Folate is a water-soluble B9 vitamin synthesised in plants from 6-hydroxymethyldihydropterin, p-aminobenzoate and glutamate and is a co-enzyme substrate involved in one-carbon transfer reactions(Reference Sid, Siow and Karmin1,Reference da Silva, Kelly and Al Rajabi2) . Natural folate exists physiologically in the form of tetrahydrofolate (THF, active form) as well as methyltetrahydrofolate (MTHF, primary form found in blood)(Reference Menezo, Elder and Clement3). Folic acid is a synthetic (stable) form commonly used in dietary supplements and food fortification(Reference Sid, Siow and Karmin1,Reference Menezo, Elder and Clement3) . At present, studies have found that folate deficiency is associated with many diseases, including macrocytic anaemia, mucositis, infertility, muscle weakness, CVD, nervous system diseases, liver diseases, cancer, etc.(Reference Shulpekova, Nechaev and Kardasheva4). Moreover, folate supplementation can reduce the occurrence of birth defects including fetal neural tube diseases, anaemia and neurological disorders in newborns(Reference Cario, Smith and Blom5–Reference Stover7). Because of these benefits, fortification of flour and grain products with folic acid has been applied in many countries(Reference Choi, Yates and Veysey8).
Although folate has an indispensable and important role in our body, there is concern for adverse effects with excessive folic acid supplementation. Currently, the RDA for folate by the US Food and Nutrition Board varies by age and sex(9). The RDA for folate in children aged 1–13 years old is 150–300 mcg of dietary folate equivalents. The RDA for folate in people aged 14 and above is 400 mcg of dietary folate equivalents, while the RDA of folate in pregnant and lactating women is 600 mcg of dietary folate equivalents and 500 mcg of dietary folate equivalents, respectively(9). Studies have found that daily intake of 400 μg of folic acid can lead to unmetabolised folic acid in plasma(Reference Sweeney, McPartlin and Scott10). However, most folic acid supplements contain more than 400 μg of folic acid(Reference Christensen, Mikael and Leung11), and some population groups have total folic acid intake that exceeds the tolerable upper limit, due to the use of fortified foods and dietary supplements(Reference Bailey, Dodd and Gahche12,Reference Bailey, McDowell and Dodd13) . Kalmbach et al. also found that folic acid fortification is related to increased exposure to circulating folic acid(Reference Kalmbach, Choumenkovitch and Troen14). The impact of folic acid on pregnant women and fetuses has always been a concern. In mice models, the recommended dose of folic acid for rodents is 2 mg/kg. The study found that a high folic acid diet (20 mg/kg) in pregnant mice is associated with embryo loss, delayed embryo growth, a higher incidence of ventricular septal defect and thinning of the left and right ventricular walls during embryonic development(Reference Mikael, Deng and Paul15). However, in human studies, the use of high doses of folic acid during pregnancy is neither harmful to fetal growth nor associated with the occurrence of oral clefts or congenital heart disease(Reference Silva, Keating and Pinto16). It’s worth noting that another observational study in humans showed that higher plasma folate was associated with higher 2-h glucose and higher odds of gestational diabetes mellitus(Reference Lai, Pang and Cai17). It is well known that gestational diabetes can lead to fetal growth restriction and an increased risk of maternal diabetes. Therefore, we believe that further clinical studies are needed to address the impact of excessive folic acid supplementation on both pregnant women and fetuses.
Moreover, high folic acid intake has been linked to an increased risk of adenomatous lesion and prostate cancer(Reference Fife, Raniga and Hider18,Reference Hultdin, Van Guelpen and Bergh19) . It has also been found that increased levels of unmetabolised folic acid in plasma can reduce cognitive test scores in seniors aged ≥ 60 years and can dampen cytotoxicity of natural killer cells in postmenopausal women(Reference Morris, Jacques and Rosenberg20,Reference Troen, Mitchell and Sorensen21) . In rats fed with a high-fat diet (HFD), it has been reported that excessive folic acid can exacerbate weight gain, fat accumulation, impaired glucose tolerance and white adipose tissue inflammation(Reference Kelly, Kennelly and Ordonez22).
The liver is an important organ of the human body, which has the functions of detoxification, metabolism, synthesis and storage(Reference Nojima, Freeman and Gulbins23), and participates in the metabolism of carbohydrates, proteins and lipids, the clearance of drugs, toxins and pathogens in the blood and the regulation of immune responses(Reference Protzer, Maini and Knolle24). However, many drugs, environmental toxins and dietary ingredients can induce liver injury, including dysregulation of lipid metabolism, degeneration and necrosis of hepatocyte and activation of immune responses(Reference Chen, Dong and Thompson25).
Folate plays an important role in maintaining methylation ability, and many methylation reactions occur in the liver(Reference da Silva, Kelly and Al Rajabi2). The liver is also the main organ for folate/folic acid storage and metabolism(Reference Sid, Siow and Karmin1), and the reduction of folic acid depends on the role of dihydrofolate reductase in the liver(Reference Menezo, Elder and Clement3). Therefore, there is an inseparable relationship between the liver and the biological effects of folate/folic acid. In order to increase people’s understanding and awareness of folate and folic acid intake, especially for people with pre-existing liver diseases, this article systematically summarises the effects and mechanisms of folate/folic acid in the liver by referring to published literature and provides theoretical support for clinical doctors and nutritionists to use folate and folic acid-related drugs and supplements reasonably.
Molecular structure, pharmacokinetics and biological function of folate/folic acid
Molecular structure and source of folate/folic acid
The chemical formula of folic acid is C19H19N7O6 (Fig. 1), and its molecular core is composed of a heterocyclic pterin structure. The methyl group at the sixth position is combined with para-aminobenzoic and glutamic acid, thus presenting pteroylglutamic acid(Reference Shulpekova, Nechaev and Kardasheva4,Reference Stover and Field26) . Pterin is composed of pyrimidine and pyrazine rings with substituting keto- and amino groups in the second and fourth positions(Reference Shulpekova, Nechaev and Kardasheva4).
Figure 1. Chemical structure of folic acid (4).
