the disappearance of lipid droplets. There is a third REH, hormone-sensitive lipase (HSL), which is dominantly expressed in adipose tissue, but is also detected in the liver [23,24]. So far, hepatic HSL is reported to be expressed only in hepatocytes, where it mainly hydrolyses cholesteryl esters to cholesterol [24,25]. Nevertheless, the role of HSL in retinoid metabolism in HSC has not been studied in detail yet.
Key factors in HSC activation and liver fibrogenesis
The extracellular matrix (ECM) is a network of proteins that provides micro-scaffolds for cells supporting cellular adhesion, migration, differentiation and proliferation. In the liver, ECM is present in the Glisson’s capsule, portal tracks, central veins and in the perisinusoidal space of Disse [26]. Collagens (I, III, IV, and V), fibronectin, laminin and proteoglycans are the predominant proteins of ECM. Collagen types I, III, and V are predominantly expressed in the portal and central areas, whereas collagen IV is localized mainly in basement membranes [27]. Fibrotic liver is highly enriched in collagen I and III (7) and about 80% is produced by HSCs, in particular collagen I [28].
Matrix metalloproteinase proteins (MMPs) are responsible for degradation of ECM proteins and, together with tissue Inhibitors of metalloproteinases (TIMPs), have fundamental roles in ECM remodeling during liver fibrosis and in fibrosis resolution. While activated HSC are the prime producers of ECM proteins, in particular collagen I, III and IV, they also express MMPs and TIMPs. Especially the expression of TIMP-1, is up-regulated in activated HSCs, thereby reducing MMP activities and promoting the accumulation of ECM (29,30). MMP-2, MMP-9, and MMP-13 are among highly-expressed proteins in activated HSCs. MMP-2, MMP-8 and MMP-9 activities are significantly elevated in alcoholic liver disease and can function as serum markers of disease severity [31]. MMP-13 activity can be used for diagnosis of alcoholic liver cirrhosis [29]. Besides induced ECM production, HSC activation is characterized by elevated expression of alpha-smooth muscle actin (α-SMA). α-SMA is a cytoskeleton protein that has a major role in mobility and contractility of activated HSC [32]. The contractile force of α-SMA is stronger than other actin isoforms in fibroblastic cells [33].
Transforming growth factor β1 (TGF-β1) is a key mediator of HSC activation upon liver. TGF-β1 is a multifunctional cytokine that regulates a variety of cellular functions, including proliferation, differentiation, apoptosis and migration [34]. During HSC activation, TGF-β1 binds to it receptors and forms a complex that directly activates Smad signalling leading to overexpression of pro-fibrotic genes. Smad2 and Smad3 are the two major downstream regulators that mediate
TGF-1
β1-induced tissue fibrosis, while Smad7 serves as a negative feedback regulator of TGF-β1/ Smad pathway and suppresses TGF-β1-mediated fibrosis [35,36].
PDGF is one of the most potent mitogens that stimulates HSC activation. Upon binding to its receptors at the membrane of HSC, PDGF triggers a signalling cascade that leads to upregulation of collagen synthesis, as well as MMP-2, MMP-9 and TIMP-1, the latter of which prevents ECM degradation [37,38]. PDGF-B and PDGF-D are the most fibrogenic isoforms of PDGF and cause PDGFR-β autophosphorylation that activates the extracellular signal-regulated kinase (ERK)1/2, C-Jun N-terminal kinase (JNK), p38 mitogen- activated protein kinase (MAPK) and protein kinase (PK)B/Akt pathways leading to further proliferation and activation of HSCs and damage to hepatocytes [37,39].
Leukotrienes are pro-inflammatory mediators that increase microvascular permeability and causes leukocyte infiltration in inflamed areas in the fibrotic liver [40]. Leukotrienes are synthesized from essential fatty acids (EFA), e.g. arachidonic acid, by the enzyme arachidonate 5-lipoxygenase, also known as 5-lipoxygenase (5-LO) (36). The inhibition of 5-LO suppresses renal fibrosis and progression of chronic kidney disease by preventing the production of leukotrienes [41]. Moreover, 5-LO, is one of the main contributors of lipid peroxidation that further increases tissue inflammation. Besides lipoxygenases, cyclooxygenases and cytochrome P450 enzymes oxidize EFAs, however, lipoxygenases contribute most prominently to the generation of lipid peroxides [42].
