Ethanol triggers unrestrained inflammation and immune turbulence via regulating key cell subsets in the hepatic immune microenvironment. Kupffer cells (KC) (liver-resident macrophages) play a crucial role in the innate immune response against infections and initiating and resolving inflammation (Yao et al., 2017; Chen M. et al., 2023). Chronic alcohol consumption impairs the intestinal mucosal barrier, leading to increase permeability and translocation of intestinal Gram-negative bacteria-derived lipopolysaccharide (LPS) to the liver. As a typical pathogen-associated molecular pattern (PAMP), LPS activates toll-like receptor 4 (TLR4) on macrophages and natural killer cells (NK cells), leading to change macrophage function and increased production of pro-inflammatory cytokines, which results in T cell activation and inflammation (Ling Yang and Lu, 2022; Fang et al., 2023).

LSECs are a highly specialized and distinctive micro-vascular cell type that regulates the hepatic micro-environment (Lafoz et al., 2020). LSEC is the first defense barrier, removing and recycling proteins, lipids, and toxins from the blood through highly permeable fenestrae when exposed to blood from the intestinal and systemic circulation (Wu et al., 2023). In addition, they are involved in response to specific injuries by regulating neighboring cells, mainly hepatic stellate cells (HSC).

With alcohol consumption, HSCs and hepatic portal fibroblasts transdifferentiate to myofibroblasts. Activated myofibroblasts produce mainly type I and type III collagen, which cross-links and accumulate in the extracellular matrix (ECM), replacing the injured liver parenchyma and forming a fibrous scar (Bellanti et al., 2023). HSCs can be directly activated by alcohol, acetaldehyde, and ROS or in a paracrine manner by various fibrogenic factors (e.g., transforming growth factor-β, platelet-derived growth factor, and inflammatory cytokines) released from damaged hepatocytes, activated Kupffer cells, and infiltrated immune cells (Ha et al., 2022).

Mitochondria are double-membrane organelles involved in various physiological functions within the cell (Park et al., 2022). Typically, mitochondrial abnormalities usually occur in the early stages of alcoholic liver injury that continually produce detrimental effects on hepatocytes. When the liver is chronically exposed to ethanol, mitochondria produce large amounts of reactive nitrogen species (RNS) and ROS, which inactivate components of the respiratory chain by destroying mitochondrial DNA (mtDNA), inducing mitochondrial permeability transition, leading to mitochondrial swelling and rupture, and ultimately hepatocyte death (Su et al., 2023). ROS secreted by mitochondria is scavenged by the cellular antioxidant system. Organisms have developed a variety of antioxidant defense systems to scavenge the accumulation of peroxides in the liver to reduce liver injury caused by oxidative stress (Molina et al., 2003).

The complex balance between parenchymal and nonparenchymal cells’ survival and death pathways is critical for regulating ALD progression. In ALD, programmed cell death (PCD), partly caused by ethanol-induced oxidative stress and innate immune responses, is thought to play a central role in the progression of the injury (Miyata and Nagy, 2020). PCD is a spontaneous and programmed cell death mode associated with multiple physiological and pathological processes (Chen Y. et al., 2023). There are four main modes of PCD: apoptosis, necroptosis, pyroptosis, and ferroptosis (Wu et al., 2023). ALD is influenced by dysregulation or hyperactivation of autophagy, which is linked to cellular death and damage.

In hepatocytes, lipid uptake, esterification, oxidation, and fatty acid secretion occur. These processes are regulated by hormones, nuclear receptors, and transcription factors to maintain hepatic lipid homeostasis (Fu et al., 2021). Alcohol affects hepatic lipid metabolism through synergistic mechanisms, accumulating fatty acids (FA) and triglycerides (Seitz et al., 2023). Ethanol intake decreases signal transducer and activator of transcription 3 (STAT3), AMP-activated protein kinase (AMPK), and peroxisome proliferative activated receptor alpha (PPARα) levels to inhibit fatty acid oxidation. In addition, alcohol intake upregulates sterol regulatory element binding protein-1 expression (SREBP-1) and lipogenic enzymes. All of these behaviors contribute to dyslipidemia and hepatic fat accumulation (Dunn and Shah, 2016; Zhao et al., 2016; Liu S. Y. et al., 2021).

