枸杞多糖通过降低氧化应激、保护线粒体功能和调节代谢途径改善犬急性肝损伤

Lycium barbarum polysaccharides ameliorate canine acute liver injury by reducing oxidative stress, protecting mitochondrial function, and regulating metabolic pathways 枸杞多糖通过降低氧化应激、保护线粒体功能和调节代谢途径改善犬急性肝损伤 1 2 first-author https://orcid.org/0000-0002-0685-3237 1 1 3 1 1 1 1 1 corresp Rongsheng SU, srsh168@163.com Rongsheng SU, srsh168@163.com srsh168@163.com https://orcid.org/0000-0003-4574-5076 华南农业大学兽医学院， 中国广州， 510642 华南农业大学兽医学院， 中国广州， 510642 College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China 广州医药研究总院有限公司， 中国广州， 510240 广州医药研究总院有限公司， 中国广州， 510240 Guangzhou General Pharmaceutical Research Institute Co., Ltd., Guangzhou 510240, China Guangzhou General Pharmaceutical Research Institute Co., Ltd., Guangzhou 510240, China 韶关大学生物与农业学院， 中国韶关， 512005 韶关大学生物与农业学院， 中国韶关， 512005 College of Biology and Agriculture, Shaoguan University, Shaoguan 512005, China College of Biology and Agriculture, Shaoguan University, Shaoguan 512005, China The development of acute liver injury can result in liver cirrhosis, liver failure, and even liver cancer, yet there is currently no effective therapy for it. The purpose of this study was to investigate the protective effect and therapeutic mechanism of Lycium barbarum polysaccharides (LBPs) on acute liver injury induced by carbon tetrachloride (CCl 4 ). To create a model of acute liver injury, experimental canines received an intraperitoneal injection of 1 mL/kg of CCl 4 solution. The experimental canines in the therapy group were then fed LBPs (20 mg/kg). CCl 4 -induced liver structural damage, excessive fibrosis, and reduced mitochondrial density were all improved by LBPs, according to microstructure data. By suppressing Kelch-like epichlorohydrin (ECH)-associated protein 1 (Keap1), promoting the production of sequestosome 1 (SQSTM1)/p62, nuclear factor erythroid 2-related factor 2 (Nrf2), and phase II detoxification genes and proteins downstream of Nrf2, and restoring the activity of anti-oxidant enzymes like catalase (CAT), LBPs can restore and increase the antioxidant capacity of liver. To lessen mitochondrial damage, LBPs can also enhance mitochondrial respiration, raise tissue adenosine triphosphate (ATP) levels, and reactivate the respiratory chain complexes I‒V. According to serum metabolomics, the therapeutic impact of LBPs on acute liver damage is accomplished mostly by controlling the pathways to lipid metabolism. 9-Hydroxyoctadecadienoic acid (9-HODE), lysophosphatidylcholine (LysoPC/LPC), and phosphatidylethanolamine (PE) may be potential indicators of acute liver injury. This study confirmed that LBPs, an effective hepatoprotective drug, may cure acute liver injury by lowering oxidative stress, repairing mitochondrial damage, and regulating metabolic pathways. 急性肝损伤的发展可导致肝硬化、肝衰竭，甚至肝癌，但目前尚无有效的治疗方法。本研究旨在探讨枸杞多糖（LBPs）对四氯化碳（CCl 4 ）诱导的急性肝损伤的保护作用及其治疗机制。为了建立急性肝损伤模型，以1 mL/kg的剂量对实验犬进行CCl 4 溶液的腹腔注射。然后给治疗组的实验犬喂食枸杞多糖（20 mg/kg）。微观结果显示，CCl 4 诱导的肝脏结构损伤、过度纤维化和线粒体密度降低都可以通过给予LBPs得到改善。LBPs可以通过抑制Kelch样ECH相关蛋白1（Keap1），促进p62、核因子E2相关因子2（Nrf2）和Nrf2下游的II期解毒基因和蛋白的产生，以及恢复过氧化氢酶（CAT）等抗氧化酶的活性，恢复并增加肝脏的抗氧化能力。LBPs还可以增强线粒体呼吸能力，提高组织ATP水平，以及重新激活呼吸链复合体I–V以减轻线粒体的损伤。血清代谢组学结果显示，LBPs对急性肝损伤的治疗作用主要通过控制脂质代谢途径实现。9-羟基十八碳二烯酸（9-HODE）、溶血磷脂酰胆碱（LPC）和磷脂酰乙醇胺（PE）可能是急性肝损伤的潜在标志物。这项研究证实，LBPs是一种有效的保肝药物，可以通过降低氧化应激、修复线粒体损伤和调节代谢途径来治疗急性肝损伤。 急性肝损伤 氧化应激 线粒体功能障碍 代谢组学 枸杞多糖 Acute liver injury Oxidative stress Mitochondrial dysfunction Metabolomics Lycium barbarum polysaccharides 1 Introduction 1 Liver illness has emerged as a major worldwide health issue, particularly acute liver injury which is associated with a high death rate ( Wu et al., 2010 Wu et al., 2010 ). The acute liver injury model induced by carbon tetrachloride (CCl 4 ) is widely used to investigate potential treatment strategies and find markers for evaluating liver injury ( Zhang et al., 2014 Zhang et al., 2014 ; Guo et al., 2017 Guo et al., 2017 ). Natural substances extracted from plants have been investigated as a reliable and safe method for treating liver disease in recent years. Lycium barbarum polysaccharides (LBPs) are a water-soluble glycoconjugate composed of seven monosaccharides isolated from the L. barbarum berries. The fundamental cause of the substantial pharmacological action of L. barbarum berries is thought to be LBPs, which comprise the principal bioactive component of the berries ( Du et al., 2022 Du et al., 2022 ). LBPs have attracted wide attention because of their potential health promotion. LBPs have pharmacological actions that include anti-tumor, immune system modulation, apoptosis inhibition, DNA damage reduction, and neuroprotection ( Tian et al., 2019 Tian et al., 2019 ). However, the therapeutic impact of LBPs and their precise mechanism of action with regard to acute liver injury are still up for debate. Kelch-like epichlorohydrin-associated protein 1 (Keap1)‍-nuclear factor erythroid 2-related factor 2 (Nrf2)‍-antioxidant response elements (AREs), one of