The protective effects of myricetin against acute liver failure via inhibiting inflammation and regulating oxidative stress via Nrf2 signaling

Abstract This study aimed to investigate the protective effects and mechanisms of myricetin on acute liver failure in mice induced by lipopolysaccharide (LPS)/D-galactosamine (D-Gal). Our results showed myricetin (25, 50 and 100 mg/kg) pretreatment significantly improved the pathological changes of liver tissues, decreased serum ALT and AST (p < 0.001) induced by LPS/D-GalN. Moreover, MDA and MPO levels were reduced (p < 0.001), CAT and SOD activities were increased (p < 0.001) with myricetin (50 and 100 mg/kg) pretreatment. Likewise, inflammatory cytokines TNF-α and IL-6 mRNA in liver tissues were markedly decreased (p < 0.001) by myricetin. Besides, Nrf2 protein expression was drastically elevated (p < 0.001) by myricetin (25, 50 and 100 mg/kg). All these findings imply that myricetin may protect against acute liver failure by suppressing inflammation and regulating oxidative stress via Nrf2 signaling, and that it may be a possible strategy to avoid liver damage. Graphical Abstract


Introduction
The liver is responsible for a variety of functions in our bodies, including digestion, metabolism, hormone production, and host-defense, and plays a critical role in maintaining normal systemic homeostasis (Yan et al. 2014). Acute liver failure (ALF) also known as fulminant hepatic failure is a fatal condition characterized by fast loss of normal liver function. It can result in severe complications such as excessive bleeding, increased cerebral pressure, lung and kidney failure, and even death (Bernal et al. 2010). ALF can be caused by a variety of viral and bacterial diseases, as well as poisons, chemicals, and medicines (Larson et al. 2005), and intraperitoneal injection of lipopolysaccharide (LPS)/D-galactosamine (D-Gal) is one of the most used experimental models for studying ALF (Mohamadi-Zarch et al. 2020). Currently, liver transplantation is the most effective treatment for ALF. Nevertheless, due to a scarcity of donor organs, high expenses, and the risk of consequences, it is critical to research alternate treatments.
In recent years, natural compounds extracted from plants or animals have been shown to possess a wide range of biological effects, such as anti-aging, anti-inflammatory, antioxidative, anti-apoptotic, anti-viral, anticancer, anxiolytic, hypoglycaemic, hypocholesterolemic, antidepressant (Alesci et al. 2021;Fumia et al. 2021;, Alesci, Nicosia, et al. 2022, which indicates its key role in drug discovery. Myricetin is a naturally occurring flavonoid that can be found in berries, fruits, vegetables, tea, red wine and many other foods . Myricetin, like other flavonoid, has a wide range of pharmacological activity, including antibacterial, antiviral, anti-diabetic, anti-tumor, antioxidant, anti-inflammatory, and anti-ulcer properties (Taheri et al. 2020). Many studies have demonstrated that myricetin has therapeutic benefits on a variety of disorders, including cancers, inflammatory diseases, allergies, cardiovascular diseases, diabetes, Alzheimer's disease and microbial infections (Gupta et al. 2020). In addition, myricetin acts by modulating inflammation, in fact inhibits Toll-like receptors, phylogenetically preserved receptors (Lauriano et al. 2021;, thus influencing the release of cytokines, playing a crucial role in the immune response (Song et al. 2021).
Though there are numerous studies on the protective role of myricetin against liver damage, the role of myricetin on ALF induced by LPS/D-GalN in BALB/c mice is still not fully known and remains to be elucidated. In this present study, we investigated the effects of myricetin on ALF induced by LPS/D-GalN, and further explored its relative mechanisms.