Therapeutic approaches to halt and/or reverse liver fibrosis
So far, the only effective way to stop or cure liver fibrosis is to eliminate the pathological stimulus that causes chronic liver damage and/or inflammation. The stimulus can be long-term exposure of a toxic agent, fatty diet, alcohol consumption, viral or bacterial infection. Clinical studies have shown that therapies aiming at elimination of the primary disease cause, e.g. antivirals in viral hepatitis, can regress liver fibrosis, even at advance stages [43–45]. Consequently, treatment against HCV infections [46–48], alcohol abstinence [49] and weight loss (50) have been reported to be effective to reverse liver fibrosis.
However, in most cases of chronic liver disease, it is challenging or even currently impossible to eliminate the primary cause of disease. As an alternative approach, anti-inflammatory drugs and/or immunosuppressants are commonly used. Inflammation is a key factor that drives liver fibrosis and is regulated by proinflammatory and anti-inflammatory factors secreted from
Kupffer cells and other recruited immune cells, such as bone marrow–derived macrophages, neutrophils and T lymphocytes. Chronic inflammation leads to progressive hepatocyte damage and activation of HSCs and portal myofibroblasts [51]. Common anti-inflammatory drugs are corticosteroids, such as prednisone and prednisolone, and are used to suppress cytokine production through transcriptional regulation [52–54]. Zileuton, a 5-lipoxygenase inhibitor, blocks leukotriene-mediated pathways and can be considered for clinical use [55– 57]. Pentoxifylline (PTX) is another anti-inflammatory drug that suppresses Tumor Necrosis Factor-alfa (TNF-α) synthesis [58]. Recent studies report celecoxib [59], azathioprine [47] and rapamycin [60,61] as suppressors of fibrogenesis through their potent immunomodulatory properties. Antioxidative agents, such as taurine [47,62] and vitamin E [63] reduce oxidative stress and thereby suppress inflammation and resultant fibrogenesis.
HSCs develop fibrotic structures by producing abnormal extra cellular matrix (ECM) dep ositions, as well by secreting inflammatory cytokines that recruit immune cells to the inflamed area and promote local inflammation. Thus, suppressing activation and promoting apoptosis or senescence of HSCs are alternative approaches to halt fibrosis [64]. Inactivation of HSCs have been achieved in experimental models by inhibiting the principal activation mechanisms, including the TGF-β1/Smad- (35,36,65) and PDGF-[37] signaling pathways. Some cytokines and growth factors, such as Insulin-like growth factor-1 [66] and interleukin-22 [67], induce senescence in HSCs while IFN-α (68) and IFN-γ [10,69] are candidates for induction of HSC apoptosis.
Promoting hepatocyte regeneration is an important strategy to preserve liver function during fibrosis. Ursodeoxycholic acid (UDCA) is a hepatoprotective bile acid used for treatment of various chronic liver diseases, including primary biliary cirrhosis (PBC), NAFLD and intrahepatic cholestasis of pregnancy (ICP) [70,71]. UDCA protects hepatocyte and cholangiocytes by replacing toxic bile acids, inhibition of apoptosis induced by toxic bile acids and stimulation of bile secretion. Hepatocyte growth factor (HGF) has been shown to reduce apoptosis and promote angiogenesis and liver regeneration by stimulating hepatocyte motility and mitogenesis [47,72,73]. Moreover, bone-marrow-derived cells and mesenchymal cells can stimulate proliferation of hepatocytes by producing mitogens, including HGF [72,74,75]. Although the impact of HGF on hepatocyte regeneration has been established in many experimental models of liver diseases, no clinical research is done to further develop HGF into treatment for liver fibrosis, in particular due to the potential risk of inducing cancer.
1
Complementary and alternative medicine for the treatment
of liver fibrosis
Besides developing novel pharmaceutical compounds to target liver fibrosis, natural products may also hold potent anti-fibrotic properties. Melatonin is a hormone produced by the pineal gland and controls the sleep-wake cycle. However, melatonin can also be produced in other tissues, including the liver, and acts as an endogenous antioxidant [76]. Recent studies have revealed that a variety foods and drinks, such as vegetables, cereals, fruits, nuts, seeds, grapes, red wine and beer, contain considerable amounts of melatonin [77–80]. Melatonin is available as supplementary medication, in particular to control the day-night sleep rhythm. Hepatoprotective effects of melatonin have been shown in various liver diseases including, hepatic steatosis [81,82], hepatitis [83], fibrosis [84] and hepatocellular carcinoma [85]. The therapeutic effects of melatonin are mostly attributed to its ability to suppress oxidative stress, inflammatory signaling, hepatocyte apoptosis, neutrophil infiltration and regulation of profibrogenic genes [86,87]. Melatonin reduces the expression of 5-LO [88] and attenuates liver fibrosis in several animal models including, bile duct ligation (BDL)-induced cirrhosis [89], thioacetamide (TAA)-induced fibrosis [90] and carbon tetrachloride (CCl4)-induced fibrosis [91]. However, a direct role of melatonin on HSC activation has not been established yet.