the most significant cell defense systems, is crucial for preserving redox homeostasis in the liver ( Jia et al., 2019 Jia et al., 2019 ). AREs with cytoprotective effect, such as superoxide dismutases (SOD1, SOD2), thioredoxins (Trx1, Trx2), heme oxygenase-1 (HO-1), catalase (CAT), glutathione reductase (GR), glutathione peroxidases (GPX1, GPX4), glucose-6-phosphate dehydrogenase (G6PD), and peroxiredoxin 3 (PRDX3), are regulated by Nrf2. Additionally, Nrf2 controls the synthesis and transcription of various drug metabolic enzymes (DMEs), transporters and cellular reduction equivalents such as glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (NADPH), as well as reactive oxygen inactivating enzymes, which together form an Nrf2-dependent antioxidant defense system ( Ma, 2010 Ma, 2010 ; Chen et al., 2020 Chen et al., 2020 ). Another protein, p62, also known as sequestosome 1 (SQSTM1), has been identified, which can activate Nrf2 by inactivating Keap1 ( Zhao et al., 2021 Zhao et al., 2021 ). When autophagy activity is impaired or oxidative stress damage occurs, p62 binds to Keap1 and transports it to autophagosomes for degradation, promoting the activation of Nrf2 and downstream AREs ( Bellezza et al., 2018 Bellezza et al., 2018 ). It is important to note that since AREs happen to be included in the promoter of p62, p62 is also the transcriptional target of Nrf2 ( Jain et al., 2010 Jain et al., 2010 ). Reactive oxygen species (ROS) are overproduced and released into the cytoplasm when mitochondrial function is compromised. Oxidative phosphorylation (OXPHOS) is hindered, the supply of adenosine triphosphate (ATP) is decreased, and these events all occur simultaneously. DNA, proteins, and lipids are then oxidized, leading to cell death ( Lustgarten et al., 2012 Lustgarten et al., 2012 ; Jardim et al., 2020 Jardim et al., 2020 ). The progression of CCl 4 -induced acute liver injury depends heavily on mitochondrial dysfunction ( Lin et al., 2019 Lin et al., 2019 ). Changes in cell metabolites are crucial for tissue homeostasis and regeneration, and can also be used as an indicator of liver illness ( Caldez et al., 2018 Caldez et al., 2018 ). Metabolomics is often used to obtain a thorough grasp of the causes of various physiological states and aberrant processes since it is a technique for identifying small alterations in biological metabolic pathways ( Johnson et al., 2016 Johnson et al., 2016 ). The information obtained by metabolomics is closer to the meaningful pathological or physiological phenotype, but no metabolomic investigations of the therapeutic impact of LBPs on canine acute liver injury have been published. To sum up, the aim of this study was to confirm the therapeutic action of LBPs, clarify the therapeutic mechanism of LBPs on acute liver injury through oxidative stress, mitochondrial function, and metabolic pathways, and provide theoretical support and references for the future clinical application of LBPs. 2 Materials and methods 1 2.1 Animals 2 At the experimental animal base in South China Agricultural University, a total of 18 healthy mongrel canines (half females, half males), ranging in age from one to two years, were reared under the standard conditions of constant temperature ((25±2) ℃). The kennel was fully cleansed and disinfected before the experiment, and all canines were reared adaptatively for a week, with unlimited access to food and water. All canines were found to be healthy before the trial based on physical, blood routine, and blood chemistry examinations. 2.2 Experimental procedure 2 The 18 mongrel canines were randomly divided into three groups: control group (CG), model group (MG), and treatment group (TG). There were six canines in each group (three females and three males). Autoclaved peanut oil was combined at 1:1 volume ratio with CCl 4 (C805332, Macklin, Shanghai, China) on the first day of the experiment. The canines in the MG and TG were intraperitoneally injected with CCl 4 plus peanut oil at a dosage of 1 mL/kg to provide a model of acute liver injury, while the CG was injected with peanut oil at the same dose. To assess whether the development of the acute liver injury model was effective or not, the blood levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured 24 h later using an automated biochemical analyzer (BS380, Mindray, Shenzhen, China). LBPs (20 mg/kg, this dose refers to the previous description by Xin et al. (2011) Xin et al. (2011) (SP9310, Solarbio, Beijing, China) were dissolved and combined with the necessary amount of distilled water, and the canines in the TG were fed once daily for 14 consecutive days after the model had been established (Fig. S1). After 14 d, all canines were anesthetized by pentobarbital sodium and euthanized, and blood and liver tissue samples were collected for further analysis. Every 3 d, all canines were weighed to ensure accurate dosage and to record statistics. 