Inhibition of Oxidative Stress and ALOX12 and NF-B Pathways

antioxidants Article Inhibition of Oxidative Stress and ALOX12 and NF-κB Pathways Contribute to the Protective Effect of Baicalein on Carbon Tetrachloride-Induced Acute Liver Injury Chongshan Dai 1,2,* , Hui Li 3, Yang Wang 1,2, Shusheng Tang 1,2, Tony Velkov 4,* and Jianzhong Shen 1,2 1 College of Veterinary Medicine, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China; [email protected] (Y.W.); [email protected] (S.T.); [email protected] (J.S.) 2 Beijing Key Laboratory of Detection Technology for Animal-Derived Food Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China 3 Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Center for Disease Prevention and Control, Beijing 100193, China; [email protected] 4 Department of Pharmacology & Therapeutics, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, VIC 3010, Australia * Correspondence: [email protected] (C.D.); [email protected] (T.V.) Abstract: This study investigates the protective effect of baicalein on carbon tetrachloride (CCl4)-induced acute liver injury and the underlying molecular mechanisms. Mice were orally administrated baicalein at 25 and 100 mg/kg/day for 7 consecutive days or ferrostatin-1 (Fer-1) at 10 mg/kg was i.p. injected in mice at 2 and 24 h prior to CCl4 injection or the vehicle. Our results showed that baicalein or Fer-1 supplementation significantly attenuated CCl4 exposure-induced elevations of serum alanine Citation: Dai, C.; Li, H.; Wang, Y.; aminotransferase and aspartate aminotransferase, and malondialdehyde levels in the liver tissues and Tang, S.; Velkov, T.; Shen, J. Inhibition unregulated glutathione levels. Baicalein treatment inhibited the nuclear factor kappa-B (NF-κB) pathway, of Oxidative Stress and ALOX12 and activated the erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway in liver tissues, and NF-κB Pathways Contribute to the Protective Effect of Baicalein on markedly improved CCl4-induced apoptosis, inflammation and ferroptosis in liver tissues exposed with Carbon Tetrachloride-Induced Acute CCl4. In vitro, baicalein treatment improved CCl4 -induced decreases of cell viabilities and knockdown Liver Injury. Antioxidants 2021, 10, of Nrf2 and arachidonate 12-lipoxygenase (ALOX12) genes partly abolished the protective effect of 976. https://doi.org/10.3390/ baicalein on CCl4 -induced cytotoxicity in HepG2 cells. In conclusion, our results reveal that baicalein antiox10060976 supplementation ameliorates CCl4-induced acute liver injury in mice by upregulating the antioxidant defense pathways and downregulating oxidative stress, apoptosis, inflammation and ferroptosis, which Academic Editors: Anna Maria Fratta involved the activation of Nrf2 pathway and the inhibition of ALOX12 and NF-κB pathways. Pasini and Luciano Cominacini Keywords: baicalein; oxidative stress; ferroptosis; acute liver injury; ALOX12 pathway; Nrf2 pathway Received: 24 April 2021 Accepted: 11 June 2021 Published: 18 June 2021 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Over the past several decades, liver disease has been consistently on the rise, so much published maps and institutional affil- so that it has become one of the leading causes of mortality and morbidity worldwide [1]. iations. Currently, approved drugs for treating liver injury usually have many side-effects and limited efficacy [2], such that to date, safer, effective hepatoprotective drugs remain an unmet medical need. Drugs, chemicals or alcohol-caused acute liver injury are common in most countries and serious liver injury often leads to liver failure, and even death [3]. The pathology of Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. acute liver injury involves necrosis, infiltration of inflammatory cells and apoptosis [4–7]. This article is an open access article Carbon tetrachloride (CCl4) is commonly used as the inducing agent in animal models of distributed under the terms and acute injury liver (e.g., mice, rats or rabbits), which are commonly employed to identify or conditions of the Creative Commons screen for hepatoprotective agents [4–7]. It is well known that CCl4 is metabolized by the Attribution (CC BY) license (https:// liver cytochrome P4502E1, which leads to the production of highly reactive trichloromethyl creativecommons.org/licenses/by/ free radicals and excessive reactive oxygen species (ROS) [8]. It is well known that excessive 4.0/). ROS production damages intracellular macromolecules (e.g., lipids, proteins, and DNA) and Antioxidants 2021, 10, 976. https://doi.org/10.3390/antiox10060976 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, x FOR PEER REVIEW 2 of 18 Antioxidants 2021, 10, 976 the