Integrating metabolomics and bioinformatics to reveal the mechanism of Epimedium-induced liver injury.

Epimedium is a traditional Chinese medicine with a wide range of clinical applications; however, there have been numerous reports of adverse reactions in recent years. The most common side effect of Epimedium is liver injury. In this study, the liquid chromatography-mass spectrometry (LC-MS) method has been established to study the components of Epimedium and to identify the components absorbed into the blood of rats. Bioinformatics was used to screen out potential toxic components, and the integrating metabolomics method was used to explore the molecular mechanism of Epimedium-induced liver injury. The chemical constituents of Epimedium were identified by LC-MS, and 62 compounds were obtained, including 57 flavonoids, four organic acids and one alkaloid. The toxicity network of "Epimedium-component-target-liver injury" was constructed using bioinformatics research methods, and then the key hepatotoxic component icaritin was identified. Integrating metabolomics was used to investigate the changes in the metabolic profile of L-02 cells with different durations of icaritin administration compared with the control group, and 106 different metabolites were obtained. A total of 14 potential biomarkers significantly associated with cell survival were screened by Pearson correlation analysis combined with the L-02 cell survival rate. Our study preliminarily revealed the mechanism of hepatotoxicity induced by Epimedium.

Protective Effect of Selenium-enriched Peptide from Cardamine violifolia on Ethanol-induced L-02 Hepatocyte Injury.

In this study, we investigated the protective effect of selenium (Se)-enriched peptide isolated from Cardamine violifolia (SPE) against ethanol-induced liver injury. Cell proliferation assays show that different concentrations of SPE protect human embryonic liver L-02 cells against ethanol-induced injury in a dose-dependent manner. Treatment with 12 µmol/L Se increases the cell survival rate (82.44%) and reduces the release of alanine aminotransferase, aspartate transaminase, lactate dehydrogenase, and apoptosis rate. SPE treatment with 12 µmol/L Se effectively reduces the concentration of intracellular reactive oxygen species and increases the contents of intracellular superoxide dismutase (51.64 U/mg), catalase (4.41 U/mg), glutathione peroxidase (1205.28 nmol/g), and glutathione (66.67 µmol/g), thereby inhibiting the effect of ethanol-induced oxidative damage. The results of the transcriptomic analysis show that the glutathione metabolism and apoptotic pathway play significant roles in the protection of L-02 hepatocytes by SPE. Real-time qPCR analysis shows that SPE increases the mRNA expression of GPX1 and NGFR. The results of this study highlight the protective effects of SPE against ethanol-induced liver injury.

[Effect of high selenium on insulin signaling pathway PI3K-AKT-mTOR in L02 cells].

OBJECTIVE: To observe the effect of high selenium on insulin signaling pathway PI3K-AKT-mTOR in L02 cells. METHODS: One group of L02 cell was treated with different concentrations of selenomethionine(SeMet, 0.001, 0.0025, 0.005, 0.0075, 0.01, 0.025, 0.05, 0.075 and 0.1µmol/L) for 48 h, then cultured with serum-free medium for 4 h and stimulated with 1 µmol/L insulin for 15 min. The insulin signaling pathway(PI3K-AKT-mTOR) was detected by WB. Another group of L02 cell was treated with the same concentrations of SeMet as above for 48 h. The cell supernatant and lysates were collected for the analysis of SELENOP and GPX1, respectively by WB. RESULTS: The expressions of P-AKT-(Ser-473), P-AKT-(Thr-308), PI3K and mTOR in L02 cells under high-Se were decreased with the increase of SeMet concentration. The expressions of GPX1 and SELENOP were enhanced with the increase of SeMet. CONCLUSION: The insulin signaling pathway, PI3K-AKT-mTOR, was damaged in L02 cell under high-Se stress.

Pharmacological Activation of AMPK Prevents Drp1-mediated Mitochondrial Fission and Alleviates Hepatic Steatosis In vitro.

