Excessive alcohol consumption can pose serious challenges to a cascade of vital organs, including the liver, brain, heart, and gastrointestinal tract (Hyun et al., 2021). Similar to chronic abuse, acute alcohol consumption is toxic to many organs, the stomach being particularly vulnerable in this process, predisposing to gastrointestinal disorders, including acute gastric mucosal lesion (AGML), gastritis, gastric ulcers, and gastric bleeding (Gonçalves et al., 2017). The role of gastroprotective effect in protecting the body against alcohol-related toxicity, oxidative stress as well as inflammation response cannot be overlooked.

Ethanol could result in severe gastric mucosal damage through direct toxic action or free radicals to activate oxidative stress, leading to acute hemorrhagic lesions, edema of the mucus membrane, epithelial exfoliation and the release of inflammatory cells (Wang et al., 2015; Li J et al., 2017). This acute gastric mucosal damage may be attributed to the low activity of defensive factors such as nitric oxide (NO), prostaglandins (PG), mucosal microcirculation, antioxidants and certain cytokines, or is due to the overproduction of reactive oxygen species (ROS), lipid peroxidation and infiltration of neutrophils (Escobedo-Hinojosa et al., 2018; Magierowska et al., 2018; Zaghlool et al., 2019). NO, working as a mixed blessing in the alimentary system, is synthesized with the participation of nitric oxide synthase (NOS) (Mohamed et al., 2022). It is widely recognized that, in the digestive system, NO generated by constitutive NOS (cNOS) is related to cytoprotective effect, whereas inducible NOS (iNOS)-produced NO, by contrast, is cytotoxic (Motawi et al., 2007). Excessive alcohol consumption may intensify gastric mucosal damage via down-modulating the levels of defensive factors, represented by NO and prostaglandin E2 (PGE2), which contribute to the improvement of gastric mucosal blood flow and mucosal microcirculation under normal conditions (Xie et al., 2019).

Several studies reported that oxidative stress and inflammatory responses play a significant role in the development and progression of alcohol-induced gastric mucosal damage(Arda-Pirincci et al., 2006; Pan et al., 2008; Wu et al., 2018). As two interplaying systems regulating the equilibrium of cellular redox status, Nrf-2/HO-1 signaling axis and NF-κB could modulate both oxidative stress and inflammatory response indeed (Casili et al., 2020; Kim et al., 2021). They express at relatively low levels normally while are upregulated under stress conditions. It is well-founded that Nrf2/HO-1 signaling are protecting factors against oxidative stress and inflammatory responses in gastric mucosal damage, while NF-κB plays an opposite role (Dimauro et al., 2021). Furthermore, Nrf2 activation and its anti-inflammatory effect are closely related to the transcription of antioxidant factor through NF-κB. Deficiency of Nrf2 could increase the activity of NF-κB, leading to increased cytokine production associated with increased oxidative stress (Sandberg et al., 2014). The production and removal of ROS equilibrates dynamically in the gastric mucosa under the physiology condition (Lebda et al., 2018). Induced by a series of factors such as ethanol exposure, inflammatory stimuli lead to the excessive production of ROS along with the decline of antioxidative enzymes, which disrupts the equilibrium of oxidation and anti-oxidation systems, thereby supervening the gastric lesions (Jeon et al., 2014). Considering the molecular crosstalk of NF-κB/Nrf-2/HO-1 is indispensable for the ROS-mediated inflammatory cascades and the regulation of ethanol-induced gastric lesions, it is fair enough to believe that mitigating the NF-κB activation or supporting Nrf2 activation may be effective strategies for treatment of ethanol-induced gastric lesions. It is well established that ethanol and metabolite acetaldehyde would attack the gastric mucosa, inducing microcirculatory disturbance and hypoxia, along with consequent propagation of the inflammatory cascade (De Araújo et al., 2018; Yu Y et al., 2020). ADH and ALDH, as crucial alcohol metabolism enzymes, could oxidize acetaldehyde to harmless acetic acid to accelerate alcohol metabolism and ameliorate the ethanol-induced gastric lesions. Some natural plant extracts have been shown to activate ADH and ALDH in previous studies (Martin and Maricle, 2015; Jang et al., 2018).