2.3 Histopathological observations 2 After fixing the liver tissue with 4% (volume fraction) paraformaldehyde for more than 48 h, the liver tissue was dehydrated with 50%, 70%, 80%, 90%, and 100% (volume fraction) alcohol, respectively, and then embedded in paraffin. The embedded liver tissue was cut into 4-‍µm-thick slices by a slicer and fixed on a glass slide treated with 3-aminopropyltriethoxysilane (APE), then stained with hematoxylin and eosin (H&E), and finally observed with an optical microscope (DM1000, Leica, Wetzlar, Germany). 2.4 Western blot analysis 2 The expression of oxidative stress-related proteins was detected by western blotting, carried out according to Liu BX et al. (2021) Liu BX et al. (2021) (supplementary materials and methods). The main antibodies used in this experiment were: p62 (1:2000 (volume ratio, the same below), PA5-27247, Thermo Fisher, Waltham, USA), Keap1 (1:2000, bs-3648R, Bioss, Beijing, China), Nrf2 (1:‍1000, bs-1074R, Bioss), HO-1 (1:‍2000, ab52947, Abcam, Cambridge, USA), Trx1 (1:‍1000, ab109385, Abcam), SOD1 (1:1000, ab13498, Abcam), GPX4 (1:‍2000, BS7323, Biogot, Nanjing, China), 8-hydroxy-2-deoxyguanosine (8-OHDG) (1:‍100 ‍000, NB600-1508, Novus, Littleton, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:‍10 000, AP0063, Biogot), β‍-actin (1:‍10 ‍000, AP0060, Biogot), and histone H3 (1:2000, T56587, Abmart, Shanghai, China). 2.5 ‍ qPCR 2 Quantitative real-time polymerase chain reaction (qPCR) was used to detect the expression levels of genes, as described previously ( Liu BX et al., 2021 Liu BX et al., 2021 ) Detailed procedures are included in the supplementary materials and methods. The sequences of relevant primers for qPCR analysis in this experiment are shown in Table S1. 2.6 Immunohistochemistry 2 The expression levels and localizations of p62, Nrf2, and 4-hydroxynonenal (4-HNE) proteins in cells were detected by immunohistochemistry (supplementary materials and methods). 2.7 Masson staining 2 A modified Masson Trichromatic Staining kit (G1346, Solarbio) was used to evaluate the extent of liver fibrosis in accordance with the manufacturer’s instructions (supplementary materials and methods). The degree of liver fibrosis in each experimental group was assessed in accordance with the collagen fiber area ratio after comparing the collagen fiber area with the total tissue area. 2.8 Electron microscopic observation 2 The liver tissues of specimens trimmed to 1 mm×1 mm×1 mm specification were fixed with 2.5% (volume fraction) glutaraldehyde (pH=7.4, 4 ℃) for 24 h, diluted overnight with phosphate-buffered saline (PBS) at 4 ℃, and then fixed in 1% (volume fraction) samarium tetroxide, dehydrated with ethanol, and finally embedded in epoxy resin. The tissue blocks were trimmed and sliced into 80-nm slices with a slicing machine. The ultrastructure was observed with a transmission electron microscope (TEM; H-7500, Hitachi, Tokyo, Japan). 2.9 Determination of parameters related to oxidative damage 2 The liver tissue stored at -80 ℃ was thawed, reagent was prepared in advance according to the manufacturer’s instructions, and an appropriate amount of tissue was taken to operate on the ice. The G6PD, thioredoxin reductase (TrxR) enzyme activity, hydrogen peroxide (H 2 O 2 ) content (BC1155, BC3595, Solarbio), the CAT, SOD1, SOD2, GPX, GR enzyme activity, and GSH and malondialdehyde (MDA) content (S0051, S0103, S0058, S0055, S0053, S0131S, Beyotime, Shanghai, China) were measured correctly. All the operations in the experiment were carried out in accordance with the kit instructions. 2.10 ‍ Determination of parameters related to mitochondrial function 2 The liver tissue (0.1 g) was weighed precisely. The ATP content was ascertained in accordance with the conditions, procedures, and instructions of the ATP Content Detection Kit (BC0305, Solarbio). The mitochondria of the liver tissue were extracted using the standard steps of differential centrifugation and at 4 ℃ ( Venâncio et al., 2013 Venâncio et al., 2013 ). The isolated mitochondria were lysed before the bicinchoninic acid (BCA) protein concentration determination kit was used to measure the concentration of mitochondrial protein, and diluted to an appropriate level. Spectrophotometry was then used to assess the respiratory chain complexes I‍–‍V activity in the isolated mitochondria in accordance with a published method ( Wang et al., 2017 Wang et al., 2017 ). Citrate synthase (CS) activity was used to adjust the measured complexes I‍–‍V activity for the potential presence of variable amounts of non-mitochondrial protein in various mitochondrial preparations, which was assessed as previously mentioned ( Frazier et al., 2020 Frazier et al., 2020 ). The mitochondrial respiration rate of liver tissue was assessed at room temperature using a mitochondrial respirometer (Oxygraph-2k, Oroboros, Innsbruck, Austria). A total of 10 mg of fresh liver tissue was placed in respiratory medium and ground with