Interplay between gut microbiota and the master iron regulator, hepcidin, in the pathogenesis of liver fibrosis

Abstract Introduction: There is a proven role for hepcidin and the composition of gut microbiota and its derivatives in the pathophysiology of liver fibrosis. Area covered: This review focuses on the literature search regarding the effect of hepcidin and gut microbiota on regulating liver physiology. We presented the regulating mechanisms of hepcidin expression and discussed the possible interaction between gut microbiota and hepcidin regulation. Furthermore, we investigated the importance of the hepcidin gene in biological processes and bacterial interactions using bioinformatics analysis. Expert Opinion: One of the main features of liver fibrosis is iron accumulation in hepatic cells, including hepatocytes. This accumulation can induce an oxidative stress response, inflammation, and activation of hepatic stellate cells. Hepcidin is a crucial regulator of iron by targeting ferroportin expressed on hepatocytes, macrophages, and enterocytes. Various stimuli, such as iron load and inflammatory signals, control hepcidin regulation. Furthermore, a bidirectional relationship exists between iron and the composition and metabolic activity of gut microbiota. We explored the potential of gut microbiota to influence hepcidin expression and potentially manage liver fibrosis, as the regulation of iron metabolism plays a crucial role in this context.


Introduction
The liver is a heterogeneous tissue composed of various cells such as he patocytes, he patic stellate cells (HSCs), resident macr opha ges (Kupffer cells), infiltr ated imm une cells, and Liv er Sinusoidal Endothelial Cells (LSECs).These cells undergo morphological and phonotypical changes during liver injury to participate in the liver fibrosis process by over-production of Extracellular Matrix (ECM).Also, the crosstalk between the putative cells is disrupted to induce pr ofibr otic r esponses under liv er injury.The normal function and structure of the liver depend on the desired functioning of cells, especially hepatocytes, HSCs, Kupffer cells, and their cross-talk, to maintain liver structure and restore liver damage.Under pathophysiological condition, the normal function of liver is disrupted and could be follo w ed b y pathological fibrosis, cirrhosis and hepatocellular carcinoma (HCC) (Aydin and Akcali 2018 ).It has been reported the important role of hepcidin, as a systemic iron regulator, and gut microbiota on the onset and de v elopment of liv er fibr osis whic h we discussed their molecular signaling and also possible interactions here.

Li v er fibrosis and Hepcidin
One of the primary mechanisms for re pairing li ver injury is hepatic fibrosis mediated by activated HSCs and ECM proteins (Lu et al. 2016 , Kissele v a andBr enner 2021 ).HSCs ar e r esident hepatic cells in quiescent phenotype, which transdifferentiate to activated cells in m yofibroblast-lik e phenotype after exposure to liver injury.Also, myofibroblast cells could originate from portal fibroblasts and fibroblasts derived from bone marrow.Activated HSCs are the primary fibrogenic cells that restore injured tissue by producing cr osslinked colla gen types I and III (Tsuchida and Friedman 2017 ).This prosses is a normal hepatic fibrosis that could be r e v ersed by a poptosis of activ ated HSCs, r e v ersed tr ansdifferentiation to inactivated form, and activity of Matrix Metalloproteinases (MMPs) after elimination of liver injury (Fallowfield et al. 2007 ).In contrast, pathogenic liver fibrosis results from chronic he patoto xic damages (such as hepatitis B virus (HBV), hepatitis C virus (HCV), alcoholic liver disease (ALD), and non-alcoholic steatohepatitis (NASH)) or bile flow obstruction, and cholestatic injury (such as biliary cholangitis, primary sclerosing cholangitis (PSC) and biliary atresia) (Lu et al. 2016, Weiskirchen et al. 2018 ).
It has been demonstrated the main features of pathogenic fibrosis and cirrhosis include disruption of epithelial and endothelial barriers and inflammatory mediators ov er pr oduction, suc h as TGF-β, by macr opha ges and infiltr ated bone marr ow-deriv ed immune cells and accumulation of ECM proteins (Bataller and Brenner 2005 ).Also, TGF-β, mainly produced by bone marrowderiv ed macr opha ges, is the most potent fibr ogenic cytokine inducing ECM production through activating the main transcriptional factors, Sma and Mother Against Deca penta plegic (SMADs) ( Khedr and Khedr ).Unlike bone marr ow-deriv ed macr opha ges, hepatic resident macrophages (Kupffer cells) dually act in liver fibrosis pathology due to anti-inflammatory activity and production of MMPs such as MMP2 and MMP9 to r esolv e ECM in r ev ersible fibr osis .T her efor e, inflammation has been consider ed as critical inducer of HSCs activation and fibrotic scar formation (Ramac handr an et al. 2012 , Tsuchida andFriedman 2017 ).