Current common-used treatments for ethanol-induced gastric lesions treatment largely include potentiating the protective mechanism of gastric mucosa and improving the gastric mucosal microcirculation to repair of gastric mucosa (Association, 2015). H2 receptor antagonists, such as cimetidine, roxatidine, ranitidine, and famotidine, continue to be the first-line therapy for peptic ulcer disease, which has been proven effective for AGML through suppressing gastric acid secretion. However, in ethanol-induced AGML, some H2 receptor antagonists could block gastric first-pass ethanol metabolism, resulting in slightly higher blood alcohol levels than normal after consuming alcohol (Weathermon and Crabb, 1999; Breslow et al., 2015). Colloid Bismuth Pectin (CBP) could protect the gastric mucosa by reacting with the complexes of biOCl and bismuth citrate to form a protective film (Li and Sun, 2012; Adeyemi and Onwudiwe, 2020). Nevertheless, the side effect of CBP is not to be ignored that grumbles from the users about constipation and other adverse reactions have gained prominence. Therefore, it is of great significance to screen out better drugs, particularly extracted from plants or herbs which display higher efficiency and lower toxicity.

The research on natural products is all the rage for their great efficacy and low toxicity, and the natural compounds they contain have been regarded as superior compatibility with the human body (Ashktorab et al., 2019). The first appearance of Lvdou Gancao decoction (LGD), a famous traditional Chinese medicine prescription, was found in Wen Cheng’s Jijiu Bianfang in the Qing Dynasty. LGD exerts a good detoxification effect in traditional use and has a reliable therapeutic effect against acute organophosphorus pesticide poisoning (AOPP) (Cailin, 1999; Wang, 2002), drug poisoning (Zhou, 2009; Wenxue, 2018), mushroom poisonings (Zhang, 2000) and digestive system diseases such as toxic hepatitis (Li and Tang, 1997; Wenquan, 2007; Weifeng and Jia, 2022) and acute pancreatitis (Wei et al., 2013). Based on this classic LGD, the modified Lvdou Gancao decoction (MLG) is made of several components and could be effective in gastropathy (Minqing and Mingwei, 2002; Dan et al., 2018; Yuewen and Qiang, 2018), liver complaints (Wang, 2017; Xie et al., 2022) and alcohol-induced conditions (Sun, 2015; Takei et al., 2015; Zhang and Zhang, 2017). Many of the herbs applied by MLG have been put into clinical use to treat the diseases of the digestive system for a long run and have shown their faculty in gastric protection and anti-inflammatory (Wang et al., 2012; Lv et al., 2018; Cao et al., 2020; Meng et al., 2020). Although their therapeutic mechanisms have not yet been known, their therapeutic efficacy has been speculated to be largely related to their antioxidant effect, for example, Dangshen(Wang et al., 1997; Li L et al., 2017), Shanyao (Qiao et al., 2018), Dingxiang (Agboola et al., 2022), Banxia (Yu L et al., 2020; Fu et al., 2021), Tianma(Dong et al., 2021), and Shengjiang (Haniadka et al., 2013). Previous studies indicated these herbs can effectively remove free radicals and inhibit the occurrence of lipid peroxidation, so as to protecting gastrointestinal mucosa. Our previous study has confirmed that MLG can efficaciously ameliorate alcohol-induced hepatotoxicity, accelerate the metabolism of alcohol and weaken inflammatory and oxidative stress responses in mice liver (Xie et al., 2022). The current study shifted its focus towards demonstrating the gastroprotective effect and mechanisms of MLG to further explore the value of clinical applications of MLG.

Ethanol-induced gastric lesion is a ubiquitous emergency worldwide, and the request for more effective therapeutic drugs still keeps impending. Although MLG shows great competence in treating alcohol-induced symptoms and gastric Lesions in clinical practice, the exact mechanism remains ambiguous.