Folate and folic acid are different forms of vitamin B9, and the main difference between these two is their sources. Folate is a natural form and can be present in dark green leaves, mushrooms, animal liver, yeast and so on(Reference Shulpekova, Nechaev and Kardasheva4,Reference Zhao, Matherly and Goldman27) . Mammals lack the enzymatic capacity to synthesise folates, therefore, it is essential to obtain folate through dietary intake(Reference Zhao, Matherly and Goldman27). Folic acid is a synthetic and stable form of folate that is normally used for extra oral supplementation and fortification(Reference Zhao, Matherly and Goldman27–Reference Wright, Dainty and Finglas29). Therefore, in some countries, such as the USA, Canada, etc., cereals, grains and breads fortified with folic acid are an important source of folate/folic acid(Reference Zhao, Matherly and Goldman27). In addition, studies have found that folate-producing bacteria in the cecum, colon (mainly including Lactobacillus and Bifidobacterium) and proximal small intestine can also serve as a source of folate(Reference Camilo, Zimmerman and Mason30–Reference Rossi, Amaretti and Raimondi32).
Absorption, bioavailability and pharmacokinetics of folate/folic acid
Dietary folate is mainly absorbed by the duodenum and proximal jejunum(Reference Rossi, Amaretti and Raimondi32,Reference Zhao, Diop-Bove and Visentin33) , while folate synthesised by gut bacteria may be adsorbed from the colon(Reference Shulpekova, Nechaev and Kardasheva4). The main mechanism of folate absorption is that folate (polyglutamate) is hydrolysed to monoglutamate by glutamate carboxypeptidase II (folate hydrolase) in the brush-border membrane of the small intestine. Then, monoglutamate is transported into cells by proton-coupled folate transporter and reduced folate carrier. The pH of the proximal small intestine is 5·8–6·0, while the reduced folate carrier performs its optimal transport function at pH 7·4, and the activity of the reduced folate carrier also decreases as the pH decreases. Therefore, the contribution of a reduced folate carrier to folate absorption is much less than that of a proton-coupled folate transporter(Reference Shulpekova, Nechaev and Kardasheva4,Reference Zhao, Diop-Bove and Visentin33,Reference Visentin, Diop-Bove and Zhao34) . After absorption by enterocytes, folic acid undergoes reduction to dihydrofolate, THF and 5,10-methylene-THF, ultimately converting to biologically active 5-methyltetrahydrofolate (5-MTHF), which is the dominant physiological form in the blood(Reference Menezo, Elder and Clement3,Reference Shulpekova, Nechaev and Kardasheva4,Reference Lucock35) . 5-MTHF is then transported to the blood through the basolateral membrane under the action of multi-drug resistance-associated protein 3, thus achieving rapid and efficient transepithelial transport(Reference Zhao, Diop-Bove and Visentin33,Reference Visentin, Diop-Bove and Zhao34) . In terms of bioavailability, folic acid is generally believed to be more bioavailable than natural folate in food(Reference Iyer and Tomar28). Because folic acid is a monoglutamate, it can be directly absorbed in the intestine without the hydrolysis step, and the bioavailability is very high, which can be approximately up to 100 %(9,Reference Iyer and Tomar28) . Afterwards, 5-MTHF enters the hepatic portal system through the bloodstream and is transported to the hepatic sinuses. There are three main metabolic routes for 5-MTHF after entering the liver. First, it can be converted to polyglutamate for storage. Second, it can be secreted into the bile, returned to the duodenum and jejunum and subsequently reabsorbed, thus completing the enterohepatic circulation. Third, it can enter the hepatic vein and eventually reach the systemic circulation, where it is taken up by peripheral tissues, converted to THF, and participates in one-carbon transfer reactions(Reference Sid, Siow and Karmin1,Reference Zhao, Matherly and Goldman27,Reference Lucock35) . 5-MTHF that does not bind to serum protein is reabsorbed by the proximal tubules after glomerular filtration(Reference Zhao, Matherly and Goldman27,Reference Lucock35) . According to previous studies, 5-MTHF can be detected in the liver, kidneys, bile acid, etc., while the liver is the main organ for storing and metabolising folate/folic acid(Reference Zhao, Matherly and Goldman27,Reference Wright, Dainty and Finglas29) . The liver plays a central role in the homeostasis of folate/folic acid(Reference Lucock35). Therefore, studying the role of folate/folic acid in the liver is crucial for exploring the systemic effects of folate/folic acid(Reference Steinberg, Campbell and Hillman36).