There is a crucial interaction between iron homeostasis and o xidati v e str ess due to the fa vor of F e + 2 in the chemistry of the F enton-Haber-Wiess reaction.T his r eaction yields extr emel y r eacti ve o xygen species (ROS) fr om hydr ogen per o xide by Fe + 2 o xidation (Sousa et al. 2020 ).In a physiologic state, the deleterious effects of ROS which is released normally during cellular metabolisms, ar e counter acted by the activity of the cellular antioxidant system.Furthermore, to inhibit the toxic potential of iron (Fe + 2 ) to induce o xidati ve stress by the Fenton reaction, it is tightly bound to ferritin and transferrin in the intracellular and extr acellular spaces, r espectiv el y (Dongiov anni et al. 2011 ).Under the ir on ov erloading condition, the acceler ated Fenton r eaction and unquenchable consequence radicals occur by the availability of Fe + 2 due to the ele v ated ir on le v el of circulation r esulting from hepcidin downregulation and also increased free iron (nontr ansferrin bind ir on, NTBI) and loosel y ir on bonded to a gents suc h as albumin, citr ate and acetate in serum.The excessive ROS generation causes damage to the cellular macromolecules , lipids , protein, and nucleic acids, cell apoptosis follo w ed b y c ytoc hr ome c release resulting from mitochondrial membrane damage, and also ferr optosis, an ir on-dependent r egulated cell death.It has been demonstrated the pathological role of ROS and iron overload in the liver diseases (Capelletti et al. 2020 ).Indeed, iron overload and its accumulation in hepatocytes have recently been reported as common features of liver fibrosis, which could induce o xidati ve str ess by pr oceeding of Fenton r eaction, inflammation, and activ ation of HSCs.Also, ir on deposition in Kupffer cells results in the pr oduction of pr o-inflammatory cytokines, leading to liv er fibr osis (Mehta et al. 2019 ).Ir on ov erload occurs due to reducing or suppressing the maser systemic iron regulator, hepcidin.Iron plays a vital role in the viability of organisms due to its involvement in biological processes such as respiration, cell growth, and differentiation (Lu et al. 2016 ).Hence, a regulating iron level mechanism is mediated by hepcidin to contr ol ir on load via interaction with ferr oportin (FPN), expr essed by the leading ir on stor es suc h as en-ter ocytes, macr opha ges, and hepatocytes (Atanasiu et al. 2007 ).This evidence implies that the altered gene expr ession pr ofile of he patocytes, especially he pcidin, can induce fibrogenic processes (such as activation of HSCs) beyond the inflammatory stimuli.
Hepcidin is a master ir on r egulator he patokine deri v ed mainl y from hepatocytes, first identified by antimicrobial activity as Hepcidin Antimicrobial Peptide (HAMP) or Liv er-expr essed Antimicrobial Pe ptide-1 (LEAP-1).He pcidin can inhibit Esc heric hia coli , Salmonella typhimurium, and Mycobacterium tuberculosis growth (Park et al. 2001, Sow et al. 2007, Nairz et al. 2008 ).It has been reported that a decrease in iron level in serum resulting from hepcidin induction, reduces the expression of outer membrane protein A (OmpA) of Aeromonas hydrophila in zebrafish model.OmpA which is induced by iron acts as a strategy to escape from the complement system in A. hydrophila in vasion.T hese findings implicated the importance of iron to bacterial growth and expression of virulence factors such as OmpA.T herefore , the combined antibacterial and iron regulatory effects of hepcidin on bacterial defense against innate immunity can reduce bacterial invasion (Smith et al. 2007, Xiong et al. 2010, Michels et al. 2015, Jiang et al. 2017 ).
Furthermor e, hepcidin le v els ar e inv ersel y r elated to ir on load due to its ability to degrade ferroportin (FPN), encoded by SLC40A1 , the principal iron efflux pump expressed on enterocytes, macr opha ges, and hepatocytes (Rice et al. 2009 ).
Hepcidin could regulate iron absorption mediated by enterocytes, primaril y thr ough r egulating FPN stability on these cells.Enter ocytes mediate ir on absor ption and circulation via se v er al pr oteins, including duodenal cytoc hr ome B (DCYTB) to r educe ferric iron to ferrous, divalent metal transporter 1 (DMT1) to transfer ferr ous ir on to enter ocytes, FPN to export ferr ous ir on to circulation, hephaestin (HEPH) to oxidize ferr ous ir on to ferric, and transferrin (TF) as an iron plasma carrier (Fuqua et al. 2012 ).It is noticed that FPN is necessary for iron absorption.It has been sho w ed that iron body load affects FPN expression so that it is inversely expr essed in r esponse to ir on deficiency in enter ocytes .Meanwhile , the expression of FPN in other tissues is differ entl y r egulated by the iron load.For example, liver-expressed FPN is decreased and increased by iron deficiency and iron o verload, respectively.T his is a pr otectiv e r egulatory mec hanism a gainst intr acellular toxic ir on accum ulation (De Domenico et al. 2006, Bogdan et al. 2016, Link et al. 2021 ).
Macr opha ges and hepatocytes are also the targets of hepcidin to control iron efflux due to their roles in hemoglobin recycling and primary stor a ge of ir on, r espectiv el y.The intr acellular ov erload of iron in hepatocytes results from the downregulation of hepcidin, which is follo w ed b y the incensement of iron efflux from macr opha ges and enter oc ytes to cir culation due to lacking inhibitory function on FPN activity (Kessler et al. 2015, Mehta et al. 2019 ).