Anti-fibrotic compounds may also be found in plants used in traditional medicine. Esculetin is present in many herbs, including Cortex fraxini, Artemisia capillaries, Citruslimonia, Euphorbia
lathyris and Fraxinus rhynchophylla [92,93]. Esculetin has been used in China for treatment of
liver and gallbladder diseases for centuries. Esculetin has anti-inflammatory and anti-tumor properties. It down-regulates the expression of MMP-1 in cartilage, while it also inhibits the production of pro-inflammatory cytokines, such as TNF-α, in co-cultured adipocytes and macrophages [93]. Furthermore, esculetin is a potent anti-oxidant that ameliorates mitochondrial damage induced by nitric oxide radicals [94], hepatic apoptosis induced by CCl4 [92] and DNA damage caused by lipid hydroperoxide-induced oxidative stress [95]. Moreover, esculetin has anti-hepatitis B virus activity (96) and inhibits 5-LO and 12-LO in leukocytes [97–99]. Other herbal compounds that exhibit anti-fibrotic activities include salvianolic acid B [100], oxymatrine [101], tetrandrine [102], glycyrrhetinic acid [103] and silymarin [104–106]. In all these examples, the pharmacological effects include scavenging free radicals and modulating the inflammatory pathways. In addition to natural compounds, there are drugs that are not preliminary developed for targeting liver fibrosis, yet could theoretically have such therapeutic effect. Verapamil for instance, a calcium channel blocker that is used to treat high blood pressure, has hepatoprotective properties. In NAFLD, verapamil reduces hepatic lipid droplet accumulation, insulin resistance and
steatohepatitis [107]. The combination of verapamil and silymarin improves liver fibrosis and significantly reduces mRNA and protein level of α-SMA [6]. Hydroxycarbamide, also known as hydroxyurea, is an antiproliferative agent, which inhibits DNA synthesis and cell cycle replication by blocking the enzyme ribonucleotide reductase [108]. Hydroxyurea is widely used to treat chronic myelogenous leukemia [109], cervical cancer [109], polycythemia [110] and sickle-cell disease [111]. Cellular absorption of hydroxyurea is an active transport carried out by solute care transporters including multi-specific organic cation transporter (OCTN1), which is expressed by HSCs[112]. Hydroxyurea has not yet been explored as anti-fibrotic agent.
The aim of this thesis
In this thesis, we explored the therapeutic effects of melatonin, esculetin and hydroxyurea on HSC activation in order to establish their potential as anti-fibrotic drugs. Additionally, we investigated the role of HSL in vitamin A homeostasis and activation of HSCs.
HSL is the dominant retinyl ester hydrolyser in adipose tissue. However, its expression and function in liver are less known and the current data is limited to its cholesterol hydrolysing activity in hepatocytes. Giving the fact that HSC lipid droplets abundantly accumulate retinyl esters, in Chapter 2, we analysed HSL expression and function in quiescent and activated HSCs. Numerous studies have examined the therapeutic properties of melatonin in a variety of liver injury models. Yet, its direct effect on HSC activation has not been explored. For possible use of melatonin in a treatment for liver fibrosis, it is essential to fully understand its potential impact on the main contributors of fibrogenesis, the activated HSC. Thus, Chapter 3 of this thesis explores the direct impact of melatonin on HSC activation and its 5-LO- associated inhibitory mechanism.
As stated earlier, the contribution of 5-LO in promoting inflammation is well-established. We hypothesized that inactivating 5-LO using esculetin reduces HSC activation and ameliorates liver fibrosis. In Chapter 4, we therefore studied the direct effect of esculetin on HSC activation and proliferation in vitro and in vivo.
In Chapter 5, we investigated the possibility of using hydroxyurea as an anti-fibrotic drug. For this, the effect of hydroxyurea on HSC activation and hepatocyte preservation and regeneration was investigated. Finally, Chapter 6 provides a summary of relevant findings and results of all experimental chapters followed by a general discussion that highlights possibilities for future research and perspectives of therapeutics for liver fibrosis.
1
References
1. Stanaway JD, Afshin A, Gakidou E, Lim SS, Abate D, Abate KH, et al. Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Stu. Lancet. 2018;392(10159):1923–94.