BACKGROUND: The incidence of non-alcoholic fatty liver disease (NAFLD) is increasing worldwide. Adenosine monophosphate-activated protein kinase (AMPK) activation is beneficial for NAFLD treatment. Recent studies show the excessive fission of mitochondria during NAFLD progression, so targeting mitochondria dynamics may be a possible target for NAFLD. Still, little is known about whether AMPK regulates mitochondrial dynamics in hepar. OBJECTIVE: This study investigated whether AMPK activation alleviates hepatic steatosis by regulating mitochondrial dynamics mediated by GTPase dynamin-related protein 1 (Drp1). METHODS: Human hepatocyte line L-02 cells were cultured and subjected to palmitic acid (PA) treatment for 24 h to establish a hepatic steatosis model in vitro, which was pre-treated with different tool drugs. Hepatocyte function, hepatocyte lipid content, mitochondrial reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) were examined. The expression levels of genes and proteins associated with mitochondrial dynamics were assessed using reverse transcription-quantitative PCR and western blotting. RESULTS: The results indicated that 5-Aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR), an AMPK activator, improved hepatocyte function, as demonstrated by decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity (P＜0.05 or P＜0.01). In addition, AICAR decreased total cholesterol (TC) and triglyceride (TG) content and lipid deposition in hepatocytes (P＜0.01); decreased ROS production; improved MMP (P＜0.01); reduced fission-1 (Fis1) and mitochondrial fission factor (Mff) mRNA expression; and downregulated p-Drp1 (Ser 616) protein expression. In contrast, AICAR increased mitochondrial fusion factor mitofusin-1 (Mfn1) and mitofusin-2 (Mfn2) mRNA expression and upregulated p-Drp1 (Ser 637) protein expression. Mdivi-1, a Drp-1 inhibitor, was used to confirm whether mitochondrial dynamics regulated by Drp1-mediated the role of AICAR. Similar to AICAR, Mdivi-1 improved hepatocyte function and MMP significantly, decreased ROS production and lipid deposition, downregulated Fis1 and Mff mRNA expression, downregulated p-Drp1 (Ser 616) protein expression, and enhanced Mfn1 and Mfn2 mRNA and p-Drp1 (Ser 637) protein expression. However, Compound C, an AMPKspecific inhibitor, had less impact on the protective effect of Mdivi-1. CONCLUSION: The results demonstrated that AMPK activation has a protective effect on hepatic steatosis in vitro, largely dependent on the inhibition of Drp1-mediated mitochondrial fission.

Hepatic effects of acetochlor chiral isomers in zebrafish and L02 cells.

The pesticide acetochlor (ACT) is a chiral isomer commonly detected in the global environment, yet its specific impacts on liver function remain poorly understood. We utilized zebrafish and L02 cells as research models to comprehensively investigate how ACT and its chiral isomers affect the liver. Our investigations unveiled that the R, Rac, and S isomers of ACT disrupt hepatic lipid transport, catabolism, and synthesis, leading to delayed yolk sac absorption and the accumulation of lipids in zebrafish embryos. These isomers induce oxidative stress in the liver of zebrafish embryos, reducing antioxidant levels and enzyme activity. The accumulated lipids in the liver render it susceptible to oxidative stress, further exacerbating hepatocyte damage. Hepatocyte damage manifests as extensive vacuolization of liver cells and alterations in liver morphology, which are induced by R, Rac, and S. Furthermore, we elucidated the molecular mechanisms underpinning the disturbance of hepatic lipid metabolism by R, Rac, and S in L02 cells. These compounds stimulate lipid synthesis through the upregulation of the AMPK/SREBP-1c/FAS pathway while inhibiting lipolysis via downregulation of the PPAR-α/CPT-1a pathway. Remarkably, our results highlight that S exhibits significantly higher hepatotoxicity in comparison to R. This study provides valuable insights into the hepatic effects of ACT chiral isomers.

[Optimization of three-dimensional culture conditions of L02 cells with response surface methodology based on VitroGel system].

OBJECTIVE: This study optimizes three-dimensional(3D)culture conditions of L02 cells using response surface methodology(RSM) based on the VitroGel system to construct the hepatocytes model in vitro. METHODS: L02 cells were 3D cultured by the VitroGel system. The appropriate level of three key factors(concentration of inoculated cells, culture time and dilution degree of the hydrogel) was determined by single-factor experiment, and the optimal conditions of 3D culture of L02 cells based on the VitroGel system were determined by RSM. During the detection process, the optical density(OD) value of cell viability was used as the detection index, and the cell viability was detected using the cell counting kit-8(CCK-8) assay. The proliferative performance and viability of L02 cells was measured by fluorescent staining assay. RESULTS: The selected optimal culture conditions by RSM were as follows: concentration of inoculated cells was 1.1 × 10~5/mL, culture time was 9.5 days, and dilution degree of hydrogel was 1∶3.7. The result shows that under optimal conditions, the predicted OD value of cell viability was 2.17 and measured 2.13 with a relative error of 1.84%, indicating that the condition was suitable and reliable. The fluorescent staining and dead and live cells detection results showed the 3D hepatocytes model was successfully constructed. CONCLUSION: The optimal conditions for 3D culture of L02 cell based on the VitroGel system were determined by RSM, and a hepatocytes model with high cellular activity was successfully constructed.

Folic Acid Protects against Ethanol-Induced Hepatic Mitophagy Imbalance by ROS Scavenging and Attenuating the Elevated Hcy Levels.