Furthermor e, the r ole of hepcidin in the intercellular communication of liv er cells, especiall y hepatocytes and HSCs, was identified in a fantastic study.Han Yeob Ch et al. sho w ed the inhibitory role of hepcidin in the activation of HSCs by degradation of FPN (expressed on HSCs) and improvement of liver fibrosis.Indeed, FPN deficiency in activated HSCs suppresses the phosphorylation of SMAD3, induced by TGF-β signaling via hepcidin ov er expr ession in CCl4-induced fibrotic mice.As a result, the suppression of HSC activity disrupts the production of ECM and inflammatory cytokines, ele v ating hepatocytes' injury and fibrosis (Han et al. 2016 ).On the other hand, disrupted iron homeostasis contributes to HSC acti vation.Unlik e quiescent, acti vated HSCs express two ironr elated r eceptors, including a specific r eceptor for the H-ferritin Figur e 1. T he effects of downregulated hepcidin in induction of liver fibrosis.
receptor and transferrin receptor-1 (TFR-1) (Mehta et al. 2019 ).Hferritin, r eleased fr om Kupffer cells after hemoglobin recycling fr om senescent RBC (erythr opha gocytosis), binds to the H-ferritin receptor and internalizes to activated HSCs (Mao et al. 2015 ).Subsequentl y, pr oinflammatory and pr ofibr ogenic effects are exerted by activating nuclear factor-kappa B (NF-κB) and elevated IL-6 and IL-1 β due to free radical production.Also, TFR-1 activation of activated HSCs with transferrin increases the transcripts of ECM components, such as α-smooth muscle actin ( α-SMA) and procollagen α1 (Bridle et al. 2003, Mehta et al. 2018 ).Inter estingl y, HSCs pr oduce excessiv e ECM and cr oss-linked matur e colla gens during liv er fibr osis, whic h ar e mor e r esistant to MMP degr adation.It has been demonstrated that iron overload may enhance fibrogenesis due to acting as a cofactor for hydroxylase enzymes catalyzing cr osslinking colla gens (Risteli and Kivirikk o 1974 ).Gener all y, we presented the main effects of downregulated of hepcidin in liver fibrosis (Fig. 1 ).

Regulation of Hepcidin Expression
According to the pivotal role of hepcidin in mana ging ir on homeostasis and pathological consequences, its gene expression is tightl y contr olled by se v er al mec hanisms, including ir on load and inflammation.Iron deficiency and increased erythropoiesis can decrease hepcidin expression, which is follo w ed b y the ele v ation of iron absorption and efflux of iron from storing cells to circulation.In contr ast, ir on ov erload upr egulates hepcidin expr ession, decr easing ir on absor ption and k ee ping excess iron in macr opha ges and hepatocytes (Ganz and Nemeth 2012 ).Hereditary hemoc hr omatosis is a known ir on loading disorder due to HAMP mutations, emphasizing hepcidin's crucial role in responding to iron status .T he severe form of iron overload is called juvenile hemochromatosis, resulting from mutation in the hemojuvelin (HJV) gene.HJV mediates one of the main inducing hepcidin expression signaling by bone morphogenetic proteins (BMP)/SMAD pathway a gainst ir on load.HJV acts as a BMP coreceptor (Xia et al. 2008 ).BMP6 is pr edominantl y r eleased by Liver Sinusoidal Endothelial Cells (LSECs) and HSCs in response to iron overload for the regulation of iron hemostasis through HAMP induction in hepatocytes.After BMP6 binds to its receptor and coreceptor, a complex of phosphorylation of SMAD 1/5/8 incor por ated with SMAD4 is formed and translocated to the nucleus to induce HAMP pr omoter (P arr ow and Fleming 2014 , Xiao et al. 2020 ).In addition, Hemoc hr omatosis Pr otein (HFE) and Tr ansferrin Receptor 2 (TFR2), whic h ar e expr essed on the plasma membrane of hepa-tocytes, sense plasma iron levels .Iron o verload induces HFE and TFR2 to upregulate HAMP by influencing the BMP/SMAD signaling for controlling the iron circulation.The hemochromatosis may result from HFE and TFR2 mutations in relatively mild phenotypes (Chen et al. 2016 ).
Another main regulatory hepcidin expression is mediated by JAK (Janus kinase)-ST A T3 (signal tr ansducer and activ ator of tr anscription 3) pathway activated via inflammatory stimuli for reducing serum iron levels.Since iron is a pivotal element for the surviv al and r eplication of micr oor ganisms, its suppr ession during infection is a defense strategy to sequester iron from microbial agents (Wang and Babitt 2016 ).In the inflammatory state, macr opha ges , monocytes , and Dendritic Cells (DCs) pr eserv e intr acellular ir on as a form binding to ferritin and inhibiting iron export meditated by hepcidin (Cairo et al. 2011 ).T herefore , hepcidin acts as innate immunity and is categorized as a type II acutephase pr otein.Ele v ated hepcidin during infection causes anemia of inflammation (AI) to diminish the viability of infectious agents.Hence, patients with a high iron level have a poor disease prognosis and a higher mortality rate (Lan et al. 2018 ).The main inducer cytokine to hepcidin expression is IL-6, which interreacts with IL-6 receptor GP-130 and induces phosphorylation and nucleus translocation of ST A T-3.This role of IL-6 depends on macrophages expr essing toll-like r eceptors (TLRs) but not r eleased fr om Kupffer cells since depletion of the liver from Kupffer cells does not affect hepcidin expression by IL-6 exposure (Lou et al. 2005 ).Inter estingl y, hepcidin expr ession r eduction was r eported in biliary atr esia (c holestatic injury).Hydr ophobic bile acid disrupted the IL-6-ST A T3 pathway to induce HAMP in hepatoc ytes b y inhibiting ST A T-3 phosphorylation (Huang et al. 2009 ).
