Protection of taraxasterol against acetaminophen-induced liver injury elucidated through network pharmacology and in vitro and in vivo experiments.

BACKGROUND: Drug-induced liver injury (DILI) is primarily caused by drugs or their metabolites. Acetaminophen (APAP) is an over-the-counter antipyretic analgesic that exhibits high hepatotoxicity when used for long-term or in overdoses. Taraxasterol is a five-ring triterpenoid compound extracted from traditional Chinese medicinal herb Taraxacum officinale. Our previous studies have demonstrated that taraxasterol exerts protective effects on alcoholic and immune liver injuries. However, the effect of taraxasterol on DILI remains unclear. HYPOTHESIS/PURPOSE: This study aimed to elucidate the effects and mechanisms of action of taraxasterol on APAP-induced liver injury using network pharmacology and in vitro and in vivo experiments. METHODS: Online databases of drug and disease targets were used to screen the targets of taraxasterol and DILI, and a protein-protein interaction network (PPI) was constructed. Core target genes were identified using the tool of Analyze of Cytoscape, gene ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses were performed. Oxidation, inflammation and apoptosis were evaluated to determine the effect of taraxasterol on APAP-stimulated liver damage in AML12 cells and mice. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to explore the potential mechanisms of taraxasterol against DILI. RESULTS: Twenty-four intersection targets for taraxasterol and DILI were identified. Among them, 9 core targets were identified. GO and KEGG analysis showed that core targets are closely related to oxidative stress, apoptosis, and inflammatory response. The in vitro findings showed that taraxasterol alleviated mitochondrial damage in AML12 cells treated with APAP. The in vivo results revealed that taraxasterol alleviated pathological changes in the livers of mice treated with APAP and inhibited the activity of serum transaminases. Taraxasterol increased the activity of antioxidants, inhibited the production of peroxides, and reduced inflammatory response and apoptosis in vitro and in vivo. Taraxasterol promoted Nrf2 and HO-1 expression, suppressed JNK phosphorylation, and decreased the Bax/Bcl-2 ratio and caspase-3 expression in AML12 cells and mice. CONCLUSION: By integrating network pharmacology with in vitro and in vivo experiments, this study indicated that taraxasterol inhibits APAP-stimulated oxidative stress, inflammatory response and apoptosis in AML12 cells and mice by regulating the Nrf2/HO-1 pathway, JNK phosphorylation, and apoptosis-related protein expression. This study provides a new evidence for the use of taraxasterol as a hepatoprotective drug.

Research Note: Taraxasterol alleviates aflatoxin B1-induced oxidative stress in chicken primary hepatocytes.

Aflatoxin B1 (AFB1) is the most toxic subtype of aflatoxin in feed. Poultry is sensitive to AFB1, and the liver is the main target organ of AFB1. Our previous studies have shown that taraxasterol isolated from the traditional Chinese medicinal herb Taraxacum has protective effects against immune-mediated and alcoholic-induced liver injuries. This study aimed to investigate whether taraxasterol has the protective effect and its mechanism against AFB1-induced injury in chicken primary hepatocytes in vitro. The chicken primary hepatocytes were induced with AFB1 (0.05 µg/mL), and treated with taraxasterol (5, 10, and 20 µg/mL). The results showed that taraxasterol increased superoxide dismutase (SOD) and glutathione (GSH) activity and decreased malondialdehyde (MDA) and reactive oxygen species (ROS) production in AFB1-induced hepatocytes. Moreover, taraxasterol up-regulated the mRNA and protein expression of antioxidant-related factors heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1) and nuclear factor erythroid E2-related factor 2 (Nrf2), while down-regulated the expression of oxidant-related factor Kelch-like ECH-associated protein 1 (Keap1) in Nrf2/Keap1 signaling pathway. In addition, taraxasterol effectively reduced AFB1-induced hepatocyte autophagy and inhibited the mRNA expression of autophagy-related genes Beclin-2, LC3-I, LC3-II, and ATG-5. Taraxasterol also inhibited AFB1-induced hepatocyte apoptosis and decreased the mRNA expression of apoptosis-related genes Caspase3 and Caspase9. These findings indicates taraxasterol alleviates oxidative stress in AFB1-induced chicken hepatocytes by activating Nrf2/Keap1 signaling pathway, and regulating the cell autophagy and apoptosis.