In our study, we mainly discussed that: 1) MLG could prevent acute alcohol intoxication and promote wakefulness after acute ethanol exposure; 2) MLG showed a significant gastric protection effect, which is able to drastically abate the gastric ulcer index, raise the ulcer inhibition rate and alleviate the pathological changes of gastric tissues in mice effectively; 3) ADH and ALDH levels in gastric tissue of the MLG treated groups showed a upward remarkably, which revealed a significant effect of MLG on accelerating the alcohol metabolism; 4) MLG could reduce the oxidative damage for gastric tissues by decreasing the production of oxidative damage products and increasing the levels of antioxidant enzymes; 5) MLG might aid the repair of the damaged gastric mucosa by inhibiting the release of inflammatory mediators and accelerating microcirculation of the gastric mucosa, NO and PGE2 possibly involved in the process; 6) MLG could attenuate the oxidative damage and inflammatory response and improve the defensive factors, which might be associated with the activation of the Nrf2/HO-1 signaling and the repression of the NF-κB signaling pathway. In general, MLG could significantly accelerate the metabolism of alcohol and attenuate ethanol-induced gastric mucosal lesions in mice, which might be put down to the activation of alcohol metabolizing enzymes, the inhibition of oxidative damage and the maintenance of gastric mucosa involving the regulation of Nrf-2/HO-1/NF-κB signaling pathway. It is worth mentioning that the protection effects of MLG for ethanol-induced gastric lesions appears to be comparable to the classical gastric mucosal protection agent Colloidal Bismuth Pectin (CBP). Moreover, MLG could significantly accelerate the metabolism of alcohol and reduce the toxicity of alcohol, a property not shared by CBP.

ADH and ALDH are crucial enzymes involved in alcohol metabolism, which convert about 90% of ethanol to acetaldehyde, and the resulting acetaldehyde is further oxidized into harmless acetic acid. This biological process plays a critical role in the rate of alcohol metabolism and the detoxification of alcohol (Gizer et al., 2011; Meng et al., 2019). We recorded the elevation of the activities of ADH and ALDH in gastric tissues after alcohol administration in mice, which could be considered as an adaptive response to alcohol stimulation. In comparison, the raise of ADH and ALDH levels in gastric tissues were more significant in mice treated with MLG, and a dose-dependent manner is showed with the increment of MLG-M and MLG-H groups being especially arresting. The marvelous results revealed that MLG can stimulate alcohol metabolism, which is convincingly supported by the enhancive ADH and ALDH levels in the gastric tissues of MLG-treated group.

Ethanol, a recognized necrotizing agent, could directly irritate and damage the gastric mucosa, making the gastric mucosal epithelium more susceptible to degeneration and necrosis. We observed that, compared with the model group, MLG-treated groups exhibited lower gastric ulcer index, higher ulcer inhibition rate and milder gastric tissue injury, suggesting that MLG could efficaciously ameliorate ethanol-induced gastric lesions in mice. The histopathological examination of gastric tissues shown a similar tendency. A series of pathological changes that indicated severe gastric lesions, including epithelial cell loss, hemorrhage, structural disorders of glandular tissues, submucosal edema, and inflammatory cells infiltration, could be seen in the model group. The administration of MLG certainly mitigated the situation in a dose-dependent manner. The pathological changes including epithelial cell loss, structural disorder of glandular tissues, submucosal edema and hemorrhage were observed to a milder extent within all MLG-treated group, and, strikingly, the effect of MLG-H group is comparable (and even better) to CBP.