Tight junctions (TJ), sticky mucus gel layers, and intestinal cells work together to form the intestinal barrier, which prevents bacteria from passing through (Suzuki, 2020). The intestinal barrier also depends on commensal bacteria, sIgA antibodies, and mucins (Guohua Li and Chen, 2022). In comparison to individuals in good health, patients with alcohol-associated liver disease exhibit a decrease in intestinal bacilli and probiotics (e.g., Lactobacillus) and increased E. coli and Enterococcus faecalis (Dunn and Shah, 2016; Shang Zeng and Li, 2022). Furthermore, studies have demonstrated that augmenting the proportion of intestinal bacilli and firmicutes can serve as a preventive measure against alcoholic liver injury and other chronic liver diseases (Qiu et al., 2019).

Patients with AUD are often accompanied by Alcohol withdrawal syndrome (AWS). AWS is caused by a dramatic decrease in GABA activity and glutamate overactivity a few hours after cessation of alcohol consumption (Airagnes et al., 2019). Benzodiazepines (BZDs) are the generalized medications used to treat alcoholic AWS, including chlordiazepoxide, oxazepam, diazepam, and lorazepam (Holleck et al., 2019). BZDs act therapeutically by stimulating γ-aminobutyric acid type A (GABA-A) receptors, mimicking their stimulation by alcohol (Airagnes et al., 2019). Phenobarbital (PB) is a standard alternative or adjunct to BZDs and can reduce AWS complications and ICU admissions. PB exerts its effects through the activation of GABA-A receptors and inhibition of N-methyl-D-aspartate (NMDA) receptors (Mo et al., 2016). However, high doses of BZDs induce liver disease and dependence (Singal et al., 2018). PB also has a low margin of safety (Mo et al., 2016). Therefore, there is a need to explore safer medications for use in AWS. Other detrimental conditions may occur during AWS, such as Wernher’s disease, Wernicke-Korsakoff syndrome (WKS) (Airagnes et al., 2019), and alcohol withdrawal delirium (AWD) (Mayo-Smith et al., 2004).

Malnutrition is one of the main adverse consequences of alcohol-associated liver disease, especially in those with severe alcoholic hepatitis. Sufficient protein and vitamins must be administered orally or intravenously as part of nutritional treatment (Griffith and Schenker, 2006). In the case of alcoholism, even if sufficient protein, vitamins, and other nutrients are consumed, the body does not use them effectively because they are not sufficiently absorbed from the intestines into the bloodstream, resulting in malnutrition (Lieber, 2003). Zinc, magnesium, and vitamin A deficiencies are also standard in ALD (McClain et al., 2011).

Corticosteroids are used for the treatment of alcoholic hepatitis (AH). One example is prednisolone which is prescribed at a therapeutic dose of 40 mg daily for 4 weeks (Singal et al., 2018). Ramond et al. found that patients treated with prednisolone had a death rate of 12.5%, whereas those given a placebo had a mortality rate of 55% (Ramond et al., 1992). Additionally, prednisolone was found to lower short-term mortality in individuals with severe AH in a clinical investigation (Mathurin et al., 1996). To predict the effect of corticosteroid therapy, the Lille Model was introduced. The Lille Model measures were age, renal insufficiency, albumin, prothrombin time, bilirubin and day 7 bilirubin levels. The model measures from the seventh day of treatment, and patients with scores >0.45 indicate a lack of response to corticosteroids; directing the need for changing the AH treatment to another suitable option (Louvet et al., 2007).

Pentoxifylline is a non-specific phosphodiesterase inhibitor with anti-inflammatory qualities (Tyagi et al., 2011). It is administered at a dose of 400 mg thrice daily to treat severe AH (Akriviadis et al., 2000). Pentoxifylline attenuates the development of hepatic fibrosis by inhibiting TNF-α, which reduces mortality in AH and may decrease the incidence of hepatorenal syndrome (Tyagi et al., 2011). However, in patients with severe AH, there is no additional survival advantage of using pentoxifylline alone compared to corticosteroids (Sidhu et al., 2012). Pentoxifylline is ineffective in patients who are also unresponsive to corticosteroid therapy (Louvet et al., 2008). According to the European Association for the Study of the Liver (EASL), pentoxifylline is a second-line therapeutic agent for patients with severe AH who have contraindications to corticosteroid therapy (Liver, 2012).

