Human Liver Cell Line L02 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.

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.

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.

Desmocollin-2 inhibits cell proliferation and promotes apoptosis in hepatocellular carcinoma via the ERK/c-MYC signaling pathway.

Hepatocellular carcinoma (HCC) is one of the most common cancers around the world with a poor prognosis. The main reason for poor prognosis is early stage HCC is inconspicuous so it is difficult to detect and effective treatment strategies are lacking for advanced HCC. In this context, novel molecular targets are urgently needed for the diagnosis and therapy of HCC. In this study, we investigated the expression level, biological function, and relative mechanism of Desmocollin-2(DSC2) in HCC. DSC2 expression levels were decreased significantly in HCC cell lines SMMC-7721(7721), Huh7, HCC-LM3(LM3), and MHCC-97H(97H), especially in LM3 cells, compared with human liver cell line L02(L02). DSC2 overexpression in LM3 cells could inhibit the proliferation (in vitro and in vivo), colony formation, migration, and invasion abilities of HCC cells, and promote cell apoptosis, while DSC2 inhibition in 7721 cells performed the opposite effect. Consistent with these results, regulating DSC2 expression in 7721 and LM3 cells could affect the expression levels of apoptosis-related proteins (Bax, Bcl-2, c-Caspase-3, Caspase-3, Caspase-8, and Survivin) and cell cycle-related proteins (Cyclin D1, Cyclin B1, CDK1, and CDK2). Furthermore, DSC2 expression was significantly negatively correlated with the levels of p-ERK and c-MYC in both LM3 and 7721 cell lines. These findings confirmed that DSC2 overexpression could inhibit the proliferation, migration, and invasion abilities while promoting apoptosis of HCC cells via the ERK/c-MYC signaling pathway. In a conclusion, DSC2 was a tumor suppressor with low expression in liver cancer.

Design and synthesis of novel 7-ethyl-10-fluoro-20-O-(cinnamic acid ester)-camptothecin derivatives as potential high selectivity and low toxicity topoisomerase I inhibitors for hepatocellular carcinoma

A series of new 7-ethyl-10-fluoro-20-O-(cinnamic acid ester)-camptothecin derivatives were synthesized and evaluated for cytotoxicity against four human tumor cell lines including HepG2 (hepatocellular carcinoma), SW480 (colorectal cancer), A2780 (ovarian cancer), and Hucct1 (intrahepatic cholangiocarcinoma). The results of cytotoxic activities in vitro showed that most of the camptothecin derivatives harbor promising cytotoxic activity against tested tumor cell lines. Among them, compound XJS-11 exhibited broad-spectrum inhibitory activities against HepG2, SW480, A2780, and Hucct1 cell lines with IC50 values of 0.03, 0.09, 0.22, and 0.32 μM, respectively. Further investigation demonstrated that compound XJS-11 exhibited more effective growth inhibition against a variety of human hepatoma cells (Sk-hep-1, Hep3B and Huh7) and lower cytotoxicity against immortalized normal human liver cell line L02 than the positive control topotecan. Especially, XJS-11 showed higher selective toxicity in two kinds of human hepatoma cells and immortalized normal human liver cell line (IC50(L-02)/IC50(HepG2) = 113.20; IC50(L-02)/IC50(Hep3B) = 85.60) than topotecan (IC50(L-02)/IC50(HepG2) = 9.45; IC50(L-02)/IC50(Hep3B) = 8.52). Mechanistically, XJS-11 induced cell cycle arrest and cell apoptosis in HepG2 and Hep3B cells by inhibiting Top I activity in a manner similar to that of topotecan. Meanwhile, XJS-11 could attenuate the tumor growth in both xenograft and primary HCC mouse models. In addition, the acute toxicity assay showed that XJS-11 did not cause lethality or significant body weight loss with a single intraperitoneal dose at 100 mg/kg or with an intraperitoneal dose at 25 mg/kg for 7 days. Moreover, unlike topotecan, XJS-11 had no apparent toxicity to the mouse liver, kidney, and hemopoietic system of the C57BL/6 mice. Taken together, XJS-11 merits further development as a new generation of the camptothecin-derived drug candidate.