Furthermor e, a macr opha ge-independent manner has been described to induce hepcidin expression in hepatocytes.In this way, the activation of TLR-4 expressed on hepatocytes (instead of macr opha ges) induces the HAMP pr omotor.Lipopol ysacc haride (LPS) has a proinflammatory activity on macr opha ges expr essing TLR-4 to induce NF-kB and its tar geted pr oinflammatory genes, suc h as IL-6, whic h is the main inflammatory inducer of hepcidin expression as a macrophage-mediated pathway (Lee et al. 2017 ).Also, LPS could interact with hepatocyte-expressed TLR-4 to induce the myeloid differentiation factor 88 (MyD88) pathway by activating c-Jun N-terminal kinase (JNK) and activator protein-1 (AP-1).The binding site to AP-1 is identified on the HAMP promotor.Ther efor e, LPS induces HAMP via the TLR-4-JNK-AP-1 axis in hepatocytes.In contrast, the suppression of li ver he pcidin expression was reported by alcohol through TLR-4 and activation of NF-kB e v en in the presence of inflammatory stimuli (Zmijewski 2014 ).Also, LPS can ele v ate SMAD signaling to induce HAMP pr omoters by activating SMAD4, followed by the TLR-4-MyD88 pathway (Kowdley et al. 2021 ).Differ ent ada ptor molecules and tr anscription factors located downstream of TLR-4 explain different signaling pathways by various stimuli.

Hepcidin Role in Chronic Li v er Disease and Hepatocellular Carcinoma
Chr onic Liv er Disease (CLD) includes alcoholic/non-alcoholic liv er disease and vir al hepatitis, r esulting in the dysfunction of inflammatory response and liver structure and progressing into fibrosis , cirrhosis , and Hepatocellular Carcinoma (HCC).Hepatic iron overloading is a common feature of CLD (Milic et al. 2016 ).Alcoholic Liver Disease (ALD) is a threat to global public health.In ALD, ethanol abuse promotes hepatic iron overload by inhibiting hepcidin gene expression.This inhibition is caused by inhibiting enhancer-binding pr otein (C/EBP), whic h r esults fr om alcohol and iron-mediated o xidati ve stress (Ferrao et al. 2022, Li et al. 2022 ).Also, alcohol can upregulate enterocyte DMT-1 and FPN, increasing serum iron and promoting liver fibrosis (Harrison-Findik et al. 2007 ).
Non-alcoholic Fatty Liver Disease (NAFLD), characterized by incr eased liv er fat accum ulation, is the most common liver disease worldwide.It consists of intracellular fat accumulation and steatosis, from simple to progressive, called non-alcoholic steatohepatitis (NASH), possibly progressing into liver fibrosis , cirrhosis , HCC, and death.There are pathogenic factors to the onset and development of NAFLD, including metabolic syndrome-related featur es suc h as obesity, insulin resistance, h ypertension, h yperlipidemia, and hepatic iron overload (Younossi et al. 2020, Pantano et al. 2021 ).Ir on ov erload can pr omote hepatic fibr osis, whic h is a NASH c har acter.According to the complexity of NASH pathogenies and the pr ov en r ole of ir on load in liv er fibr osis, r educing ir on load can be a strategy to reduce steatosis, inflammation, and fibrosis in NASH treatment.In this regard, Chen et al. demonstrated a potential ther a peutic r ole for hepcidin in alleviating steatohepatitis and fibrosis in NASH-induced animal models via recombinant adeno-associated virus genome 2 serotype 8 vector expressing Hamp (rAAV2/8-Hamp)-mediated hepcidin intervention.They reported that the overexpression of HAMP significantly improved liv er fibr osis by suppr essing pr oinflammatory r esponse, infiltr ation of macr opha ges, and HSCs activ ation in a mouse model of N ASH induced b y a choline-deficient l-amino acid-defined (CDAA) diet (Chen et al. 2022 ).Furthermor e, se v er al studies demonstrated a significant correlation between serum hepcidin level, hepatic ir on, and fibr osis in N AFLD/N ASH patients (Valenti et al. 2010, Nelson et al. 2011, Nelson et al. 2012 ).Lu et al. emphasized the role of iron and hepcidin in the severity of NAFLD.They reported the role of hepcidin in regulating metabolic processes and lipid and carbohydrate metabolism in high fat-fed and high sucrose-fed Hamp1 knoc k out mice (Lu 2016 ).
Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) are causative infectious factors in liver fibrosis, demonstrating disrupted hepcidin le v els.Inter estingl y, HCV is a stronger oxidative stress inducer than other viral hepatitis agents.Also, HCV-induced liv er fibr osis inhibits hepcidin expr ession by binding impairing C/EBP and ST A T3 hepcidin pr omoters.Furthermor e, HCV-induced hepcidin suppression is due to the antiviral activity inhibiting HCV r eplication.Ther efor e, antivir al ther a py, whic h incr eases ST A T3 expr ession le v el, could effectiv el y r estor e hepcidin le v els and decr ease vir al loads (Barry et al. 2009, Vela 2018 ).
