Normal Human Liver Cell Line Research Articles

Processing-induced reduction in dianthrones content and toxicity of Polygonum multiflorum: Insights from ultra-high performance liquid chromatography triple quadrupole mass spectrometry analysis and toxicological assessment.

Polygonum multiflorum-induced liver injury (PM-DILI) has significantly hindered its clinical application and development. This study investigates the variation in content and toxicity of dianthrones, the toxic components of P. multiflorum, during different processing cycles. We employed the ultra-high-performance liquid chromatography triple quadrupole mass spectrometry method to quantify six dianthrones in raw P. multiflorum and formulations processed with a method called nine cycles of steaming and sunning. Additionally, toxicity assessments were conducted using human normal liver cell line L02 and zebrafish embryos. Results indicate a gradual reduction in dianthrones content with increasing processing cycles. Processed formulations exhibited significantly reduced cytotoxicity in L02 cells and hepatotoxicity in zebrafish embryos. Our findings elucidate the relationship between processing cycles and P. multiflorum toxicity, providing theoretical support for its safe use.

The Cytoprotective Effect of C60 Derivatives in the Self-Microemulsifying Drug Delivery System against Triptolide-Induced Cytotoxicity In Vitro.

The aim of this study was to optimize the formulation of a C60-modified self-microemulsifying drug delivery system loaded with triptolide (C60-SMEDDS/TP) and evaluate the cytoprotective effect of the C60-SMEDDS/TP on normal human cells. The C60-SMEDDS/TP exhibited rapid emulsification, an optimal particle size distribution of 50 ± 0.19 nm (PDI 0.211 ± 0.049), and a near-neutral zeta potential of -1.60 mV. The release kinetics of TP from the C60-SMEDDS/TP exhibited a sustained release profile and followed pseudo-first-order release kinetics. Cellular proliferation and apoptosis analysis indicated that the C60-SMEDDS/TP (with a mass ratio of TP: DSPE-PEG-C60 = 1:10) exhibited lower toxicity towards L02 and GES-1 cells. This was demonstrated by a higher IC50 (40.88 nM on L02 cells and 17.22 nM on GES-1 cells) compared to free TP (21.3 nM and 11.1 nM), and a lower apoptosis rate (20.8% on L02 cells and 26.3% on GES-1 cells, respectively) compared to free TP (50.5% and 47.0%) at a concentration of 50 nM. In comparison to the free TP group, L02 cells and GES-1 cells exposed to the C60-SMEDDS/TP exhibited a significant decrease in intracellular ROS and an increase in mitochondrial membrane potential (ΔψM). On the other hand, the C60-SMEDDS/TP demonstrated a similar inhibitory effect on BEL-7402 cells (IC50 = 28.9 nM) and HepG2 cells (IC50 = 107.6 nM), comparable to that of the free TP (27.2 nM and 90.4 nM). The C60-SMEDDS/TP group also exhibited a similar intracellular level of ROS and mitochondrial membrane potential compared to the SMEDDS/TP and free TP groups. Fullerenol-Grafted Distearoyl Phosphatidylethanolamine-Polyethylene Glycol (DSPE-PEG-C60) was synthesized and applied in the self-microemulsifying drug delivery system. The C60-SMEDDS/TP was formulated using Cremophor EL, medium-chain triglycerides (MCT), PEG-400, and DSPE-PEG-C60, and loaded with triptolide (TP). The toxicity and bioactivity of the C60-SMEDDS/TP were assessed using normal human liver cell lines (L02 cells), normal human gastric mucosal epithelial cell lines (GES-1 cells), and liver cancer cell lines (BEL-7402 cells and HepG2 cells). The production of reactive oxygen species (ROS) after the C60-SMEDDS/TP treatment was assessed using 2',7'-dichlorofluorescein diacetate (DCFDA) staining. The alterations in mitochondrial membrane potential (ΔψM) were assessed by measuring JC-1 fluorescence. The cytoprotection provided by the C60-SMEDDS/TP favored normal cells (L02 and GES-1) over tumor cells (BEL-7402 and HepG2 cells) in vitro. This suggests a promising approach for the safe and effective treatment of TP.