Functional mechanism and signalling pathways
Folate plays an important role in one-carbon transfer reactions involved in nucleic acid biosynthesis, methylation reactions and sulphur-containing amino acid metabolism(Reference Sid, Siow and Karmin1). The one-carbon metabolism of folate is mediated by THF and is a metabolic network of interdependent biosynthetic pathways, which mainly occurs in mitochondria, cytoplasm and nucleus(Reference Fox and Stover37). In the mitochondria, it is mainly the interconversion of activated one-carbon units carried by THF(Reference Tibbetts and Appling38). First, serine, glycine, dimethylglycine and sarcosine undergo catabolism under the action of mitochondrial serine hydroxymethyltransferase (SHMT), aminomethyltransferase, dimethylglycine dehydrogenase and sarcosine dehydrogenase, respectively(Reference Stover and Field26,Reference Fox and Stover37) . This process depends on THF and produces 5, 10-methylene-THF(Reference Stover and Field26). Then, 5, 10-methenylTHF is formed under the action of 5,10-methylenetetrahydrofolate dehydrogenase and further oxidised to 10-formyl-THF by 5, 10-methyltetrahydrofolate cyclohydrolase(Reference Fox and Stover37). Finally, formate and free THF are formed under the action of methylenetetrahydrofolate dehydrogenase 1 like (MTHFD1L)(Reference Stover and Field26,Reference Fox and Stover37,Reference Froese, Fowler and Baumgartner39) . In addition, Met-tRNA forms fMet-tRNA under the cofactor 10-formyl-THF to initiate mitochondrial protein synthesis(Reference Stover and Field26). Serine, glycine and formate are transported to the cytoplasm through the mitochondrial membrane and become carbon donors in the cytoplasm(Reference Tibbetts and Appling38,Reference Pike, Rajendra and Artzt40) . In addition, one-carbon can also be directly produced in the cytoplasm through the catabolism of histidine, purine and serine, but formate from mitochondrial one-carbon metabolism is the main source(Reference Stover and Field26,Reference Fox and Stover37) . In the cytoplasm, folate-mediated one-carbon metabolism involves three interdependent biosynthetic pathways, including de novo synthesis of purine nucleotides, thymidylate (dTMP) and remethylation of homocysteine to methionine(Reference Stover and Field26). The pathway involved is that serine transfers a carbon unit to THF under the action of SHMT to form 5,10-methylene-THF and glycine(Reference Tibbetts and Appling38). 5,10-Methylene-THF can synthesise dTMP directly under dTMP synthase(Reference Fox and Stover37,Reference Tibbetts and Appling38,Reference Pike, Rajendra and Artzt40) . While 5,10-methylene-THF can be oxidised to 10-formyl-THF for purine synthesis, formate can also produce 10-formyl-THF(Reference Fox and Stover37,Reference Tibbetts and Appling38,Reference Pike, Rajendra and Artzt40) . Conversely, 5,10-methylene-THF can be reduced to 5-MTHF, which is a carbon donor for the remethylation of homocysteine. Methionine is synthesised by homocysteine under the action of methionine synthase and then enters the methyl cycle by S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH)(Reference Stover and Field26,Reference Fox and Stover37,Reference Tibbetts and Appling38,Reference Pike, Rajendra and Artzt40) . In the nucleus, 5,10-methylene-THF, as a carbon donor, catalyses the methylation of deoxyuridylate to dTMP under dTMP synthase. Moreover, the nucleus also contains the mutual conversion of THF and dihydrofolate and the process of serine acting as a carbon donor through the serine hydroxymethyltransferase reaction, which involves three enzymes (SHMT, dTMP synthase and dihydrofolate reductase) constituting the dTMP synthesis cycle(Reference Fox and Stover37,Reference Tibbetts and Appling38) .
Both dTMP synthesis and homocysteine remethylation are sensitive to folate deficiency(Reference Stover7). Folate deficiency reduces dTMP levels, which increases errant incorporation of uracil into DNA, leading to abnormalities in DNA and chromosome structure(Reference Kim, Jang and Sauer41). The previous study has found that folate deficiency can induce DNA deletion, cytochrome c oxidase dysfunction, membrane depolarisation and superoxide overproduction in rat liver, thereby promoting mitochondrial oxidative decay. Folic acid supplementation can ameliorate this defect(Reference Chang, Yu and Lu42). In a carbon tetrachloride-induced rat model of liver injury, reduced folate levels interfered with DNA synthesis and prevented or delayed liver regeneration(Reference Leevy, Ten-Hove and Frank43). Alcoholics have lower levels of folate(Reference Wu, Chanarin and Slavin44), and folate deficiency may contribute to alcoholic liver disease (ALD) through epigenetic effects that reduce methyl supply for silencing gene expression and/or DNA stability(Reference Medici and Halsted45). Folate deficiency also can promote alcoholic liver injury by disrupting liver methionine metabolism and DNA strand break in a micropig experiment(Reference Halsted, Villanueva and Devlin46). In addition, there is a strong negative correlation between folate and plasma homocysteine levels(Reference Krishnaswamy and Madhavan Nair47,Reference Chen, Yang and Peng48) . Increased homocysteine leads to increased serum and cellular SAH levels, which is a potent inhibitor of SAM-dependent methylation reactions including protein and DNA methylases(Reference Stover7). Hyperhomocysteinaemia can cause the activation of multiple transcription factors including sterol regulatory element-binding protein-2, cAMP response element-binding protein and nuclear factor Y in the liver and lead to an increase in HMG-CoA (3-hydroxy-3-methylglutaryl co-enzyme A) reductase and cholesterol biosynthesis, resulting in hepatic lipid accumulation and hypercholesterolaemia(Reference Woo, Siow and Pierce49). Combined treatment with folic acid and vitamin B12 can normalise plasma homocysteine levels and reduce oxidative stress to attenuate alcohol-induced liver injury(Reference Chen, Yang and Peng48). Moreover, folate deficiency is associated with impaired antioxidant enzyme activity, increased production of reactive oxygen species and lipid peroxidation(Reference Chang, Yu and Lu42,Reference Chern, Huang and Chen50,Reference Huang, Hsu and Lin51) . A study has shown that folate may play a direct effect on free-radical-induced oxidation of LDL(Reference Nakano, Higgins and Powers52). In conclusion, folate-mediated one-carbon metabolism plays a crucial role in liver diseases. Studying the role of folate in liver disease and optimal folic acid dose has the potential to guide clinical treatment. The next section addresses the role and mechanism of folate/folic acid in different liver diseases.
The role of folate/folic acid in liver diseases
Folate/folic acid and non-alcoholic fatty liver disease/non-alcoholic steatohepatitis
Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease characterised by steatosis, inflammation, fibrosis and liver injury. There is currently no effective treatment for this disease, apart from strengthening exercise and controlling diet(Reference Sid, Siow and Karmin1). NAFLD can develop into non-alcoholic steatohepatitis (NASH), a more severe inflammatory and hepatocyte damage process typically accompanied by pericellular fibrosis, which may develop into cirrhosis(Reference Kim and Min53,Reference Friedman, Neuschwander-Tetri and Rinella54) . A study has shown that low serum folate level is an independent risk factor for NAFLD in the Chinese population and is associated with the severity of hepatic steatosis(Reference Xia, Bian and Zhu55). The hepatic steatosis is often observed in disorders of one-carbon metabolism(Reference da Silva, Kelly and Al Rajabi2). It has been reported that patients with NAFLD have lower circulating folate levels, but it is unclear whether folate deficiency is a cause or a consequence of NAFLD. In addition, although serum folate levels may be associated with the development of NAFLD(Reference Sid, Siow and Karmin1), there is evidence that a moderate amount of folic acid can prevent NAFLD, while excessive folic acid may be counterproductive.