2. Ginès P, Graupera I, Lammert F, Angeli P, Caballeria L, Krag A, et al. Screening for liver fi brosis in the general population : a call for action. Lancet Gastroenterol Hepatol [Internet]. 2016;1(3):256–60. Available from: http://dx.doi.org/10.1016/S2468-1253(16)30081-4
3. Tsertsvadze T, Gamkrelidze A, Nasrullah M, Sharvadze L, Morgan J, Gvinjilia L, et al. Treatment outcomes of patients with chronic hepatitis C receiving sofosbuvir-based combination therapy within national hepatitis C elimination program in the country of Georgia. J Hepatol. 2017;66(1):S743. 4. Hayashi T, Takatori H, Horii R, Nio K, Terashima T, Iida N, et al. Danaparoid sodium-based anticoagulation
therapy for portal vein thrombosis in cirrhosis patients. BMC Gastroenterol. 2019;19(1):217. 5. Campana L, Iredale JP. Regression of Liver Fibrosis. Semin Liver Dis. 2017;37(1):1–10.
6. Shafik AN, Khodeir MM, Gouda NA, Mahmoud ME. Improved antifibrotic effect of a combination of verapamil and silymarin in rat-induced liver fibrosis. Arab J Gastroenterol [Internet]. 2011;12(3):143– 9. Available from: http://dx.doi.org/10.1016/j.ajg.2011.07.001
7. Roehlen N, Crouchet E, Baumen TE. Liver Fibrosis : Mechanistic Concepts and. Cells. 2020. 1–43 p. 8. Lee YA, Wallace MC, Friedman SL. Pathobiology of liver fibrosis: a translational success story. Gut,
2015;64(5):830–41.
9. Mederacke I, Hsu CC, Troeger JS, Huebener P, Mu X, Dapito DH, et al. contributors to liver fibrosis independent of its etiology. 2014;
10. Higashi T, Friedman SL, Hoshida Y. Hepatic stellate cells as key target in liver fibrosis. Adv Drug Deliv Rev. 2017;121:27-42.
11. Friedman SL. Hepatic Stellate Cells: Protean, Multifunctional, and Enigmatic Cells of the Liver. Physiol Rev. 2010;88(1):125–72. doi: 10.1152/physrev.00013.2007
12. Blaner WS, O’Byrne SM, Wongsiriroj N, Kluwe J, D’Ambrosio DM, Jiang H, et al. Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage. Biochim Biophys Acta [Internet]. 2009 Jun [cited 2014 Jun 19];1791(6):467–73. Available from: http://www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=2719539&tool=pmcentrez&rendertype=abstract
13. Saeed A, Dullaart RPF, Schreuder TCMA, Blokzijl H, Faber KN. Disturbed Vitamin A Metabolism in Non-Alcoholic Fatty Liver Disease ( NAFLD ). :1–25.
14. Zhong M, Kawaguchi R, Kassai M, Sun H. Retina, Retinyl, Retinal and the Natural History of Vitamin A as a Light Sensor. 2012;2069–96.
15. Review BVA, Tanumihardjo SA, Russell RM, Stephensen CB, Gannon BM, Craft NE, et al. Biomarkers of Nutrition for Development. 2016;
16. Grumet L, Taschler U, Lass A. Hepatic retinyl ester hydrolases and the mobilization of retinyl ester stores. Nutrients. 2017;9(1):1–21.
17. Ajat M, Molenaar M, Brouwers JFHM, Vaandrager AB, Houweling M, Helms JB. Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT. Biochim Biophys Acta - Mol Cell Biol Lipids [Internet]. 2017;1862(2):176–87. Available from: http://dx.doi.org/10.1016/j.bbalip.2016.10.013
18. 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. 19. Zhang Z, Guo M, Shen M, Li Y, Tan S, Shao J, et al. Oroxylin A regulates the turnover of lipid droplet
via downregulating adipose triglyceride lipase (ATGL) in hepatic stellate cells. Life Sci [Internet]. 2019;238(October):116934. Available from: https://doi.org/10.1016/j.lfs.2019.116934
20. Dongiovanni P, Meroni M, Longo M, Fargion S, Fracanzani AL. miRNA Signature in NAFLD : A Turning Point for a Non-Invasive Diagnosis. 2018;3.
21. 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.
22. Amengual J, Golczak M, Palczewski K, Von Lintig J. Lecithin: Retinyl acyltransferase is critical for cellular uptake of vitamin A from serum retinyl-binding protein. J Biol Chem. 2012;287(29):24216–27. 23. 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.
24. 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.
25. Mello T, Nakatsuka A, Fears S, Davis W, Bosron WF, Sanghani SP. types. 2009;374(3):460–4. 26. Bedossa P, Paradis V. Liver extracellular matrix in health and disease. J Pathol. 2003;200(4):504–15. 27. Woalder. 乳鼠心肌提取 HHS Public Access. Physiol Behav. 2017;176(1):139–48.