In-vitro hepatotoxic activity of mitragynine and paynantheine isolated from the leaves of Mitragyna speciosa Korth. (Kratom)

Mitragynine, a primary alkaloid found in kratom leaves has been reported to have a broad range of pharmacological and toxicological properties while its congener, paynatheine has comparatively less information available on these aspects. Mitragynine and its congener, paynantheine, were isolated from the ethanol kratom leaves extract using gravity column chromatography techniques. Our study evaluated the cytotoxicity potential of mitragynine and paynantheine on normal human liver cell line, HL-7702, and human hepatoma cell line, HepG2. Mitragynine exhibited a moderate inhibitory effect on the HepG2 cell line with IC50 value of 42.11 ± 1.31 μM in comparison with vinblastine (IC50: 15.45 ± 0.72 μM) while it showed non-cytotoxic properties towards the HL-7702 cell line with concentrations ranging below 200 μM. In contrast, paynantheine exhibited weak cytotoxic properties towards HepG2 and HL-7702 cell lines. Further comprehensive evaluations of both compounds are needed to establish more details on the cytotoxicity potential of kratom alkaloids.

Baicalin attenuates aflatoxin B1-induced hepatotoxicity via suppressing c-Jun-N-terminal kinase-mediated cell apoptosis.

Aflatoxin B1 (AFB1) is classified as a Class I carcinogen and common pollutant in human and animal food products. Prolonged exposure to AFB1 can induce hepatocyte apoptosis and lead to hepatotoxicity. Therefore, preventing AFB1-induced hepatotoxicity remains a critical issue and is of great significance. Baicalin, a polyphenolic compound derived from Scutellaria baicalensis Georgi, has a variety of pharmacodynamic activities, such as antiapoptotic and anticancer activities. This study systematically investigated the alleviating effect of baicalin on AFB1-induced hepatotoxicity from the perspective of apoptosis and explored the possible molecular mechanism. In the normal human liver cell line L02, baicalin treatment significantly inhibited AFB1-induced c-Jun-N-terminal Kinase (JNK) activation and cell apoptosis. In addition, the in vitro mechanism study demonstrated that baicalin alleviates AFB1-induced hepatocyte apoptosis through suppressing the translocation of phosphorylated JNK to the nucleus and decreasing the phosphorylated c-Jun/c-Jun ratio and the Bax/Bcl2 ratio. Molecular docking and drug affinity responsive target stability assays demonstrated that baicalin has the potential to target JNK. This study provides a basis for the therapeutic effect of baicalin on hepatocyte apoptosis caused by AFB1, indicating that the development of baicalin and JNK pathway inhibitors has broad application prospects in the prevention of hepatotoxicity, especially hepatocyte apoptosis.

Paraoxonase-2 agonist vutiglabridin promotes autophagy activation and mitochondrial function to alleviate non-alcoholic steatohepatitis.

Only limited therapeutic agents have been developed for non-alcoholic steatohepatitis (NASH). Glabridin, a promising anti-obesity candidate, has only limited druggability due to its low in vivo chemical stability and bioavailability. Therefore, we developed vutiglabridin (VUTI), which is based on a glabridin backbone, and investigated its mechanism of action in treating NASH in animal models. Anti-NASH effects of VUTI were determined in in vitro fatty liver models, spheroids of primary human hepatocytes and L02 normal liver cell lines. To identify VUTI possible cellular target/s, biotin-labelled VUTI was synthesized and underwent chemical proteomic analysis. Further, the evaluation of VUTI therapeutic efficacy was carried out using an amylin-NASH and high-fat (HF) diet-induced obese (DIO) mouse models. This was carried out using transcriptomic, lipidomic and proteomic analyses of the livers from the amylin-NASH mouse model. VUTI treatment markedly reduces hepatic steatosis, fibrosis and inflammation by promoting lipid catabolism, activating autophagy and improving mitochondrial dysfunction, all of which are hallmarks of effective NASH treatment. The cellular target of VUTI was identified as paraoxonase 2 (PON2), a newly proposed protein target for the treatment of NASH, VUTI enhanced PON2 activity. The results using PON2 knockdown cells demonstrated that PON2 is important for VUTI- activation of autophagy, promoting mitochondrial function, decreasing oxidative stress and alleviating lipid accumulation under lipotoxic condition. Our data demonstrated that VUTI is a promising therapeutic for NASH. Targeting PON2 may be important for improving liver function in various immune-metabolic diseases including NASH.