Studies have found that folic acid supplementation can inhibit the NF-κB pathway by reducing the concentration of reactive oxygen species and homocysteine. At the same time, folic acid can also inhibit the expression of pro-inflammatory cytokines (IL-6, TNF-α, IL-1β), thereby improving liver inflammation in mice fed HFD and HepG2 cells treated with palmitic acid or homocysteine(Reference Sid, Shang and Siow56,Reference Bagherieh, Kheirollahi and Zamani-Garmsiri57) . Folic acid can improve HFD-induced steatohepatitis in rats, partly because it can increase PPARα levels through a silence information regulation factor 1-dependent manner, thereby improving liver lipid metabolism. Another reason is that folic acid administration can also restore depleted liver one-carbon metabolism and gut microbiota diversity(Reference Xin, Zhao and Zhang58). During HFD feeding, folic acid supplementation restores AMP-activated protein kinase activation by increasing AMP levels and liver kinase B1 phosphorylation in the liver, which contributes to ameliorating glucose and cholesterol metabolism impaired by high-fat dietary intake(Reference Sid, Wu and Sarna59). Further research in high-fructose-fed rats revealed that folic acid enhanced the levels of phosphorylated AMP-activated protein kinase and liver kinase B1 and increased phosphorylation (inactivation) of acetyl co-enzyme A carboxylase in the liver, thereby inhibiting hepatic lipogenesis and ameliorating hepatic steatosis(Reference Kim and Min53). The activity of NADPH oxidase in the liver of HFD-fed mice significantly increased. However, supplementation with folic acid can downregulate the gene expression of NADPH oxidase subunits (including gp91phox, p22phox and p47phox) and inhibit the activation of NADPH oxidase by inhibiting NF-κB pathway, thus improving liver oxidative stress(Reference Sarna, Wu and Wang60). Moreover, folic acid can also increase the activities of the antioxidant enzymes superoxide dismutase and catalase and correct the equilibrium between reduced glutathione (GSH) and oxidised glutathione, suggesting a protective role of folic acid against HFD-induced oxidative damage in the liver(Reference Sarna, Wu and Wang60). These results illustrate the mechanism and therapeutic role of folic acid in NAFLD/NASH, suggesting that folic acid may become a therapeutic drug for NAFLD/NASH in the future. However, in a randomised controlled trial of the effects of folic acid supplementation on liver enzymes, lipids and insulin resistance in patients with NAFLD, folic acid supplementation (1 mg/d) for 8 weeks was able to prevent an increase in homocysteine but did not significantly alter serum liver enzyme levels, degree of liver steatosis, insulin resistance and lipid levels. Therefore, the course and dosage of folic acid supplementation in NAFLD patients in the future are also the focus of exploration(Reference Molaqanbari, Zarringol and Talari61). The trial included only sixty-six patients and needs to be confirmed in larger trials (Fig. 2).
Figure 2. Folic acid supplementation and NAFLD/NASH. Folic acid inhibits the NF-κB pathway by decreasing ROS and Hcy concentrations and inhibits IL-6, TNF-α and IL-1β to improve liver inflammation. Folic acid improves hepatic lipid metabolism by increasing PPARα levels in a SIRT1-dependent manner and restores hepatic single-carbon metabolism and gut microbiota diversity. Folic acid restores AMPK activation by increasing AMP and LKB1 phosphorylation levels, thus ameliorating glucose and cholesterol metabolism. Folic acid inhibits hepatic steatosis by increasing the phosphorylation of AMPK and LKB1 and ACC. Folic acid improves liver oxidative stress by inhibiting the activation of NADPH oxidase, increasing the activities of SOD and catalase and correcting the equilibrium between reduced GSH and GSSG. ROS, reactive oxygen species; Hcy, homocysteine; SIRT1, silence information regulation factor 1; p-, phosphorylation; LKB1, liver kinase B; AMPK, AMP-activated protein kinase; ACC, acetyl co-enzyme A carboxylase; SOD, superoxide dismutase; GSH, glutathione; GSSG, oxidised glutathione; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis. ↑increase; ↓decrease; 
alleviate.
Although most studies have shown the protective effect of folic acid on NAFLD/NASH, detrimental roles due to its excessive dosage have also been indicated. When detecting the cell viability of HepG2 cells exposed to folic acid at different gradient concentrations, treatment with 5–75 μg/ml of folic acid had no statistics effect on HepG2 cell viability, whereas a high concentration of folic acid (higher than 100 μg/ml) appeared to be toxic and reduced the cell viability(Reference Bagherieh, Kheirollahi and Zamani-Garmsiri57). In addition, excessive intake of folic acid can affect the metabolic processes of glucose and lipids, exacerbating metabolic syndrome. In HFD rats, feeding excess folic acid (7·5 mg/kg, 12 weeks) exacerbates weight gain, fat mass and glucose intolerance(Reference Kelly, Kennelly and Ordonez22). The increased adipose size and mass have been proven to be induced by increased key transcriptional regulators of lipid metabolism (PPARγ, sterol regulatory element-binding transcription factor 1, sterol regulatory element-binding transcription factor 2, nuclear receptor subfamily 1 group H member 2, nuclear receptor subfamily 1 group H member 3 and lipogenic genes. Excess folic acid can increase TAG accumulation by upregulating the expression of PPARγ-2 in 3T3-L1 cells. Moreover, excessive folic acid increases the inflammation of white adipose tissue in rats by increasing the levels of monocyte chemoattractant protein-1, TNF-α, NADPH oxidase 1 and binding Ig protein (BiP)(Reference Kelly, Kennelly and Ordonez22). High dose of folic acid intake (20 mg/kg folic acid, 6 months) reduces the level of methylenetetrahydrofolate reductase protein and activity, resulting in methylenetetrahydrofolate reductase deficiency, as well as the reduction of MTHF and methylation capacity. Methylenetetrahydrofolate reductase-deficient hepatocyte cannot alleviate the effects of phospholipid and lipid disorders, thereby leading to hepatocyte damage and liver injury(Reference Christensen, Mikael and Leung11). It has also been found that excessive folic acid supplementation (40 mg/kg diet) in pregnant mice will lead to reduced islet β-cell mass and insulin synthesis in their offspring. At the same time, excessive folic acid also can result in increased expression of fat metabolism related genes Pparγ2 and cell death-inducing DFF45-like effector c (Cidec) and higher liver TAG content in the offspring(Reference Kintaka, Wada and Shioda62). These suggest that excessive folic acid may lead to glucose intolerance, fat accumulation and adipose tissue inflammation, thereby exacerbating the development of metabolic syndrome. Excessive folic acid supplementation may interfere with lipid metabolism, promoting changes in one-carbon metabolic pathways and gene expression patterns, thereby leading to liver injury and harmful effects (Fig. 3).