28. Zhang CY, Yuan WG, He P, Lei JH, Wang CX. Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets. World J Gastroenterol. 2016;22(48):10512–22.
29. 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
30. Robert S, Gicquel T, Bodin A, Lagente V, Boichot E. Characterization of the MMP / TIMP Imbalance and Collagen Production Induced by IL-1 β or TNF- α Release from Human Hepatic Stellate Cells. 2016;1–14.
31. Prystupa A, Boguszewska-czubara A, Bojarska-junak A, Toruń-jurkowska A, Roliński J, Załuska W. Activity of MMP-2 , MMP-8 and MMP-9 in serum as a marker of progression of alcoholic liver disease in people from Lublin Region , eastern Poland. 2015;22(2):325–8.
32. Liu Z, Chang AN, Grinnell F, Trybus KM, Milewicz DM, Stull JT, et al. Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts. Proc Natl Acad Sci U S A. 2017;114(28):E5569–78.
33. Hinz B, Phan SH, Thannickal VJ, Prunotto M, Desmoulire A, Varga J, et al. Recent developments in myofibroblast biology: Paradigms for connective tissue remodeling. Am J Pathol. 2012;180(4):1340– 55.
34. Kim JY, An HJ, Kim WH, Gwon MG, Gu H, Park YY, et al. Anti-fibrotic Effects of Synthetic Oligodeoxynucleotide for TGF-β1 and Smad in an Animal Model of Liver Cirrhosis. Mol Ther - Nucleic Acids. 2017;8(September):250–63.
35. Wang K, Fang S, Liu Q, Gao J, Wang X, Zhu H, et al. EBioMedicine TGF- β 1 / p65 / MAT2A pathway regulates liver fi brogenesis via intracellular SAM. EBioMedicine [Internet]. 2019;1–12. Available from: https://doi.org/10.1016/j.ebiom.2019.03.058
36. Dai J, Xu M, Zhang X, Niu Q, Hu Y, Li Y, et al. Bi-directional regulation of TGF- β / Smad pathway by arsenic : A systemic review and meta-analysis of in vivo and in vitro studies. 2019;220(November
1
2018):92–105.
37. Ying HUAZ, Chen QIN, Zhang WENYOU, Zhang HH. PDGF signaling pathway in hepatic fibrosis pathogenesis and therapeutics ( Review ). 2017;7879–89.
38. Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev. 1999;79(4):1283–316.
39. Dijk F Van. Targeted therapies in liver fibrosis : combining the best parts of platelet-derived growth factor BB and interferon gamma. 2015;2(October).
40. El-Swefy S, Hassanen SI. Improvement of hepatic fibrosis by leukotriene inhibition in cholestatic rats. Vol. 8, Annals of Hepatology. 2009. p. 41–9.
41. Montford JR, Bauer C, Dobrinskikh E, Hopp K, Levi M, Weiser-Evans M, et al. Inhibition of 5-lipoxygenase decreases renal fibrosis and progression of chronic kidney disease. Am J Physiol - Ren Physiol. 2019;316(4):F732–42.
42. Sun QY, Zhou HH, Mao XY. Emerging Roles of 5-Lipoxygenase Phosphorylation in Inflammation and Cell Death. Oxid Med Cell Longev. 2019;2019.
43. Zimmermann HW, Tacke F. In search of the magic bullet: Can liver inflammation and fibrosis be reversed with medications? Expert Rev Gastroenterol Hepatol. 2015;9(9):1139–41.
44. Jung YK, Yim HJ. Reversal of liver cirrhosis: Current evidence and expectations. Korean J Intern Med. 2017;32(2):213–28.
45. Arthur MJP. Reversibility of liver fibrosis and cirrhosis following treatment for hepatitis C. Gastroenterology. 2002;122(5):1525–8.
46. Berenguer M, Schuppan D. Progression of liver fibrosis in post-transplant hepatitis C: Mechanisms, assessment and treatment. J Hepatol [Internet]. 2013;58(5):1028–41. Available from: http://dx.doi. org/10.1016/j.jhep.2012.12.014
47. Zhou WC, Zhang QB, Qiao L. Pathogenesis of liver cirrhosis. World J Gastroenterol. 2014;20(23):7312– 24.
48. T. W, M. A, E. G, C. J, J. R. Diagnosis and management of hepatitis C. Am Fam Physician [Internet]. 2015;91(12):835–42. Available from: http://www.embase.com/search/results?subaction=viewrecord &from=export&id=L604876189%5Cnhttp://ak7rt6cb3z.search.serialssolutions.com?sid=EMBASE &issn=15320650&id=doi:&atitle=Diagnosis+and+management+of+hepatitis+C&stitle=Am.+Fam. +Ph ys.&title=American+Famil
49. Stickel F, Datz C, Hampe J, Bataller R. Pathophysiology and management of alcoholic liver disease: Update 2016. Gut Liver. 2017;11(2):173–88.