Discovery of a novel DYRK1A inhibitor with neuroprotective activity by virtual screening and in vitro biological evaluation.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is implicated in accumulation of amyloid β-protein (Aβ) and phosphorylation of Tau proteins, and thus represents an important therapeutic target for neurodegenerative diseases. Though many DYRK1A inhibitors have been discovered, there is still no marketed drug targeting DYRK1A. This is partly due to the lack of effective and safe chemotypes. Therefore, it is still necessary to identify new classes of DYRK1A inhibitors. By performing virtual screening with the workflow mainly composed of pharmacophore modeling and molecular docking as well as the following DYRK1A inhibition assay, we identified compound L9, ((Z)-1-(((5-phenyl-1H-pyrazol-4-yl)methylene)-amino)-1H-tetrazol-5-amine), as a moderately active DYRK1A inhibitor (IC50: 1.67μM). This compound was structurally different from the known DYRK1A inhibitors, showed a unique binding mode to DYRK1A. Furthermore, compound L9 showed neuroprotective activity against okadaic acid (OA)-induced injury in the human neuroblastoma cell line SH-SY5Y by regulating the expression of Aβ and phosphorylation of Tau protein. This compound was neither toxic to the SH-SY5Y cells nor to the human normal liver cell line HL-7702 (IC50: >100μM). In conclusion, we have identified a novel DYRK1A inhibitor with neuroprotective activity through virtual screening and in vitro biological evaluation, which holds the promise for further study.

Antibacterial insights into alternariol and its derivative alternariol monomethyl ether produced by a marine fungus.

Alternaria alternata FB1 is a marine fungus identified as a candidate for plastic degradation in our previous study. This fungus has been recently shown to produce secondary metabolites with significant antimicrobial activity against various pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and the notorious aquaculture pathogen Vibrio anguillarum. The antibacterial compounds were purified and identified as alternariol (AOH) and its derivative, alternariol monomethyl ether (AME). We found that AOH and AME primarily inhibited pathogenic bacteria (MRSA or V. anguillarum) by disordering cell division and some other key physiological and biochemical processes. We further demonstrated that AOH could effectively inhibit the unwinding activity of MRSA topoisomerases, which are closely related to cell division and are the potential action target of AOH. The antibacterial activities of AOH and AME were verified by using zebrafish as the in vivo model. Notably, AOH and AME did not significantly affect the viability of normal human liver cells at concentrations that effectively inhibited MRSA or V. anguillarum. Finally, we developed the genetic operation system of A. alternata FB1 and blocked the biosynthesis of AME by knocking out omtI (encoding an O-methyl transferase), which facilitated A. alternata FB1 to only produce AOH. The development of this system in the marine fungus will accelerate the discovery of novel natural products and further bioactivity study.IMPORTANCEMore and more scientific reports indicate that alternariol (AOH) and its derivative alternariol monomethyl ether (AME) exhibit antibacterial activities. However, limited exploration of their detailed antibacterial mechanisms has been performed. In the present study, the antibacterial mechanisms of AOH and AME produced by the marine fungus Alternaria alternata FB1 were disclosed in vitro and in vivo. Given their low toxicity on the normal human liver cell line under the concentrations exhibiting significant antibacterial activity against different pathogens, AOH and AME are proposed to be good candidates for developing promising antibiotics against methicillin-resistant Staphylococcus aureus and Vibrio anguillarum. We also succeeded in blocking the biosynthesis of AME, which facilitated us to easily obtain pure AOH. Moreover, based on our previous results, A. alternata FB1 was shown to enable polyethylene degradation.