Figure 3. Excessive folic acid and NAFLD/NASH. High doses of folic acid can cause cytotoxicity and decrease cell viability. Excessive folic acid promotes the increase of Pparg, Srebf1, Srebf2, Nr1h2 and Nr1h3 and induces the increase of fat size and mass. Excess folic acid upregulates PPARγ to increase TAG accumulation. Excessive folic acid increases the levels of monocyte chemoattractant protein-1, TNF-α, NADPH oxidase 1 and BiP to promote inflammation in white adipose tissue. High-dose folic acid intake causes MTHFR deficiency and reduces MTHF and methylation capacity, which leads to liver damage. Excessive supplementation of folic acid during pregnancy can reduce insulin synthesis and increase TAG content by raising the expression of Pparγ2 and Cidec in the offspring. Pparg, PPARγ, peroxisome proliferator-activated receptor γ; Srebf, sterol regulatory element-binding transcription factor; Nr1h, nuclear receptor subfamily 1 group H member; MCP-1, monocyte chemoattractant protein-1; NOX1, NADPH oxidase 1; BiP, binding Ig protein; MTHFR, methylenetetrahydrofolate reductase; MTHF, methyltetrahydrofolate; Cidec, cell death-inducing DFF45-like effector c. ↑increase; ↓decrease; 
aggravate.
In conclusion, the decrease of folate level can aggravate the steatosis of the liver. Moderate supplementation of folic acid may alleviate NAFLD/NASH by improving the oxidative stress and lipid metabolism of the liver and reducing the inflammation level of the liver, but excessive supplementation of folic acid may aggravate NAFLD/NASH. Unfortunately, how to determine the ‘moderate’ and ‘excessive’ level of folic acid is currently unknown in human experimental studies because most of the research is based on cells and animals. So it is necessary to explore the appropriate clinical dose for NAFLD/NASH in future study.
Folate/folic acid and alcoholic liver disease
The report states that about 69–80 % of alcoholics have low serum folate levels(Reference Zhao, Gao and Zhang63), which may be due to multiple reasons. People who are chronically dependent on excessive alcohol may develop folate deficiency due to poor diet, reduced folate absorption and liver uptake and increased renal excretion. Moreover, folate deficiency may promote the development of ALD by exacerbating abnormal methionine metabolism(Reference Halsted, Villanueva and Devlin64). Ethanol can change liver methionine metabolism, resulting in the interference of SAM-dependent transmethylation(Reference Schalinske and Nieman65). Folate deficiency increases plasma homocysteine levels, decreases SAM/SAH ratio and GSH levels and increases cell apoptosis and DNA strand breaks. Therefore, folate deficiency promotes alcohol-induced liver damage by exacerbating liver methionine metabolism disorders and DNA damage(Reference Halsted, Villanueva and Devlin46,Reference Halsted, Villanueva and Devlin64) . In addition, cytochrome P-450 2E1 and endoplasmic reticulum (ER) stress signals including glucose-regulated protein 78 (GRP78), caspase 12 and sterol regulatory element-binding protein-1c are activated in response to folate deficiency. Their expression levels are positively correlated with SAH and/or homocysteine levels in the liver and negatively correlated with the SAM/SAH ratio. These results indicate that abnormal hepatic methionine metabolism induced by a combination of ethanol and insufficient folate is closely associated with cytochrome P-450 2E1 activation and enhanced ER stress pathway, which aggravated steatosis and apoptosis(Reference Esfandiari, Villanueva and Wong66).
Therefore, folic acid supplementation may become a key method for the treatment of ALD. Folic acid supplementation can reduce alanine transaminase and aspartate transaminase activities, decrease lipid and DNA oxidation, increase GSH levels, decrease homocysteine and ameliorate oxidative stress to reduce hepatotoxicity caused by chronic alcohol intake(Reference Lee, Kang and Min67,Reference Ojeda, Rua and Nogales68) . Folic acid inhibits the elevation of TAG, total cholesterol and LDL and liver fat deposition caused by ethanol. The potential mechanism is that folic acid supplementation ameliorates hepatic Th17/Treg imbalance in mice with long-term alcohol exposure by reducing DNA methyltransferase 3α levels and then downregulating the methylation levels of carboxypeptidase G2 and carboxypeptidase G3 in Forkhead box P3 promoter region, thereby improving ethanol-induced inflammatory damage(Reference Zhao, Guo and Zuo69). In addition, folic acid can improve mitochondrial function and inhibit mitophagy and mitochondrial fission by reducing the expression of PTEN-induced putative kinase 1-parkin and dynamin-related protein 1, thereby preventing hepatocyte apoptosis(Reference Zhao, Gao and Zhang63). In summary, folic acid can improve ALD by regulating oxidative stress, lipid metabolism and mitophagy. Considering the harm of folate deficiency in ALD patients and the benefits of supplementing folic acid, taking folic acid is recommended for ALD patients (Fig. 4).