Ample evidence indicates that ethanol-induced oxidative stress and mitochondrial dysfunction are central to the pathogenesis of alcoholic liver disease (ALD). As an adaptive quality control mechanism, mitophagy removes dysfunctional mitochondria to avert hepatic lesions in ALD. Folic acid exhibits potential radical scavenging properties and has been proven to ameliorate mitochondrial disorder in oxidative stress-related diseases. In this study, we aimed to uncover the mitophagy regulatory effects of folic acid in a 10w alcohol C57BL/6J mice feeding model (56% v/v) and L02 cells model cultured with ethanol (2.5% v/v). The results showed that folic acid alleviates ethanol-induced liver injury, decreasing oxidative stress and restoring liver enzyme. Furthermore, folic acid improved the mitochondrial function and inhibited ethanol-activated mitophagy through decreasing PINK1-Parkin and Drp1 expression, which inhibited the release of mitochondrial cytochrome C to the cytoplasm, preventing hepatocyte apoptosis. Intriguingly, folic acid attenuates the elevated hepatic homocysteine (Hcy) level. Additionally, the pretreatment of L02 cells with folic acid also ameliorated Hcy-induced oxidative damage, mitochondrial fission, and mitophagy. In summary, these results suggest that folic acid has beneficial effects in mitophagy remodeling by ROS scavenging and facilitating Hcy metabolism and could be developed as a potential therapeutic agent against ALD.

The Preventive Mechanism of Anserine on Tert-Butyl Hydroperoxide-Induced Liver Injury in L-02 Cells via Regulating the Keap1-Nrf2 and JNK-Caspase-3 Signaling Pathways.

Anserine is a naturally occurring histidine dipeptide with significant antioxidant activities. This study aimed to investigate the preventive mechanism of anserine on tert-butyl hydroperoxide (TBHP)-induced liver damage in a normal human liver cell line (L-02 cells). The L-02 cells were pretreated with anserine (10, 20, and 40 mmol/L) and then induced with 400 µmol/L of TBHP for 4 h. The results showed that the survival rates of L-02 cells and the contents of GSH were significantly increased with the pretreatment of anserine; the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the extracellular fluid were sharply decreased; and the formation of reactive oxygen species (ROS), nuclear fragmentation, and apoptosis were significantly inhibited. In addition, anserine could bind to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) with a binding force of -7.2 kcal/mol; the protein expressions of nuclear factor-erythroid 2-related factor-2 (Nrf2), quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and Bcl-2 were upregulated by anserine in TBHP-induced L-02 cells, with the downregulation of p-JNK and caspase-3. In conclusion, anserine might alleviated liver injury in L-02 cells via regulating related proteins in the Keap1-Nrf2 and JNK-Caspase-3 signaling pathways.

α-amanitin induces autophagy through AMPK-mTOR-ULK1 signaling pathway in hepatocytes.

Amanitin poisoning is one of the most life-threatening mushroom poisonings. α-Amanitin plays a key role in Amanita phalloides intoxication. α-Amanitin shows toxic effects on the liver. However, the mechanism by which α-amanitin induces liver injury has not been elucidated. Autophagy plays a crucial role in maintaining cellular homeostasis and is closely related to the occurrence of a variety of diseases. Studies have shown that autophagy may play an important role in the process of α-amanitin-induced liver injury. However, the mechanism of α-amanitin-induced autophagy remains unclear. Thus, this study aimed to explore the mechanisms of α-amanitin in inducing hepatotoxicity in Sprague Dawley (SD) rats and the normal human liver cell line L02 cells. The SD rats and L02 cells exposed to α-amanitin were observed to determine whether α-amanitin could induce the autophagy of rat liver and L02 cells. The regulatory relationship between autophagy and the AMPK-mTOR-ULK pathway by exposing the autophagy agonist (rapamycin (RAPA)), autophagy inhibitor (3-methylademine (3-MA)), and AMPK inhibitor (compound C) was also explored. Autophagy-related proteins and AMPK-mTOR-ULK pathway-related proteins were detected using Western blot. The results of the study indicated that exposure to different concentrations of α-amanitin led to morphological changes in liver cells and significantly elevated levels of ALT and AST in the serum of SD rats. Additionally, the expression levels of LC3-II, Beclin-1, ATG5, ATG7, AMPK, p-AMPK, mTOR, p-mTOR, and ULK1 were significantly increased in the rat liver. And we found that L02 cells exposed to 0.5 µM α-amanitin for 6 h significantly induced autophagy and activated the AMPK-mTOR-ULK1 pathway. Pretreated with RAPA, 3-MA, and compound C for 1 h, the expression levels of autophagy-related proteins and AMPK-mTOR-ULK pathway-related proteins significantly changed. Our results indicates that autophagy and the AMPK-mTOR-ULK pathway are involved in the process of α-amanitin-induced liver injury. This study may foster the identification of actionable therapeutic targets for A. phalloides intoxication.