a tissue grinder. The mixture was then added to the chamber of the respiratory apparatus to measure the basic oxygen consumption rate of mitochondria and the steady-state oxygen flux obtained by adding specific substrates: 10 mmol/L glutamic acid and 2 mmol/L malic acid were used to evaluate complex I, and 10 µmol/L cytochrome C was used to verify the integrity of liver cell membranes. Totally, 4 mmol/L adenosine diphosphate (ADP) was used to evaluate the phosphorylation process of synthetic ATP. The maximum uncoupling of mitochondria could be obtained by titrating 2 µmol/L carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP), and then 0.5 µmol/L rotenone (complex I inhibitor) and 2.5 µmol/L antimycin A (complex III inhibitor) were added to completely inhibit respiration. Finally, the data were derived and analyzed by DatLab7 software. 2.11 Non-targeted metabolomics detection 2 The main process and data analysis were as previously described ( Qiao et al., 2021 Qiao et al., 2021 ). Detailed procedures are included in the supplementary materials and methods. The selected metabolites were analyzed by multivariate statistical analysis with SIMCA 14.1 (Figs. S2–‍S7). 2.12 Statistical analysis 2 The results are expressed as mean±standard deviation (SD). GraphPad Prism 7.0 ( https://www. https://www. ‍graphpad.com/scientific-software/prism) was used for statistical analysis and image drawing, while Student’s t -test was used to evaluate whether there were significant differences between the two groups. The metabolites were screened and analyzed by SIMCA 14.1. The Human Metabolome Database (HMDB; http://www. http://www. ‍ hmdb.‍ca) was used to identify metabolites, Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg http://www.genome.jp/kegg ) analysis was used to find metabolic pathways, and MetaboAnalyst 5.0 ( http://www.metaboanalyst. http://www.metaboanalyst. ‍ca) was used to enrich pathways and construct heat maps. 3 Results 1 3.1 ‍ Reduction of the liver dysfunction and pathological injury of CCl 4 -induced acute liver injury by LBPs 2 On the days following CCl 4 injection, canine ALT and AST levels in the MG and TG were considerably higher than those in the CG ( Fig. 1 Fig. 1 a). The blood AST and ALT levels of the MG had dropped, but were still high on Day 14 after the model was formed and stable. In contrast, those in the TG were significantly lower than those in the MG ( P < 0.01), and the serum AST levels of the TG had reverted to the normal range. Fig. 1 Fig. 1 b depicts abnormal alterations to the liver tissue. Compared to the CG, the hepatic tissue in the MG showed a significant area of fragmented necrosis, virtually no liver cord structure, hepatocytes with abnormal morphology and fatty degeneration, nuclear pyknosis and dissolution, dilated and clogged hepatic sinuses, and a significant amount of hemoglobin being broken down and converted into hemosiderin adhesion. Although the hepatic cord structure of the TG was obvious and cell morphology was complete, there were still some incomplete hepatocytes with steatosis and congested hepatic sinusoids. The liver ultrastructure is shown in Fig. 1 Fig. 1 c. The density of mitochondria in the MG was substantially lower than that in the CG (there were few mitochondria in a single hepatocyte), and there was vacuolation within the mitochondria as well as partial nuclei that were distorted and cleaved as the nucleolus disintegrated and vanished. Unlike in the MG, the density of mitochondria in the TG was increased, the nucleus and nucleolus were relatively normal, mitochondria were normal in morphology and structure, but the density of mitochondria in the TG was still lower than that in the CG. The level of liver fibrosis in each experimental group is shown in Figs. 1 1 d and 1 1 e. The liver in MG had extensive fibrosis, a high collagen fiber area ratio, and damage to the hepatic cable structure. The degree of fibrosis in the TG was much lower than that in the MG, while the collagen fiber area ratio was nevertheless larger than that in the CG. 3.2 Improvement of the antioxidant capacity of the liver by regulating the p62-Keap1-Nrf2 pathway with LBPs 2 The p62 , Keap1 , and Nrf2 gene transcription levels in the MG were lower than those in the CG ( Fig. 2 Fig. 2 a). In contrast to the MG, the expression of the p62 and Nrf2 genes in the TG was clearly enhanced, whereas the downward trend of Keap1 was consistent with that of the MG. Additionally, there was no discernible difference between the MG and CG in terms of the expression of the protein p62 or Keap1, but there was a clear down-regulation of Nrf2 in both the nucleus and the cytoplasm in the MG. In contrast to the MG and CG, Keap1 and cytoplasmic Nrf2 (Cyto-Nrf2) protein expression levels showed a significant negative trend, while p62 and nuclear Nrf2 (Nucl-Nrf2) in the TG were noticeably up-regulated (Figs. 2 2 b and 2 2 c). The immunohistochemistry analysis of p62 revealed no discernible differences between the MG and CG, except that the TG had a much greater positive expression rate of p62 than that in the CG ( Fig. 2 Fig. 2 d). According to the Nrf2 immunohistochemistry findings, the Nrf2-positive signal was weaker in the MG than in the CG, and the brown signal was primarily found in the cytoplasm, indicating that most Nrf2 did not enter the nucleus, whereas there were a lot of brown and yellow Nrf2-positive signals in the TG liver nucleus, and significantly more than those in the CG ( Fig. 2 Fig. 2 e). 