The pathogenesis for ethanol-induced gastric lesions is manifold and complicated, but chief among them is oxidative stress-mediated aggravated inflammatory response. It is well established that ethanol and its metabolite acetaldehyde attack the gastric mucosa causing microcirculatory disturbance and hypoxia, along with the propagation of the inflammatory cascade due to the depletion of mucus and bicarbonate, eventually resulting in cellular necrosis and the excessive production of ROS (Périco et al., 2020). It is ROS that is responsible for the pathogenesis of ethanol-induced gastric lesions mediated by oxidative stress (Sistani Karampour et al., 2019), which interferes with the antioxidant systems of endogenous cells in mucosa, induces leukocyte recruitment and boosts inflammatory response. In the gastric mucosa, the relation, between ROS generation and antioxidant protection mediated through antioxidant enzymes, maintains homeostasis. Oxidative stress triggers a cascade, bringing on the excessive production of ROS and the accumulation of malondialdehyde (MDA). MDA is a significant marker of ROS peroxidation due to its role as a primary lipid peroxidation end product, which manifests the damage of ROS on gastric mucosal directly and is mediated by the development of gastric lesions (Yuksel et al., 2012). ROS also stimulates inhibitor Kappa B (IκB) kinase to trigger the proteasomal breakdown of IκBα, the release of NF-κB p65, and, at length, the activation of NF-κB. NF-κB serves as a catalyst for the transcription of a host of inflammatory cytokines and chemokines, being responsible for the high expression of inflammatory factors including TNF-α, IL-6, and IL-1β (Wardyn et al., 2015; Mitchell et al., 2016; Akanda and Park, 2017). TNF-α and IL-1β, multifunctional pro-inflammatory cytokines, conduce to the activation and migration of inflammatory cells into the gastric mucosa as well as the gastric inflammatory process (Sugimoto et al., 2009). The secretion of inflammatory mediators, including TNF-α and IL-1β, is consistent with the activation of polymorphonuclear neutrophil leukocytes, inflammatory infiltration of lymphocytes, and macrophages in gastric tissue after acute ethanol exposure (Bagheri et al., 2015).

When oxidative stress transpires, antioxidant defense system acts actively as well to scavenge ROS, defend against oxidative stress in cell and guard the body. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial transcriptional regulator for cellular defenses against oxidant-associated damage. Excessive ROS phosphorylates Nrf2 and dissociates it from its inhibitor, Kelch-like epichlorohydrin-associated protein 1 (Keap1); the activated Nrf2 will translocate to the nucleus and bind with Maf proteins, to stimulate antioxidant response elements (AREs) and activate downstream antioxidant enzymes such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) (Takei et al., 2015; Kim et al., 2022). HO-1, a stress-inducible enzyme, is considered as a reliable anti-oxidative and cytoprotective agent, which catalyzes the breakdown of heme into equimolar amounts of biliverdin, ferrous iron and carbon monoxide (Puentes-Pardo et al., 2020). Signaling pathways associated with inflammation and oxidative stress, mainly including Nrf2 and NF-κB, control the expression of HO-1. SODs are universal and essential enzymes for organisms that live in the presence of oxygen, and they are widely recognized as the first hurdle in the fight against oxygen free radicals (Wang et al., 2018). Characterized by requiring for different catalytic metal ions, SODs are divided into three classes in various organisms including copper/zinc SOD (Cu/Zn SODs), manganese/iron SOD (Mn SOD/Fe SODs), and nickel SOD (Ni SODs) (Wang et al., 2018). GSH-Px is highly concentrated in gastric tissue, which is also a vital member of antioxidant system, and gastric mucosa GSH loss may further exacerbate lipid peroxidation along with cell membrane and gastric mucosa damage (Zhang et al., 2013).