50. Ellis EL, Mann DA. Clinical evidence for the regression of liver fibrosis. J Hepatol [Internet]. 2012;56(5):1171–80. Available from: http://dx.doi.org/10.1016/j.jhep.2011.09.024
51. Koyama Y, Brenner DA, Koyama Y, Brenner DA. Liver inflammation and fibrosis Find the latest version : Liver inflammation and fibrosis. 2017;127(1):55–64.
52. Xue J, Feng R, Fu H, Jiang Q, Jiang H, Lu J, et al. Combined prednisone and levothyroxine improve treatment of severe thrombocytopenia in hepatitis B with compensatory cirrhosis accompanied by subclinical and overt hypothyroidism. 2018;61(8):924–5.
53. Kawaratani H, Tsujimoto T, Douhara A, Takaya H, Moriya K, Namisaki T, et al. The Effect of Inflammatory Cytokines in Alcoholic Liver Disease. 2013;2013.
54. Beretta-piccoli BT, Mieli-vergani G, Vergani D, Beretta-piccoli BT, Ticino E. Autoimmune hepatitis : standard treatment and systematic review of alternative treatments. 2017;23(33):6030–48.
55. Abueid L, Uslu Ü, Cumbul A, Öğünç AV, Ercan F, Alican İ. Inhibition of 5-lipoxygenase by zileuton in a rat model of myocardial infarction. Anatol J Cardiol. 2017;17(4):269–75.
56. Gounaris E, Heiferman MJ, Heiferman JR, Shrivastav M, Vitello D, Blatner NR, et al. Zileuton, 5-lipoxygenase inhibitor, acts as a chemopreventive agent in intestinal polyposis, by modulating polyp and systemic inflammation. PLoS One. 2015;10(3):1–13.
57. Dahlin A, Qiu W, Litonjua AA, Lima JJ, Tamari M, Kubo M, et al. The phosphatidylinositide 3-kinase (PI3K) signaling pathway is a determinant of zileuton response in adults with asthma. Pharmacogenomics J [Internet]. 2018;18(5):665–77. Available from: http://dx.doi.org/10.1038/s41397-017-0006-0 58. Alam S, Hasan SKMN, Mustafa G, Alam M. Effect of pentoxifylline on histological activity and fibrosis
of nonalcoholic steatohepatitis patients : A one year randomized control trial. 2017;5(3):155–63. 59. Zhang J, Wang M, Zhang Z, Luo Z, Liu F, Liu J. Celecoxib derivative OSU-03012 inhibits the proliferation
and activation of hepatic stellate cells by inducing cell senescence. Mol Med Rep. 2015;11(4):3021–6. 60. Wei R, Liu H, Chen RU, Sheng Y, Liu TAO. Astragaloside Ⅳ combating liver cirrhosis through the PI3K /
Akt / mTOR signaling pathway. Exp Ther Med. 2019;393–7.
61. Liu J, Jiang C, Feng Y, Zhu W, Jin B, Xia Y, et al. Rapamycin inhibits peritoneal fibrosis by modifying lipid homeostasis in the peritoneum. 2019;11(3):1473–85.
62. Yu Y, Ni X, Huang J, Zhu Y, Qi Y. International Journal for Parasitology : Drugs and Drug Resistance Taurine drinking ameliorates hepatic granuloma and fi brosis in mice infected with Schistosoma japonicum. Int J Parasitol Drugs Drug Resist [Internet]. 2016;6(1):35–43. Available from: http://dx.doi. org/10.1016/j.ijpddr.2016.01.003
63. Wang X, Zhang R, Du J, Hu Y, Xu L, Lu JUN, et al. Vitamin E reduces hepatic fibrosis in mice with Schistosoma japonicum infection. 2012;465–8.
64. Zhang Z, Yao Z, Zhao S, Shao J, Chen A, Zhang F, et al. Interaction between autophagy and senescence is required for dihydroartemisinin to alleviate liver fibrosis. Cell Death Dis [Internet]. 2017;8(6). Available from: http://dx.doi.org/10.1038/cddis.2017.255
65. Deng K. Ferulic acid attenuates liver fibrosis and hepatic stellate cell activation via inhibition of TGF- β / Smad signaling pathway. 2018;4107–15.
