The liver is considered a vital organ of the body with critical functions in protein synthesis, glucose homeostasis, detoxification, and nutrient utilization (Gao et al., 2008; Al-Seeni et al., 2016). The high levels of environmental toxins, alcohol, heavy metals, and metabolic dysfunction of the liver are among various insults that may lead to acute or chronic hepatic injury, followed by progressive fibrosis, cirrhosis, and even carcinoma (Sun et al., 2011; Nwokocha et al., 2012; Su et al., 2019; Chan et al., 2020).

Due to the implication of oxidative stress and inflammation in the etiology of liver injury, carbon tetrachloride (CCl₄), a typical environmental toxicant, is widely used in experimental hepatopathy (Zou et al., 2016). CCl₄-induced hepatotoxicity is attributed to reductive dehalogenation reactions catalyzed by the hepatic cytochrome P450, producing unstable trichloromethyl (CCl₃•). Trichloromethyl peroxyl (Cl₃COO•) radicals subsequently bind to proteins or lipids initiating lipid peroxidation and culminate in liver injury due to depletion of antioxidant enzymes (McGill and Jaeschke, 2013; Dong et al., 2015). During CCl₄-induced oxidative damage, various inflammatory mediators, including interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), are released (Ashrafullah et al., 2018). Hepatic injury is further aggravated by the feedback mechanism.

Although some medicines have been developed to protect the liver, hepatoprotective drugs have potentially adverse side effects (Wang et al., 2019). Hence, finding natural plant extracts effective for the protection of liver injury is of intense interest.

Medicinal mushrooms have a long history of application in traditional oriental therapies, and fungal extracts are increasingly being employed to treat a wide range of diseases (Nakahara et al., 2020; Tung et al., 2020). Morchella importuna is a rare edible and medicinal mushroom species of true morels (the genus Morchella), highly appreciated for its commercial value, potent bioactivities, culinary qualities, and taste (He et al., 2019; Wang et al., 2020). Polysaccharide, an important active constituent of M. importuna, possesses various bioactivities, including antibacterial, antioxidant, anti-inflammatory, and antitumor properties, and a cardiovascular protective effect (Meng et al., 2018; Hou et al., 2020; Mu et al., 2020; Tang et al., 2020; Xue et al. 2019). This study has indicated that some plant polysaccharides also have hepatoprotective activity against drug- and toxicant-induced liver injuries. The polysaccharide of wild edible mushroom Russula vinosa protects the liver from CCl₄-induced oxidative stress injury (Liu et al., 2014). Similarly, the antioxidant activities of Cyclocarya paliurus polysaccharide might be of therapeutic value in ameliorating the hepatic and renal oxidative stress caused by CCl₄ (Wu et al., 2020b).

Morchella importuna polysaccharides (MIPs), with a molecular weight (Mw) of 28.5 kDa, are composed of N-acetylglucosamine, galactose, glucose, and mannose with molar ratios of 1.00:14.95:1.53:10.51 (Wen et al., 2019). These MIPs show strong antioxidant, immune-enhancing, hepatoprotective, and lipid-lowering activities. Therefore, it is attractive to further investigate their bioactivities, which provides feasible ideas and insights for the potential curative application of MIPs.

Accordingly, this study was performed to explore the effects of MIP administration on alleviating CCl₄-induced oxidative stress and inflammation in mice. Subsequent metabolomic analysis was performed to uncover the potential pathways that were disturbed by CCl₄ intoxication. These results provide partial theoretical evidence about the MIPs in preventing and treating oxidative injury in the liver.

Finding natural sources of hepatoprotective medicines is of keen research interest in ensuring liver protection and thereby its roles in regulating numerous physiological processes, including metabolism, synthesis, and detoxification (Bing-Ya et al., 2015; Lu et al., 2016; Yang et al., 2019). M. importuna mushrooms are rich sources of many bioactive components, such as polysaccharides, dietary fiber, and proteins (Gursoy et al., 2009). In particular, polysaccharides from M. importuna have shown multiple pharmacological and biological activities, including antioxidant, antimicrobial, antiviral, and hepatoprotective activities (Heleno et al., 2013). However, how MIPs confer protection against oxidative injury remains unknown. The animal model of acute liver injury induced by CCl₄ is one of the classical models used to screen antioxidative medicines. Therefore, we aimed to evaluate the suitability as potent medicinal foods by examining the effects of LMIPs and HMIPs on CCl₄-induced acute liver damage in mice.