Figure 4. The mechanism of folate deficiency and folic acid supplementation in ALD. Folate deficiency increases Hcy levels, decreases SAM/SAH ratio and GSH levels and increases apoptosis and DNA strand breakage. Folate deficiency leads to the activation of CYP2E1 and ER stress signals including GRP78, caspase 12 and SREBP-1c, thereby increasing levels of SAH and homocysteine, reducing the SAM/SAH ratio and exacerbating steatosis and apoptosis. Folic acid supplementation can lower ALT and AST, reduce lipid and DNA oxidation and improve oxidative stress by increasing GSH and decreasing Hcy levels. Folic acid can improve Th17/Treg imbalance by decreasing DNMT3a level, and downregulating CPG2 and CPG3 methylation levels in the Foxp3 promoter region. Folic acid can reduce the expression of PINK1-parkin and Drp1, improve mitochondrial function, inhibit mitochondrial autophagy and mitochondrial division and thus prevent hepatocyte apoptosis. Hcy, homocysteine; GSH, glutathione; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; GRP78, glucose-regulated protein 78; CYP2E1, cytochrome P-450 2E1; SREBP-1c, sterol regulatory element-binding protein-1c; ER, endoplasmic reticulum; ALT, alanine transaminase; AST, aspartate transaminase; DNMT3a, DNA methyltransferase 3 alpha; Foxp3, Forkhead box P3; CPG, carboxypeptidase G; PINK1, PTEN-induced putative kinase 1; Drp1, dynamin-related protein 1. ↑increase; ↓decrease; 
aggravate; 
alleviate.
Folic acid and drug-induced liver injury
Most studies have shown that folic acid has a protective effect on drug-induced liver injury (DILI) induced by different aetiologies. In a randomised clinical trial, folic acid can better reduce alanine transaminase and aspartate transaminase levels in response to antiepileptic DILI compared with silymarin, suggesting an advantage of folic acid in the treatment of DILI(Reference Asgarshirazi, Shariat and Sheikh70). Folic acid can significantly increase the activities of superoxide dismutase and catalase, as well as GSH levels, thereby alleviating oxidative stress caused by acetaminophen and reducing the area of liver necrosis(Reference Akgun, Boyacioglu and Kum71). In liver injury induced by homocysteine, on the one hand, folic acid supplementation can effectively inhibit the generation of superoxide anion mediated by NADPH oxidase, prevent oxidative stress and thus reduce liver lipid peroxidation(Reference Woo, Prathapasinghe and Siow72). On the other hand, folic acid can inhibit the expression of DNA methyltransferase 1 (DNMT1) and enhancer of zeste homolog 2, decrease levels of SAM and SAH and increase the intracellular ratio of SAM/SAH. These result in the inhibition of homocysteine-induced DNA methylation and trimethylation of lysine 27 on histone H3 of cystic fibrosis transmembrane conductance regulator promoter, thereby upregulating cystic fibrosis transmembrane conductance regulator expression, accelerating homocysteine metabolism and alleviating ER stress and hepatocyte apoptosis(Reference Yang, Sun and Mao73). In CCL4-induced liver injury, folic acid can restore the activities of catalase and GSH, downregulate the mRNA expression levels of programmed cell death-receptor and TNF-α and upregulate the concentration of cell survival signals protein kinase B, interferonγ, so as to prevent lipid peroxidation, reduce inflammation and improve liver injury. Additionally, the combination of folic acid and melatonin has a better effect in protecting liver function(Reference Ebaid, Bashandy and Alhazza74). In a rat model of liver injury induced by antituberculosis drugs (isoniazid and rifampicin), folic acid supplementation can alleviate liver injury caused by antituberculosis drugs. The potential mechanism may be associated with regulating n-acylethanolamine metabolism, enhancing the detoxification and clearance of isoniazid and rifampicin, promoting liver regeneration, downregulating inflammation and so on(Reference Jiang, Gai and Ni75). Folic acid combined with vitamin B12 can increase the activities of superoxide dismutase, catalase and the level of non-protein-soluble thiol in the liver, inhibit the increase of thiobarbituric acid reactive substances and conjugated dienes and thus relieve liver tissue degeneration and DNA damage and prevent liver injury caused by arsenic exposure(Reference Chattopadhyay, Deb and Maiti76). However, there is no study on the improvement of arsenic-induced liver injury by folic acid supplementation alone, which still needs deeper exploration. Therefore, the efficacy, side effects and optimal dose of folic acid in the treatment of DILI need to be further studied (Fig. 5).
Figure 5. Folic acid and DILI. Folic acid supplementation can lower ALT and AST levels. Folic acid can increase the activities of SOD and catalase and the level of GSH, thereby alleviating oxidative stress and reducing the area of liver necrosis. Folic acid supplementation can inhibit the generation of superoxide anions mediated by NADPH oxidase and prevent oxidative stress, thereby reducing hepatic lipid peroxidation. Folic acid can restore the activities of catalase and GSH, downregulate the levels of Fas and TNF-α and upregulate the concentrations of Akt1 and IFN-γ, so as to prevent lipid peroxidation and reduce liver inflammation. Folic acid can inhibit DNMT1 and EZH2, decrease SAM and SAH levels, increase SAM/SAH ratio and inhibit DNA methylation and H3K27me3, thereby upregulating CFTR expression and alleviating ER stress and hepatocyte. Folic acid can regulate n-acylethanolamine metabolism, enhance the detoxification and clearance of INH and RIF, promote liver regeneration, downregulate inflammatory response and thus improve liver injury. Folic acid combined with vitamin B12 can increase SOD, catalase and NPSH levels and reduce TBARS and CDs, thus alleviating liver tissue degeneration and DNA damage. ALT, alanine transaminase; AST, aspartate transaminase; SOD, superoxide dismutase; GSH, glutathione; Fas, programmed cell death-receptor; Akt1, protein kinase B; IFN-γ, interferon γ; DNMT1, DNA methyltransferase 1; EZH2, enhancer of zeste homolog 2; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; H3K27me3, trimethylation of lysine 27 on histone H3; CFTR, cystic fibrosis transmembrane conductance regulator; ER, endoplasmic reticulum; INH, isoniazid; RIF, rifampicin; NPSH, non-protein-soluble thiol; TBARS, thiobarbituric acid reactive substances; CDs, conjugated dienes; DILI, drug-induced liver injury; VitB12, vitamin B12. ↑increase; ↓decrease; 
alleviate.
