The protective effect of Veronica ciliata Fisch. Extracts on relieving oxidative stress-induced liver injury via activating AMPK/p62/Nrf2 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Veronica ciliata Fisch. existed in various Tibetan medicine prescriptions, which was recorded to treat liver diseases in the Tibetan medicine roll of Chinese materia medica. HYPOTHESIS/PURPOSE: The current study aimed to examine the effect of active constituents from V.ciliata relieving oxidative stress-mediated liver injury and clarify the underlying mechanism. MATERIALS AND METHODS: tert-Butyl hydroperoxide (BHP) induced liver injury in mice model was established to evaluate the hepatoprotective effect of ethyl acetate extract of V. ciliata (EAFVC). Serum and liver indicators, as well as the histopathological change of liver were examined. Next, the constituents of EAFVC were separated and characterized by high-speed countercurrent chromatography (HSCCC) and Ultra performance liquid chromatography-mass spectrometer (UPLC-MS), respectively. Based on the above, the antioxidant activity of EAFVC and two fractions was evaluated using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2, 2'-azino-bis (3-ethylbenzothiazoli- ne-6-sulfonic acid) (ABTS) free radical scavenging assays. The hepatoprotective activity of EAFVC and its fractions/compounds attenuating ethanol-induced hepatocyte damage in BRL-3A cells was evaluated using the MTT method. The effect of the fraction and compounds with the strongest protective activity on ethanol-induced cytotoxicity, reactive oxygen species (ROS) accumulation, and glutathione (GSH) depletion was investigated. mRNA expression of nuclear factor-E2-related factor 2 (Nrf2) and nuclear factor of κB (NF-κB), as well as their downstream target genes, was determined by RT-qPCR. Finally, the potential mechanism of fraction 1 and luteolin on the AMPK/p62/Nrf2 signal pathway was studied using western blotting. RESULTS: Firstly, EAFVC could relieve liver impairment induced by t-BHP in mice. Next, fraction 1 enriched with polyphenolic compounds and luteolin derived from EAFVC were screened to yield the highest hepatoprotective activity against ethanol-induced hepatocyte damage. Further study demonstrated that fraction 1 and luteolin relieved BRL-3A cells damage by decreasing the aspartate aminotransferase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH) activities, ROS accumulation, as well as the depletion of GSH. Also, we determined that fraction 1 and luteolin suppressed inflammation and apoptosis of BRL-3A cells. The mechanistic studies indicated that fraction 1 could attenuate oxidative stress, inflammation, and apoptosis by activating AMPK phosphorylation, which promotes autophagy associated protein expression (LC3-B, Beclin1 and p62) as well as promote phosphorylation of p62 -dependent autophagic degradation of Keap1, to induce Nrf2 dissociation from Keap1 and translocate to nuclear. Nrf2 in the nuclear activate cytoprotective related genes to exert hepatoprotective function. Finally, we found that luteolin activated the protein expression of p-AMPK, p-p62, p62, Nrf2, and its downstream target genes. CONCLUSIONS: This study clarified that fraction 1 enriched phenolic compounds could attenuate ethanol-induced liver injury in BRL-3A cells via activating AMPK/p62/Nrf2 pathway. Luteolin could serve as the major bioactive component in the therapeutic effect of fraction 1. These active constituents in V. ciliata could be used as the potential drugs targeted activation of AMPK or p62 for relieving oxidative stress-mediated liver disorders.