Design, Synthesis, and Biological Evaluation of a Class of TEMPOmodified Naphthalimide Derivatives with Good Selectivity for Tumor Cells

Background: Naphthalimides have been receiving continuous attention for their outstanding performance in treating cancer. However, as a class of cytotoxic anti-tumor agents, their toxic side effects greatly limit their clinical application. Therefore, developing naphthalimide reagents with good selectivity for tumor cells is of great importance. Methods: A class of TEMPO-modified naphthalimide derivatives substituted with oxygen at the 6- position were designed, synthesized, and evaluated for the anti-tumor activity in vitro against three cancer cell lines (A549, MCF-7, and HeLa) as well as one normal human liver cell line (QSG-7701) by MTT assay. HRMS (or MS) and EPR spectra were used to identify the structures of target compounds. The UV–vis titration absorption spectra were performed to study the interaction between compounds and DNA. Results: The target compounds NT3, NT9, NT10, NT11, and NT12 exhibited moderate inhibition to all the three tested tumor cell lines, while NT1, NT2, and NT4 showed selectivity against A549, All the target compounds did not show measurable activities in QSG-7701, which imply the target compounds are likely to overcome the nonspecific toxicity against normal cells. UV-vis spectra showed this class of compounds could effectively intercalate into DNA and selectively bind to G-C base pairs. Conclusion: A novel class of TEMPO-modified naphthalimide derivatives substituted with oxygen at 6- position and the preliminary discussion on their SARs provide promising opportunities to guide further research on naphthalimide derivatives as anti-tumor agents.

Harnessing the potential of de-sulfated heparin for targeted drug delivery: A three-component approach exemplified by conjugation with galactose and paclitaxel

Heparin, an anticoagulant with a century-long history of use, has been investigated over the past decade as a potential drug delivery vehicle. Despite its safety and efficacy, its interactions with many proteins through specific sulfate patterns can complicate drug delivery by mediating diverse biological functions. Here, we present the synthesis of a three-component drug delivery system comprising de-sulfated heparin as the carrier, galactose as the targeting moiety, and paclitaxel as the therapeutic drug. Removal of sulfates eliminated most of its anticoagulant effects in all intermediates. Through coupling with galactose and paclitaxel, the system improved the solubility of the drug and achieved selective targeting and efficient drug delivery to HepG2 cells, a liver carcinoma cell line with high galactose receptor expression. While the three-component system exhibited a slightly higher IC50 value than native paclitaxel, demonstrating its efficacy as a drug carrier, the IC50 value for the normal human liver cell line QSG7701 was significantly higher, indicating its selectivity and safety. Our study introduces a novel approach utilizing desulfated heparin as a carrier, warranting further investigation to unlock its potential in targeted drug delivery strategies.

In vitro α-amylase/α-glucosidase, cytotoxicity and radical scavenging potential of Hypoxishemerocallidea synthesized magnesium oxide nanoparticles