[Intervention study to inhibit the glucose metabolic remodeling in hepatocytes induced by high-selenium in vitro].

OBJECTIVE: To observe the effect of exogenous serine or glycine on the synthesis of selenoprotein and endogenous serine and the expression of metabolic enzymes in hepatocytes cultured with high-selenium in vitro and its dose-response relationship. METHODS: The experiment was divided into two parts, namely a inhibition experiment and a dose-response experiment, using L02 cells as the intervention target. In the inhibition experiment, the blank control group, high-Se(SeMet) group, serine intervention group and high-Se+serine intervention group were set up. Both SeMet and serine were given at a level of 0.05 µmol/L, and the blank control group was given the same volumes of saline. In the dose-response experiment, the concentration of SeMet was 0.05 µmol/L, and the intervention concentration gradients of serine or glycine were 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 and 500 µmol/L. The expression of phosphoglycerate dehydrogenase(PHGDH)、serine hydroxymethyltransferase 1(SHMT1)、methylenetetrahydrofolate reductase(MTHFR)、selenoprotein P(SELENOP) and glutathione peroxidase 1(GPX1)was detected by Western Blot(WB). RESULTS: (1)In the inhibition experiment, compared with the blank control group, the expression of selenium proteins(GPX1 and SELENOP) in L02 cells of the other three groups were significantly increased(P<0.05). Compared with the high expression of PHGDH in L02 cells of high-Se group, the expressions of PHGDH, SHMT1 and MTHFR in high-Se + serine group were significantly decreased(P<0.05). (2) In the dose-response experiment, the expression of PHGDH enzyme in L02 cells gradually decreased with the increase of the concentration of exogenous serine or glycine, showing an obvious dose-dependent effect. In contrast, none of the other metabolic enzymes(SHMT1 and MTHFR) showed similar trends in protein expression. CONCLUSION: The upregulated expression of PHGDH, the key enzyme in the de novo synthesis pathway of serine in hepatocytes cultured with high-selenium can be inhibited feedback by exogenous serine or endogenous serine transformed from exogenous glycine directly.

Structure characterization of pectin from the pollen of Typha angustifolia L. and the inhibition activity of lipid accumulation in oleic acid induced L02 cells.

The pollen of Typha angustifolia L. decoction was clinically used to treat hyperlipidemia in China. A pectin polysaccharide (PTPS-2-2) was obtained from T. angustifolia pollen through water extraction, ion-exchange chromatography, and gel chromatography. Structural characterization showed that PTPS-2-2 had a molecular weight of 54 kDa and was composed of rhamnose, arabinose, xylose, galactose, and galacturonic acid with a molar ratio of 11.5: 36.5: 4.1: 36.7: 11.2. PTPS-2-2 consisted of rhamnogalacturonan I (RG-I) and arabinogalactan II (AG-II) domains. Its backbone was predominantly composed of →4-α-D-GalpA-(1 â†’ 2)-α-L-Rhap-(1→, with branches of 1,3-Galp, 1,6-Galp, 1,3,6-Galp, T-Araf, 1.5-Araf and T-Xylp, connected to the 4-position of 1,2-Rhap and the 3-position of 1,4-GalpA. The inhibitory effect of PTPS-2-2 on lipid accumulation was studied in vitro, using L02 cells induced by oleic acid. This experiment shows that PTPS-2-2 treatment at 100-400 µg/mL dose-dependently reduce cellular triglycerides (TG), cholesterol (TC), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and malondialdehyde (MDA) levels, while elevated superoxide dismutase (SOD) levels. This indicated that PTPS-2-2 potentially ameliorated oleic acid-induced hepatic steatosis by inhibiting lipid accumulation and oxidative stress.

The protective effect of trigonelline on H / 中国药理学通报

Aim To study the protective effect of trigonelline on H

Ros-mediated mitochondrial oxidative stress is involved in the ameliorating effect of ginsenoside GSLS on chlorpyrifos-induced hepatotoxicity in mice.

Chlorpyrifos (CPF), as an extensively used organophosphorus pesticide, often remains on food surfaces or contaminates water sources. CPF can cause many toxic effects on human production and life. As an additional product of non-medicinal parts of ginseng, the pharmacological activity of ginseng stem and leaf total saponin (GSLS) has been verified and applied in recent years. This study aimed to evaluate the protective effect of GSLS on CPF-induced liver damage in mice. Experimental results in vivo demonstrate that GSLS can reduce the accumulation of oxidation product MDA by relieving CPF-induced liver function indicators in mice and enhancing the antioxidant enzyme SOD and CAT activities of mice. With the decrease in mRNA expression of BAX, NF-KB, and TIMP in liver tissues, the mRNA expression of Nrf-2, HO-1, and XIAP increased. Through anti-inflammatory, antioxidant, anti-inflammatory and other effects, cpf-induced hepatotoxicity can be alleviated by GSLS. In vitro experiments have proved that GSLS can show the ability to scavenge DPPH free radicals and hydroxyl radicals. In addition, GSLS can alleviate chlorpyrifos-induced ROS accumulation in L02 cells, alleviating cytokinetic potential reduction. In summary, by fighting oxidative stress, GSLS can alleviate liver damage caused by CPF.