Cytotoxicity and Apoptosis Induced by Chenopodium ambrosioides L. Essential Oil in Human Normal Liver Cell Line L02 via the Endogenous Mitochondrial Pathway Rather Than the Endoplasmic Reticulum Stress.

Chenopodium ambrosioides L. (C. ambrosioides) has been used as dietary condiments and as traditional medicine in South America. The oil of Chenopodium ambrosioides L. (C. ambrosioides) can be used as a natural antioxidant in food processing. It also has analgesic, sedating, and deworming effects, and can be used along with the whole plant for its medical effects: decongestion, as an insecticide, and to offer menstruation pain relief. This study was conducted to investigate the cytotoxicity and apoptosis effects of an essential oil from C. ambrosioides in vitro. The cytotoxicity evaluation of the essential oil from C. ambrosioides on human normal liver cell line L02 was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. AO/EB dual fluorescent staining assay and Annexin V-FITC were used for apoptosis analysis. The changes in mitochondrial membrane potential (MMP) were analyzed with 5,5,6,6′-tetrachloro-1,1,3,3,-tetraethyl-imidacarbocyanine iodide (JC-1) dye under a fluorescence microscope. The level of apoptosis related protein expression was quantified by Western blot. The L02 cells were treated with the essential oil from C. ambrosioides at 24, 48, and 72 h, and the IC50 values were 65.45, 58.03, and 35.47 μg/mL, respectively. The AO/EB staining showed that viable apoptotic cells, non-viable apoptotic cells, and non-viable non-apoptotic cells appeared among the L02 cells under the fluorescence microscope. Cell cycle arrest at the S phase and cell apoptosis increased through flow cytometry in the L02 cells treated with the essential oil. MMP decreased in a concentration-dependent manner, as seen through JC-1 staining under the fluorescence microscope. In the L02 cells as shown by Western blot and qPCR, the amount of the apoptosis-related proteins and the mRNA expression levels of cytochrome C, Bax, Caspase-9, and Caspase-3 increased, Bcl-2 decreased, and Caspase-12, which is expressed in the endoplasmic reticulum, showed no obvious changes in protein amount or mRNA expression level. The essential oil form C. ambrosioides had a cytotoxic effect on L02 cells. It could inhibit L02 cell proliferation, arrest the cell cycle at the S phase, and induce L02 cell apoptosis through the endogenous mitochondrial pathway.

3D organization profiling of human hepatocellular carcinoma cell line PLC/PRF/5 in comparison with normal human liver cell line L02 by in situ Hi-C

Genetic and epigenetic alterations accumulate in the process of hepatocellular carcinogenesis, but the role of genomic spatial organization in HCC is still unknown. Here, we performed in situ Hi-C in HCC cell line PLC/PRF/5 compared with normal liver cell line L02, together with RNA-seq and ChIP-seq of SMC3/CTCF/H3K27ac. The results indicate that there were significant compartment switching, TAD shifting and loop pattern altering in PLC/PRF/5. These spatial changes are correlated with abnormal gene expression and more opening promoter regions of the HCC cell line. Thus, the 3D genome organization alterations in PLC/PRF/5 are important in epigenetic mechanisms of HCC tumorigenesis.