The prevalence of diabetes has continued to increase over the past decade. Medicinal extract-synthesized nanoformulations incorporating trace elements found in the body have emerged as alternative therapeutic technology for the treatment of diabetes. In this study, magnesium oxide (MgO) nanoparticles were prepared using Hypoxishemerocallidea (HH) and investigated for their cytotoxicity, antioxidant, and antidiabetic potentials. MgO NPs were characterized by transmission electron microscopy (TEM), Fourier-transform infrared (FTIR) spectroscopy, and zeta-potential techniques. TEM analysis confirmed the 2D nanosheet-like morphology of the nanoparticles with particle size (diameter; 300 nm and length of about 1 µm) while the FTIR spectra showed functional groups correlating to the bioactive compounds of HH in the MgO NPs. Moderate antioxidant activity of MgO NPs was observed against DPPH (IC50 = 57.35 ± 0.28 µg/mL) and ABTS (IC50 = 52.08 ± 0.24 µg/mL). The nanoparticles were shown to be less toxic to normal embryonic (HEK293) and human liver (HEPG2) cell lines, with IC50 of 48.63 ± 0.73 and 32.39 ± 0.95 µg/mL, respectively compared to a known cytotoxic drug, doxorubicin which exhibited IC50 of 2.70 ± 0.32 and 8.62 ± 0.57 µg/mL respectively. Besides, the anti-hyperglycemic potential of the MgO NPs as demonstrated by α-amylase and α-glucosidase activities were significantly high with IC50 values of 33.03 ± 1.43 and 52.38 ± 3.06 µg/mL, respectively. These results were compared with an FDA-approved antidiabetic reference standard Acarbose, which exhibited IC50 values of 24.54 ± 1.55 and 6.54 ± 0.27 µg/mL. The results indicated that the HH bioinspired MgO NPs are capable of inhibiting meditators of diabetes and oxidative stress. This study further suggests that the MgO NPs synthesized using HH could be a good candidate for the management of diabetes and other inflammatory diseases due to their strong enzyme inhibition, efficient antioxidant properties, and biocompatibility.

Bioassay-guided isolation of anti-leukemic steroids from Aglaia abbreviata by inducing apoptosis

The strategy of bioactivity-guided isolation is widely used to obtain active compounds as quickly as possible. Thus, the inhibitory effects on human erythroleukemia cells (HEL) were applied to guide the isolation of the anti-leukemic compounds from Aglaia abbreviata. As a result, 19 compounds (16 steroids, two phenol derivatives, and a rare C12 chain nor-sesquiterpenoid), including 13 new compounds, were isolated and identified based on spectroscopic data analysis, single-crystal X-ray diffraction data, and electronic circular dichroism (ECD) calculations. Among them, 9 steroids exhibited good selective anti-leukemic activity against HEL and K562 (human chronic myeloid leukemia cells) cells with IC50 values between 2.29 ± 0.18 μM and 19.58 ± 0.13 μM. Notably, all the active compounds had relatively lower toxicity on the normal human liver cell line (HL-7702). Furthermore, five compounds (1, 4, 8, 10, and 19) displayed good anti-inflammatory effects, with IC50 values between 7.15 ± 0.16 and 27.1 ± 0.37 μM. An α,β-unsaturated ketone or a 5,6Δ double bond was crucial for improving anti-leukemic effect from the structure–activity relationship analysis. The compound with the most potential, 14 was selected for the preliminary mechanistic study. Compound 14 can induce apoptosis and cause cell cycle arrest. The expression of the marker proteins, such as PARP and caspase 3, were notably effected by this compound, thus inducing apoptosis. In conclusion, our investigation implied that compound 14 may serve as a potential anti-leukemia agent.

Structure characterization and cryoprotective activity of an exopolysaccharide from Pseudoalteromonas sp. LP6-12-2

Non-toxic natural antifreeze agents are urgently needed in many fields. Exopolysaccharides produced by psychrophilic bacteria from harsh habitats, such as Antarctica, to battle cold environments are a significant source of biological antifreeze agents. In this study, a homogeneous exopolysaccharide (EPS0-1) was extracted from Antarctica Pseudoalteromonas sp. LP6-12-2. Using High-performance gel permeation chromatography and High-efficiency anion chromatogram, respectively, the homogeneity, molecular weight, and monosaccharide composition of EPS0-1 were examined. EPS0-1 with a molecular weight (Mw) of 4351.31 kDa was an α-configuration polysaccharide containing uronic acid, amino and sulfuric acid groups, and composed of galactosamine hydrochloride, rhamnose, glucosamine hydrochloride, galactose, glucose, mannose, galacturonic acid, and glucuronic acid with rates of 1.40:9.40:4.90:32.0:37.2:6.50:4.10:4.40, respectively. Congo red test and I2-KI reaction found that the triple helix structure of EPS0-1 polysaccharide chain was weakly bound internally, with a longer side chain and/or more branches. The smooth and dense surface of EPS0-1 under Atomic force microscopy and Scanning electron microscopy revealed that it was large polysaccharide particles. Freeze-thaw cycle and freezing point determination experiment showed that EPS0-1 may significantly increase E. Coli's freezing resistance and decrease the solution's freezing point, respectively. EPS0-1 was capable of increasing the viscosity of the solution and displaying a high degree of thermal stability, which was proved by the rheological properties. And it was not hazardous to human normal liver cell line (LO2) in a specific concentration range. Therefore, the findings of this investigation established the basis for EPS0-1 as a potential antifreeze and food functional component.