3.3 Promotion of the expression of downstream antioxidant genes and proteins of Nrf2 by LBPs for reduction of oxidative damage 2 SOD1, SOD2, Trx1, Trx2, HO-1, CAT, GR, GPX1, GPX4, G6PD, and PRDX3 have been confirmed to be transcriptional sites of Nrf2 and regulated by Nrf2 ( Myers, 2012 Myers, 2012 ; Shelton and Jaiswal, 2013 Shelton and Jaiswal, 2013 ; Raghunath et al., 2018 Raghunath et al., 2018 ; Tu et al., 2019 Tu et al., 2019 ; Gou et al., 2020 Gou et al., 2020 ). Fig. 3 Fig. 3 illustrates how LBPs affect the expression of downstream antioxidant genes and proteins. According to the findings, all antioxidant genes found in the MG had lower expression levels than those in the CG. However, the expression of genes SOD1 , HO-1 , Trx1 , SOD2 , CAT , PRDX3 , and G6PD increased evidently in the TG, but the expression of the genes GPX4 , Trx2 , GR , and GPX1 was restored to normal levels ( Fig. 3 Fig. 3 a). The information on expression levels of these genes was gathered to create a hierarchical clustering heat map, which more clearly illustrates the inhibition of protective genes by acute liver injury and the repair and promotion of protective genes by LBPs ( Fig. 3 Fig. 3 b). Protein expression analysis revealed that SOD1, SOD2, Trx1, GPX4, and HO-1 expression levels in the MG were lower than those in the CG. GPX4 expression was higher in the TG than in the MG, but was still only at a low level (Figs. 3 3 c and 3 3 d). 3.4 Reduction of oxidative damage by repairing or increasing the activity of antioxidant enzymes and increasing the reserve of reduction equivalent with LBPs 2 Except for CAT activity, the results demonstrated that the antioxidant enzyme activity in the MG was decreased to variable degrees compared to that in the CG, but the CAT results also suggested that not all of the antioxidant systems in the MG had collapsed. The results of the TG showed that LBPs could successfully increase the enzyme activity of G6PD, CAT, and SOD1, and repair the enzyme activity of GR, GPX, and SOD2. However, TrxR activity had a limited potential to be saved ( Fig. 4 Fig. 4 a). The GSH contents of the MG and TG were decreased while that of the TG was still much higher than that of the MG ( Fig. 4 Fig. 4 a). Cytochrome P450 family 2 subfamily E member 1 (CYP2E1) is an efficient generator of ROS. The MG had a considerably greater level of CYP2E1 gene expression than did the CG and TG ( Fig. 4 Fig. 4 b). 8-OHDG is widely used as a biomarker of DNA oxidative damage. The level of 8-OHDG in the MG was clearly higher than that in the CG, suggesting that the DNA oxidative damage was more serious. The amount of 8-OHDG in the TG was much lower than that in the MG, while still being greater than that in the CG. The findings revealed that both the MG and TG had greater H 2 O 2 content than the CG, although the H 2 O 2 concentration in the TG was much lower than that in the MG ( Fig. 4 Fig. 4 c). The MDA level in the MG was substantially greater than that in the CG, indicating that the lipid peroxidation of liver tissue had undergone significant modification ( Fig. 4 Fig. 4 c). A biomarker for oxidative/nitrosative stress is 4-HNE. The brownish-yellow signal of 4-HNE protein expression in the MG was substantially greater than that in the CG and TG. The expression signal of 4-HNE in the TG was much lower than that in the MG, despite the brownish-yellow signal still being greater than that in the CG ( Fig. 4 Fig. 4 d). 3.5 Improvement of the respiratory function of damaged mitochondria and increase of the content of tissue ATP by LBPs 2 The oxygen consumption rate (OCR, also known as the respiratory rate or oxygen flux) as it relates to complex I is shown in Fig. 5 Fig. 5 a. The overall OCR of the MG was obviously lower than that of the CG. Compared with the CG, the routine respiration, spare respiratory capacity, maximal respiration, and respiratory control ratio of the MG were impaired, while in the TG, the routine respiration and mitochondrial respiratory control ratio were restored to the normal level. The maximal respiration and spare respiratory capacity had not completely recovered, but had improved ( Fig. 5 Fig. 5 b). The results of ATP revealed that the OXPHOS process of the MG was compromised and that its ATP content was reduced, whereas the ATP content of the TG appeared to have increased and exceeded that of the CG ( Fig. 5 Fig. 5 a). The consumption of mitochondrial respiratory substrates (nicotinamide adenine dinucleotide (NADH) and flavine adenine dinucleotide, reduced (FADH2)) and the effectiveness of electron transport are represented by the activity of the respiratory chain complex. Compared with the CG, the CS and complexes I‍–‍V activity of the MG was damaged to variable degrees, and tricarboxylic acid cycle and proton transport were disturbed. In the interim, complexes I, III, and IV activity matched those of the CG, and complex V activity was even enhanced, with the exception of CS and complex II activity, which did not recover to the usual level in the TG. However, CS and complex II activity was still much better than those of the MG ( Fig. 5 Fig. 5 c). 