Phytochemical characterization and hepatoprotective effect of active fragment from Adhatoda vasica Nees. against tert-butyl hydroperoxide induced oxidative impairment via activating AMPK/p62/Nrf2 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Adhatoda vasica Nees., which existed in a large; number of Tibetan medicine prescriptions for hepatopathy, used as an adjuvant to treat liver diseases. HYPOTHESIS/PURPOSE: Oxidative stress is the key player in the development and progression of liver pathogenesis. In recent years, research is increasingly being focused on exploitation of the active components from medicinal plants to combat the liver oxidative injury. In our study, we aimed to screen the active principles from A. vasica and clarify whether they could relieve oxidative damage induced by tert-Butyl hydroperoxide (t-BHP) and its potential mechanism via activating AMPK/p62/Nrf2 pathway. MATERIALS AND METHODS: Ultra performance liquid chromatography (UPLC) was adopted for analysis of chemical composition in the extracts. Furthermore, the antioxidant activity of the fractions was evaluated using DPPH, ABTS and reducing power assay. Along with this, the compounds in this fraction with highest antioxidant activity were analyzed using UPLC-MS. Based on this, the condition for extracting flavonoids of this subfraction was optimized via response surface method. CCK-8 assay was used to detect cell viability. Detection kits were used to measure the activity changes of AST, ALT, LDH and CAT as well as MDA and GSH levels induced by t-BHP. Detection of reactive oxygen species (ROS) production was used DCFH-DA probe. DAPI staining and flow cytometry was used to detect cell apoptosis. In terms of the mechanistic studies, the expression of proteins involved in AMPK/p62/Nrf2 pathway was measured using western blotting. RESULTS: Eventually, 70% ethanol extract from leaf of A. vasica was chosen due to its highest active components compared with other extracts. Further, ethyl acetate fraction derived from 70% ethanol extract in A. vasica (AVEA) possess highest ability for scavenging DPPH and ABTS free radicals as well as strongest reducing power than other fractions. Chemical composition analysis showed that AVEA contained 17 compounds, including 1 quinazoline alkaloid, 12 flavonoid-C-glycosides and 4 flavonoid-O-glycosides. In addition, the conditions (ratio of solid-liquid 1:14, the concentration of ethanol 73%, and the temperature 65 °C) were selected to enrich the flavonoids in AVEA. Furthermore, AVEA could attenuate t-BHP induced hepatocyte damage via increasing the cell viability, restoring abnormal the activities of AST, ALT, LDH and CAT as well as the levels of MDA and GSH. ROS fluorescence intensity was reduced by AVEA. Meanwhile, it could inhibit the cell apoptosis of BRL 3 A cells, as evidenced by restoration of cell morphology and decreasing the number of apoptotic cells. Further mechanistic studies indicated AVEA could promote p-AMPK expression to further induce autophagy adaptor-p62 protein expression, which could autophagic degradation of Keap1, leading to Nrf2 release and translocation into nucleus to induce antioxidant genes (HO-1, NQO-1, GCLC and GCLM) expression. CONCLUSION: In our study, AVEA was first to screen as the active fraction in A. vasica with alkaloids and abundant flavones. Moreover, the fraction potentiates its beneficial aspect by displaying the protective role on relieving t-BHP induced oxidative stress and activating AMPK/p62/Nrf2 pathway. AVEA helps maintain the redox homeostasis of hepatic cells and could be considered as an effective candidate against oxidative stress related liver disorders.

Activation of p62-Keap1-Nrf2 Pathway Protects 6-Hydroxydopamine-Induced Ferroptosis in Dopaminergic Cells.

Parkinson's disease (PD) is a common neurodegenerative disorder primarily caused by the death of dopaminergic neurons in the substantia nigra pars compacta (SNpc). However, the manner of death of dopaminergic neurons remains indistinct. Ferroptosis is a form of cell death involving in the iron-dependent accumulation of glutathione depletion and lipid peroxide. Besides, previous studies indicated that ferroptosis might be involved in the death of dopaminergic neurons. In this study, we aim to explore the protective effect of the p62-Keap1-Nrf2 pathway against 6-hydroxydopamine (6-OHDA)-induced ferroptosis in dopaminergic cells. Firstly, our results demonstrated that 6-OHDA-induced ferroptosis could be observed in vivo zebrafish and in vitro human dopaminergic cell line (SH-SY5Y cells) model. Moreover, ferroptosis induced by 6-OHDA mitigates in SH-SY5Y cells upon ferrostatin-1 (Fer, an inhibitor of ferroptosis) treatment via upregulating the protein expression of glutathione peroxidase 4 (GPX4). Then, we found that high p62/SQSTM1 (p62) expression could protect SH-SY5Y cells against ferroptosis through promoting Nrf2 nuclear transfer and upregulating the expression of the antioxidant protein heme oxygenase-1 (HO-1). Ultimately, high p62 expression activates the Nrf2/HO-1 signaling pathway through binding to Kelch-like ECH-associated protein 1 (Keap1). Collectively, the activation of the p62-Keap1-Nrf2 pathway prevents 6-OHDA-induced ferroptosis in SH-SY5Y cells, targeting this pathway in combination with a pharmacological inhibitor of ferroptosis can be a potential approach for PD therapy.

