The Regulatory Roles of Polysaccharides and Ferroptosis-Related Phytochemicals in Liver Diseases

Liver disease is a global health burden with high morbidity and mortality worldwide. Liver injuries can develop into severe end-stage diseases, such as cirrhosis or hepatocellular carcinoma, without valid treatment. Therefore, identifying novel drugs may promote liver disease treatment. Phytochemicals, including polysaccharides, flavonoids, alkaloids, and terpenes, are abundant in foods and medicinal plants and have various bioactivities, such as antioxidation, immunoregulation, and tumor killing. Recent studies have shown that many natural polysaccharides play protective roles in liver disease models in vitro and in vivo, such as fatty liver disease, alcoholic liver disease, drug-induced liver injury, and liver cancer. The mechanisms of liver disease are complex. Notably, ferroptosis, a new type of cell death driven by iron and lipid peroxidation, is considered to be the key mechanism in many hepatic pathologies. Therefore, polysaccharides and other types of phytochemicals with activities in ferroptosis regulation provide novel therapeutic strategies for ferroptosis-related liver diseases. This review summarizes our current understanding of the mechanisms of ferroptosis and liver injury and compelling preclinical evidence of natural bioactive polysaccharides and phytochemicals in treating liver disease.


Overview of Liver Diseases and Polysaccharides
Chronic liver disease (CLD) is an important public health problem in the world, which is a major cause of morbidity and mortality worldwide. There are many types of CLDs, mainly including alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), viral hepatitis, cirrhosis, and hepatocellular carcinoma (HCC), etc. [1]. The causes of CLDs are complex, including hepatic viruses, excessive alcohol consumption, metabolic syndrome, and drug toxicity, which are the major risk factors resulting in chronic liver injury [2]. CLD patients always have persistent inflammation, massive cell death (such as apoptosis and ferroptosis), and abnormal hepatocyte regeneration in the liver, which develop to end-stage liver pathologies, such as cirrhosis and HCC [3]. Due to the increase in hospitalized CLD patients, the economic and social burden has significantly increased, especially in developing countries [4].
Phytochemicals refer to active substances derived from plants, such as polysaccharides, polyphenols, and alkaloids. Many studies have shown that many phytochemicals, such as baicalin and curcumin, have remarkable anti-tumor efficacies with lower side effects compared to other chemotherapeutic drugs [5]. Some phytochemicals have advantageous effects on obesity, cardiovascular diseases, neurological diseases, and cancer by alleviating oxidative stress due to their antioxidative activity [6]. Meanwhile, various natural antioxidants protect against the hepatotoxicity induced by the chemotherapeutic drug cisplatin via antioxidant, anti-inflammatory, and anti-apoptosis activities [7].
Many polysaccharides could play hepatoprotective roles in NAFLD via moderating glucose metabolism, such as Angelica sinensis polysaccharide (ASP), SCP, and FFM. The PI3K/Akt pathway mediates glucose metabolism to decrease lipid accumulation in the liver [44]. ASP reduced blood glucose levels and ameliorated insulin resistance by activating the PI3K/Akt pathway in high-fat-diet-fed mice [45]. SCP alleviated insulin resistance by regulating the metabolism of ascorbic acid and uronic acid as well as the transformation pathway of pentose and glucuronic acid [29]. FFM has the potential to reduce insulin resistance in patients with NAFLD in a clinical trial [32].

Hepatic Fibrosis
Hepatic fibrosis is an outcome of wound healing in response to chronic liver injury. Without timely and valid treatment, liver fibrosis might finally develop into end-stage cirrhosis. The mechanisms of liver fibrosis are complex, consisting of inflammation, hepatic stellate cell (HSC) activation, extracellular matrix (ECM) production, and the deposit of collagen in liver [61,62]. Therefore, liver fibrosis can be reversed via ceasing chronic liver damage, blocking inflammation, deactivating HSCs, and degrading ECM [63]. The progression of hepatic fibrosis could be blocked by polysaccharides (Table 1) via these anti-fibrosis pathways.