3.6 Active regulation of the metabolic pathways of acute liver injury canines by LBPs 2 A total of 141 different metabolites that were enriched by MetbraAnalyst 5.0 were tested. A total of 10 metabolic pathways were involved in the process of liver injury caused by CCl 4 and the therapeutic action of LBPs, according to a path enrichment analysis ( Fig. 6 Fig. 6 a). Glycerophospholipid metabolism, linoleic acid metabolism, α‍-linolenic acid metabolism, glycosylphosphatidylinositol (GPI)‍-anchor biosynthesis, and arachidonic acid metabolism were the five metabolic pathways with the greatest pathway effect value. The cluster heat map was created by MetbraAnalyst 5.0 using the first 38 metabolites with the greatest variable important in projection (VIP) value ( P < 0.05). The findings demonstrated that these metabolites significantly varied amongst the three experimental groups ( Fig. 6 Fig. 6 b). The expression levels of the metabolites in the TG and MG were compared to those of the CG, and the log 2 (fold change) value for each metabolite was then calculated. After that, the 12 metabolites with the biggest variation in expression between the TG and MG were chosen ( Table 1 Table 1 ). The findings demonstrated that eight metabolite levels declined in the TG but increased in the MG most significantly, whereas four increased in the TG but reduced in the MG to a greater extent. HMDB: the Human Metabolome Database; ID: identity; CG: control group; MG: model group; TG: treatment group; LysoPC: lysophosphatidylcholine; PE: phosphatidylethanolamine; 9-HODE: 9-hydroxyoctadecadienoic acid. 2 HMDB ID 2 Metabolite 2 log 2 (fold change) MG TG HMDB0034757 4-Methoxybenzene propanol 1-(2-sulfoglucoside) -1.749 ‍465 ‍525 2.063 ‍044 ‍499 HMDB0010402 LysoPC (22:5 (4Z, 7Z, 10Z, 13Z, 16Z)) -1.872 ‍372 ‍824 1.770 ‍261 ‍707 HMDB0061883 Caproate (6:0) -1.972 ‍595 ‍645 1.349 ‍482 ‍079 HMDB0010393 LysoPC (20:3 (5Z, 8Z, 11Z)) -1.648 ‍135 ‍217 1.085 ‍991 ‍362 HMDB0042040 Tipredane 1.327 ‍642 ‍219 -1.113 ‍751 ‍673 HMDB0009185 PE (18:4 (6Z, 9Z, 12Z, 15Z)/14:1 (9Z)) 1.224 ‍684 ‍429 -2.182 ‍379 ‍043 HMDB0032844 4,5-Dihydroniveusin A 1.249 ‍274 ‍922 -2.531 ‍588 ‍018 HMDB0009181 PE (18:3 (9Z, 12Z, 15Z)/P-18:0) 1.856 ‍621 ‍306 -2.619 ‍365 ‍474 HMDB0010223 9-HODE 1.335 ‍135 ‍457 -2.812 ‍219 ‍681 HMDB0030767 11,12-Dimethoxydihydrokawain 2.319 ‍154 ‍115 -2.999 ‍131 ‍712 HMDB0002429 (3a,5b)-24-Oxo-24-[(2-sulfoethyl)amino]cholan-3-yl-b-D-glucopyranosiduronic acid 3.639 ‍143 ‍103 -3.053 ‍959 ‍516 HMDB0038158 Jangomolide 1.399 493 203 -3.095 ‍003 ‍637 4 Discussion 1 Although numerous hepatoprotective medications have been researched and used in clinical settings, some may have negative side effects ( Liu et al., 2014 Liu et al., 2014 ). Finding “liver protecting agents” that are efficient and safe for both animals and humans is thus important. According to the findings of the experiments, LBPs can prevent oxidative damage by decreasing the expression of Keap1, increasing the expression of p62, and activating Nrf2. The increased expression levels of p62 and Nucl-Nrf2 represent an increase in cell survival rate ( Lewis et al., 2015 Lewis et al., 2015 ; Haga et al., 2017 Haga et al., 2017 ), which may be one of the key mechanisms by which LBPs lessen hepatocyte damage and boost hepatocyte lifespan. Some antioxidant genes, such as Trx1 , GPX2 , and nicotinamide adenine dinucleotide phosphate (NAD(P)H):‍quinone oxidoreductase 1 ( NQO1 ), are highly dependent on the regulation of Nrf2 ( Xu et al., 2019 Xu et al., 2019 ). As a result, these antioxidant genes and proteins in the TG were expressed at levels higher than those of the CG due to the increased expression of Nrf2. Even while certain antioxidant genes were up-regulated following LBP treatment, they were still suppressed compared to those in the CG. This may be because these genes are either not sensitive to Nrf2 or are only partially dependent on Nrf2, and are controlled by other genes. The main reaction substrate for GPX4 is GSH. Toxic peroxides may be converted by GPX4 into non-toxic hydroxyl compounds using two equivalents of GSH ( Liu WW et al., 2021 Liu WW et al., 2021 ). To counter the ongoing creation of harmful peroxides, many of the GPX4 