liver cytochrome P4502E1, which leads to the production of highly reactive trichloro-2 of 18 methyl free radicals and excessive reactive oxygen species (ROS) [8]. It is well known that excessive ROS production damages intracellular macromolecules (e.g., lipids, proteins, and DNA) and leads to lipid peroxidation and oxidative stress; the two biochemical hall leads to lipid peroxidation and oxidative stress; the two biochemical hall marks of acute marks of acute liver injury [9–12]. CCl4 exposure induces necrosis, apoptosis and inflam- liver injury [9–12]. CCl exposure induces necrosis, apoptosis and inflammatory responses matory responses in hepatocytes4 via several pathways, including the activation of autoph- in hepatocytes via several pathways, including the activation of autophagy, p53, toll-like agy, p53, toll-like receptor 4 (TLR4), mitogen-activated protein kinase (MAPK), and trans- receptor 4 (TLR4), mitogen-activated protein kinase (MAPK), and transforming growth forming growth factor-β1 (TGF-β1) pathways [13–15]. CCl4 exposure can also up-regulate factor-β1 (TGF-β1) pathways [13–15]. CCl exposure can also up-regulate the nuclear factor- the nuclear factor-kappaB (NF-κB)-mediated4 inflammatory response, which leads to the kappaB (NF-κB)-mediated inflammatory response, which leads to the release and expression release and expression of tumor necrosis factor-alpha (TNF-α), high mobility group box- of tumor necrosis factor-alpha (TNF-α), high mobility group box-1 protein 1 (HMGB1), interleukin-11 protein 1 β(HMGB1(IL-1β),) IL-6,, interleukin and inducible-1β (IL nitric-1β), oxide IL-6, synthaseand inducible (iNOS) nitric [13–16 oxide]. Notably, synthase we have(iNOS previously) [13–16]. shownNotably, that we the have up-regulation previously shown of cellular that anti-oxidant the up-regulation systems of (superoxidecellular anti- dismutaseoxidant systems [SOD], (superoxide and catalase dismutase [CAT] and [SOD production], and catalase of glutathione [CAT] and [GSH]), production ameliorates of glutathione [GSH]), ameliorates CCl4-induced acute liver injury [13,15,17]. Recent studies re- CCl4-induced acute liver injury [13,15,17]. Recent studies revealed that ferroptosis, a form of regulatedvealed that cell ferroptosis, death, may a be form a key of player regulated in acute cell liverdeath, injury. may Ferroptosis be a key player is characterized in acute liver by theinjury. iron-dependent Ferroptosis accumulationis characterized of lipidby the hydroperoxides iron-dependent to accumulation lethal cellular of levels lipid [18 hydrop-]. The up-regulatederoxides to lethal expression cellular of cyclooxygenase-2 levels [18]. The (COX-2),up-regulated increased expression of malondialdehyde of cyclooxygenase (MDA)-2 levels(COX and-2), increased depletion of of malondialdehyde glutathione (GSH) (MDA levels) are levels considered and depletion as the majorof glutathione biomarkers (GSH of) levels are considered as the major biomarkers of ferroptosis [18]. CCl4 exposure-induced ferroptosis [18]. CCl4 exposure-induced acute liver injury involves a similar pathology as well asacute increased liver injury levels ofinvolves monounsaturated a similar pathology and polyunsaturated as well as increased fatty acids levels (PUFA) of monounsatu- [19]. In line withrated these and findings,polyunsaturated ferroptosis fatty inhibitors acids (PUFA (e.g.,) N[19-acetylcysteine]. In line with (NAC)these findings, and deferoxamine ferroptosis (DFO)inhibitors were ( showne.g., N- toacetylcysteine effectively improve (NAC) CCl and4-induced deferoxamine acute liver(DFO injury) were or shown liver fibrosis to effec- in ratstively [20 improve,21]. CCl4-induced acute liver injury or liver fibrosis in rats [20,21]. HerbalHerbal antioxidantsantioxidants areare attractive attractive therapeutic therapeutic alternativesalternatives for for preventing preventing or or treating treating acuteacute liverliver injuryinjury duedue toto theirtheir higherhigher efficiencyefficiency andand lowlow incidenceincidence ofof adverseadverse effectseffects [[22].]. BaicaleinBaicalein (5,6,7-trihydroxy-2-phenylchromen-4-one;(5,6,7-trihydroxy-2-phenylchromen-4-one; FigureFigure1 1),), is is a a nature nature product product from from thethe rootroot ofof Scutellaria baicalensis baicalensis. .It Ithas has many many beneficial beneficial pharmacological pharmacological activities, activities, includ- in- cludinging anti anti-inflammatory,-inflammatory, anti anti-oxidative,-oxidative, anti anti-macrobiotic-macrobiotic and andimmuno immuno-regulatory-regulatory [22]. [ 22Bai-]. Baicaleincalein has has a time a time-honored-honored place place

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