Inhibitory Effects of Inonotus obliquus Polysaccharide on Inflammatory Response in Toxoplasma gondii-Infected RAW264.7 Macrophages.

Our previous reports have shown that Inonotus obliquus polysaccharide (IOP) has protective effects against Toxoplasma gondii (T. gondii) infection in vivo. The aim of the present research is to explore the in vitro anti-inflammatory effects of IOP and its mechanism in RAW264.7 macrophages infected by T. gondii. In this study, it is indicated that IOP decreased the excessive secretion of inflammatory cytokines tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1ß (IL-1ß), IL-4, and IL-6 in T. gondii-infected RAW264.7 macrophages. IOP effectively suppressed the mRNA expression of these cytokines and chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α). Moreover, IOP inhibited the phosphorylation of inhibitor kappa B kinase α/ß (IKKα/ß), inhibitor κBα (IκBα), p65 in nuclear factor-kappa B (NF-κB) signaling pathway and p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) in mitogen-activated protein kinases (MAPKs) signaling pathway. Meantime, IOP prevented NF-κB p65 and c-Jun translocation from the cytoplasm to the nucleus. Further, IOP downregulated the protein expression of toll-like receptor 2 (TLR2) and TLR4 in T. gondii-infected RAW264.7 macrophages. The above results suggest that IOP can inhibit the inflammatory response infected with T. gondii via regulating TLR2/TLR4-NF-κB/MAPKs pathways and exerting its anti-T. gondii role in vitro.

Taraxacum mongolicum protects against Staphylococcus aureus-infected mastitis by exerting anti-inflammatory role via TLR2-NF-κB/MAPKs pathways in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional Chinese medicine, Taraxacum mongolicum has been widely used for the prevention and treatment of a variety of inflammatory and infectious diseases, and also clinically used as a remedy for mastitis. However, the scientific rationale and mechanism behind its use on mastitis in vivo are still unclear. AIM OF THE STUDY: This study aimed to investigate the protective effect and potential mechanism of Taraxacum mongolicum Hand.-Mazz. (T. mongolicum) on mastitis infected by Staphylococcus aureus (S. aureus). MATERIALS AND METHODS: Female ICR mice were given intragastrically 2.5, 5 and 10 g/kg of T. mongolicum extract twice per day for 6 consecutive days, and infected with S. aureus via teat canal to induce mastitis. Pro-inflammatory cytokine tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) levels were determined by ELISA. Myeloperoxidase (MPO) activity and distribution were measured by reagent kit and immunohistochemistry. Histopathological changes of mammary gland tissues were observed by H&E staining. Toll-like receptor 2 (TLR2) expression, phosphorylations of related proteins in nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways were detected by western blot. RESULTS: T. mongolicum decreased TNF-α, IL-6 and IL-1ß levels, and reduced MPO activity and distribution in sera and mammary glands with S. aureus-infected mastitis. In addition, T. mongolicum effectively attenuated histopathological damages and cell necrosis of mammary gland tissues infected by S. aureus. Moreover, T. mongolicum inhibited the expression of TLR2, and the phosphorylations of inhibitor κBα (IκBα), p65, p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) proteins in mammary glands with S. aureus-infected mastitis. CONCLUSIONS: This study suggests that T. mongolicum protects against S. aureus-infected mastitis by exerting anti-inflammatory role, which is attributed to the inhibition of TLR2-NF-κB/MAPKs pathways.

Protective Effects of Taraxasterol against Ethanol-Induced Liver Injury by Regulating CYP2E1/Nrf2/HO-1 and NF-κB Signaling Pathways in Mice.