proteins that have been translated interact with GSH. This may explain the regular expression of the GPX4 gene, but reduced protein expression in the TG, as well as the decrease in GSH concentration. According to the relevant research, Nrf2-mediated genes must control liver functions such as GSH synthesis, binding, detoxification, and transport ( Ma, 2010 Ma, 2010 ; Chen et al., 2020 Chen et al., 2020 ). If Nrf2 transcriptional activity decreases, GSH production will be lost ( Jin et al., 2019 Jin et al., 2019 ). Therefore, the partial recovery of GSH stores in the TG may be connected to Nrf2 activation by LBPs. SOD2, PRDX3, and Trx2 regulated by Nrf2 are specifically located in mitochondria ( Sun et al., 2015 Sun et al., 2015 ; Gao et al., 2018 Gao et al., 2018 ; Hu et al., 2018 Hu et al., 2018 ). After activation by mitochondrial deacetylase sirtuin 3 (Sirt3), SOD2 converts locally produced free radicals into H 2 O 2 ( Sharma et al., 2020 Sharma et al., 2020 ), while Trx2 can provide electrons for PRDX3 to scavenge H 2 O 2 from SOD2 conversion ( Chen et al., 2019 Chen et al., 2019 ). Therefore, part of the reason for the decrease of H 2 O 2 content in the TG compared with that in the MG may be that LBPs stimulate the expression of SOD2 , PRDX3 , and Trx2 genes through Nrf2, while one of the important reasons for the high H 2 O 2 content in the TG may be that LBPs did not fully restore the activity of TrxR. Therefore, through restoring and boosting Nrf2 expression in injured hepatocytes, LBPs are expected to partially repair mitochondrial redox equilibrium. The reduction of routine respiration corresponds to a decrease in cell quantity ( Kandel et al., 2016 Kandel et al., 2016 ). The finding of H & E staining in the MG indicated that a significant number of damaged hepatocytes help to corroborate this theory. Although LBPs still partially repaired this damaged feature, the spare respiratory capacity of both the TG and MG was compromised, indicating that both groups of hepatocytes were less able to handle the abrupt rise in ATP demand. The decline in maximum respiratory rate is a significant sign of possible mitochondrial malfunction ( Brand and Nicholls, 2011 Brand and Nicholls, 2011 ). Although symptoms in the TG were less severe, the reduction in maximum respiratory rates of the MG and TG suggested that mitochondrial malfunction existed. The respiratory control ratio represents the efficiency of OXPHOS of mitochondria. The findings demonstrated that LBPs may effectively repair the OXPHOS system of mitochondria. OXPHOS is closely related to the formation of ATP. When the respiratory control ratio of the TG was not significantly enhanced, the ATP level of the TG was increased, and the enhanced activity of ATP synthase may be the reason for this phenomenon. The rise in ATP levels demonstrated the increased energy need of TG hepatocytes. We speculate that LBPs raise the ATP level to provide hepatocytes more energy to cope with their own damage. The activity of CS is regarded as a significant biomarker that reflects the density of mitochondria ( Larsen et al., 2012 Larsen et al., 2012 ), and the results of electron microscopic observation correspond to the results of CS activity. Ralph et al. (2011) Ralph et al. (2011) made the observation that the functional impairment of complex II might lead to the production of ROS and the accumulation of succinate in cells. We thus hypothesize that the imbalance in redox homeostasis of the MG and TG may be caused by the impairment of the activity of complex II (such as a result of the increase in H 2 O 2 content). The outcomes of pathway enrichment demonstrated the critical role that lipid metabolism plays in the management of CCl 4 -induced acute liver injury by LBPs. The results of this experiment were congruent with serum metabolomics research on liver injury, which found that serum lysophosphatidylcholine (LysoPC/LPC) was reduced in the liver injury group ( Ma et al., 2014 Ma et al., 2014 ). The cause of the drop-in serum LPC levels is unknown. According to the study by Hinkovska-Galcheva et al. (2021) Hinkovska-Galcheva et al. (2021) , several medications can suppress lysosomal enzymes like phospholipase A2, which causes phospholipids to build up in lysosomes. At the same time, the secretion of LPC from the liver is quantitatively important for plasma LPC levels ( Balogun et al., 2013 Balogun et al., 2013 ). Therefore, some scholars suggested that the inhibition of hepatic phospholipase A2 might result in a decrease in the levels of LPC in the plasma ( Saito et al., 2014 Saito et al., 2014 ), which may be the cause of the decrease of serum LPC observed in the MG. Phosphatidylethanolamine (PE) is a fundamental component of mammalian biological membranes and is involved in a multitude of cell physiological functions ( Vance, 2015 Vance, 2015 ). PE can also trigger OXPHOS ( Calzada et al., 2019 Calzada et al., 2019 ), which is crucial