Results and discussion
As shown in Figure S1, when compared to that of control group, LPS/D-GalN injection obviously destroyed the structure of liver tissues, leading to severe hemorrhagic necrosis and a mass of infiltration of inflammatory cells in portal area. Myricetin pretreatment considerably decrease histological alterations, resulting in less hemorrhage and cell infiltration, which suggested that myricetin had a protective impact on liver tissues in LPS/D-GalN-induced ALF. Furthermore, considering ALT and AST are often used to assess the hepatotoxicity, we investigated the effects of myricetin on these two indicators. Similarly, myricetin pretreatment inhibited the rise in serum ALT and AST caused by LPS/D-GalN, demonstrating its liver-protection ( Figure S2).
Oxidative stress is defined as an imbalance in the generation of ROS production and antioxidants, which is implicated in tissue damage in various diseases (Betteridge 2000). MDA, MPO, CAT and SOD are common oxidative stress indicators (Ho et al. 2013). In this present study, myricetin reversed the alterations in these indicators caused by LPS/D-GalN injection, which is consistent with other natural antioxidants (Wang et al. 2012), showing its critical role in controlling oxidative stress in liver. When compared to that of control group, LPS/D-GalN significantly increased MDA and MPO levels, and myricetin pretreatment reduced this rise ( Figure S3). MDA level was only slightly decreased by 25 mg/kg myricetin, but 50 mg/kg and 100 mg/kg myricetin obviously decreased MDA level. Moreover, three concentrations of myricetin significantly reduced the increase of MPO level induced by LPS/D-GalN. These results suggested that myricetin pretreatment could possibly regulate LPS/D-GalN induced oxidative stress via decreasing MDA and MPO levels. CAT and SOD are the components of intrinsic antioxidant defense system, and play a vital role during oxidative stress. When compared to the control group, LPS/D-GalN injection significantly lowered CAT and SOD activities, while myricetin pretreatment substantially increased the activities of these two enzymes ( Figure S4), suggesting that myricetin has potent antioxidant effects in liver via elevating CAT and SOD enzyme activities.
TNF-a and IL-6 are two important inflammatory cytokines during inflammation, which are implicated in LPS/GalN-induced ALF. Compared to that of control group, the levels of TNF-a and IL-6 mRNA in liver tissues was dramatically boosted with LPS/ D-GalN injection, whereas myricetin pretreatment significantly reduced their levels ( Figure S5). Given that numerous studies have shown that controlling inflammation can help with ALF , myricetin may protect mice against LPS/D-GalNinduced ALF by suppressing inflammation.
Studies from several animal models showed that Nrf2 pathway exhibits a variety of biological functions against many liver diseases via targeting gene expression. During oxidative stress, Nrf2 detaches from Keap 1, translocates to the nucleus, and binds to the antioxidant response element resulting in the expression of antioxidant genes (Vomhof-Dekrey and Picklo 2012). As shown in Figure S6, compared to that of control group, LPS/D-GalN increased Nrf2 protein expression, while myricetin pretreatment further elevated its expression obviously. Besides, myricetin alone also could improve Nrf2 expression. It should be noted that there were no statistical differences in Nrf2 protein expression between 50 mg/kg and 100 mg/kg myricetin groups (p > 0.05), even though it appears a decrease in 100 mg/kg myricetin group in comparison to 50 mg/kg myricetin group. Similar to other antioxidants, myricetin increased Nrf2 protein expression in LPS/D-GalN induced ALF, suggesting that myricetin could activate Nrf2 signaling pathway to mitigate liver damage caused by LPS/D-GalN.

Conclusions
We found that myricetin protected mice from LPS/D-GalN-induced ALF, displaying myricetin pretreatment alleviated the histological alterations in liver tissues caused by LPS/D-GalN and decreased serum ALT and AST levels. Moreover, mRNA expression of inflammatory cytokines TNF-a and IL-6 in liver tissues were reduced with myricetin pretreatment. Further investigations revealed that myricetin pretreatment modulated oxidative responses in liver tissues by lowering MDA and MPO levels while enhancing CAT and SOD activity. Additionally, Nrf2 protein expression was drastically increased by myricetin pretreatment. All these data pointed to the possibility that myricetin protected mice from LPS/D-GalN-induced ALF by reducing inflammation and regulating oxidative stress. Nevertheless, in this present study, we only investigated the protective effects of myricetin on LPS/D-GalN-induced ALF in vivo, further work is needed to ensure the underlying mechanism of myricetin on ALF in vitro.

Funding
This work was supported by grants from the National Natural Science Foundation of China (No. 31772721).