Periploca forrestii Schltr. ameliorate liver injury caused by fluorosis in rat

To investigate the impact of the ethanoic fractions of Periploca forrestii Schltr. (P. forrestii) in ameliorating the liver injury caused by fluoride ingestion and to explore the potential mechanisms. Initially, an in vitro fluorosis cell model was constructed using the human normal liver cell line (L-02) induced by fluoride. Cell viability was assessed using the CCK-8 assay kit. The lactate dehydrogenase (LDH) assay kit was utilized to measure LDH content in the cell supernatant, while the malonic dialdehyde (MDA) assay kit was employed to determine MDA levels within the cells. Subsequently, a fluorosis rat model was established, and LDH content in the cell supernatant was measured using the LDH assay kit. Various parameters, including MDA, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and reactive oxygen species (ROS) content within the cells, were detected using appropriate assay kits. Additionally, cell apoptosis rate was determined using the Annexin V-FITC/PI cell apoptosis assay kit. The protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3, Cleaved Caspase-3, Caspase-9, and Cleaved Caspase-9 were analyzed through Western blotting. Compared to the model group, the ethanolic fraction D of P.forrestii (Fr.D) increased cell viability (P < 0.01) and decreased LDH and MDA levels (P < 0.01). In the high-dose Fr.D treatment group of fluoride-poisoned rats, serum ALT, AST, LDH and MDA levels significantly decreased (P < 0.01). Results from rat primary cells exhibited that the Fr.D administration group exhibited significantly higher cell survival rates than the fluoride group (P < 0.01). Similarly, primary rat cells treated with Fr.D showed enhanced cell viability (P < 0.05) and reduced apoptosis rate, LDH, MDA, SOD, GSH-Px, CAT, and ROS levels (P < 0.05) compared to the model group. Western blot analysis indicated that the Fr.D treatment group elevated the Bcl-2/Bax protein expression ratio and reduced Caspase-3 and Caspase-9 activation levels (P < 0.01) compared to the model group. The results suggest that components within the Fr.D from Periploca forrestii may alleviate fluoride-induced liver injury by potentially counteracting oxidative stress and cell apoptosis.

Synthesis, characterization, antiproliferative activity and DNA binding calculation of substituted-phenyl-terpyridine copper(II) nitrate complexes

Ten 4′- (R-phenyl) -2,2′: 6′, 2′ – terpyridine ligands (R = hydrogen (L1), hydroxyl (L2), methoxyl (L3), methylsulfonyl (L4), methyl (L5), phenyl (L6), fluoro (L7), chloro (L8), bromo (L9), and iodo (L10)) were synthesized. The reaction of these ligands with copper(II) nitrate led to complexes 1–10. The characterization of 1–10 was carried out by means of mass spectrometry, elemental analysis, infrared spectroscopy and X-ray single crystal diffraction. Four cell lines including esophageal cancer cell line (Eca-109), human liver cancer cell line (Bel-7402), human breast cancer cell line (SIHa) and human normal liver cell line (HL-7702) were selected to carry out antiproliferation and cytotoxicity experiments in vitro. The results showed that the complexes have strong inhibitory ability on the growth of tumor cells. In order to study the anticancer mechanism of the complexes, the binding mode and binding ability of the complexes with DNA were further determined and discussed with UV–Vis spectroscopy and circular dichroism. The effects of the lowest binding energy and hydrogen bond on the binding were studied using molecular docking calculation.