[Preparation and characterization of fenugreek leaf flavonoids and their protective effects against oxidative damage to hepatocytes].

The present study investigated the main components of fenugreek(Trigonella foenum-graecum L.) leaf flavonoids(FLFs) and their antioxidant activity. FLFs were prepared and enriched by solvent extraction, and the flavonoids were characterized by high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS). The protective effect of FLFs against H_2O_2-induced stress damage to L02 hepatocytes was also investigated. Firstly, the cell viability was measured by MTT assay. The oxidative stress injury model was induced by H_2O_2 in L02 cells. The release of lactate dehydrogenase(LDH), the content of reduced glutathione(GSH) and malondialdehyde(MDA), and the activities of superoxide dismutase(SOD) and catalase(CAT) were measured by assay kits. Hoechst fluorescence staining was performed to observe the cell apoptosis. The expression levels of c-Jun N-terminal kinase(JNK), extracellular signal-regulated kinase 1/2(ERK1/2), nuclear factor erythroid-2 related factor 2(Nrf2), heme oxygenase 1(HO-1), and their phosphorylated proteins were detected by Western blot. Based on the MS fragment ion information and data in databases, FLFs contained eight flavonoids with quercetin and kaempferol as the main aglycons. The cell viabi-lity assay revealed that as compared with the conditions in the H_2O_2 treatment group, 3.125-25 µg·mL~(-1) FLFs could increase the viability of L02 cells, reduce LDH release and MDA content in a dose-dependent manner, potentiate the activities of SOD, CAT, and GSH, decrease the phosphorylation of JNK and ERK1/2 proteins, and up-regulate the expression of Nrf2 and HO-1. The results of fluorescence staining showed that the nucleus of the H_2O_2 treatment group showed concentrated and dense strong blue fluorescence, while the blue fluorescence intensity of the FLFs group decreased significantly. FLFs showed a protective effect against H_2O_2-induced oxidative damage in L02 cells, and the underlying mechanism is associated with the enhancement of cell capability in clearing oxygen free radicals and the inhibition of apoptosis by the activation of the MAPKs/Nrf2/HO-1 signaling pathway. The antioxidant effect of fenugreek leaf is related to its rich flavonoids.

Crocetin ameliorates non-alcoholic fatty liver disease by modulating mitochondrial dysfunction in L02 cells and zebrafish model.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine considers that the etiology and pathogenesis of non-alcoholic fatty liver disease (NAFLD) are related to liver depression and qi stagnation. Saffron and its active ingredient, crocetin (CCT), are used for the treatment of metabolic diseases owing to their "Liver deobstruent" and "Liver tonic" effects. However, the effect of CCT on NAFLD has not been fully elucidated. In the present study, the effect and potential molecular mechanism of CCT were explored in both in vivo and in vitro models of NAFLD. MATERIALS AND METHODS: CCT was isolated from saffron and purity and structure characterization were performed using HPLC, MS, 1H-NMR, and 13C-NMR. The effect of CCT on the viability of L02 cells and its maximum tolerable concentration (MTC) in zebrafish were investigated. Free fatty acids (FFA) and thioacetamide (TAA) were used to induce lipid accumulation in L02 cells and steatosis in zebrafish, respectively. The effects of CCT on indexes related to lipid metabolism, oxidative stress, and mitochondrial function in NAFLD models were explored using biochemical assay kits, Western blot analysis, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), histopathology analysis, and determination of mitochondrial membrane potential (ΔΨm). Morphological analysis of mitochondria was performed using transmission electron microscopy (TEM). RESULTS: The levels of triglyceride (TG), total cholesterol (TC), malondialdehyde (MDA), and alanine/aspartate aminotransferases (ALT/AST) activities in FFA treated L02 cells were significantly reduced after CCT treatment. CCT treatment significantly increased ATP concentration, ΔΨm, and activities of superoxide dismutase (SOD), catalase (CAT), and cytochrome c oxidase (COX IV) in FFA treated L02 cells. TEM images showed restoration of mitochondrial morphology. CCT decreased ATP concentration and upregulated expression of B-cell lymphoma-2 (Bcl-2) and COX IV, whereas, CCT downregulated expression of BCL2-Associated X (Bax) and cleaved caspase-3 in TAA treated zebrafish. These findings indicated that mitochondrial dysfunction was alleviated after CCT treatment. Oil Red O staining of L02 cells and zebrafish showed that CCT treatment reversed the accumulation of lipid droplets. CONCLUSION: In summary, CCT treatment effectively alleviated the symptoms of NAFLD and restored mitochondrial function in L02 cells and zebrafish NAFLD model.