Folic acid and liver fibrosis/cirrhosis
Plasma folate concentrations are low in patients with cirrhosis, and low folate levels are significantly associated with the severity of fibrosis(Reference Ho, Tsai and Hsieh77,Reference Chen, Luo and Li78) . The role of folic acid in liver fibrosis or cirrhosis is still controversial. Ho et al. found that in cirrhotic rats (with or without hyperhomocysteinaemia), folic acid (100 mg/kg per d) could significantly reduce the shunting degree and mesenteric vascular density, thereby decreasing splanchnic blood flow and mitigating the stress to form portosystemic collaterals. In addition, the pathological angiogenesis was reduced by downregulating the protein expression of mesenteric vascular endothelial growth factor and phosphorylated endothelial nitric oxide synthase(Reference Ho, Tsai and Hsieh77). In addition, a cross-sectional study of 5417 participants showed that individuals with higher serum folate levels had a lower chance of developing advanced liver fibrosis(Reference Chen, Luo and Li78). However, another study confirmed that moderately high folic acid (25 mg/kg) administration aggravated the development of CCL4-induced liver fibrosis in rats. The underlying mechanism was proven that folic acid significantly decreased the expression of the inhibitor of apoptosis 2 (IAP2 or Birc2) and the B-cell leukaemia/lymphoma 2 (Bcl-2) genes, increased the expression of procollagen type I α2 (Col1α2) and matrix metalloproteinase 7. In addition, a higher hepatic collagen content, serum aspartate transaminase and bilirubin and a lower serum albumin concentration were detected in rats receiving folic acid supplements (25 mg/kg)(Reference Marsillach, Ferré and Camps79). A recent study also showed that dietary restriction of folate can promote the regression of liver fibrosis in NASH mice. Folate can shift to mitochondrial metabolism during hepatic stellate cell activation and result in the depletion of α-linolenic acid, which maintains transforming growth factor β1 signalling and leads to fibrosis. However, the restriction of folate can block SHMT2/MTHFD2-mediated mitochondrial folate metabolism, prevent depletion of α-linolenic acid, increase the biological transformation of α-linolenic acid to DHA and thus inhibit transforming growth factor β1 signalling by downregulating TGFBR1 mRNA expression. Therefore, blocking mitochondrial folate metabolism is expected to be an important step in improving liver fibrosis in NASH mice(Reference Gao, Zheng and Xu80). In view of this, further studies are needed to confirm whether folate/folic acid improves or worsens liver fibrosis and cirrhosis and to explore the therapeutic or pathogenic dosage of folic acid.
Folate/folic acid and hepatocellular carcinoma
Studies have shown a negative correlation between folate level and the development of hepatocellular carcinoma (HCC)(Reference Welzel, Katki and Sakoda81,Reference Cui, Quan and Piao82) , and low blood folate status can promote the progression of HCC(Reference Kuo, Lin and Wu83). This may be due to several mechanisms. First, folate deficiency affects epithelial-to-mesenchymal transition. Su et al. found that folate deficiency could significantly upregulate mesenchymal markers such as Snail, zinc finger E-box binding homeobox 2 and vimentin in HCC cells and downregulate E-cadherin to promote epithelial-to-mesenchymal transition. In addition, cancer stem-like cell markers including octamer-binding transcription factor 4, β-catenin and CD133 increased, and paired-related homeobox 1 decreased in the folate deficiency group, indicating promoted tumour stem-like phenotype and metastatic potential(Reference Su, Huang and Huang84). However, another study suggested that epithelial markers (Syndecan-1, e-cadherin) did not change significantly under folate deficiency and mesenchymal marker (vimentin) significantly decreased. Therefore, folate deficiency was shown to reduce the transition of epithelial cells to mesenchymal cells and inhibit the invasion and migration of cancer cells, but this study was not confirmed in vivo(Reference Goyal, Sharma and Lamba85). Second, folate deficiency can induce apoptosis(Reference Goyal, Sharma and Lamba85). It was found that folate deficiency in HepG2 cells could lead to S-phase cell accumulation and G2/M phase block, thus inducing apoptosis(Reference Huang, Ho and Lin86). This may be due to the increased accumulation of homocysteine caused by folate deficiency, which leads to the overproduction of hydrogen peroxide and the overactivation of the redox-sensitive transcription factor NF-κB(Reference Chern, Huang and Chen50). Folic acid administration during cell culture can save apoptotic culture and restore the cell cycle to normal(Reference Huang, Ho and Lin86). Third, folate deficiency can lead to genomic instability. In a folate/methyl-deficient rodent model of hepatocarcinogenesis, DNA methyltransferase activity was increased, and p53 gene expression was decreased in the HCC group compared with the control and preneoplastic group. These changes can induce genomic instability and clonal neoplastic phenotype expansion in the liver(Reference Pogribny, Miller and James87). Moreover, previous study also proved DNA damage, DNA hypomethylation and tumour progression in folate/methyl-deficient rodent model(Reference James, Pogribny and Pogribna88). Fourth, folate deficiency can induce ER stress. Goyal et al. found that folate deficiency could induce ER stress by activating protein kinase R-like endoplasmic reticulum kinase/activating transcription factor 4/lysosome-associated membrane glycoprotein 3 pathway, and protein kinase R-like endoplasmic reticulum kinase inhibitors could inhibit the migration and invasion of HepG2 cells and further lead to the reduction of epithelial-to-mesenchymal transition and apoptosis(Reference Goyal, Sharma and Lamba85). In addition, there is overexpression of folate-related enzymes in HCC, including methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (MTHFD1), SHMT2, methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and MTHFD1L, which is associated with low survival rate, short recurrence time, poor prognosis and metastasis in HCC patients(Reference Quevedo-Ocampo, Escobedo-Calvario and Souza-Arroyo89). Among them, MTHFD1L contributes to the production and accumulation of NADPH, and MTHFD1L knockdown can increase oxidative stress and make cancer cells sensitive to sorafenib(Reference Lee, Xu and Chiu90).