As mentioned abo ve , hepatic iron overload could lead to fibrosis , cirrhosis , and HCC due to the hepatocarcinogenic potential of ir on.Contr ary to other types of cancer in whic h the hepcidin le v el ele v ates, hepcidin is downr egulated in HCC (Fan et al. 2021 , Joachim andMehta 2022 ).Evidence shows that an iron supplementation diet correlates with neoplastic hepatic nodules and HCC in animal and clinical models (Bothwell et al. 1964, Asare et al. 2006 ).Although the increased level of hepcidin inducer is seen in HCC, various mechanisms can be considered for downr egulated HAMP expr ession in HCC.T hey ma y include the hypermethylation of HAMP promoters and BMP6 as potent hepcidin inducers, whic h ar e down-r egulated in HCC (He et al. 2014, Udali et al. 2018 ).Additionall y, ther e may be downregulation of TFR2 and HJV as iron sensing mediators that induce hepcidin (Maegdefrau et al. 2011 , Joachim andMehta 2022 ).Ir on ov erload can lead to the downregulation of the tumor suppressor P53, which in turn decr eases HAMP expr ession due to the presence of P53 response on HAMP promoters.Another possible factor is a mutation on TP53, the gene encoding P53, which activates HAMP transcription (Hussain et al. 2007, Shen et al. 2014 ).Also, ther e may be an ele v ation of matriptase-2 expr ession, whic h acts as a negativ e r egulator for hepcidin expression (Lofft et al. 2020 ).
The downregulation of hepcidin in HCC can affect HCC pathogenesis by promoting the growth of cancerous cells by activating the cyclin-dependent kinase-1/ST A T3 (CDK1/ST A T3) pathway (Shen et al. 2019 ).Liv er fibr osis and cirrhosis ar e significant factors in HCC de v elopment.Ther efor e, the downr egulation of hepcidin in HCC can eliminate the pr otectiv e effect of hepcidin because it lacks the inhibitory effect on TGF-β-induced smad3 phosphorylation, whic h inhibits HSC activ ation (Joac him and Mehta 2022 ).Also, incr eased BMP expr ession and activ ated BMP-SMAD signaling can influence HCC metastasis and invasion by promoting cancer cell migration (Maegdefrau and Bosserhoff 2012 ).The potential of hepcidin as a dia gnostic, pr ognostic, and ther a peutic factor has been suggested (Joachim and Mehta 2022 ).There is a challenge concerning HCC diagnosis, which affects prognosis, treatment, and cost burden.HCC is mainly diagnosed by assessing serum Alpha-fetopr otein (AFP) le v els and ima ging tec hniques.Appr oximatel y half of HCC patients exhibit AFP-negative tests, emphasizing the lack of comprehensive HCC biomarkers (Wang and Zhang 2020 ).Joachim JH et al. highlighted the potential of hepcidin as a new diagnostic biomarker for HCC (Joachim and Mehta 2022 ).Nahon P et al. reported an association between low le v els of hepcidin and higher risks for HCC and poor prognosis in alcoholic cirrhotic patients (Nahon et al. 2016 ).Furthermore, a study reported the beneficial role of defer asir ox (DFX) ir on c helating a gainst HCC (Saeki et al. 2016 ).Ther efor e, the modulation of iron level and hepcidin could be a target for HCC treatment.

Microbiota-Gut-li v er Axis and Li v er Fibrosis
Gut microbiota is a co-evolved complex microbial population that colonizes the gastrointestinal (GI) tract.Currently, the pivotal role of gut microbiota in determining health and disease state has well been known (Fan and Pedersen 2021 ).Many studies implicate the involvement of the dysbiotic gut microbiota in the pathology of a wide range of diseases, including the most common liver diseases such as chronic hepatitis B (CHB), chronic hepatitis C (CHC), alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohe patitis (NASH), li ver cirrhosis, and hepatocellular carcinoma (HCC) (Lin et al. 2014, Wieland et al. 2015, Wang et al. 2018, Álv ar ez-Mercado et al. 2019 ).Besides the regulatory role of gut microbiota in the modulation of various mec hanisms, suc h as imm une and metabolism pathways in the GI tract, it bi-directionally communicates with extraintestinal organs, especially the liver named the gut-liver axis.According to the anatomical and functional similarities between the GI tract and liver, their bidirectional interaction has been established to be primarily mediated via portal circulation (Adolph et al. 2018 ).The role of gut microbiota in the maintenance of normal liver function could be summarized in se v er al main mec hanisms, including (i) the modulation of systemic inflammatory responses due to the presence of nearly 70% of immune cells in lamina propria that is educated by lumen sampling of micr obiota-deriv ed immunological components (Ahmadi Badi et al. 2021 ), (ii) regulation of permeability of intestinal epithelium layer that determines pathogenic and symbiotic bacteria and their immunological components' translocation to lamina propria and circulation (Régnier et al. 2021 ), (iii) the influence on the bile acids (BAs) composition and pool size through deconjugation of primary to secondary B As and B As-farnesoid X receptor (FXR) interaction modulating metabolic and immune pathways and BAs production (Sayin et al. 2013 ) and (iv) the control of HSCs transdifferentiation by modulating the intr ahepatic imm une micr oenvir onment (composition of immune cells and cytokines/chemokines profile) (Liang et al. 2020 ).
