Rhoifolin Alleviates Alcoholic Liver Disease In Vivo and In Vitro via Inhibition of the TLR4/NF-κB Signaling Pathway

Abstract

Background: Alcoholic liver disease (ALD) is a common chronic liver disorder worldwide, which is detrimental to human health. A preliminary study showed that the total flavonoids within Citrus grandis “Tomentosa” exerted a remarkable effect on the treatment of experimental ALD. However, the active substances of Citrus grandis “Tomentosa” were not elucidated. Rhoifolin (ROF) is a flavonoid component present in high levels. Therefore, this research aimed to evaluate the hepatoprotective effects of ROF and its possible mechanisms.Methods: Molecular docking was performed to analyze the binding energy of ROF to the main target proteins related to ALD. Subsequently, mice were fed ethanol (ETH) for 49 days to establish the chronic alcoholic liver injury models. The liver pathological injury, serum aminotransferase levels, and oxidative stress levels in the liver tissue were measured. Human normal hepatocytes (LO2 cells) were incubated with ETH to construct the alcoholic liver cell model. The inflammatory markers and apoptosis factors were evaluated using real-time PCR and flow cytometry. Finally, the effects of ROF on the CYP2E1 and NF-κB signaling pathways were tested in vitro and in vivo.Results: Molecular docking results demonstrated that ROF was able to successfully dock with the target proteins associated with ALD. In animal studies, ROF attenuated ETH-induced liver damage in mice by decreasing the serum concentrations of AST and ALT, reducing the expression of inflammatory cytokines, and maintaining antioxidant balance in the liver tissue. The in vitro experiments demonstrated that ROF suppressed ETH-induced apoptosis in LO2 cells by promoting Bcl-2 mRNA and inhibiting Bax mRNA and caspase 3 protein expression. ROF decreased the level of LDH, ALT, AST, ROS, and MDA in the supernatant; induced the activity of GSH and SOD; and inhibited TNF-α, IL-6, and IL-1β expression levels. Mechanistically, ROF could significantly downregulate the expression levels of CYP2E1, TLR4, and NF-κB phosphorylation.Conclusion: This study indicates that ROF is the active component within the total flavonoids, which may alleviate ETH-induced liver injury by inhibiting NF-κB phosphorylation. Therefore, ROF may serve as a promising compound for treating ALD.

The antioxidant peptides from walnut protein hydrolysates and their protective activity against alcoholic injury

“…54 TLR4 triggers the production of multiple inflammatory factors through activation of the NF-κB pathway, which is closely related to the inflammatory response in alcoholic liver injury. 53,55 Molecular docking experiments revealed that the docking binding energies of HGEPGQQQR, VAPFPEVFGK and HNVADPQR with TLR4 () were −6.5 kcal mol −1 , −6.7 kcal mol −1 , and −5.9 kcal mol −1 , respectively. HGEPGQQQQR forms hydrogen bonds with Glu142, Gln163, Ser140, Asn143, Ala139, Leu117, Thr136, Gln115, and Ile114 residues of TLR4 (Fig.…”

“…52 CYP2E1 catalyzes the oxidation of ethanol to acetaldehyde, and its metabolites can produce hepatocytotoxicity and increase the risk of developing ALD. 53 Molecular docking revealed that the docking binding energies of HGEPGQQQR, VAPFPEVFGK and HNVADPQR with CYP2E1 (3E6I) were −7.7 kcal mol −1 , −8.2 kcal mol −1 , and −8.2 kcal mol −1 , respectively, suggesting that all three peptides could effectively bind to CYP2E1 and form stable conformations. As can be seen in Fig.…”

Melosira nummuloides Ethanol Extract Ameliorates Alcohol-Induced Liver Injury by Affecting Metabolic Pathways

“…Subsequently, the mRNA expression of inflammatory cytokines, including TNF-α, IL-1β, and IL-6, was notably suppressed in alcohol-treated HepG2 cells and the livers of chronic-binge alcohol-fed mice. Previous studies have suggested that NFκB activation is responsible for regulating the expression of TNF-α, IL-6, and IL-1β in the livers of alcohol-treated mice . Inflammatory stimuli further enhance COX-2 expression, contributing to liver injury.…”

“…Previous studies have suggested that NFκB activation is responsible for regulating the expression of TNF-α, IL-6, and IL-1β in the livers of alcohol-treated mice. 30 Inflammatory stimuli further enhance COX-2 expression, contributing to liver injury. Additionally, multiple studies have demonstrated that exposure to alcohol increases the levels of P-Akt, which plays a crucial role as an upstream activator in the inflammatory response involving NFκB in the liver.…”

Sirtuin 1 alleviates alcoholic liver disease by inhibiting HMGB1 acetylation and translocation

“…Ethanol metabolism generates acetaldehyde, disrupting the equilibrium between the body’s oxidative and peroxide systems, resulting in oxidative stress. This imbalance contributes to increased levels of cytotoxic oxidative product MDA in liver tissues, increased ROS content, compromised antioxidant capacity, reduced free-radical scavenging ability, and lipid peroxidation of liver cell membranes, collectively causing liver cell damage ( Mai et al, 2022 ). In the present study, the EtOH group showed significantly increased ALT and AST levels in HepG2 cells compared to the control group, indicating liver cell damage.…”