Chronic alcohol consumption can damage the intestinal mucosal barrier leading to an increase in its permeability. Lipopolysaccharide (LPS) from intestinal Gram-negative bacteria translocates to the liver, where it binds to Toll-like receptor 4 (TLR4) on kupffer cells, causing its activation and subsequent release of a series of pro-inflammatory factors, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1β), which result in hepatic inflammation and systemic injury (Y, 2014; Shang Zeng and Li, 2022). Nuclear factor κB (NF-κB) is a key transcription factor in regulating inflammatory responses and a key component of intracellular signaling cascades (Xing et al., 2021). Naringenin, Lutein, Myricetin, and Apigenin reduce ethanol-induced levels of the hepatocyte inflammatory factor NF-κB (Jayaraman et al., 2012; Wang et al., 2017; Guo et al., 2019; Zhao et al., 2023). Activation of NF-κB stimulates the production of various inflammatory mediators, such as IL-1β, IL-6, TNF-α, COX-2, and inducible nitric oxide synthase (iNOS) (Jayaraman et al., 2012; Du et al., 2015; Zhao et al., 2016). Among them, IL-1β, IL-6, and TNF-α are synthesized and released upon activation of hepatic stellate cells (Li et al., 2013). These are critical inflammatory factors in alcoholic liver injury and induce further necrosis, apoptosis, and exacerbation of inflammatory response in hepatic cells (Yuan et al., 2022). Quercetin reduces the levels of TNF-α, IL-1β, and IL -6 levels (Chen, 2010); DHM and lutein can reduce TNF-α, and IL-1β (Shen et al., 2012; Zhao et al., 2023), and myricetin can also reduce IL-6, and TNF-α levels (Guo et al., 2019; Ahmad et al., 2022). Naringenin, and apigenin can only reduce the concentration of TNF-α (Jayaraman et al., 2012; Wang et al., 2017). The essential enzyme that starts inflammation, cyclooxygenase-2 (COX-2), is one of NF-κB’s target genes and is expressed by kupffer cells. Normal tissues have low levels of Cox-2 expression; however, an inflammatory stimulation increases Cox-2 expression with subsequent production of prostaglandins (Jayaraman et al., 2012). The inflammatory protective mechanism of naringenin and lutein mainly consists of inhibiting the overexpression of Cox-2 induced by alcohol exposure (Jayaraman et al., 2012; Zhao et al., 2023). Inducible nitric oxide synthase (iNOS) is the enzyme responsible for NO production and is not present in most cells under normal conditions. Its expression is inducible, often associated with inflammation and malignant diseases (Kielbik et al., 2019), and is a downstream gene of NF-κB (Du et al., 2015). Lutein supplementation reverses alcohol-induced changes in iNOS, an indicator of inflammation (Zhao et al., 2023).

Interleukin 10 (IL-10), a robust anti-inflammatory factor, lessens liver damage (Chen, 2010). Quercetin has been shown to positively affect the liver by up-regulating the expression of Heme Oxygenase-1(HO-1) and IL-10, inhibiting the NOD-like receptor thermal protein domain associated protein 3(NLRP3) inflammatory vesicles (Liu et al., 2018).

In addition to ADH, the MEOS metabolizes ethanol to acetaldehyde in ALD. The primary components of MEOS are the cytochrome P450 (CYP450) enzymes found in the smooth endoplasmic reticulum (SER), including CYP2E1 and CYP3A, among others (Lu and Cederbaum, 2018). Prolonged alcohol intake leads to the activation of the MEOS system, which can affect NADPH oxidation and ATP consumption, leading to oxidative stress in hepatocytes (Chen et al., 2013). Within the cytochrome P450 family, CYP2E1 is considered the most relevant to ALD due to its high degree of induction and catalytic activity in response to alcohol (Leung and Nieto, 2013). Heat shock protein 70 (HSP70) facilitates the synthesis of CYP2E1 proteins in the endoplasmic reticulum (ER) and their transfer into mitochondria (Zhou et al., 2018). SP1, a ubiquitously expressed transcription factor, transcribes and activates CYP2E1 and HSP70 (Morgan, 1989; Tang et al., 2019). Upon beta-sitosterol, DHM, naringenin, myricetin, kaempferol, emodin, and apigenin treatment, the levels of endogenous CYP2E1 were reduced in liver tissues, which attenuated hepatocyte injury (Jayaraman and Namasivayam, 2011; Liu et al., 2014; Wang et al., 2017; Zhou et al., 2018; Chen Z. et al., 2020; Silva et al., 2020; Ahmad et al., 2022). Kaempferol reduced ethanol-induced protein levels of CYP2E1 in microsomes and mitochondria and decreased the expression of HSP70 and SP1, reducing oxidative stress and increasing cell viability (Zhou et al., 2018).