Effects of microRNA-106b on hepatic gluconeogenesis and its underlying mechanism

Objective: To investigate the potential effects of microRNA-106b (miR-106b) on gluconeogenesis in normal human liver cell line L02 and its underlying mechanisms. Methods: Normal human liver L02 cells were cultured in DMEM containing 10% FBS and transfected with 20 nmol/L of miR-106b mimic or antagomiR-106b, respectively. Twenty-four hours later after transfection, Western blot was performed to detect the levels of proteins or phosphorylated proteins. Quantitative RT-PCR was carried out to measure the mRNA expressions of gluconeogenesis-related genes. Glucose Assay Kit was used to detect the glucose contents in the medium. Results: MiR-106b mimic significantly increased the protein abundances of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase)(P＜0.01,P＜0.01, respectively), enhanced the mRNA expression of phosphoenolpyruvate carboxykinase 1 (PCK1) (P＜0.01), and decreased the mRNA level of glucokinase (GCK) (P＜0.01). AntagomiR-106b dramatically reduced the protein levels of PEPCK and G6Pase (P＜0.01,P＜0.01, respectively), suppressed the mRNA level of PCK1 (P＜0.01), and increased the mRNA level of GCK(P＜0.01). In addition,miR-106b mimic or antagomiR-106b significantly reduced or enhanced the protein levels of signal transducer and activator of transcription 3 (STAT3)(P＜0.01,P＜0.01, respectively).The inhibition of STAT3 by its specific inhibitor abolished the inhibitory effects of antagomiR-106b on hepatic gluconeogenesis. Conclusion: miR-106b increases hepatic gluconeogenesis by inhibiting the STAT3 signaling pathway.

A milbemycin compound isolated from Streptomyces Sp. FJS31-2 with cytotoxicity and reversal of cisplatin resistance activity in A549/DDP cells

Streptomyces Sp FJS31-2 is a strain isolated from special habitat soils in the early stage of our laboratory for producing a new type of halogenated type II polyketide antibiotic with good anti-MRSA activity. In this experiment, a variety of chromatographic and spectroscopic methods was used to isolate and identify a milbemycin compound VM48130 from the ethyl acetate extract of the fermentation products. To investigate its bioactivity, Cell Counting Kit-8 (CCK-8) assay was used to test the cytotoxic activity of the compound against a variety of cancer cells (human liver cancer cell line MHCC97H and SK-Hep1, human nasopharyngeal carcinoma cell line CNE1, mouse melanoma cell line B16, human colon cancer cell line LOVO, human lung adenocarcinoma cell line A549) and normal cells (human bronchial epithelial cell line 16HBE, human normal liver cell line L02, human nasopharyngeal epithelial cell line NP69). The results showed that the compound had significant cytotoxic activity against the above cancer cells, and the IC50 values were 21.96 ± 1.45, 22.18 ± 0.55, 19.42 ± 0.71, 18.61 ± 1.68, 18.62 ± 0.67, 18.52 ± 0.64 μM, respectively. Furthermore, the CCK-8 method was used to evaluate the compound's reversal of cisplatin resistance in multidrug resistant cisplatin-resistant human lung adenocarcinoma (A549/DDP) cells. The results indicated that when the compound concentration was 0.5 μM, the reversal fold (RF) reached 6.25 and showed a dose-dependent effect. At 5 μM, the RF reached 8.35, which was approximately equivalent to the reversal effect of the positive drug verapamil at the same concentration. The expression of MDR1, MRP1, LRP, MAST1 resistance genes and the corresponding proteins were analyzed by quantitative RT-PCR and Western blot assay, and found that the compound could significantly down-regulate the expression of these genes and proteins. These results indicated that VM48130 had the potential of being a lead compound for the treatment or adjuvant treatment of cancer.

PP2Ac upregulates PI3K-Akt signaling and induces hepatocyte apoptosis in liver donor after brain death

Multiple research groups have demonstrated that the outcome of patients receiving liver grafts from brain death donors (DBD) is poorer when compared with patients receiving grafts from living donors. This might be due to an increased hepatocyte apoptosis induced after brain death (BD). In this work, we found that the activity of PP2A-Akt pathway is significantly increased in clinical donor ex vivo hepatocytes after BD by iTRAQ protein quantification analysis. The same results were confirmed in animal models. A time-dependent promotion of apoptosis was also found in DBD rabbit liver, as demonstrated by the increased levels of cleaved Caspase 3 and the decreased of Bcl-2. To further investigate the roles of PP2A and Akt in regulating apoptosis of hepatocytes after BD, we cultivated human liver cell line L02 with serum deprivation and hypoxia, to simulate the ischemic and hypoxic conditions of hepatocytes in DBD. Increased apoptosis and decreased viability were observed during the time in this model. Meanwhile PP2A activity and Akt activity were respectively increased and decreased. Notably, the proportion of Akt phosphorylation at Ser473 decreased, while other known targets of PP2A (p38, JNK and ERK) were not affected in terms of protein levels or phosphorylation. These results suggested that PP2A is involved in apoptotic induction of hepatocytes after brain death by specific suppression of Akt. This discovery was further confirmed with pharmaceutical and genetic methods. Our work implied potential targets for reducing liver cell apoptosis and improving organ donor quality after BD.