The li ver contin uously is exposed to antigen-derived nutrients , pathogens , microbiota, and their metabolites (such as shortchain fatty acids (SCFAs)), which reach the liver by the portal vein.In normal conditions, LPS derived from Gram-negative members of gut microbiota is translocated to the liver (by portal vein) and detoxified and cleared through the phagocytic activity of Kupffer cells in the reticuloendothelial system (RES) (Leber et al. 2012 ).Under liver injury conditions, the mentioned mechanism is disrupted due to the dysfunction of the gut barrier and the reduction of RES activity, finally resulting in the dominancy of pr oinflammatory r esponses.On the other hand, incr eased gut micr obiota-deriv ed LPS pr omotes HSCs tr ansdiffer entiation by activ ating TLR-4 expr essed on HSCs, follo w ed b y the incensement of cytokines and chemokines (Zheng and Wang 2021 ).In addition, in the normal state, SCFAs deriv ed fr om gut micr obiota help maintain the normal liver function through several routes including the reinforcement of gut barrier integrity and control of bacterial translocation to the liver, acting as a signaling molecule to interact with G-protein-coupled receptor (GPR) 41, GPR43 and peroxisome pr olifer ator-activ ated r eceptor γ (PPAR-γ ), whic h mediate immune and metabolic homeostasis , ha ving the epigenetic potential to the impr ov ement of r egulatory T-cells (Tr eg) by inhibiting histone deacetylase (HDAc) (Koh et al. 2016 , Zheng andWang 2021 ).In liver injury, the optimal concentration of SCFAs is disrupted, follo w ed b y the dysbiosis of gut microbiota composition, especiall y Bacter oidetes and Firmicutes phyla, including Ruminococcaceae, Lac hnospir aceae, and Clostridiales, which are the main SCFAs producers (Furusawa et al. 2013, Zhang et al. 2021 ).Ther efor e, the gut-liv er axis is negativ el y influenced by a liver injury that induces dysbiosis of gut microbiota and vis v ersa, whic h, in turn dysbiosis of gut microbiota could cause liver damage.

Interplay between Gut Microbiota and Iron Homeostasis from Intestinal Iron absorption to Hepatic Hepcidin Expression
Ir on deriv ed fr om diet and hem could influence gut micr obiota composition and metabolic activity.It has been demonstrated that very low iron condition negatively affects gut microbiota health by decr easing butyr ate-pr oducing bacteria, suc h as Roseburia spp.and Bacteroides spp.because Fe acts as a cofactor for enzyme involvement in the fermentation pathway (Dostal et al. 2013 ).Furthermore , Firmicute abundance , the main gut microbiota phylum, has been reported to increase and decrease under iron supplementation and iron deficiency conditions, respectively (Dostal et al. 2012, Dostal et al. 2013 ).Also, hem enriched intestinal lumen resulting from a hem-rich diet or intestinal bleeding may induce dominancy of bacteria containing the heme-uptake coding genes (Constante et al. 2017 ).There is an ongoing competition for iron acquisition between microbes and hosts mainly mediated by the release of iron-chelating proteins to the intestinal lumen, including sider ophor e and lipocalin-2, r espectiv el y (Chieppa and Giannelli 2018 ).Ther efor e, ir on av ailability may be a pivotal regulator in preserving the symbiotic relationship between gut microbiota and the host.
Evidence emphasizes the role of gut microbiota in liver pathophysiology concentrated on iron homeostasis mediated by hepcidin.Hence, it is necessary to consider the mechanisms influ-enced by gut microbiota to modulate iron metabolism and hepcidin regulation for liver fibrosis control.On the one hand, the gut microbiota could affect iron metabolism due to the absorption of dietary iron in the GI tract colonized by this microbial community.Although the duodenum is the primary site for iron absor ption (nearl y 15% absor ption r ate), the r est of the ir on r eac hes the colon, which is the leading site for the gut microbiota population (Yilmaz and Li 2018 ).T he a vailability and valency of iron could be influenced by microbiota composition via the production of sider ophor es (c helating molecules for the acquisition of iron for bacterial cells) and SCFAs that can incr ease ir on absor ption by se v er al mec hanisms, including the suppl y of ener gy for the pr olifer ation of epithelial cells, r eduction of ir on to the ferr ous form, pH dr opping of GI tr act, and incr ement of ir on solubility (Salov aar a et al. 2003 , Yilmaz andLi 2018 ).There is a correlation between gut micr obiota-deriv ed vitamins including vitamin B12 (cobalamin) and folate with erythropoiesis since their deficiency can lead to anemia (Koury and Ponka 2004 ).Furthermore, it has been reported that Vitamin B such as cobalamin affects gut microbiota composition and its capacity to produce SCFAs (Wan et al. 2022 ).Ther efor e, gut micr obiota-deriv ed vitamins such as vitamin B12 can influence the crosstalk between iron homeostasis and gut microbiota by the mentioned effects .T he iron storage and expression of intestinal iron absorption/exporter-mediated proteins, such as DMT1, DCYTB, and FPN, could be controlled by gut microbiota.A study demonstrated that germ-free (GF) mice had significantly higher and lo w er levels of absorptive iron proteins (DMT1 and DCYTB) and iron efflux FPN than those colonized with microbiota.Also, Bacteroides thetaiotaomicron , Faecalibacterium prausnitzii, and pr obiotic str ains ( Streptococcus thermophilus LMD-9) could induce ferritin stor a ge in the colon (Desc hemin et al. 2016 ).Furthermore, it has been reported that Lactobacillus species are the main gut microbiota to sense iron levels and reduce host iron absorption.Gut micr obiota metabolites, suc h as 1,3-diaminopr opane (DAP) and r euterin, r egulate ir on homeostasis and amelior ate tissue ir on ov erload by acting as a suppr essor of hypoxia-inducible factor 2a (HIF-2a), a transcription factor of three k e y intestinal ir on tr ansporters (DMT1, DCYTB, and FPN), and increament of iron storage ferritin (Das et al. 2020 ).Interestingly, intestinal HIF-2a activation has been shown that is in association with the hepcidin/FPN axis to control iron absorption (Schwartz et al. 2019 ).