The mitogen-activated protein kinases (MAPKs) are important cellular signaling molecules transferring various extracellular signals to intracellular responses by sequential phosphorylation cascades. Several distinct but parallel subgroups have been identified, including C-Jun N-terminal kinase (JNK), Extracellular signal-regulating kinases (ERKs) and p38 MAPK(Bak et al., 2016). MAPKs are involved in a variety of cell biological processes, such as cell proliferation, differentiation, stress response, apoptosis, cell migration, and survival. p38 MAPK is closely related to multiple pathways related to oxidative stress, which is activated and phosphorylated under environmental influence. It is well founded that p38 MAPK is activated and phosphorylated by oxidative stress. Tissue damage and other external stimuli will trigger the secretion of multiple pro-inflammatory cytokines such as TNF-α, IL-1β, as well as IL-6, and subsequently, the activation of p38(Chaparro Huerta et al., 2008). The relation between p38 MAPK and NF-κB has been reported that a variety of pro-inflammatory factors can give rise to the modified IκBs degradation and the p65 translocation into the nucleus directly or via the activated p38-MAPK to activate NF-κB pathway (Xie et al., 2019). Recent studies have found that high expression of P38 can threaten the integrity of gastrointestinal mucosa, and, in contrast, the inhibition of p38 MAPK phosphorylation can regulate claudin expression, thereby improving the function of gastrointestinal mucosal epithelial barrier (Carrozzino et al., 2009). Increased TNF-α level leads to the disruption of tight epithelial cells and the phosphorylation of P38. HO-1 can keep the barrier intact by blocking tight junction disruption caused by TNF-α and phosphorylation of ERK, P38, and JNK(Zhang Z et al., 2021). Furthermore, MAPK cascades are found to be involved in HO-1 activation. It has been reported that p38 MAPK takes part in the protein synthesis of HO-1 as well as the activation and translocation of Nrf2 to help the body resist oxidative stress (Bak et al., 2016).

The destruction of gastric mucosal integrity can be considered as the disequilibrium between multiple endogenous aggressive factors (such as hydrochloric acid, leukotrienes, and ROS) and defensive factors, including a functional mucus-bicarbonate barrier, nitric oxide (NO), prostaglandins (PG), mucosal microcirculation, antioxidants and some growth factors (Ibrahim et al., 2016). Prostaglandin E2 (PGE2) and NO are postulated to be important defenders of the gastric epithelial mucosal barrier. PGE2, a production of arachidonic acid, is well recognized as a protective factor essential for the repair of damaged gastric mucosa. PGE2 synthesis involves two steps: arachidonic acid is converted into prostaglandin H2 (PGH2) by cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), and then PGH2 is isomerized to PGE2 by prostaglandin E synthases (Kloska et al., 2020). As a recognized vasodilator factor, PGE2 has been shown to inhibit platelet aggregation and thrombosis, as well as increase blood flow to the mucosa surface of the stomach, restrain gastric acid secretion, regenerate cells, mediate adaptive immune protective functions and promote mucosal repair (Chen et al., 2016; Fang et al., 2019). COX isoenzymes mainly exist in two isoforms: the constitutive form (COX-1) and the inducible form (COX-2). COX-2 is restricted at the site of inflammation, acting as a proinflammatory enzyme, and its expression could vary drastically as reacting to inflammatory stimuli or growth factors (Zhang J et al., 2021). It is well known that NO plays an indispensable role in maintaining the integrity of the gastric mucosal, which can repair damaged gastric mucosa by regulating gastric mucosal blood flow, acid and alkaline secretion as well as mucus secretion (Ismail Suhaimy et al., 2017). Paradoxically, NO also triggers mucosal damage. NOS, playing a pivotal role in the synthesis of NO by transforming l-arginine to l-citrulline, mainly can be divided into three forms including neuronal NOS (nNOS, type I), inducible NOS (iNOS, type II), and endothelial NOS (eNOS, type III), whereby each of them participates in multiple biological processes. nNOS and eNOS can both be grouped into constitutive NOS (cNOS), characterized by calcium dependence, while iNOS is calcium independent (Kumar and Chanana, 2017; Mohamed et al., 2022). In the digestive system, cNOS-generated NO and iNOS-produced NO are related to contradictory outcomes, with the former one displaying the cytoprotective effect and the latter being cytotoxic (Motawi et al., 2007). eNOS-generated NO facilitates the healing of ulcer via scavenging the damaging free radicals, eliciting angiogenesis, increasing vasodilation, and attenuating leukocyte infiltration, thereby aiding mucous secretion and epithelial tissue integrity restoration (Khattab et al., 2001; Abd El-Rady et al., 2021). iNOS usually generates NO in response to a wide range of stimuli, such as the production of inflammatory cytokines (IL-1β, TNF-α). iNOS-produced NO is found to be highly involved in gastric damage, which reacts with superoxide directly to form a potent cytotoxic oxidant, peroxynitrite (Abdel-Raheem, 2010; Kumar and Chanana, 2017). Somatostatin (SS), an important gastrointestinal hormone, could directly inhibit the secretion of acid and indirectly suppress the secretion of histamine and gastrin to protect the gastric mucosa (El-Salhy et al., 2014). Myeloperoxidase (MPO) is mainly found in neutrophils and therefore becomes the specific marker of neutrophils. During the initial inflammatory response, the accumulation of neutrophils results in high expression of MPO, and the activity of MPO can in turn reflect the severity of inflammation in tissues (Al-Quraishy et al., 2017). Moreover, MPO can induce oxidative stress by stimulating the production of reactive oxygen species (ROS) and active nitrogen (RNS) (Chen et al., 2020).