The P2X7 receptor (P2X7R), is a critical receptor for oxidative stress and inflammation in ALD, induces the generation of activated ROS and imbalance of the antioxidant defense system. P2X7R is an adenosine triphosphate (ATP)-gated ion channel, that can be activated by high levels of ATP to exacerbate oxidative stress through the PI3K/Keap1/Nrf2 pathway. Quercetin treatment eliminates ethanol-induced overexpression of P2X7R and exerts a cytoprotective effect (Zhao et al., 2021).

The antioxidant defense system can be used to scavenge the accumulation of peroxides in the liver to reduce hepatic injury caused by oxidative stress. The major endogenous antioxidant enzyme systems include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione reductase (GR); non-enzymatic endogenous antioxidants are mainly glutathione (GSH) and vitamin E (Molina et al., 2003). Alcohol intake leads to increased levels of MDA and decreased GSH, SOD, and CAT, leading to liver injury. GSH is the first line of defense against free radicals and is the key to antioxidant activity (Vidhya and Indira, 2009; Tang et al., 2012). Treatment with DHM, quercetin, lutein, and apigenin increases the levels of the antioxidant GSH (Donglin Su and Yao, 2009; Vidhya and Indira, 2009; Sindhu et al., 2010; Wang et al., 2017; Hou et al., 2020). In addition, enzymes such as SOD and GPX are essential in scavenging free radicals to prevent their formation (Jayaraman and Namasivayam, 2011). Alcohol use has been linked to a drop in the liver’s MDA and SOD and CAT levels. Treatment with beta-sitosterol, lutein, and apigenin promoted the elevation of SOD and GPX (Sindhu et al., 2010; Wang et al., 2017; Zhao et al., 2018; Chen Z. et al., 2020; Zhao et al., 2023). However, quercetin only promoted the increase of SOD (Vidhya and Indira, 2009). The activity of CAT responds to the cell’s ability to scavenge ROS and its resistance to oxidative damage (Hao and Liu, 2019). The reduction in CAT levels brought on by alcohol can be reversed by beta-sitosterol and apigenin (Wang et al., 2017; Chen Z. et al., 2020). Apigenin treatment can potentially elevate GR levels in alcoholic liver injury (Wang et al., 2017). In the study by Liu et al., the effects of quercetin on GSH, SOD, and CAT levels were guided by pretreatment rather than post-treatment (Liu et al., 2010). Quercetin has also been demonstrated to scavenge oxygen-free radicals more effectively than conventional vitamin C and E forms.