On the other hand, a stud y re ported the inducing effect of gut microbiota on hepatocyte hepcidin expression.It identified the ability of Bifidobacterium longum and Bacteroides fragilis to upregulate hepcidin in a macr opha ge-stim ulated manner whic h is mediated by activation of BMP/SMAD signaling with IL-1 β.Also, the novel association between IL-1 β and BMP/SMAD signaling to induce hepcidin and possibly different hepcidin induction mechanisms between humans and mice (the role of IL-1 β) were suggested (Shanmugam et al. 2015 ).
Also, a fantastic study reported the effect of gut microbiota on the induction of hepcidin produced by non-hepatocyte sources.Bessman NJ reported a protective effect of gut microbiota on m ucosal healing thr ough the induction of hepcidin pr oduction fr om conv entional dendritic cells (cDCs), whic h ar e essential to repair tissue by hepcidin production in the inflamed intestine.The cDCs produce hepcidin in response to gut microbiota stimuli.This limits the availability of local intestinal iron by sequestering it by targeting intestinal phagocytes that express FPN.It also helps to limit tissue infiltration.This hepcidin, produced from cDC, has been c har acterized and compar ed with hepcidin deriv ed fr om the liv er, whic h is induced by inflammatory r esponses Figur e 2. T he bar plot of the HAMP gene in A) biological processes and B) pathogenic phenotypes.In biological pr ocesses, ir on metabolism, positiv e and negative self-regulation in the formation of hemoglobin by this gene are highly evident.Also, in diseases related to liver fibrosis, there has been a more significant observation of liver mass and iron metabolism in the liver.and pr ovides pr otection a gainst systemic infection (Bessman et al. 2020 ).
The bidirectional crosstalk between gut microbiota members and the host for iron acquisition and metabolism plays an important role in shaping the metabolism of both the host and the gut micr obiota.As discussed abov e, the potential crosstalk is mediated by the influence of bacterial species on iron metabolism and the impact of n utrient-deri v ed ir on on gut microbiota composition.This interaction affects iron levels and storage, the immune system, and glucose metabolism, whic h is r elated to metabolic syndr ome (Mayneris-Perxac hs et al. 2022 ).Ther efor e, ther e is a corr elation between ir on and glucose metabolism due to disrupted iron homeostasis and changes in gut microbiota composition in patients with metabolic disease and insulin resistance (Fillebeen et al. 2020 ).Se v er al studies have reported that iron overload is a risk factor for diabetes (Simcox and Mcclain 2013 ,  Fernández-Real and Manco 2014 ).There is an interconnection be-tween iron and glucose metabolism, which are hormonally regulated by hepcidin and insulin, r espectiv el y.In this r egard, documents r epr esent the dir ect induction effect of hepcidin via ST A T3 on he patocytes.Ad ditionall y, ther e is e vidence of incr eased hepcidin le v els due to glucose intake in healthy subjects (Aigner et al. 2013, Wang et al. 2014 ).Accordingly, metabolic syndr ome, suc h as type 2 diabetes, is associated with altered gut microbiota and iron metabolism.A study supports that the impr ov ement of glucose le v els may be controlled by Salidroside (SAL), a Chinese herbal compound with the potential to affect gut microbiota composition and protect against iron o verloading.T his is achieved by targeting gut microbiota and iron metabolism in diabetic mice (Shi et al. 2022 ).Ther efor e, considering the putativ e corr elation could be useful for designing ther a peutic str ategies for metabolic syndrome.
Using primary and integrated bioinformatics analysis, we investigated the HAMP gene and identified its role in biological pro-Figur e 3. T he scatter plot of the degree of accumulation and the correlation of HAMP expression with various bacterial strains and microbes.cesses and the types of diseases caused by defects in this gene.Next, we r epr esented the bacteria inter acting with the HAMP gene as a scatter dia gr am.These findings r e v ealed that this gene plays a significant role in iron-dependent metabolic processes and is associated with diseases such as thalassemia, liver fibrosis, immune system disorders in the liv er, ir on metabolism disorders, and aplasia in red blood cells (Fig. 2 ).
In the next part of the analysis, we found that the HAMP gene is significantly associated with bacterial strains such as Mycobacterium tuberculosis and Brucella suis .Additionall y, HAMP is r elated to other micr oor ganisms suc h as Coxsac kie virus, Human Imm unodeficiency Virus, and Leishmania (Fig. 3 ).
According to the crucial role of gut microbiota in driving the gut-liver axis, bacterial translocation to the liver and possible in-fluential direct and macrophage-mediated effects on hepcidin expression could be rationally based on the stimulation of systemic immunity and continued exposure of the liver to gut microbiota components and metabolites.Taken together, we summarized the possible crosstalk between gut microbiota and hepcidin expression to determine the physiology and pathophysiology of liver fibrosis (Fig. 4 ).