<p>Emodin-induced autophagy against cell apoptosis through the PI3K/AKT/mTOR pathway in human hepatocytes</p>

BackgroundEmodin, a major component of Polygonum multiflorum (PM), has been reported to exert both protective and toxic effects in several cell types. However, the effects and underlying mechanisms of action of emodin in hepatic cells are still obscure.MethodsThe present study used the normal human liver cell line L02 to investigate the effects and mechanisms of emodin in hepatic cells. After treatment with emodin, L02 cells were examined for viability, apoptosis and autophagy with the Cell Counting Kit-8 (CCK-8), annexin V/PerCP staining and GFP-LC3 plasmid transfection. The expression of proteins including cleaved caspase-3, LC3B-I/II, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR and actin was examined by using Western blot.ResultsEmodin significantly inhibited the viability of and induced apoptosis in L02 cells in a dose- and time-dependent manner. In addition, emodin increased the number of GFP-LC3 puncta in L02 cells and upregulated the expression of LC3B-II compared to those in control cells. Furthermore, emodin significantly decreased the expression of p-PI3K, p-AKT and p-mTOR in a dose-dependent manner compared to that in control cells without altering the expression of PI3K, AKT and mTOR. Notably, cotreatment with emodin and 3-methyladenine (3-MA) or rapamycin significantly increased and decreased the apoptosis rate of L02 cells, respectively, compared to that of cells treated with emodin alone.Conclusion In conclusion, emodin exhibited cytotoxicity in the L02 human hepatic cell line by promoting apoptosis, and it also induced autophagy through the suppression of the PI3K/AKT/mTOR signalling pathway. The autophagy could play a protective role following emodin treatment.

Genotoxic and mutagenic properties of atmospheric pressure plasma jet on human liver cell line L02

In clinical plasma medicine, the long-term safety is an important issue which needs to be addressed. In this study, normal human liver cell line L02 was chosen as an in vitro model to investigate the genotoxic and mutagenic properties of the atmospheric pressure plasma jet. The normal human liver cell line L02 was treated by the plasma first for different times, and then, the survived progeny cells from 30 min plasma treatment time after seven passages (approximately 20 generations) were treated by the plasma again. Apoptosis assay, micronucleus formation assay, and the hypoxanthine phosphoribosyl transferase (HPRT) gene mutation assay were performed on both L02 cells and the progeny of surviving cells after the first 30 min plasma treatment. It is found that plasma treatment induces cell death and micronucleus formation in a time dependent manner. However, no delayed genomic instability like delayed reproductive cell death and micronucleus formation was found in the progeny cells. Besides, it is found that the plasma treatment did not induce increased HPRT mutation frequency either in the L02 cells or the progeny of surviving cells.

Simultaneous quantitation of 14 DNA alkylation adducts in human liver and kidney cells by UHPLC-MS/MS: Application to profiling DNA adducts of genotoxic reagents