66. Takahashi Y. The Role of Growth Hormone and Insulin-Like Growth Factor-I in the Liver. Int J Mol Sci. 2017 Jul 5;18(7):1447. doi: 10.3390/ijms18071447.
67. Sziksz E, Pap D, Lippai R, et al. Fibrosis Related Inflammatory Mediators: Role of the IL-10 Cytokine Family. Mediators Inflamm. 2015;2015:764641. doi:10.1155/2015/764641
68. Chang X, Chang Y, Jia A. Effects of interferon-alpha on expression of hepatic stellate cell and transforming growth factor- 1 and -smooth muscle actin in rats with hepatic fibrosis. 2005;11(17):2634–6. 69. Horras CJ, Lamb CL, Mitchell KA. IFN-γ inhibits liver progenitor cell proliferation in HBV-infected
patients and in 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet-fed mice, Journal of Hepatology, 2012;22(1):35–43. Doi: 10.1016/j.jhep.2013.05.041.
70. Santiago P, Scheinberg AR, Levy C. Cholestatic liver diseases : new targets , new therapies. 2018;1–15. 71. Ikebuchi Y, Shimizu H, Ito K, Yoshikado T, Yamanashi Y, Takada T, et al. Ursodeoxycholic acid stimulates the formation of the bile canalicular network. Biochem Pharmacol [Internet]. 2012;84(7):925–35. Available from: http://dx.doi.org/10.1016/j.bcp.2012.07.008
72. Physiology C. Human Adipose Derived Stem Cells Exhibit Enhanced Liver Regeneration in Acute Liver Injury by Controlled Releasing Hepatocyte Growth Factor. 2019;190(32):935–50.
73. Moon SH, Lee CM, Park SH, Jin Nam M. Effects of hepatocyte growth factor gene-transfected mesenchymal stem cells on dimethylnitrosamine-induced liver fibrosis in rats. Growth Factors
1
[Internet]. 2019;37(3–4):105–19. Available from: https://doi.org/10.1080/08977194.2019.1652399 74. Tricot T, De Boeck J, Verfaillie C. Alternative Cell Sources for Liver Parenchyma Repopulation: Where
Do We Stand? Cells. 2020;9(3):566.
75. Montanari E, Pimenta J, Szabó L, Noverraz F, Passemard S, Meier RPH, et al. Beneficial Effects of Human Mesenchymal Stromal Cells on Porcine Hepatocyte Viability and Albumin Secretion. J Immunol Res. 2018;2018.
77. Iriti M. Melatonin in grape, not just a myth, maybe a panacea. J Pineal Res. 2009;46(3):353. 78. Maldonado MD, Moreno H, Calvo JR. Melatonin present in beer contributes to increase the levels
of melatonin and antioxidant capacity of the human serum. Clin Nutr. 2009;28(2):188–91. Available from: http://dx.doi.org/10.1016/j.clnu.2009.02.001
79. Manchester LC, Tan D-X, Reiter RJ, Park W, Monis K, Qi W. High levels of melatonin in the seeds of edible plants. Life Sci. 2000;67(25):3023–9.
80. Arnao MB, Hernández-Ruiz J. The potential of phytomelatonin as a nutraceutical. Molecules. 2018;23(1):1–19.
81. Mahmoud GS, El-Deek HE. Melatonin modulates inflammatory mediators and improves olanzapine-induced hepatic steatosis in rat model of schizophrenia. Int J Physiol Pathophysiol Pharmacol. 2019;11(3):64–75.
82. Stacchiotti A, Grossi I, García-Gómez R, et al. Melatonin Effects on Non-Alcoholic Fatty Liver Disease Are Related to MicroRNA-34a-5p/Sirt1 Axis and Autophagy. Cells. 2019;8(9):1053. Published 2019 Sep 8. doi:10.3390/cells8091053
83. Oleshchuk O, Ivankiv Y, Falfushynska H, Mudra A, Lisnychuk N. Hepatoprotective effect of melatonin in toxic liver injury in rats. Med. 2019;55(6):1–11.
84. 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.
85. 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. 86. 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.
87. 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.
88. Radogna F, Albertini MC, De Nicola M, Diederich M, Bejarano I, Ghibelli L. Melatonin promotes Bax sequestration to mitochondria reducing cell susceptibility to apoptosis via the lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid. Mitochondrion [Internet]. 2015;21:113–21. Available from: http:// dx.doi.org/10.1016/j.mito.2015.02.003
89. 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.