Conclusion
We can illustrate the potential pathways for their interactions based on the role of gut microbiota in liver pathophysiology and iron metabolism, as well as the complexity of iron homeostasis and its pr ominent r ole in liv er fibr osis.Gut micr obiota may modulate hepatocyte hepcidin gene expr ession, especiall y via direct and macrophage-mediated effects .Furthermore , the intesti-Figur e 4. T he possible interaction between gut microbiota and hepatic hepcidin expression in the determination of normal liver function and pathological fibrosis; in a symbiosis state, the liver has normal functioning due to fine-tuning of hepcidin gene expression in he patocytes.He pcidin contr ols liv er physiology by suppr essing HSCs activ ation (by degr ading FPN and suppr essing SMADs signaling to pr oduce ECM) and inhibiting intracellular hepatic iron overload through inhibiting iron over-efflux from enterocytes and macrophages to circulation.Also, intestinal iron absorption could be desired by the normal function of DMT-1 and DCYTB and an intact gut barrier to inhibit the over-translocation of immunological components to lamina propria and circulation, which control inflammatory signals in the liver.Gut microbiota components, such as LPS, could regulate hepcidin expression by direct (stimulation of TLR-4) and macrophage-mediated (activation of IL-6 receptor) effects in hepatocytes.Under a dysbiotic state, the perturbed gut barrier function increases bacterial translocation, inducing pro-inflammatory responses, and exacerbating hepatic fibr ogenic r eactions .T he inhibitory he pcidin effect on the acti v ated HSCs is disrupted whic h r esults to ele v ated activ ation of HSCs and excessiv e accumulation of ECM.On the other hand, the plasma iron level and hepatic intracellular iron overload could be affected by gut microbiota, their components, and metabolites via influencing the expression of iron abortive proteins, such as DMT-1 and DCYTB, and iron efflux pump, FPN.nal expression of proteins mediating iron absorption and exportation may be regulated by gut microbiota composition.These data suggest the potential of the gut microbiota as hepatoprotective bacteria in controlling and modulating liver fibrosis by regulating hepcidin, which controls iron homeostasis.Ho w ever, it is necessary to elucidate the molecular signaling pathways related to the fine-tuning control of iron levels and preserve the symbiotic relationship between the host and gut microbiota in liver fibrosis.

Expert Opinion
P athological liv er fibr osis occurs due to c hr onic liv er injury.Hepatotoxic and c holestatic liv er injuries resulting from chronic viral infections , metabolic syndrome , and obstructive bile flow, such as biliary atr esia, can pr ogr ess to fibr osis , cirrhosis , and Hepatocellular Carcinoma (HCC).One of the main features of liver fibrosis is iron accumulation in hepatic cells, including hepatocytes.This accumulation can induce an o xidati ve stress response, inflammation, and activation of Hepatic Stellate Cells (HSCs), ultimately leading to the onset and development of fibrogenic re-sponses .T here is a proven role for hepcidin as the main regulator of iron in the pathophysiology of liver fibrosis by targeting FPN.The FPN activity involves the efflux of iron from the main iron donor and storage cells, including enterocytes , macrophages , and hepatocytes, into circulation.Hepatocyte iron accumulation is considered the starting point for the induction of inflammatory and fibrogenic responses resulting from decreased hepcidin le v els.
In addition, fine-tuning intercellular communication between hepatic cells, such as hepatocytes , Kupffer cells , and HSCs , preserves the liver's normal function, which could be disrupted by he pcidin d ysr egulation.In contr ast, it has been demonstr ated that gut microbiota plays a pivotal role in regulating liver function through the gut-liver axis.Also, iron levels could alter gut microbiota composition and metabolic acti vity, affecting li ver function.Some studies report the alteration of gut microbiota composition in liv er injuries, suc h as liv er fibr osis, whic h could be follo w ed b y cirrhosis and hepatocellular carcinoma.Ther efor e, the gut-liv er axis is negativ el y influenced by liv er injury, whic h can induce dysbiosis of the gut micr obiota.Conv ersel y, dysbiosis of the gut microbiota can also cause liver damage .Furthermore , the iron level, which is regulated by the hepcidin-FPN axis and proteins involved in ir on uptakes, suc h as DCYTB and DMT1, could be influenced by the composition of gut micr obiota, whic h can be altered by changes in iron levels.
Hepcidin expression is controlled by various factors, especially ir on le v els and the inflammatory r esponse, whic h ar e corr elated with maintaining the symbiotic relationship between the host and gut micr obiota.Ther efor e, we can consider a powerful interplay between hepcidin and gut microbiota as two k e y factors in liver fibrosis .T his delicate perspective could be considered when designing a gut micr obiota-tar geted interv ention for r egulating ir on le v els influenced by hepcidin activity.By considering the potential role of gut microbiota in modulating inflammation and hepatopr otectiv e activities, it is necessary to understand the interplay between gut microbiota and hepcidin during liver fibrosis.Furthermore, we should consider that antifibrotic drugs may have side effects on normal hepatic cells.On the other hand, r estor ation of intercellular crosstalk between hepatic cells through modulation of hepcidin le v els may offer a promising therapeutic strategy for pr e v enting, contr olling, and tr eating liv er fibr osis.It has been documented that gut microbiota is pivotal in determining health and disease status, such as liver complications .T herefore, we should consider gut microbiota members as important actors in liver fibrosis therapeutic strategies to restore dysbiotic gut microbiota-host interactions.Targeted interventions for the gut microbiota and the discovery of novel components derived from the gut microbiota have shown promising potential in this field.