Nrf2 is a protective factor expressed in response to oxidative stress. Excessive ROS disrupt the Keap1-Nrf2 interaction, triggering an effective atomic translocation of Nrf2. The translocated Nrf2 binds to the antioxidant response element (ARE) in blood and enhances the expression of antioxidant and detoxifying enzymes (Lee et al., 2019). Nrf2 has emerged as a potent therapeutic agent for ALD, aiming to protect downstream targets, namely, HO-1 and GSH. HO-1 is the rate-limiting enzyme for heme catabolism. It can degrade heme to ferrous ions, carbon monoxide (CO), and biliverdin, which play a vital role in combating oxidative stress (Liu et al., 2012). Biliverdin then reductase breaks down biliverdin into bilirubin. Heme has a pro-oxidant nature and increases the susceptibility of cells to oxidative stress, while biliverdin and bilirubin exhibit noteworthy anti-inflammatory, antioxidant, and anti-apoptotic properties (Yao et al., 2009). DHM and lutein can activate the antioxidant system by increasing the expression of Nrf2 and its antioxidant product, HO-1 (Zhang et al., 2018; Silva et al., 2020; Chen et al., 2021; Zhao et al., 2023). Quercetin exerts its protective effect against ALD mainly through ERK/Nrf2-mediated upregulation of HO-1 to reduce ROS production (Liu et al., 2012; Zhao et al., 2021). In the mitogen-activated protein kinase (MAPK) signaling pathway, p38 and extracellular regulated protein kinases (ERK) mediated translocation of nuclear factor-erythroid 2-related factor 2(Nrf2) into the nucleus subsequently induces HO-1 activity, with a more critical mediating role for ERK (Yao et al., 2007). It has also been shown that in addition to Nrf2, HO-1 activation is regulated by other transcription factors, including activator protein-1 (AP-1) and nuclear factor (NF-ΚB) (Lee et al., 2019).

Chronic alcohol exposure induces multiple forms of cellular stress, including oxidative stress, hypoxia, and endoplasmic reticulum (ER) stress, which activate intrinsic and extrinsic pathways of apoptosis (Miyata and Nagy, 2020). Cell-intrinsic pathways receive tight regulation from the Bcl-2 protein family (Guicciardi et al., 2013). Bax belongs to the Bcl-2 protein family, which plays an essential role in apoptosis (Bruckheimer et al., 1998). Bax appears at the cell membrane in a healthy condition, which gets transferred to the mitochondria in response to apoptotic stressors (DNA damage, misfolded proteins, etc.), increasing the permeability of the cell membrane (Matsuyama et al., 2016). Cytochrome c exists on the inner membrane and then translocates to the cytoplasm (Choi et al., 2022). Bax initiates the intrinsic apoptotic pathway and triggers the release of cytochrome c, which then causes a series of caspase cascade reactions that induce apoptosis (Matsuyama et al., 2016). Beta-sitosterol inhibits alcohol-induced increase in the expression of Bax and inhibits cytochrome c, caspase-3, and caspase-9 elevation (Chen Z. et al., 2020). However, DHM and naringenin only decrease the elevation of caspase-3 after ethanol-mediated (Zhao et al., 2018; Silva et al., 2020).

The core events of iron toxicity include increased iron accumulation, impaired lipid repair systems, and lipid peroxidation, ultimately leading to membrane destruction and cell death (Wu et al., 2021). Numerous studies have shown that alcohol consumption is associated with iron overload in the liver and decreased hepcidin (Hepc) synthesis, which is thought to be a critical iron mediator of homeostasis (Liu S. Y. et al., 2021). Hepc, a small molecule peptide secreted by the liver, plays a role in iron homeostasis by inhibiting iron uptake and mobilization from tissue stores. Alcohol stimulates iron absorption and hepatic storage by inhibiting the secretion of hepc, which releases free iron from various iron-containing proteins. Free iron in cells exists as a labile iron pool (LIP), i.e., redox-active, weakly chelated iron (Li et al., 2016); that when present in excess catalyzes the Fenton reaction to produce hydroxyl radicals (·OH), exacerbating alcoholic liver injury. Hydroxyl radicals are the most toxic component of ROS (Li et al., 2014; Li et al., 2016). The Fenton reaction occurs within lysosomal compartments, which can accumulate large amounts of redox-active iron and are the primary source of LIP (Li et al., 2016). Quercetin helps to maintain intracellular LIP levels and attenuate intracellular free iron-mediated production of hydroxy radicals (·OH), which can alleviate alcohol-induced liver injury (Li et al., 2014; Li et al., 2016). Ethanol feeding stimulated elevated levels of iron and ferritin in the liver of rats, leading to increased oxidative stress. Whereas naringenin intake significantly reduced the expression of iron and ferritin in the liver (Jayaraman et al., 2012).