As our observation in the current study, ethanol exposure induced a drastic augment in the MDA level, while GSH-Px and T-SOD concentrations suffered a dramatic decline, signifying the enhancement of oxidative stress as well as lipid peroxidation. This, in turn, serves as a catalyst in the wreck of the antioxidant defense system, making the gastric mucosa more susceptible to injury. MLG has significant antioxidant activity, represented by significantly inhibiting the elevation of MDA while promoting the T-SOD and GSH-Px levels even in a small dose (Figures 5A–C), which effectively reduced the production of oxidative damage products, inhibited the activity of antioxidant enzymes, and protected the mucosa of the stomach from alcohol-induced damage. Our results demonstrated that MLG could inhibit the secretion of inflammatory mediators (TNF-α, IL-1β, iNOS, MPO, p38-MAPK, and COX-2) and up-regulate the activities of defensive factors (PEG2, NO, eNOS, and SS) in gastric tissues after ethanol exposure, which could attenuate the inflammation response and accelerate the flow of gastric mucosal microcirculation to repair the damaged gastric mucosa. On this account, we speculated that MLG could reduce the infiltration of inflammatory lymphocyte cells in gastric mucosa, which is consistently supported by the amelioration of inflammatory cells infiltration in mice gastric tissue histomorphology examination of MLG-treated groups. We found that MLG treatment markedly blocked the NF-κB expression and suppressed the phosphorylation of p65 and IκBα indicating that MLG alleviates ethanol-induced gastric lesions by inhibition of the ROS-mediated inflammatory signaling cascade. Synchronously, Nrf2 and HO-1 levels were observed up-regulated in MLG treatment groups, which demonstrated that the cytoprotective factors were activated to defense the oxidative stress damage and improve the gastric healing of ethanol-induced gastric lesions at the same time. In other words, MLG might have defensive effects on gastric mucosa by counterbalancing ROS generation and antioxidant defense systems in the gastric mucosa, which might rely on reducing the oxidative stress and strengthening the antioxidant defense concurrently via the molecular cross-talk of NF-κB/Nrf-2/HO-1. Taken together, our results demonstrated that MLG, as natural antioxidants, could help protect the gastric mucosa from oxidative stress and improve its defenses in order to moderate gastric mucosal microcirculation and preserve gastric mucosa.

Taken together, our study delivered a point of view that MLG could effectively protect the gastric mucosa of mice against the gastric damage of ethanol-induced gastric lesions, mainly presented as the activation of alcohol metabolizing enzymes, attenuation of oxidative damage and inflammatory response, up-regulation of the defensive factors and improvement of vasodilation. Most glaring of all, MLG could remarkably accelerate the metabolism of alcohol and reduce the toxicity of alcohol, a property not shared by CBP. The gastroprotective effect of MLG on ethanol-induced gastric lesions may be achieved through the weakened of damage factors (TNF-α, IL-1β, iNOS, MPO, p38-MAPK, and COX-2) and the enhancement of defensive factors (PEG2, NO, eNOS, and SS) in gastric tissues involving NF-κB/Nrf2/HO-1 signaling pathway. We further confirmed that MLG has a strong potential in preventing and treating ethanol-induced gastric lesions.