Study on spectrum-toxicity relationship of in vitro hepatotoxicity of aqueous extract from Euodia rutaecarpa / 中国药房

OBJECTIVE To study the spectru m-toxicity relationship of in vitro hepatotoxicity of aqueous extract from Euodia rutaecarpa. METHODS The aqueous extract from 16 batches of E. rutaecarpa from different habitats were prepared. The fingerprints of aqueous extract from E. rutaecarpa were established by ultra high performance liquid chromatography （UPLC） method and Similarity Evaluation System of TCM Fingerprint （2012A edition ），and common peaks were identified and the similarity was evaluated. Using normal human hepatocytes L 02 as subject ，inhibitory effect of aqueous extract from 16 batches of E. rutaecarpa to them were investigated. The spectrum-toxicity relationship of UPLC fingerprint of aqueous extract from E. rutaecarpa with the hepatotoxicity of hepatocytes L 02 was analyzed by grey relational analysis （GRA）and partial least squares regression analysis （PLSR）. The corresponding compound of the chromatographic peak with the greatest correlation with the in vitro hepatotoxicity of E. rutaecarpa were isolated ，prepared and identified. RESULTS There were 27 common peaks in UPLC fingerprints of aqueous extract from 16 batches of E. rutaecarpa ，with similarity of 0.375-0.995. Totally 9 peaks were confirmed ，i.e. neochlorogenic acid （peak 5），chlorogenic acid （peak 9），cryptochlorogenic acid （peak 10），caffeic acid （peak 12），rutin （peak 16），hyperin（peak 17），dehydroevotarine（peak 19），evotarine（peak 24），rutecarpine（peak 25）. The aqueous extract from 16 batches of E. rutaecarpa showed significant inhibitory effect on the growth of L 02 cells（P＜0.05 or P＜0.01），and the inhibitory rate ranged from 6.68％ to 67.95％. GRA showed that there were 18 common peaks with correlation degree greater than 0.8，which were peak 8＞peak 3＞peak 23＞peak 7＞peak 4＞peak 9＞peak 12＞peak 2＞peak 19＞peak 6＞ 4928381。E-mail：799247687@qq.com peak 15＞peak 5＞peak 1＞peak 17＞peak 21＞peak 26＞ peak 20＞peak 14 in descending order of correlation degree. PLSR showed that there were 14 peaks with regression coefficient＞0 and variable importance projection value ＞1，and the order of regression coefficient was peak 8＞peak 3＞peak 23＞ peak 2＞peak 7＞peak 4＞peak 12＞peak 9＞peak 19＞peak 5＞peak 17＞peak 26＞peak 10＞peak 15. Peak 8 had the greatest correlation with in vitro hepatotoxicity，and the corresponding compound of this peak was identified as 6-O-trans caffeoyl gluconic acid. CONCLUSIONS The in vitro hepatotoxicity of aqueous extract from E. rutaecarpa is the result of multiple component interaction，among which 6-O-trans caffeoyl gluconic acid shows closest relation with in vitro hepatotoxicity.

Preparation and characterization of fenugreek leaf flavonoids and their protective effects against oxidative damage to hepatocytes / 中国中药杂志

The present study investigated the main components of fenugreek(Trigonella foenum-graecum L.) leaf flavonoids(FLFs) and their antioxidant activity. FLFs were prepared and enriched by solvent extraction, and the flavonoids were characterized by high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS). The protective effect of FLFs against H_2O_2-induced stress damage to L02 hepatocytes was also investigated. Firstly, the cell viability was measured by MTT assay. The oxidative stress injury model was induced by H_2O_2 in L02 cells. The release of lactate dehydrogenase(LDH), the content of reduced glutathione(GSH) and malondialdehyde(MDA), and the activities of superoxide dismutase(SOD) and catalase(CAT) were measured by assay kits. Hoechst fluorescence staining was performed to observe the cell apoptosis. The expression levels of c-Jun N-terminal kinase(JNK), extracellular signal-regulated kinase 1/2(ERK1/2), nuclear factor erythroid-2 related factor 2(Nrf2), heme oxygenase 1(HO-1), and their phosphorylated proteins were detected by Western blot. Based on the MS fragment ion information and data in databases, FLFs contained eight flavonoids with quercetin and kaempferol as the main aglycons. The cell viabi-lity assay revealed that as compared with the conditions in the H_2O_2 treatment group, 3.125-25 μg·mL~(-1) FLFs could increase the viability of L02 cells, reduce LDH release and MDA content in a dose-dependent manner, potentiate the activities of SOD, CAT, and GSH, decrease the phosphorylation of JNK and ERK1/2 proteins, and up-regulate the expression of Nrf2 and HO-1. The results of fluorescence staining showed that the nucleus of the H_2O_2 treatment group showed concentrated and dense strong blue fluorescence, while the blue fluorescence intensity of the FLFs group decreased significantly. FLFs showed a protective effect against H_2O_2-induced oxidative damage in L02 cells, and the underlying mechanism is associated with the enhancement of cell capability in clearing oxygen free radicals and the inhibition of apoptosis by the activation of the MAPKs/Nrf2/HO-1 signaling pathway. The antioxidant effect of fenugreek leaf is related to its rich flavonoids.