A rapid, sensitive and wide coverage ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method has been developed and validated for the simultaneous quantitation of 14 alkylation DNA adducts in cell genomic DNA, RNA and cell contents isolated from the in vitro cultured human kidney cell line 293 T and the human liver cell line L02 exposed to 3 genotoxic reagents: N-methyl-N-nitrosourea (MNU), methyl methanesulfonate (MMS) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). After exposure, DNA was isolated and directly hydrolysed under acid conditions or digested by enzymes to obtain the hydrolysates containing DNA alkylation adducts followed by optimization of the pretreatment method and chromatographic separation conditions. Quantification was performed on a Waters ACQUITY UPLC BEH Amide column (1.7 μm, 2.1 × 150 mm) using an electrospray ionization (ESI) source in positive mode by selective reaction monitoring (SRM) at the precursor to product ion transitions of 14 analytes. The method showed selectivity, good linearity (r＞0.9950), accuracy (82.1%–115%), and intra-day (RSD%＜14%) and inter-day (RSD%＜15%) precision for 14 analytes. The recoveries of two pretreatment methods were all more than 50.5%, and no relative matrix effects were observed. Additionally, the samples were stable after short-term storage at 20 ℃ for 2 h, at 4 ℃ for 48 h or one cycle of freeze-thaw at −80 ℃. The established UHPLC-MS/MS method was used to evaluate the changes in alkylation DNA adducts and epigenetic modification-related methylcytosine after exposure to genotoxic reagents. For the first time, the results demonstrated that 3 genotoxic reagents induced different total amounts of adducts in the following sequence: MMS ＞ NNK ＞ MNU, and showed significant differences in the ratios of 7MeG to 1MeA and 1MeG to 1MeA in the 293 T cell model. Meanwhile, 293 T and L02 cells revealed significantly different DNA adduct formation characteristics in the contents of 1MeG and 1MeA. The DNA adduct formation relationships between DNA, RNA, and cell contents were probed to predict cancer risk and potential genotoxic exposure. This approach could be used to investigate the DNA adducts, their formation and the relationship to the mutagenicity or carcinogenicity of genotoxic reagents in future studies.

Rosmarinic Acid Ameliorates H2O2-Induced Oxidative Stress in L02 Cells Through MAPK and Nrf2 Pathways.

Liver cells are easily damaged by oxidative stress during progression both in liver development and throughout adult life, resulting in tissue pathology that ranges from simple hepatitis to nonalcoholic fatty liver disease. In this study, we determined the attenuation of oxidative stress in liver cells with pretreatment of rosmarinic acid (RA), which is an antioxidant agent from Rosmarinus officinalis. The human liver cell line L02 was damaged by hydrogen peroxide (H2O2). In the RA treatment group, the viability of L02 cells increased and the intracellular reactive oxygen species levels decreased compared with the H2O2-induced damage group. Analysis of flow cytometry revealed that the percentage of G2/M cell cycle arrest and cell apoptosis decreased in the RA treatment group. This alteration was associated with activation of a G2/M DNA damage and oxidative stress apoptotic signal. Furthermore, we determined the redox-sensitive protein expression of mitogen-activated protein kinases (MAPKs), quinone acceptor oxidoreductase 1 (NQO1), and nuclear factor E2-related factor 2 (Nrf2), and the expression of both MAPKs and Nrf2 was activated in the RA group. Results showed that the relevant protein expression of MAPKs and Nrf2 was activated in the RA group. Thus, RA protected L02 cells from oxidative damage through suppressing cell cycle arrest and cell apoptosis with the activation of MAPK and Nrf2 signaling pathways.

Establishment and optimization of hepatocyte steatosis model

Objective: To establish a new model of hepatic steatosis cells by optimizing the original ethanol or high fat, the present study proposed an in vitro hepatocyte steatosis model for the study of fatty liver. Methods: Oil red O staining was used to observe the effects of fetal bovine serum, oleic acid and ethanol on lipid accumulation in human liver cell line L02 in a concentration- and time-dependent manner. RT-PCR was used to detect the mRNA expression levels of PPAR-γ and AP-2, and the suitable conditions for the establishment of hepatocyte steatosis model were screened out. A t-test was used for comparison between the two groups, and one-way Analysis of Variance (ANOVA) was used in more than three groups. Results: Oil red O staining showed the number of reddish-orange lipid droplets in L02 cells gradually increased with the increase of fetal bovine serum, oleic acid and ethanol in a concentration - and time-dependent manner. Compared with 0.00% oleic acid and 2% ethanol, the count value of red particle was 100.00% ± 17.63% at the beginning and after 24 h, 0.003% oleic acid and 2% ethanol jointly acted in L02 cells. After incubation for 48 hours with 2% ethanol and serum-free DMEM medium, the accumulation of lipid droplets was the highest with a count value of 802.38%+71.06%(t = 42.36, P < 0.001). RT-PCR analysis showed the lipid accumulation induced by this method was positively correlated with the mRNA expression of PPAR-γ and AP-2. Conclusion: L02 cells were successfully exposed to high fat and ethanol, and the hepatocyte steatosis model was established and optimized, suggesting that the occurrence of hepatic cell steatosis was related to the up-regulation of PPAR-γ and AP-2.