O. lanpingensis polysaccharides (OLP) and Dictyophora polysaccharides could significantly decrease the accumulation of ECM and collagen by upregulating MMPs and collagenase expression, which are essential for collagenolysis [64,65]. Quiescent HSCs play important roles in the progression of liver fibrosis because active HSC can transdifferentiate into myofibroblasts, which produce ECM [62]. ASP could alleviate liver fibrosis by activating the IL-22/STAT3 pathway in HSCs to inhibit the HSC-myofibroblast switch [66,67].
In chronic liver damage, the persistent activation of NF-κB signaling and inflammatory cytokines always results in fibrosis [68]. OLP could alleviate liver fibrosis by decreasing inflammatory cytokines and oxidative stress [64], and Pleurotus citrinipileatus polysaccharide could inhibit the progression of liver fibrosis via targeting the NF-κB pathway [69].
Intestinal dysbiosis from alcohol or a high-fat diet might result in liver inflammation and fibrosis and eventually develop to liver cirrhosis [70]. Polysaccharides could affect the development of liver fibrosis by improving gut health. LBP, Miltiorrhiza bunge polysaccharides, walnut green husk polysaccharides (WGHP), and MDG-1 were reported to alleviate hepatic steatosis via modulating gut microbiota in a high-fat-diet-induced NAFLD model [35,[71][72][73]. In a randomized controlled trial, LBP could alleviate the hepatic injury and intestinal dysbiosis in NAFLD patients [74]. WGHP and MDG-1 could moderate the intestinal microecology in mice to reduce liver lipid accumulation [35,72]. Moreover, EPP could attenuate intestinal inflammation and improve barrier integrity to protect against alcohol-induced liver damage [50]. Similarly, DOP also protected against CCl 4 -induced liver fibrosis by improving the intestinal barrier [75].

Hepatocellular Carcinoma (HCC)
Liver cancer is one of the top 10 cancer types, with the mortality of 8.2%, and it ranks fifth in terms of global cases and second in terms of deaths for males. Hepatitis viruses (such as HBV and HCV), alcohol, metabolic syndrome, diabetes, obesity, NAFLD, tobacco, aflatoxins, and other dietary factors have been consistently associated with the effected risk of liver cancer. The prevalence of NAFLD/NASH is increasing and may soon overtake viral factors as the major cause of HCC globally [76]. Several polysaccharides were reported to have therapeutic effects on HCC (Table 1).
Polysaccharides could inhibit the progression of tumors by reducing immunosuppression. The liver has a complex immune microenvironment, and immunosuppressive cells in the tumor tissue can promote HCC tolerance. Tumor-associated macrophages (TAMs), which are one of the key components maintaining the immunosuppressive microenvironment of HCC, can facilitate tumor growth [77]. Therefore, remodeling the microenvironment of tumors could be a therapeutic strategy for anti-tumor immune responses [76]. Astragalus polysaccharides (APS) and polysaccharide from Pleurotus ostreatus could inhibit HCC growth via immunoregulation with the enhanced secretion of immune-stimulating cytokines (IL-2, TNF-α, IFN-γ, etc.) [78,79]. Ganoderma lucidum spore polysaccharide (GLSP) promoted the polarization of primary macrophages into M1 type and cytokine expression (such as TNF-α, IL-1β, IL-6, and TGF-β1) [80].
HCC is highly vascularized. Polysaccharides could inhibit the invasion of HCC cells by reducing tumor angiogenesis. The initiation of angiogenesis is driven by the metabolic demands of tumor cells, such as hypoxia or nutrients. Many factors stimulate this process, including hypoxia-inducible factors (HIFs), mammalian target of rapamycin (mTOR), and PI3K/AKT signaling [81]. Several polysaccharides could block HCC angiogenesis by downregulating hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factors (VEGFs). Moreover, asparagus and dandelion polysaccharides could inhibit MAPK and PI3K signaling pathways to block tumor angiogenesis [82][83][84].