Folic acid plays a certain therapeutic role in HCC. Folic acid supplementation can depress an important oncogene-lipocalin 2 by promoting the level of histone H3 lysine 9 di-methylation in lipocalin 2 promoter, thereby inhibiting cell proliferation and cell cycle and playing a chemopreventative role in the tumorigenesis of HCC(Reference Zhang, Xue and Miao91). Folate deficiency in some poorly differentiated and invasive subclone variants, such as SK-Hep-1 cells, can promote redox adaptation and upregulate GRP78 and survivin, which act as reactive oxygen species inhibitors to induce multi-drug resistance and are conducive to the progression of HCC. So, it is a potent clue to supply folic acid to prevent its deficiency in order to achieve a more satisfactory chemotherapy effect in the treatment of HCC(Reference Ho, Shang and Chang92). Moreover, the treatment of folic acid combined with chemotherapy has been widely studied presently. Folic acid is used as a tumour targeting part because of its high affinity with folic acid receptors, which can overexpress in many tumours including liver cancer(Reference Lee, Kim and Lee93). Folate/folic acid can combine with polyethylene glycol cyclodextrin nanoparticles, paclitaxel, doxorubicin, selenium nanoparticles and other substances to improve the effectiveness of targeted nanomedicine therapy and play an important role in the treatment of HCC(Reference Li, Shi and Sun94–Reference Xia, Zhao and Chen96). However, Sharma et al. have found that excessive dietary folic acid supplementation can promote the early progression of HCC. In the rat model of establishing HCC with diethylnitrosamine, an excessive diet of folate (20 mg/kg) resulted in an increased case of HCC and a decreased case of cirrhosis when compared with folate normal group (2 mg/kg)(Reference Sharma, Ali and Negi97). Therefore, when using treatment regimens containing folic acid, it is necessary to pay attention to the dosage of folic acid. In summary, folate plays an important role in the occurrence and development of HCC. The development of targeted drugs containing folic acid has clinical value in the treatment of HCC, but the dietary or drug supplementation dosage of folic acid needs further exploration (Fig. 6).
Figure 6. The mechanism of folate deficiency and folic acid supplementation in HCC. Folate deficiency upregulates Snail, ZEB2 and vimentin and downregulates E-cadherin to promote EMT. Folate deficiency increases Oct4, β-catenin and CD133 and decreases PRRX1, indicating promoted tumour stem-like phenotype and metastatic potential. Folate deficiency leads to S-phase cell accumulation and G2/M phase arrest in the cell cycle and can promote increased Hcy accumulation, resulting in excess H2O2 production and NF-κB activation, thereby inducing apoptosis. Folate deficiency increases DNA methyltransferase activity and decreases p53 gene expression, which induce liver genomic instability and clonal neoplastic phenotype expansion. Folate deficiency can induce ER stress by activating the PERK/ATF4/LAMP3 pathway. Folate deficiency can promote redox adaptation and upregulate GRP78 and survivin to induce multi-drug resistance, which is not conducive to the treatment of HCC. Folic acid supplementation inhibits LCN2 by promoting the level of H3K9Me2, thereby inhibiting cell proliferation and cell cycle. ZEB2, zinc finger E-box binding homeobox 2; EMT, epithelial-to-mesenchymal transition; Oct4, octamer-binding transcription factor 4; PRRX1, paired-related homeobox 1; Hcy, homocysteine; H2O2, hydrogen peroxide; PERK, protein kinase R-like endoplasmic reticulum kinase; ATF4, activating transcription factor 4; LAMP3, lysosome-associated membrane glycoprotein 3; ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78; H3K9Me2, histone H3 lysine 9 di-methylation; LCN2, lipocalin 2; HCC, hepatocellular carcinoma. ↑increase; ↓decrease; 
aggravate; 
alleviate.
Folate/folic acid and viral hepatitis
Folic acid may play an active role in viral hepatitis. In the treatment of viral hepatitis, folic acid and corsal treatment can significantly increase the clearance and elimination rate of antipyrine and reduce the area under the pharmacokinetic curve, which may be due to the involvement of its derivatives in de novo synthesis of nucleotides(Reference Zviarynski and Zavodnik98). In pregnant patients with viral hepatitis E, genetic alterations in folate pathway genes, folate receptor α deficiency and decreased vitamin B12 level may cause elevated homocysteine that promotes oxidative stress, therefore increasing the risk of preterm delivery, disease severity and negative pregnancy outcomes(Reference Tiwari, Das and Sultana99). Supplementation with vitamin B12 and folic acid may be an effective treatment option to combat elevated homocysteine in viral hepatitis E-infected pregnancy cases and diminish associated fetal and maternal morbidity and mortality(Reference Tiwari, Das and Sultana99). At present, there are still few studies on folic acid and viral hepatitis, so further exploration of its therapeutic effect is needed to provide more clinical reference.
Conclusion
Folate, as a B-group vitamin, participates in various biological reactions in the body, such as lipid metabolism, oxidative stress and methionine metabolism. Therefore, it plays a crucial role in the human body. This article provides a detailed overview of the role of folate/folic acid in NAFLD/NASH, ALD, DILI, liver fibrosis/cirrhosis, HCC and viral hepatitis. By describing the association and potential pathogenesis between folate deficiency or folic acid excess and liver diseases, clinical doctors can have a more comprehensive understanding on the role of folate/folic acid. When studying the published literature, we found that the dosage of folic acid as a dietary or drug supplement still needs further exploration, especially for people who have already presented with liver diseases. We look forward to more animal and clinical studies on folic acid dosage to guide the rational clinical application. In addition, there is relatively little research on the role of folic acid in liver fibrosis/cirrhosis and viral hepatitis, despite conflicting views in current research. Therefore, further research is needed in this field. In summary, this review can help people systematically understand folate/folic acid in the context of liver diseases, which is beneficial for the clinical application and development of targeted drugs containing folic acid.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (no. 82100620) and the Doctoral Startup Research Fund of Liaoning Province (2021-BS-218).
C. M. J. and M. H. conceived and designed the article. H. M. and H. L. authored drafts of the paper. C. M. J., M. H. and H. L. corrected the content and language. H. M. and Y. T. Y. prepared pictures and references. All authors have read and approved the manuscript and agree to be accountable for all aspects of the article.
The authors declare that they have no competing interests.