Adaptive homeostasis of the vitamin D–vitamin D nuclear receptor axis in 8-methoxypsoralen-induced hepatotoxicity

8-methoxypsoralen (8-MOP) with ultraviolet A radiation therapy (PUVA) is the standard therapy for patients with psoriasis, despite the reported potential risks of 8-MOP-induced cholestatic liver injury in both humans and animals. Usually, patients with chronic cholestasis exhibit lower serum 25-hydroxy vitamin D (25(OH)D) levels. But those patients receiving PUVA for psoriasis showed an increase in serum 25(OH)D levels, probably highlighting that the vitamin D–vitamin D nuclear receptor (VD-VDR) axis play a protective role in 8-MOP-induced hepatotoxicity. The present study confirmed 8-MOP could increase serum 25(OH)D levels in conventional lighting and diet (CLD) and vitamin D deficient (VDD) Sprague-Dawley rats. Potential liver risks were also found in CLD and VDD rats after 8-MOP treatment. We proved that 8-MOP could be a potent ligand for VDR using molecular docking and luciferase report assay. Effect of 8-MOP on VDR subcellular distribution was determined using human liver cell line L02. We found 8-MOP could increase VDR protein expression in the nuclear and cytosol extracts and also total cell extracts in L02. siRNAs for VDR were used to determine the role of VDR in protecting 8-MOP-induced cholestasis and potential cellular mechanisms. The results showed 8-MOP could affect the CYP7A1, SHP and MRP3 expression via VDR, and such effects could be reversed by knockdown of VDR expression, suggesting a vital role of VDR involved in 8-MOP-regulated bile acid synthesis and transportation. In conclusion, these results revealed activation of VD–VDR axis may play a beneficial role in 8-MOP-mediated regulation of bile acid synthesis and transportation.

PBN11-8, a Cytotoxic Polypeptide Purified from Marine Bacillus, Suppresses Invasion and Migration of Human Hepatocellular Carcinoma Cells by Targeting Focal Adhesion Kinase Pathways.

The development of antitumor drugs has attracted cancer researchers and the identification of novel antitumor lead compounds is certainly of great interest. The fermentation broth of Bacillus sp. N11-8, which was isolated from the Antarctic waters, showed cytotoxicity towards different cells. A cytotoxic polypeptide, PBN11-8, was purified from the fermentation broth of Bacillus sp. N11-8 using ultrafiltration, ammonium sulfate precipitation, anion exchange liquid chromatography and high performance liquid chromatography (HPLC). Cloning and sequence analysis showed that PBN11-8 polypeptide (MW: ~19 kDa by the electrospray-ionization (ESI)) displayed high similarity with peptidase M84 from Bacillus pumilus. PBN11-8 possessed moderate cytotoxicity towards several cancer cell lines with IC50 values of 1.56, 1.80, 1.57, and 1.73 µg/mL against human hepatocellular carcinoma cell line BEL-7402, human renal clear cell adenocarcinoma cell line 786-0, human hepatocellular carcinoma cell line HepG2, and human pancreatic cancer cell line Panc-28, respectively. Moreover, the polypeptide displayed weak cytotoxicity towards normal cell line renal tubular epithelial cell line HK2 and human normal liver cell line L02 cells. Wound healing migration and Transwell experiments demonstrate that PBN11-8 could inhibit the migration and invasion of BEL-7402. Further investigation revealed that PBN11-8 suppresses focal adhesion kinase (FAK)-mediated adhesion, migration, and invasion by disturbing FAK/extracellular regulated protein kinases (ERK) signaling and matrix metalloproteinase-2(MMP-2) and matrix metalloproteinase-9 (MMP-9) in BEL-7402 cells. Thus, PBN11-8 represents a potential novel anti-cancer lead compound.