Many polysaccharides could enhance the effect of chemotherapeutics or simultaneously reduce the negative effects or toxicities of these drugs. Mannan conjugation could enhance the effect of adenovirus-mediated phosphatase and tensin homologue (PTEN) gene therapy in a murine HCC model [92]. Polysaccharides from Lachnum sp. (LSP) com-bined with 5-fluorouracil or cyclophosphamide (CTX) and polysaccharides from Lentinus edodes combined with oxaliplatin were reported to inhibit the migration and invasion of HCC in a synergistic manner in vitro or in vivo [93][94][95]. Neutral polysaccharide from Panax notoginseng combined with CTX and aconitine combined with crude monkshood polysaccharide enhanced the tumor-killing effect by immunoregulation [96,97]. Moreover, nanoparticles made by polysaccharides are also applied in chemotherapeutic drug delivery. ASP, a plant polysaccharide with good biocompatibility, aqueous solubility, and intrinsic liver-targeted capability has been developed into targeted drug delivery nanoparticles for HCC therapy [98,99].

Drug-Induced Liver Injury (DILI)
Due to the first-pass effect of the liver in gastrointestinal nutrition metabolism, the liver is more susceptible to drug toxicity during clinical treatment. The incidence of DILI was estimated to be higher in Asia than that in Western countries [100]. Polysaccharides have significant protective roles in drug-induced liver damage (Table 1). ASP, Schisandra chinensis acidic polysaccharide, Phellinus linteus mycelia polysaccharide, PNP80b-2, fucoidan, and Seabuckthorn berry polysaccharide could protect against acetaminophen (APAP)-induced acute liver injury and cell death by suppressing oxidative stress [52,[101][102][103][104][105]. Sagittaria sagittifolia L. polysaccharide and Yulangsan polysaccharide exert protective effects against isoniazid-or rifampicin-induced liver injury via Nrf2 activation and downstream antioxidant gene transcription [106,107]. Meanwhile, the administration of GLP reversed Bacillus Calmette Guérin-induced hepatic injury in vivo via inhibiting nitric oxide production and inflammation [108].

Cell Death in Liver Diseases
Cell death is a critical event for liver injury, often persisting over decades. Long-term or massive dysregulated cell death may develop into severe clinical outcomes. For example, massive hepatocellular death always results in liver failure, while hepatocyte immortalization may cause HCC. Different types of cell death (such as apoptosis, necrosis, autophagy, and ferroptosis) trigger specific pathological responses and promote the progression of liver disease through distinct mechanisms [109]. The discovery of novel modes of cell death has greatly improved our understanding of the development of liver disease.

Polysaccharides Regulating Apoptosis
Apoptosis is classic cell death, and hepatocyte apoptosis is often considered to be the major mechanism of liver injury over decades. At the molecular level, apoptosis is divided into two major branches, the intrinsic and extrinsic pathways. The extrinsic apoptosis of hepatocytes can be initiated by inflammatory cytokines, which then trigger Fasdependent death-inducing signaling complex and downstream caspase-8/9 activation [110]. The caspase-9-induced pro-death protein BID-BAX axis is the major link between the intrinsic and extrinsic pathways. In the intrinsic apoptotic pathway, the mitochondrial outer membrane permeabilization by BAX and BAK results in the release of mitochondrial pro-death effectors, such as the hemoprotein cytochrome c, which triggers the formation of the apoptosome and caspase-3/7 activation [110,111]. Cytochrome c is normally bound to cardiolipin, and therefore the oxidation of cardiolipin by ROS also triggers cytochrome c release and downstream apoptotic signaling, including caspase activation and death execution [112].
Many polysaccharides have been identified as apoptosis regulators (Table 1). Polysaccharides with activities to suppress apoptosis can protect against NASH, ALD, and APAPinduced acute liver injury. On the other hand, polysaccharides such as apoptosis agonists can inhibit HCC development by promoting tumor cells apoptosis.