Hepatic steatosis represents the liver’s earliest and most common response to acute or chronic alcohol exposure (Huo et al., 2018). During steatosis, triglycerides (TG), cholesterol (TC), and phospholipids build up in hepatocytes (Dunn and Shah, 2016). Several studies have shown that quercetin, naringenin, and apigenin reduced alcohol-induced hepatic steatosis. Moreover, these compounds can lower serum TG and TC concentrations, ultimately restoring cell viability (Seo et al., 2003; Zhao et al., 2018; Zhao et al., 2021). Lutein, kaempferol, and emodin only reduced TG levels and had no significant effect on TC (Liu et al., 2014; Wang et al., 2015; Zhao et al., 2023). High-density lipoprotein (HDL-C) is involved in the reverse transport of cholesterol from peripheral tissues and lipids from the vasculature to the liver for clearance (Wierzbicki and Mikhailidis, 2002). Naringin increases the serum HDL-C and HDL-C/total-C ratios, returning these lipid parameters to normal levels and reducing alcohol damage to the liver (Seo et al., 2003). Serum low-density lipoprotein cholesterol (LDL-C) acts oppositely to HDL-C, transporting lipids from the liver into the vasculature and inducing disease. Apigenin reduces the alcohol-induced higher serum values of LDL-C (Wang et al., 2017).

AMPK, the classical mammalian upstream serine protein kinase, induces lipolysis and suppresses lipid synthesis. Its activation can lead to a reduction in steatosis and inflammation (Silva et al., 2020; Pang et al., 2021). Ethanol and acetaldehyde inhibit fatty acid oxidation by downregulating the levels of AMPK (Dunn and Shah, 2016; Zhao et al., 2016; Liu S. Y. et al., 2021). DHM and myricetin can decrease AMPK levels (Guo et al., 2019). Increased phosphorylation of AMPK promotes phosphorylation of SREBP-1c (Zhu et al., 2019). One of the critical regulators of adipogenesis, the transcription factor SREBP-1c, plays a role in the transcriptional activation of genes that encode the rate-limiting enzymes in adipogenesis, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1) (Fang et al., 2019). Myricetin and apigenin significantly inhibited alcohol-induced levels of the liposynthesis gene SREBP-1c (Wang et al., 2017; Guo et al., 2019). FAS catalyzes the condensation of acetyl-CoA and malonyl-CoA to produce long-chain fatty acids in the cytoplasm (Ronnett et al., 2005). Myricetin and apigenin inhibit the elevation of FAS caused by alcohol exposure (Wang et al., 2017; Guo et al., 2019). ACC is the rate-limiting enzyme for the de novo synthesis of fatty acids and catalyzes acetyl-CoA conversion to malonyl-CoA (Pang et al., 2021). In the liver, phosphorylation of AMPK inactivates ACC, which decreases fatty acid synthesis and increases fatty acid oxidation (Liu Y. S. et al., 2021). DHM regulates alcohol-induced hepatic steatosis through the AMPK-ACC pathway, which reduces lipid accumulation (Chen et al., 2021).

The peroxisome proliferator-activated receptor (PPAR) belongs to the nuclear hormone receptor superfamily and plays a crucial role in glucose and fatty acid metabolism. There are three known members of this superfamily: PPAR-α, PPAR-β/δ, and PPAR-γ. Known as the “energy homeostasis receptor,” PPAR-γ is a crucial regulator in lipid metabolism. PPAR-γ regulates several downstream genes involved in fatty acid oxidation, such as carnitine palmitoyltransferase-1 (CPT-1) (Zheng and Cai, 2019). DHM and emodin decreased PPAR-γ caused by ethanol exposure (Liang and Olsen, 2014; Liu et al., 2014; Chen et al., 2021). Fatty acid catabolism occurs in mitochondria and is regulated by CPT-1, the rate-limiting step in entering fatty acids into mitochondria (Ronnett et al., 2005). Diacylglycerol acyltransferase (DGAT), a microsomal enzyme widely expressed in mammalian tissues, is also a key enzyme in triglyceride synthesis (Chen and Farese, 2000). Pre-administration of apigenin decreased hepatic DGAT protein expression in mice with alcohol-induced liver injury, while hepatic CPT-1 protein expression was inversely increased. These findings show that pretreatment with apigenin reduced hepatic lipid synthesis and increased fatty acid utilization, indicating that apigenin may be able to avoid abnormalities of hepatic lipid metabolism (Wang et al., 2017).