EXOSC10 is a novel hepatocellular carcinoma prognostic biomarker: a comprehensive bioinformatics analysis and experiment verification.

Hepatocellular carcinoma (HCC) is a common malignant tumor. There are few studies on EXOSC10 (exosome component 10) in HCC; however, the importance of EXOSC10 for HCC remains unclear. In the study, the prognosis value of EXOSC10 and the immune correlation were explored by bioinformatics. The expression of EXOSC10 was verified by tissue samples from clinical patients and in vitro experiment (liver cancer cell lines HepG2, MHCC97H and Huh-7; normal human liver cell line LO2). Immunohistochemistry (IHC) was used to detect EXOSC10 protein expression in clinical tissue from HCC. Huh-7 cells with siEXOSC10 were constructed using lipofectamine 3000. Cell counting kit 8 (CCK-8) and colony formation were used to test cell proliferation. The wound healing and transwell were used to analyze the cell migration capacity. Mitochondrial membrane potential, Hoechst 33342 dye, and flow cytometer were used to detect the change in cell apoptosis, respectively. Differential expression genes (DEGs) analysis and gene set enrichment analysis (GSEA) were used to investigate the potential mechanism of EXOSC10 and were verified by western blotting. EXOSC10 was highly expressed in tissues from patients with HCC and was an independent prognostic factor for overall survival (OS) in HCC. Increased expression of EXOSC10 was significantly related to histological grade, T stage, and pathological stage. Multivariate analysis indicated that the high expression level of EXOSC10 was correlated with poor overall survival (OS) in HCC. GO and GSEA analysis showed enrichment of the cell cycle and p53-related signaling pathway. Immune analysis showed that EXOSC10 expression was a significant positive correlation with immune infiltration in HCC. In vitro experiments, cell proliferation and migration were inhibited by the elimination of EXOSC10. Furthermore, the elimination of EXOSC10 induced cell apoptosis, suppressed PARP, N-cadherin and Bcl-2 protein expression levels, while increasing Bax, p21, p53, p-p53, and E-cadherin protein expression levels. EXOSC10 had a predictive value for the prognosis of HCC and may regulate the progression of HCC through the p53-related signaling pathway.

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.

Restoration and preservation effects of mung bean antioxidant peptides on H2O2-induced WRL-68 cells via Keap1-Nrf2 pathway.

Mung bean antioxidant peptides (MBAPs) were prepared from mung bean protein hydrolysate, and four peptide sequences including Ser-Asp-Arg-Thr-Gln-Ala-Pro-His (~953 Da), Ser-His-Pro-Gly-Asp-Phe-Thr-Pro-Val (~956 Da), Ser-Asp-Arg-Trp-Phe (~710 Da), and Leu-Asp-Arg-Gln-Leu (~644 Da) were identified. The effects of MBAPs on the oxidation-induced normal human liver cell line WRL-68 were analyzed to determine the mechanism protecting the oxidation-induced injury. The results showed that the cells were subjected to certain oxidative damage by H2O2 induction, as evidenced by decreased cell number and viability, overproduction of intracellular ROS, and decreased mitochondrial membrane potential. Compared with the H2O2-induced group, the MBAP-treated oxidation-induced group exhibited significantly higher cell number and viability, and the intracellular ROS was similar to that of the control group, suggesting that MBAP scavenges excessive intracellular free radicals after acting on the oxidation-induced cells. Combined with Western blotting results, it was concluded that the MBAP-treated oxidation-induced group also significantly promoted the expression of proteins related to the kelch-like ech-related protein 1 (Keap1)/ nuclear factor e2-related factor 2 (Nrf2) signaling pathway, which resulted in an approximately 2-fold increase in antioxidant enzymes, and a decrease in malondialdehyde content of approximately 55% compared to oxidatively-induced cells, leading to the recovery of both cell morphology and viability. These results suggest that MBAPs scavenge intracellular free radicals and improve oxidative stress in hepatocytes through the expression of Keap1/Nrf2 pathway-related protein, thereby reducing oxidative attack on the liver. Therefore, MBAP is applied as a nutritional ingredient in the functional food field, and this study provides a theoretical basis for the high utilization of mung bean proteins.