Polysaccharides and Other Phytochemicals Regulating Ferroptosis
Ferroptosis is a new type of cell death that was identified in 2012 [113]. Ferroptosis was observed in RAS-mutated tumor cells treated with the lethal compound erastin or RSL3. RAS mutations always result in apoptosis resistance, indicating ferroptosis is morphologically, biochemically, and genetically distinct from other forms of cell death. In the discovery of ferroptosis, either lipid peroxidation scavengers (i.e., ferrostatin-1) or iron chelators (i.e., deferoxamine) could specifically inhibit ferroptosis agonist (erastin or RSL3)-induced cell death, and therefore ferroptosis was characterized as a lipid-peroxidation-induced and iron-dependent cell death [113][114][115]. Ferroptosis serves as a major pathological mechanism in a wide range of organs, including the liver, heart, brain, and kidney [115][116][117]. In the past decade, the regulatory mechanisms of ferroptosis have been revealed (Figure 1) but not fully elucidated. Iron homeostasis is tightly maintained in the body, including iron absorption, storage, and utilization. Dysregulated iron metabolism is the key trigger of ferroptosis. In previous studies, an iron overload resulting from a high-iron diet or hereditary hemochromatosis was shown to cause hepatic ferroptosis, and an iron-deficient diet challenge or ferrostatin-1 treatment could rescue iron-overload-induced ferroptosis and liver damage [118,119]. Moreover, in normal cells, excessive iron is stored in ferritin, and the deletion of ferritin H in cardiomyocytes could increase the liable iron pool and result in ferroptotic heart injury [120]. Lipid peroxidation, the oxygenation of polyunsaturated phosphatidylethanolamines (PEs) in the cytoplasm membrane or mitochondrial membrane, is considered to be the executor of ferroptosis by decreasing the membrane integrity [117,121]. Lipid peroxidation is mediated by PE-binding protein 1 (PEBP1), a scaffolding protein that binds with both PEs and lipoxygenases and allows them to generate lipid peroxides [115]. The antioxidant glutathione (GSH)-glutathione peroxidases (GPXs) axis is a major mechanism for cleaning lipid peroxidation. The cystine/glutamate antiporter xc − is essential for cellular GSH, and its subunit SLC7A11 mediates cystine uptake, which is then reduced into cysteine for GSH synthesis [115]. The genetic deletion or mutation of SLC7A11 inhibited GSH synthesis and resulted in increased tissue lipid peroxidation and ferroptotic injury [118], while overexpressing SLC7A11 could increase the GSH content and rescue ferritin H knockout-induced ferroptotic heart damage [120]. GPX4, an enzyme that catalyzes GSH reacting with lipid peroxidation, plays critical roles in blocking ferroptosis. Therefore, inducing ferroptosis by the pharmacological inhibition of SLC7A11 and GPX4 provides novel strategies for tumor chemotherapy [113,[122][123][124]. Nrf2 is the key transcription factor of many antioxidant genes involved in ferroptosis, including SLC7A11 and GPXs. Besides GSH, other antioxidants, including NADPH and reduced thioredoxin (Trx), can also inhibit ferroptosis by reducing lipid peroxidation [125][126][127]. Recently, the FDA-approved anti-rheumatoid arthritis drug auranofin was identified as a a novel ferroptosis agonist by pan-inhibiting thioredoxin reductases (TXNRDs), which could refresh reduced Trx after reacting with lipid peroxidation. Therefore, auranofin and ferroptosis inhibitor (i.e., ferrostatin-1) combined treatment was suggested to be a safer strategy in the clinic to avoid ferroptotic toxicity from high-dose auranofin [127]. Moreover, polyunsaturated fatty acids (PUFAs) are essential for ferroptosis due to their sensitivity to lipid peroxidation [128,129]. ACSL4, an enzyme that catalyzes arachidonic acids synthesizing into PUFAs, could drive ferroptosis via oxidized phospholipids accumulating in the cell membrane [130][131][132][133].