for the healthy operation of mitochondria. In disease processes, exposure to PE has been used as a biomarker of cell death ( Elvas et al., 2017 Elvas et al., 2017 ). PE is exposed and released into the blood when a significant number of hepatocytes die, leading to an increase of PE in the blood of the MG, which is compatible with the concept put forward in the research by Gonzalez et al. (2012) Gonzalez et al. (2012) . A ligand for the G protein-coupled receptor G2A (GPR132), 9-hydroxyoctadecadienoic acid (9-HODE), is a cyclooxygenase product of linoleic acid ( Osthues et al., 2020 Osthues et al., 2020 ). Under oxidative stress, 9-HODE produces and activates G2A through a non-enzymatic reaction, which activates the mitogen-activated protein kinase (MAPK) pathway and mediates intracellular calcium mobilization to promote the inflammatory process ( Rolin et al., 2013 Rolin et al., 2013 ). The level of serum 9-HODE dramatically rises during the time of liver fibrosis and inflammation caused by illness, according to previous research that has established a positive association between the concentrations of 9-HODE in the liver and serum ( Maciejewska et al., 2020 Maciejewska et al., 2020 ). Therefore, the high levels of oxidative stress, inflammation, and potential liver fibrosis may be indicated by the elevated 9-HODE in the MG. The study by Zhang et al. (2021) Zhang et al. (2021) indicated that G2A functions as a proton-sensing G protein-coupled receptor, while LPC is an antagonist for this receptor that controls such proton-dependent G2A activation. The therapeutic mechanism of LBPs on acute liver injury may be as follows: relieving the inhibition of hepatic phospholipase A2, increasing the LPC level, inhibiting G2A, and reducing the 9-HODE level, thereby reducing the level of inflammation and oxidative stress in liver tissue, intervening in cell death pathways such as iron death or apoptosis, lowering hepatocyte mortality, promoting the entry of PE in blood into the liver as raw materials for hepatocyte and mitochondrial regeneration, and then ultimately reducing liver injury. Three metabolites are undoubtedly candidates for use as blood indicators of acute liver injury. This study validated the protective effect of LBPs on the liver and revealed the therapeutic mechanism of LBPs on acute liver injury. However, note that the treatment of liver injury by LBPs is not comprehensive. To enhance the therapeutic effects, LBP therapy of acute liver injury patients should be supplemented with medications targeting mitochondria (such as mitoquinone (MitoQ)) or other hepatoprotective agents (such as glucuronolactone). There were some shortcomings in our study: we did not set up an LBP control group and could not exclude some positive or negative effects of LBPs on the body of healthy canines. We will continue to conduct LBP-related experiments in canines to gain more insight into the effects of LBPs on the organism. 5 Conclusions 1 Overall, the therapeutic effect of LBPs on acute liver injury was supported: LBPs can eliminate excessive oxidants through the p62-Keap1-Nrf2 pathway, reduce oxidative damage, and improve the dysfunction of mitochondria. Lipid metabolism is the primary metabolic pathway involved in both the development of acute liver injury and the therapeutic role of LBPs. Additionally, three key possible biomarkers are LPC, PE, and 9-HODE. 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All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data. This work was supported by the Science and Technology Project of Shaoguan Science and Technology Bureau (No. 200811104530939). In addition, we would like to thank Na QIAO and Hanming CHEN from the Veterinary Medicine of South China Agricultural University for their help in the experiment and professional advice in the process of writing this manuscript. 10.1631/jzus.B2200213 1673-1581（2023）02-0157-15 黄健佳,白雨曼,谢文婷等.枸杞多糖通过降低氧化应激、保护线粒体功能和调节代谢途径改善犬急性肝损伤[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(02):157-171. 黄健佳,白雨曼,谢文婷等.枸杞多糖通过降低氧化应激、保护线粒体功能和调节代谢途径改善犬急性肝损伤[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2023,24(02):157-171. This work was supported by the Science and Technology Project of Shaoguan Science and Technology Bureau (No. 200811104530939). 2022-04-11 2022-09-25 2023-02-15 2023-02-09 2022-12-19 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2023 浙江大学出版社 Jianjia HUANG, Yuman BAI, Wenting XIE, Rongmei WANG, Wenyue QIU, Shuilian ZHOU, Zhaoxin TANG, Jianzhao LIAO, and Rongsheng SU declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. All experimental procedures and animal care were carried out in accordance with pertinent guidelines and regulations approved by the Institutional Animal Care and Use Committee of South China Agricultural University (No. 2020A132). Compliance with ethics guidelines Supplementary information 1 Table S1; Figs. S1–S7; Materials and methods 浙江大学学报（英文版）（B辑：生物医学和生物技术） 2 24