Role of NRF2 signaling pathway in trichloromethane-induced oxidative stress in L02 cells / 中国职业医学

@#Objective - （ ） To investigate the effects of nuclear factor erythroid 2 related factor 2 NRF2 on the oxidative stress （ ） Methods ） ，， induced by trichloromethane TCM in human normal hepatocyte L02 cells. i L02 cells were stimulated with 1 2 ， ， ， （ ）， 4 8 12 16 and 20 mmol/L TCM solution dissolved in dimethyl sulfoxide and the control group and blank group were set ， - ， up. After culturing for 24 hours the cell viability was detected by CCK 8 colorimetric method and the concentration of TCM ） -， - stimulation was screened. ii L02 cells in logarithmic growth phase were randomly divided into control group and low medium - ， ， ， and high dose groups. After 24 hours of exposure to 0 4 8 and 12 mmol/L TCM the cells were collected. The activity of （ ）， （ ）， （ - ） （ ） superoxide dismutase SOD catalase CAT glutathione peroxidase GSH Px and the level of malondialdehyde MDA NRF2， - （HO-1）， were detected by colorimetric analysis. The mRNA expression levels of heme oxygenase 1 glutamate cysteine （GCLC） （） （NQO1） - ligase catalytic subunit and NAD P H quinone dehydrogenase 1 were detected by real time fluorescence ， - ， polymerase chain reaction. The protein levels of NRF2 HO 1 GCLC and NQO1 were detected by Western blotting.Results ） ， ， ， ， i When the concentration of TCM was 4 8 12 16 and 20 mmol/L the survival rate of L02 cells decreased （ P ） ， ， significantly compared with the control group all <0.05 . The concentration of 0 4 8 and 12 mmol/L were selected as the ） ， - stimulation doses for subsequent experiments. ii Compared with the control group the activities of SOD and GSH Px in L02 （ P ） （ P ）， - cells in the three doses groups decreased all <0.05 and the levels of MAD increased all <0.05 with a dose effect - （P ）， relationship. The CAT activity of L02 cells in the medium dose group was lower than that in the control group <0.05 and the - （ P ） CAT activity of L02 cells in the high dose group was lower than that in the others three groups all <0.05 . Compared with the ， NRF2 - （P ），NRF2 control group the relative expression levels of mRNA in L02 cells in the low dose group decreased <0.05 - （P ）， NRF2 mRNA in L02 cells in the medium dose group increased <0.05 mRNA and NRF2 protein expression in L02 cells in （ P ） HO-1，GCLC， NQO1 ， the highdose group increased both <0.05 . The relative expression level of mRNA and GCLC NQO1 （ P ） protein expression in L02 cells in the three doses groups increased compared with the control group all <0.05 . The relative NRF2 - - - expression level of mRNA in L02 cells in the high dose group was higher than that in the low and medium dose groups （ P ）， - （P ）， both <0.05 and the relative expression of NRF2 protein was higher than that in the low dose group <0.05 but the HO-1 GCLC - - （ relative expression levels of and mRNA and HO 1 protein level were lower than those in the medium dose group all P ）Conclusion - <0.05 . TCM exposure can inhibit the proliferation of L02 cells by inducing oxidative stress with a dose effect ， relationship. In this process the antioxidant mechanism mediated by NRF2 was activated. The expression of antioxidant defense ， - ， and detoxification related target genes downstream of NRF2 signaling pathway was activated and the expression of HO 1 - GCLC and NQO1 was up regulated to alleviate the oxidative damage caused by TCM.