The liver is one of the most important organs for iron storage. The hepatic iron and ROS burden are greater in the diseased liver than in the normal liver, suggesting that ferroptosis may be associated with chronic liver diseases [116]. Currently, ferroptosis has been identified as the key mechanism in NASH, ALD, ischemia/reperfusion, and iron overload (hemochromatosis)-related liver injury [118,[140][141][142][143]. However, polysaccharides have not been reported to regulate ferroptosis in liver diseases, while many phytochemicals of other types, such as terpene, alkaloid, and flavonoid, can alleviate the pathogenesis of liver diseases via regulating ferroptosis (Table 2). For one thing, phytochemicals could induce ferroptosis to suppress the progression of liver fibrosis and HCC. For instance, magnesium isoglycyrrhizinate, derivatives of artemisinin (such as artemether, artesunate, and dihydroartemisinin (DHA)), wild bitter melon extracts, chrysophanol, and zalkaloid berberine could block the development of liver fibrosis by triggering HSC ferroptosis [144][145][146][147][148][149][150][151][152]. Be-sides, several studies revealed that DHA could trigger ferroptosis to block HCC growth by promoting PEBP1/15LO formation or an unfolded protein response [153,154]. Moreover, DHA and artesunate could enhance the anti-tumor efficacy of sorafenib on HCC by inducing ferroptosis [155,156]. In addition, alkaloid solasonine promotes the ferroptosis of HCC cells via inhibiting GPX4 and GSH synthetase [157]. Meanwhile, heteronemin, a natural marine product isolated from Hippospongia sp., was reported to trigger HCC cell ferroptosis and apoptosis by increasing intracellular ROS and inhibiting MAPK signaling [158].
Nutrients 2022, 14, x FOR PEER REVIEW 10 of 21 essential for ferroptosis due to their sensitivity to lipid peroxidation [128,129]. ACSL4, an enzyme that catalyzes arachidonic acids synthesizing into PUFAs, could drive ferroptosis via oxidized phospholipids accumulating in the cell membrane [130][131][132][133]. Figure 1. Regulatory pathways of ferroptosis. Iron metabolism is tightly regulated in transport and storage. Cellular iron overload can trigger ferroptosis. Cellular iron uptake is mediated by TfR1, which imports transferrin-binding iron, and by DMT1 and SLC39A14, which import non-transferrin-binding iron. Ferroportin1 (Fpn1) is the only known iron exporter to date. Heme can be degraded by HO-1 to release free iron. Cellular excess iron is stored in ferritin, while ferritin can be degraded by NCOA4-mediated ferritinophagy in an iron-deficiency condition. System xc − , a heterodimer composed of SLC7A11 and SLC3A2, is a cystine/glutamate antiporter that mediates the efflux of glutamate and the influx of cystine at a 1:1 ratio. After entering the cell, cystine is reduced to cysteine and then synthesized into GSH. GPX4 scavenges lipid ROS via GSH. Lipid ROS derives from PUFAs-PE oxidation by lipoxygenases. The scaffolding protein PEBP1 can bind PE on the cell membrane and then recruit the lipoxygenase 15LO to generate lipid ROS. ACSL4 can increase lipid ROS by producing PUFAs-PE. Moreover, TCA cycle disorder or iron overload in mitochondria can also increase ROS, which results in ferroptosis. The CoQ/FSP1 and Trx/TXNRD axes inhibit ferroptosis in a GSH-independent manner. The Keap1/NRF2, p53, and YAP/TAZ signaling are necessary for the transcription of ferroptosis regulators, such as SLC7A11 and ACSL4. Erastin, RSL3, and auranofin are ferroptosis agonists by targeting SLC7A11, GPX4, and TXNRD, respectively. Ferroptosis inhibitors include iron chelators and lipid ROS scavengers (ferrostatin-1, liproxstatin-1, vitamin E, etc.).