Phenols fragment of Veronica ciliata Fisch. ameliorate free radical-induced nonalcoholic fatty liver disease by mediating PI3K/Akt signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Veronica ciliata Fisch. is used in numerous of Tibetan medicine prescriptions because of its hepatoprotective effect. AIMS OF THIS STUDY: Here, we aimed to investigate the hepatoprotective effect and mechanism of phenolic fraction (PF) of V. ciliata Fisch. on liver injury induced by free radical. MATERIALS AND METHODS: BRL 3A cells were pre-treated with PF and luteolin (Lut) following tert-butyl hydroperoxide (t-BHP) treatment. The cell viability, lactate dehydrogenase (LDH) levels, reactive oxygen species (ROS) generation, apoptosis, cell cycle and autophagy were analyzed. Apoptotic, inflammatory, and autophagy,- related proteins were analyzed using Western blotting. The combination of molecular docking and drug affinity targeting experiments (DARTS) were first utilized to analysis the target protein of Lut. RESULTS: PF effectively suppressed t-BHP-induced apoptosis caused by mitochondrial dysfunction, which were associated with inhibiting ROS generation. Further investigation indicated that PF significantly suppressed apoptosis, inflammation, and autophagy by regulating the expression of related proteins. The results of molecular docking and drug affinity targeting experiments (DARTS) revealed that PI3K was the target protein of PF and Lut. Further studies have shown that PF relieved liver injury induced by t-BHP via suppressing phosphorylated expression of PI3K. CONCLUSION: Our results indicate that PF effectively protect against hepatotoxicity induced by t-BHP through inhibiting the abnormal activation of PI3K-Akt signaling pathway and highlight the health benefits of PF regarding oxidative stress, proving it to be an important source of bioactive compounds associated with Nonalcoholic fatty liver disease (NAFLD).

Phytochemical composition, isolation and hepatoprotective activity of active fraction from Veronica ciliata against acetaminophen-induced acute liver injury via p62-Keap1-Nrf2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Veronica ciliata Fisch, a traditional Tibetan medicine, used to cure hepatitis and existed in lots of Tibetan medicine prescriptions owing to its hepatoprotective activity. AIMS OF THIS STUDY: In this study, we are aimed to systematically analysis and isolate the chemical constituents of the ethyl acetate fraction from V. ciliata (EAFVC), and test the hepatoprotective effect and mechanism of EAFVC and its compounds on attenuating the liver injury induced by acetaminophen (APAP) in vivo and vitro. MATERIALS AND METHODS: UPLC-PDA-ESI-MS method was established for the analysis of the components in EAFVC, which was further separated using multiple chromatographic techniques. The MS, 1H and 13C NMR were applied to elucidate their structures. UPLC-PDA method was applied for the simultaneous quantification of major compounds of EAFVC. Furthermore, the protective effect of the EAFVC was determined using APAP-induced acute hepatotoxicity in mice and BRL-3A cells model, respectively. In addition, the hepatoprotective activity of two main compounds in EAFVC on relieving APAP-induced liver injury was further evaluated. Finally, we have some concerns about the protective mechanism of EAFVC via enzyme-linked immunosorbent assay (ELISA), reactive oxygen species (ROS) detection, quantitative real-time PCR (qPCR), western blot analysis and molecular docking. RESULTS: Thirteen compounds were successfully identified using UPLC-PDA-ESI-MS for the first time. Meanwhile, other twelve compounds were separated from EAFVC. Eventually, twenty-five compounds were successfully identified from the EAFVC. Among these compounds, fourteen compounds (3, 8, 10, 14-17, 19-25) were separated from V.ciliata for the first time. In addition, UPLC-PDA analysis method was first to establish for simultaneous determination of the main compounds (1, 2, 4, 5, 7, 9, 12). Further assay indicated that the liver injury in mice induced by APAP showed a significant reversal by EAFVC, as evidenced by reducing the activities of liver function enzymes, suppressing the lipid peroxidation as well as increasing the serum total antioxidant capacity (T-AOC) and the activities of antioxidant enzymes. Pathological sections showed that the liver in the high dose has significant improvement in mice. In vitro experiment also showed that EAFVC elevate the viability, inhibiting the activities of liver function enzymes as well as the generation of ROS of BRL-3A cells. In addition, Catalposide and verproside could reverse the low cell viability of BRL-3A cells induced by APAP. The mechanism research in vitro demonstrated that EAFVC could promote the mRNA and protein expression of heme oxygenase-1 (HO-1), NAD(P) H dehydrogenase quinone 1 (NQO-1) and catalytic or modify subunit of glutamate-cysteine ligase (GCLC/GCLCM) via enhancing nuclear factor-E2-related factor 2 (Nrf2) and p62/SQSTM1 (p62) expression in protein level. Molecular docking results demonstrated that catalposide and verproside have strong affinity to the kelch-like ECH-associated protein-1(Keap1) Kelch domain. CONCLUSION: This research is the first to clarify the substance basis of the hepatoprotective activity of the EAFVC and provide the further scientific data for the traditional use of this Tibetan Medicine. EAFVC is valuable to be further investigated as active preparations for application in liver protection via activating p62- Keap1-Nrf2 pathway.