90. Czechowska G, Celinski K, Korolczuk A, Wojcicka G, Dudka J, Bojarska A, et al. Protective effects of melatonin against thioacetamide-induced liver fibrosis in rats. J Physiol Pharmacol. 2015;66(4):567– 79.
91. 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.
92. 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.
93. Liang C, Ju W, Pei S, Tang Y, Xiao Y. Pharmacological Activities and Synthesis of Esculetin and Its Derivatives: A Mini-Review. Molecules. 2017;22(3):387. Published 2017 Mar 2. doi:10.3390/ molecules22030387
94. 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
95. Kaneko T, Tahara S, Takabayashi F. Suppression of lipid hydroperoxide-induced oxidative damage to cellular DNA by esculetin. Biol Pharm Bull. 2003;26(6):840–4.
96. 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.
97. Neichi T, Koshihara Y, Murota S.Inhibitory effect of esculetin on 5-lipoxygenase and leukotriene biosynthesis. Biochim Biophys Acta. 1983 Aug 29;753(1):130-2.
98. 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.
99. Tomohiro N, Yasuko K, Sei-Itsu M. Inhibitory effect of esculetin on 5-lipoxygenase and leukotriene biosynthesis. Biochim Biophys Acta (BBA)/Lipids Lipid Metab. 1983;753(1):130–2.
100. Yu F, Lu Z, Chen B, Wu X, Dong P. Salvianolic acid B-induced microRNA-152 inhibits liver fibrosis by attenuating DNMT1-mediated Patched1 methylation. 2015;19(11):2617–32.
101. Wu J, Pan L, Jin X, Li W, Li H, Chen J, et al. The role of oxymatrine in regulating TGF-β1 in rats with. 33(3):207–15.
102. Yin M, Lian L, Piao D, Nan J. Tetrandrine stimulates the apoptosis of hepatic stellate cells and ameliorates development of fibrosis in a thioacetamide rat model. 2007;13(8):1214–20.
103. Alho DPS, Salvador JAR, Cascante M, Marin S. Synthesis and Antiproliferative Activity of Novel Heterocyclic Glycyrrhetinic Acid Derivatives. 2019;1–21.
104. Jakub Š, Ja J, Biedermann D, Petrásková L, Vladimír K, Muchová L, et al. Isolated Silymarin Flavonoids Increase Systemic and Hepatic Bilirubin Concentrations and Lower Lipoperoxidation in Mice. 2019;2019.
105. Kheiripour N. Hepatoprotective Effects of Silymarin on Liver Injury via Irisin Upregulation and Oxidative Stress Reduction in Rats with Type 2 Diabetes. 2019;44(2).
106. Federico A, Dallio M, Loguercio C. Silymarin/Silybin and chronic liver disease: A marriage of many years. Molecules. 2017;22(2).
107. Xu D, Wu Y, Liao ZX, Wang H. Protective effect of verapamil on multiple hepatotoxic factors-induced liver fibrosis in rats. Pharmacol Res. 2007;55(4):280–6.
108. Nazaretyan SA, Savic N, Sadek M, Hackert BJ, Courcelle J, Courcelle CT. Replication Rapidly Recovers and Continues in the Presence of Hydroxyurea in Escherichia coli. J Bacteriol. 2018;200(6):e00713-17. Published 2018 Feb 23. doi:10.1128/JB.00713-17
109. Pongudom S, Phinyo P, Chinthammitr Y, Charoenprasert K, Kasyanan H, Wongyai K, et al. Efficacy and Safety of Metronomic Chemotherapy Versus Palliative Hydroxyurea in Unfit Acute Myeloid Leukemia Patients: A Multicenter, Open-Label Randomized Controlled Trial. Asian Pac J Cancer Prev. 2020;21(1):147–55.
1
110. Yacoub A, Mascarenhas J, Kosiorek H, Prchal JT, Berenzon D, Baer MR, et al. Pegylated interferon alfa-2a for polycythemia vera or essential thrombocythemia resistant or intolerant to hydroxyurea. Blood. 2019;134(18):1498–509.
111. Garadah T, Jaradat FMA, Thani K Bin. The effects of hydroxyurea therapy on the six-minute walk distance in patients with adult sickle cell anemia: An echocardiographic study. J Blood Med. 2019;10:443–52. 112. Tang Y, Masuo Y, Sakai Y, Wakayama T, Sugiura T, Harada R, et al. Localization of Xenobiotic Transporter
OCTN1/SLC22A4 in Hepatic Stellate Cells and Its Protective Role in Liver Fibrosis. J Pharm Sci [Internet]. 2016;105(5):1779–89. Available from: http://dx.doi.org/10.1016/j.xphs.2016.02.023