Weight loss after CCl₄ intoxication is considered to be caused by the direct toxicity of CCl₄ or the indirect toxicity associated with liver damage (Yang et al., 2010). However, BW was not decreased by CCl₄ intoxication and marginally increased by MIP administration in this study. Marked swelling of the CCl₄-treated liver occurred, as evidenced by the enhancement of liver weight. In comparison, a significant decrease in liver weight was observed in all mice infused with HMIPs. Clinical assessment of liver injury involves measurement of the ALT, AST, TG, TC, and TBA, which in part, reflect the degree of hepatocyte damage and necrosis. This study demonstrated that the levels of these markers were elevated by CCl₄ intoxication. MIPs inhibited the leakage of these enzymes from the liver tissue into the bloodstream. CCl₄ is decomposed into numerous free radicals, such as trichloromethyl, after its metabolization by cytochrome P450 in hepatocytes (He et al., 2016). Due to the free radical-mediated destruction and increased permeability of the cellular membrane structure, high levels of ALT and AST enzymes infiltrate into the bloodstream, significantly promoting the activity of these enzymes in the serum. Concurrently, the trichloromethyl peroxyl radical is generated by the reaction of trichloromethyl radical with molecular oxygen and can attack liver microsomal lipid and hepatocyte membrane phospholipid molecules, leading to the initiation of the lipid peroxidation reaction (Bruinsma et al., 2015; Shi-Bin et al., 2015). High levels of TG and TC were deposited in hepatocytes, which was concomitant with an increase in the serum levels of TG and TC (Lykkesfeldt, 2007).

Lipid peroxidation resulting from oxidative stress produces a large amount of MDA, which can also contribute to hepatocyte damage, thereby aggravating the lesions (Tsikas, 2017). PC is the product of damage to amino acid side chains by oxygen-free radicals. With the aggravation of oxidative stress, the protein carbonylation becomes more severe, which impedes protein folding and promotes protein–protein cross-linking. Thus, PC is considered an effective index to evaluate the degree of protein oxidation. In contrast, SOD and CAT are important antioxidant enzymes, responsible for scavenging free radicals and inhibiting lipid peroxidation, thus protecting the hepatocytes against CCl₄-mediated toxicity (Dmitry et al., 2017). In this study, the excessive increase in the hepatic MDA and PC contents in the CCl₄ group were markedly attenuated by HMIP administration. Furthermore, MIPs dose-dependently alleviated the significantly decreased concentration of SOD caused by CCl₄ intoxication. Hence, MIP administration preserved hepatic health by attenuating the oxidative damage in the liver.

In addition, the inflammatory response observed during CCl₄-induced hepatic damage may be accompanied by the release of several cytokines (i.e., IL-1, IL-6, and TNF-α), contributing to a degenerative scenario (Sha et al., 2020). The MPO activity serves as a marker for inflammatory cell infiltration and indicates the degree of inflammation (Chen et al., 2020). Our data showed a dramatic increase in the levels of IL-1β, IL-6, TNF-α, and MPO by CCl₄ intoxication. However, MIP administration led to a marked decrease in pro-inflammatory cytokines and MPO compared with the CCl₄ group. Moreover, H&E staining confirmed that CCl₄ intoxication induced acute liver damage, as indicated by vacuolation, the ballooning of hepatocytes, and inflammatory cell infiltration. HMIP administration mitigated these detrimental injuries, which further supported the aforementioned hepatoprotective effects. These results supported that MIPs possess potent hepatoprotective activity by inhibiting oxidative stress and inflammation.