Taraxasterol, a pentacyclic-triterpene compound, is one of the main active components isolated from the traditional Chinese medicinal herb Taraxacum. The objective of this study is to evaluate the protective effects of taraxasterol and its possible underlying mechanisms against ethanol-induced liver injury in mice. ICR mice were fed with Lieber-DeCarli diet containing 5% ethanol for 10 d and then challenged with a single dose of 20% ethanol (5 g/kg BW) by intragastric administration. The mice were intragastrically treated daily with taraxasterol (2.5, 5, and 10 mg/kg). Tiopronin was used as a positive control. The liver index was calculated, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in sera were detected. The contents of reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) and the activity of superoxide dismutase (SOD) in the livers were measured. The histopathological changes of liver tissues were observed by hematoxylin and eosin (H&E) staining. The protein expression levels of hepatic cytochrome P450 2E1 (CYP2E1), nuclear factor erythroid 2-related factor 2 (Nrf2), antioxidant protein heme oxygenase-1 (HO-1), and nuclear factor-kappa B (NF-κB) signaling pathway in liver tissues were detected by immunohistochemistry and Western blot methods. Taraxasterol significantly reduced the ethanol-induced increases of liver index, ALT, AST, and TG levels in sera and TG and MDA contents in the livers and hepatic ROS production and suppressed the ethanol-induced decreases of hepatic GSH level and SOD activity. Taraxasterol also significantly inhibited the secretion of proinflammatory cytokines TNF-α and IL-6 induced by ethanol. In addition, taraxasterol improved the liver histopathological changes in mice with ethanol-induced liver injury. Further studies revealed that taraxasterol significantly inhibited the ethanol-induced upregulation of CYP2E1, increased the ethanol-induced downregulation of Nrf2 and HO-1, and inhibited the degradation of inhibitory kappa Bα (IκBα) and the expression level of NF-κB p65 in liver tissues of ethanol-induced mice. These findings suggest that taraxasterol possesses the potential protective effects against ethanol-induced liver injury in mice by exerting antioxidative stress and anti-inflammatory response via CYP2E1/Nrf2/HO-1 and NF-κB signaling pathways.

Immunomodulatory and anti-inflammatory effects of total flavonoids of Astragalus by regulating NF-ΚB and MAPK signalling pathways in RAW 264.7 macrophages.

Astragalus membranaceus Bunge has long been used to improve immune function in traditional Chinese medicine. The total flavonoids of Astragalus (TFA) are the main active components isolated from Astragalus membranaceus Bunge. Our recent study has shown that TFA has in vivo and in vitro immunomodulatory and anti-inflammatory effects; however, its potential mechanisms have not yet been elucidated. The present study aims to confirm the immunomodulatory and anti-inflammatory mechanisms of the action involved. Murine RAW 264.7 macrophages were treated with 10, 25 and 100 µg/ml of TFA. The mRNA expression levels of the tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-1ß, IL-10, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were examined by RT-PCR in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The protein expression levels of iNOS and COX-2, in addition to the phosphorylations of proteins in the mitogen activated protein kinase (MAPK) and nuclear factor (NF)-κB signalling pathways were measured by Western blot in LPS-stimulated RAW 264.7 macrophages. The results showed that TFA significantly inhibited TNF-α, IL-1ß, IL-6, iNOS and COX-2 mRNA levels and increased IL-10 mRNA level in LPS-stimulated RAW 264.7 cells in a dose-dependent manner. Further studies revealed that TFA significantly inhibited iNOS and COX-2 protein levels, the phosphorylations of p38 and JNK in MAPKs pathway and IKKα/ß, IκBα and the expression of nuclear NF-κB p65 in NF-κB pathway in LPS-stimulated RAW 264.7 cells. It suggests that TFA possesses immunomodulatory and anti-inflammatory effects by regulating MAPK and NF-ΚB signalling pathways in RAW 264.7 macrophages.