Phillygenin Mitigates LPS/ATP-induced L02 Cell Inflammation by Regulating P2X7R/NF-κB/NLRP3 Inflammasome Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effects of phillygenin （PHI） on the inflammation in L02 cells induced by lipopolysaccharide （LPS） and adenosine triphosphate （ATP） and the expression of purinergic 2X7 receptor （P2X7R）， NOD-like receptor family pyrin domain containing 3 （NLRP3）， and nuclear factor kappa B （NF-κB） expression. MethodIn this study， the inflammation model was induced in L02 cells by 100 μg·L-1 LPS treatment for 24 h and 5 mmoL·L-1 ATP treatment for 5 h. The cells in the PHI groups were cultured with PHI （100， 50， 25 mg·L-1） for 6 h in the LPS treatment period， followed by LPS treatment for another 18 h. After ATP treatment for 5 h， the mRNA and protein expression of interleukin-1β （IL-1β）， interleukin-18 （IL-18）， P2X7R， NLRP3， Caspase-1 precursor （pro-Caspase-1）， cleaved Caspase-1， NF-κB， and NF-κB inhibitor protein α （IκBα） in L02 cells was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. Molecular docking was used to predict whether P2X7R could bind to PHI， and DCFH-DA was employed to detect the accumulation of reactive oxygen species （ROS） in cells. P2X7R was silenced by small interfering ribonucleic acid （siRNA）， and then the mRNA expression of IL-1β， IL-18， P2X7R， NLRP3， Caspase-1， NF-κB， and IκBα was detected by Real-time PCR. ResultReal-time PCR and Western blot showed that compared with the normal group， the model group showed increased expression of IL-1β and IL-18 （P<0.05）， and compared with the model group， the PHI groups showed down-regulated IL-1β， IL-18 mRNA and protein expression （P<0.05）. Molecular docking suggested a good binding effect of PHI to P2X7R. Real-time PCR and Western blot analysis showed that the expression of P2X7R in the model group was significantly up-regulated compared with that in the normal group （P<0.05）， and compared with the model group， the PHI groups showed down-regulated mRNA and protein expression of P2X7R （P<0.05）. DCFH-DA results showed that compared with the normal group， the model group showed increased content of ROS （P<0.05）， and compared with the model group， the PHI groups decreased the accumulation of ROS （P<0.05）. As demonstrated by Real-time PCR and Western blot， compared with the normal group， the model group showed increased expression of NLRP3 inflammasome and NF-κB （P<0.05）， and compared with the model group， the PHI groups significantly decreased the mRNA and protein expression of NLRP3 and cleaved Caspase-1， and up-regulated the mRNA and protein expression of NF-κB and IκBα （P<0.05）. Real-time PCR analysis showed that compared with the results in the model group， after silencing P2X7R by siRNA， the mRNA expression of IL-1β， IL-18， P2X7R， NLRP3， Caspase-1， NF-κB， and IκBα was decreased （P<0.05）. PHI exerted the same effect， and the mRNA expression was further reduced after the combination of them. ConclusionPHI is presumed to suppress the expression of the NLRP3/NF-κB signaling pathway by down-regulating upstream P2X7R to alleviate the LPS/ATP-induced inflammation in L02 cells， suggesting that P2X7R may be the target of PHI against inflammation.

The role of PKA/PP2B-mediated Drp1 phosphorylation and the subsequent EGFR inhibition in Cr(VI)-induced premature senescence.

In recent years, frequent hexavalent chromium [Cr(VI)] pollution incidents have severely damaged the ecology and endangered the public health. It is well known that cell senescence could promote the carcinogenesis, thus the related research on the occurrence of premature senescence is of great significance to the elucidation of the carcinogenic mechanism of Cr(VI). We previously confirmed that long-term low-dose Cr(VI) exposure induced premature senescence, but the key molecular events that determine the occurrence of premature senescence are still unclear. In the present study, we found that Cr(VI) induced phosphorylation of dynamin-relatedprotein 1 (Drp1)-S637 site in premature senescent cells, which was accompanied with the decrease of mitochondrial fission. We also demonstrated that the phosphorylation status of Drp1-S637 after Cr(VI) exposure was related to the antagonism of PKA/PP2B, and continuous dephosphorylation of Drp1-S637 attenuated premature senescence caused by Cr(VI). The epidermal growth factor receptor (EGFR) overexpression significantly alleviated the occurrence of premature senescence, and the expressions of EFGR and its downstream molecules were related to the phosphorylation status of Drp1-S637. In brief, we revealed the role of PKA/PP2B-mediated Drp1 phosphorylation and the subsequent EGFR inhibition in Cr(VI)-induced premature senescence. This study is the first time to link the phosphorylation of Drp1 with Cr(VI)-induced premature senescence, in order to find the key molecular events that determine the occurrence of premature senescence and demonstrate the molecular mechanism of abnormal elongated mitochondria formation in the senescence process. The significance of this study is to explore the carcinogenesis of Cr(VI) and provide new ideas and strategies for the targeted treatment of Cr(VI)-related cancers.