Several polysaccharides have been identified as ferroptosis regulators to date, consisting of ferroptosis agonists and inhibitors (Table 2). Red ginseng polysaccharide and LBP exhibited anti-tumor efficacy by triggering ferroptosis [134][135][136]. Fucoidans, APS, Figure 1. Regulatory pathways of ferroptosis. Iron metabolism is tightly regulated in transport and storage. Cellular iron overload can trigger ferroptosis. Cellular iron uptake is mediated by TfR1, which imports transferrin-binding iron, and by DMT1 and SLC39A14, which import non-transferrinbinding iron. Ferroportin1 (Fpn1) is the only known iron exporter to date. Heme can be degraded by HO-1 to release free iron. Cellular excess iron is stored in ferritin, while ferritin can be degraded by NCOA4-mediated ferritinophagy in an iron-deficiency condition. System xc − , a heterodimer composed of SLC7A11 and SLC3A2, is a cystine/glutamate antiporter that mediates the efflux of glutamate and the influx of cystine at a 1:1 ratio. After entering the cell, cystine is reduced to cysteine and then synthesized into GSH. GPX4 scavenges lipid ROS via GSH. Lipid ROS derives from PUFAs-PE oxidation by lipoxygenases. The scaffolding protein PEBP1 can bind PE on the cell membrane and then recruit the lipoxygenase 15LO to generate lipid ROS. ACSL4 can increase lipid ROS by producing PUFAs-PE. Moreover, TCA cycle disorder or iron overload in mitochondria can also increase ROS, which results in ferroptosis. The CoQ/FSP1 and Trx/TXNRD axes inhibit ferroptosis in a GSH-independent manner. The Keap1/NRF2, p53, and YAP/TAZ signaling are necessary for the transcription of ferroptosis regulators, such as SLC7A11 and ACSL4. Erastin, RSL3, and auranofin are ferroptosis agonists by targeting SLC7A11, GPX4, and TXNRD, respectively. Ferroptosis inhibitors include iron chelators and lipid ROS scavengers (ferrostatin-1, liproxstatin-1, vitamin E, etc.).
For another thing, some phytochemicals also inhibit hepatic ferroptosis to protect against NASH, drug-induced liver injury, and acute liver failure (Table 2). For example, some investigations discovered that some natural products, such as ginkgolide B and dehydroabietic acid, could alleviate NASH pathology by activating Nrf2 signaling to inhibit ferroptosis [159,160]. Clausenamide could prevent drug-induced hepatocyte ferroptosis via the activation of the Keap1-Nrf2 pathway [161]. Glycyrrhizin significantly reduced the degree of ferroptosis in acute liver failure by enhancing glutathione synthesis [162]. Holly (Ilex latifolia Thunb.) polyphenol extracts are able to relieve hepatic ferroptosis by inhibiting iron transport and enhancing GPX4 expression [163]. Moreover, baicalein supplementation ameliorates CCl 4 -induced acute liver injury in mice by inhibiting ferroptosis and inflammation, which involves the activation of Nrf2 and the inhibition of lipoxygenases and the NF-kB pathway [164].

Conclusions and Future Directions
Liver disease is a global health burden that has complex mechanisms and needs effective therapeutics in the early stage of liver injury. Given a growing number of polysaccharides with bioactivities (such as antioxidant, immunoregulation, and tumor killing activities) have been identified, the polysaccharide may provide a promising therapeutic strategy for liver diseases. Recently, various natural polysaccharides have been reported to possess protective roles in several liver diseases resulting from fatty liver, alcohol, drug toxicity, or HCC. Moreover, angelica polysaccharides can be developed into a hypoxia-responsive nano-drug delivery system that facilitated HCC chemotherapy. However, studies about polysaccharides on virus hepatitis have been reported less than other liver diseases, suggesting polysaccharides with anti-virus bioactivity need to be identified. Currently, the majority of data are collected from in vitro and animal experiments. Therefore, further studies in humans are needed in order to evaluate the efficacy of these polysaccharides in the clinic.
Cell death, including apoptosis or ferroptosis, is a double-edged sword for health. Therefore, phytochemicals, such as cell death agonists or inhibitors, may play different roles in treating liver diseases. For example, some phytochemicals that inhibit cell death could alleviate ALD, DILI, or chemotherapeutic toxicity in the liver, while lethal phytochemicals may serve as chemotherapeutics in HCC. Ferroptosis is a new type of cell death with features of iron and lipid ROS accumulation that is different from other types of cell death. The discovery of ferroptosis has greatly improved the understanding and therapeutic strategies of liver disease. Several compounds and phytochemicals could alleviate liver injury by targeting ferroptosis, while inhibitors of other cell death (such as apoptosis) could not. Moreover, phytochemicals with ferroptosis-inducing activities might be effective and promising drugs for HCC because ferroptosis agonists can evade the drug resistance of classic chemotherapeutics (e.g., cisplatin, which kills tumors by apoptosis). Therefore, elucidating the mechanisms of ferroptosis and identifying more ferroptosis-regulatory phytochemicals may provide novel therapeutic strategies for liver diseases in the future.