Based on the more favorable inhibitory effects of HMIPs over LMIPs on CCl₄-induced liver injury, a UHPLC–MS/MS-based metabolomic analysis was performed to evaluate the protective effect of HMIPs against CCl₄-induced hepatic damage. The observed trends in serum biomarkers following CCl₄ intoxication or HMIP administration are shown in Tables 1, 2. These metabolites were mainly involved in flavonoid biosynthesis, amino acid metabolism, energy metabolism, and toxicant degradation. Numerous studies have indicated multifunctional flavonoids isolated from Chinese medicines and plants as potential therapeutic agents for the improvement of the liver tissue via antioxidant, anti-inflammatory, and antifibrotic mechanisms (Gou et al., 2020; Wu et al., 2020a). Serum metabolomic analysis revealed that metabolites (i.e., isoliquiritigenin and prunin) related to flavonoid biosynthesis were significantly reduced by CCl₄ intoxication. However, the ratio of prunin between the HMIP and CCl₄ groups was lower than 1.0, perhaps indicating that HMIP administration did not affect flavonoid biosynthesis.

The liver is a vital organ in regulating amino acid metabolism, and any dysfunction in the liver could cause a disturbance in the amino acid concentrations (Waters et al., 2005). The decreased ratio of γ-aminobutyric acid observed between the control and CCl₄ groups in this study indicated that CCl₄ infusion might disturb the alanine, aspartate, and glutamate metabolism because γ-aminobutyric is one of the metabolites in this pathway. Tryptophan is metabolized along the kynurenine and serotonin pathways, while phenylacetylglycine participates in phenylalanine metabolism (Kamiguchi et al., 2017; Li et al., 2017). In this study, amino acids in the serum were promoted by CCl₄ intoxication, perhaps because of decreased protein synthesis and increased protein catabolism (Guo et al., 2017). CCl₄ intoxication can disturb methylation pathways such as the hypomethylation of ribosomal RNA, leading to defective protein synthesis (Clawson et al., 1987). Moreover, protein catabolism plays an important role in regulating lipid and glucose metabolism, which could induce cytokine secretion under abnormal conditions (Sinha et al., 2020).

Trigonelline (N-methylnicotinic acid) and 6-hydroxynicotinic acid are intermediates of nicotinate and nicotinamide metabolism, respectively. Nicotinamide has been used as a metabolic precursor of nicotinamide adenine dinucleotide (NAD⁺) biosynthesis to raise the intracellular NAD⁺ pool for improvement of metabolic diseases involving glycolysis (French, 2015; Wan et al., 2019). Trigonelline showed a hepatoprotective effect against non-alcoholic fatty liver diseases by reducing oxidative stress and pro-apoptosis (Zhang et al., 2015). In this study, we discovered that trigonelline was impeded by CCl₄ intoxication as evidenced by the ratio between the control and CCl₄ groups (Table 1). However, the reduced levels of trigonelline could not be reversed by HMIP infusion. Additionally, it was reported that some commonly used medications, such as p-aminobenzoate, an antifibrotic agent, may unpredictably be the underlying cause of liver injury (Al Attar and Kilgore, 2018). In our study, catechol, a strong toxicant with carcinogenic properties, was decreased by HMIP administration. Thus, we speculated that HMIPs might beneficially facilitate toxicant degradation to protect the liver tissues. In brief, HMIPs showed hepatoprotective activity against liver injury by affecting amino acid metabolism, energy metabolism, and toxicant degradation. However, the underlying mechanisms for these findings remain unclear and require elucidation.

Taken together, our findings demonstrated that MIP administration alleviated CCl₄-induced hepatic injury by promoting antioxidant and anti-inflammatory activities. Furthermore, the metabolomic analysis showed that HMIPs regulate the pathways of amino acid metabolism, energy metabolism, and toxicant degradation to subvert the disturbances induced by CCl₄. This study provides a scientific basis for MIPs as a curative treatment for hepatic injury.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by the Ethics Review Committee for Animal Experimentation of Sichuan Academy of Agricultural Sciences.

YX, BG, and WP conceived and designed the experiments. YX, LX, JT, and YC conducted the experiments. YX, XH, ZZ, and JZ analyzed the data. YX and WP wrote the article. All authors have read and agreed to the published version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.