Geniposide and Gentiopicroside Suppress Hepatic Gluconeogenesis via Regulation of AKT-FOXO1 Pathway

Hepatic gluconeogenesis plays an important role in regulating fasting plasma glucose levels and is a target of anti-diabetic drugs. Several kinds of iridoid glucosides exhibit hypoglycemic effect, whereas the mechanism was not clear. In this study, the effects of geniposide and gentiopicroside, two natural iridoid glucosides, on hepatic gluconeogenesis were investigated. Glucose uptake assay, MTT assay, q-PCR, luciferase assay and western blot assay were performed to investigate the pharmacological effect of geniposide and gentiopicroside on human liver cell line L02. Thereby the fast blood glucose and intraperitoneal glucose tolerance were measured in high fat diet induced hyperglycemic mice after geniposide or gentiopicroside administration. The results showed that geniposide and gentiopicroside inhibited the transcription of G6PC and PEPCK in L02cells and in mice. Additional experimental data indicated that these two compounds were able to inhibit the transcriptional activity of FOXO1 by inducing phosphorylation of AKT at Ser473. Furthermore, we found that these two compounds alleviated high fat diet induced hyperglycemia in mice. Geniposide and gentiopicroside might reduce blood glucose and suppress hepatic gluconeogenesis by regulating the AKT-FOXO1 pathway, and the potential use of these two iridoid glucosides as anti-diabetic agents merits further in-depth exploration.

Seasonal variations of tris (2-chloroethyl) phosphate and cytotoxicity of organic extracts in water samples from Wuhan, China.

Tris (2-chloroethyl) phosphate (TCEP) is a typical phosphate flame retardant. Its potential adverse health effects have recently aroused concern. We investigated the seasonal variations of TCEP concentrations in the raw, finished and tap water samples from two drinking water treatment plants (DWTPs) in China, and evaluated the cytotoxicity and apoptosis/necrosis of organic extracts (OEs) in water samples. We enriched TCEP and OEs in water samples by solid-phase extraction method. The TCEP concentrations in water samples were determined by gas chromatography-mass spectrometry. Normal human liver cell line L02 was treated with OEs in the water samples, and then the cytotoxicity and apoptosis/necrosis were measured by 3-(4, 5-dimethyithiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and flow cytometry, respectively. The results showed that cytotoxicities of OEs in raw water samples from both DWTPs in summer and winter were stronger than those in spring and autumn, cytotoxicity of OEs in finished and tap water samples from both DWTPs in summer and autumn were stronger than those in spring and winter. In all seasons, the maximal concentrations (100 mL water/mL cell culture) of OEs in the raw water samples from both DWTPs induced late apoptosis/necrosis. The reasons for seasonal variations of TCEP in water samples and potential toxic effects of other pollutants in the water samples need to be further investigated.

Alleviating the hepatotoxicity of trazodone via supramolecular encapsulation

In order to develop a novel strategy to alleviate the inherent hepatotoxicity of antidepressant trazodone (TZ), Cucurbit[7]uril (CB[7]) was adopted as pharmaceutical excipients and was studied for its capability to reduce the hepatotoxicity of TZ via supramolecular encapsulation. CB[7] was found to form strong 1:1 host-guest complexes with TZ and its metabolite m-chlorophenyl piperazine (mCPP), with binding constants of 1.50 (±0.13) × 106 M−1 and 6.90 (±0.49) × 105 M−1, respectively. The supramolecular complexations were examined by 1H NMR and UV-visible spectroscopic titrations, ESI-MS and ITC. In the presence of 0.5 mM CB[7], the IC50 values of TZ and mCPP on a human normal liver cell line L02 were increased from 215.5 ± 3.3 μM to 544.1 ± 51.2 μM, and from 166.8 ± 3.8 μM to 241.7 ± 6.8 μM, respectively. Evaluation on a zebrafish model demonstrated that CB[7] (0.1 mM) significantly alleviated the TZ induced liver toxicity, as shown by the level of liver degeneration, liver size and yolk sac retention. Our study may provide a supramolecular strategy to alleviate the hepatotoxicity induced by TZ and its metabolite mCPP, and this strategy may be extendable to other drugs that have inherent hepatotoxicity or other adverse effects.