Discovery of diverse sesquiterpenoids from Magnolia grandiflora with cytotoxic activities by inducing cell apoptosis

Phytochemical study of Magnolia grandiflora led to the isolation of 39 sesquiterpenoids, including 15 new compounds (1–15). Compounds 1 and 2 are discovered to be the first 13-norgermacrane type sesquiterpenoids in natural products. Compound 15 is a rare 5,6-seco-guaiane type sesquiterpene and its possible biogenic precursor is presumed to be compound 20. Subsequent structural modification for compound 28 led to 21 derivatives, among which 15 derivatives were new compounds. All compounds were tested for the inhibitory effects on three tumor cell lines, and 17 compounds were active with the IC50 values ranging from 1.91 ± 0.39 μM to 12.29 ± 1.68 μM. The structure–activity relationships implied that an α, β-unsaturated lactone group was an important active group for the cytotoxicity. Two most active compounds (19 and 29) with low toxicity on normal human liver cell line were selected for further mechanism study. Compound 29 could induce apoptosis on Colo320DM cells through influencing the key apoptotic related proteins, such as PARP, Cleaved PARP, cleaved Caspase-3, and pro-Caspase 3. In addition, compound 19 with the best cytotoxic activity on HEL cells also could induce the apoptosis in dose- and time-dependent manners. In summary, our investigation implied that compounds 19 and 29 are two new potential anti-cancer candidates for ongoing study in the future.

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.

Design, synthesis, and biological evaluation of artemyrianolide H derivatives as potential antihepatoma agents

Artemyrianolide H (AH) is a germacrene-type sesquiterpenolid isolated from Artemisia myriantha, and showed potent cytotoxicity against three human hepatocellular carcinoma cell lines HepG2, Huh7, and SK-Hep-1 with IC50 values of 10.9, 7.2, and 11.9 µM, respectively. To reveal structure−activity relationship, 51 artemyrianolide H derivatives including 19 dimeric analogs were designed, synthesized, and assayed for their cytotoxicity against three human hepatoma cell lines. Among them, 34 compounds were more active than artemyrianolide H and sorafenib on the three cell lines. Especially, compound 25 exhibited the most promising activity with IC50 values of 0.7 (HepG2), 0.6 (Huh7), and 1.3 µM (SK-Hep-1), which were 15.5, 12.0, and 9.2-fold higher than that of AH and 16.4, 16.3 and 17.5-fold higher than that of sorafenib. Cytotoxicity evaluation on normal human liver cell lines (THLE-2) demonstrated good safety profile of compound 25 with SI of 1.9 (HepG2), 2.2 (Huh 7) and 1.0 (SK-Hep1). Further studies revealed that compound 25 dose-dependently arrested cells at G2/M phase which was correlated with the up-regulation of both cyclin B1 and p-CDK1, and induced apoptosis through the activation of mitochondrial pathways in HepG2 cells. In addition, the migratory and invasive abilities in HepG2 cells after treatment with 1.5 μM of compound 25 were decreased by 89% and 86% with the increase of E-cadherin expression accompanied by the decrease of N-cadherin, vimentin expression. Bioinformatics analysis based on machine learning predicted that PDGFRA and MAP2K2 might be acting targets of compound 25, and SPR assays demonstrated compound 25 were bound with PDGFRA and MAP2K2 with KD value of 0.168 nM, and 8.49 µM, respectively. This investigation proposed that compound 25 might be considered as a promising lead compound for the development of antihepatoma candidate.