LO2 Cells Research Articles

LR12 Promotes Liver Repair by Improving the Resolution of Inflammation and Liver Regeneration in Mice with Thioacetamide- (TAA-) Induced Acute Liver Failure.

Background Triggering receptor expressed on myeloid cells-1 (TREM-1) controls the mobilization of inflammatory cells in response to injury and consequently enhances liver damage. LR12 is a TREM-1 inhibitory peptide. However, the role of LR12 in acute liver failure (ALF) has remained elusive. This study was aimed at indicating whether LR12 could promote liver repair in mice with thioacetamide- (TAA-) induced ALF. Methods BALB/c mice were intraperitoneally injected with TAA, followed by intravenous injection of LR12. Damage and regeneration of the liver were assessed. LO2 cells and macrophages were used to assess the therapeutic effects of LR12. Results Mice treated with TAA for 24 h developed ALF, while liver inflammation was alleviated after LR12 treatment. Moreover, LR12 promoted hepatocyte regeneration in mice with TAA-induced ALF. In vitro, the supernatant from TAA+LR12-treated macrophages promoted the proliferation of LO2 cells. Cytokine protein microarray analysis suggested that LR12 promoted the secretion of C-C chemokine ligand 20 (CCL20) from macrophages. Besides, neutralization of CCL20 blocked the effects of LR12, thus inhibited the proliferation of LO2 cells in vitro, aggregated the liver inflammation, and restrained hepatocyte regeneration in ALF mice in vivo. Furthermore, we also found that LR12 activated the p38 mitogen-activated protein kinase (MAPK) pathway in hepatocytes through promoting the secretion of CCL20 from macrophages. Conclusions LR12 could improve the resolution of inflammation and liver regeneration in mice with TAA-induced ALF by promoting the secretion of CCL20 from macrophages and activating the p38 MAPK pathway. Therefore, LR12 could be an attractive therapeutic target for the treatment of ALF.

Dapagliflozin Alleviates Hepatic Steatosis by Restoring Autophagy via the AMPK-mTOR Pathway.

As a newly approved oral hypoglycaemic agent, the sodium-glucose cotransporter 2 (SGLT2) inhibitor dapagliflozin, which is derived from the natural product phlorizin can effectively reduce blood glucose. Recent clinical studies have found that dapagliflozin alleviates non-alcoholic fatty liver disease (NAFLD), but the specific mechanism remains to be explored. This study aimed to investigate the underlying mechanism of dapagliflozin in alleviating hepatocyte steatosis in vitro and in vivo. We fed the spontaneous type 2 diabetes mellitus rats with high-fat diets and cultured human normal liver LO2 cells and human hepatocellular carcinoma HepG2 cells with palmitic acid (PA) to induce hepatocellular steatosis. Dapagliflozin attenuated hepatic lipid accumulation both in vitro and in vivo. In Zucker diabetic fatty (ZDF) rats, dapagliflozin reduced hepatic lipid accumulation via promoting phosphorylation of acetyl-CoA carboxylase 1 (ACC1), and upregulating lipid β-oxidation enzyme acyl-CoA oxidase 1 (ACOX1). Furthermore, dapagliflozin increased the expression of the autophagy-related markers LC3B and Beclin1, in parallel with a drop in p62 level. Similar effects were observed in PA-stimulated LO2 cells and HepG2 cells. Dapagliflozin treatment could also significantly activated AMPK and reduced the phosphorylation of mTOR in ZDF rats and PA-stimulated LO2 cells and HepG2 cells. We demonstrated that dapagliflozin ameliorates hepatic steatosis by decreasing lipogenic enzyme, while inducing fatty acid oxidation enzyme and autophagy, which could be associated with AMPK activation. Moreover, our results indicate that dapagliflozin induces autophagy via the AMPK-mTOR pathway. These findings reveal a novel clinical application and functional mechanism of dapagliflozin in the treatment of NAFLD.

Shikonin treatment ameliorates Lipopolysaccharides (LPS) induced acute liver failure in mice via regulating the miR-106/MCL1 and miR-34a/SIRT1/TP53 signaling

IntroductionThe treatment with shikonin (SKN) suppresses the expression of miR-106 and miR-34a. Furthermore, SIRT1 and MCL1 are targets of miR-34a and miR-106, respectively. In this study, we treated an animal model of ALF with high dose (1.0 mg/kg) and low dose (0.5 mg/kg) of SKN to investigate its effect on liver functions and signaling pathways of SKN/miR-106/MCL1 and SKN/miR-34a/SIRT1/TP53.Material and methodsALF animal model was established and the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed to evaluate the effects of different doses of SKN. TUNNEL was performed to assess hepatocyte apoptosis. Luciferase assay, RT-qPCR and Western blot analysis were performed to measure the relationship between miR-106, miR-34a, SIRT1 and MCL1.ResultsIn the ALF mice models, the administration of SKN decreased the levels of ALT and AST in a dose-dependent manner, along with a significantly decreased number of apoptotic hepatocytes. And SKN may protect liver during ALF via reducing the level of inflammation. Luciferase assay showed that the co-transfection of wild-type MCL1/SIRT1 and miR-106/miR-34a significantly decreased the luciferase activity of LO2 cells, thus indicating that MCL1 and SIRT1 are identified as targets of miR-106 and miR-34a, respectively, while SIRT1 could act as a regulator of TP53. Moreover, the expression of miR-106, miR-34a and TP53 was decreased over an increasing concentration of SKN, along with the increasing mRNA and protein levels of MCL1 and SIRT1.ConclusionsIn this study, we showed that SKN alleviated ALF in a dose-dependent manner via regulating the signaling pathways of SKN/miR-106/MCL1 and SKN/miR-34a/SIRT1/TP53.

Pulchinenosides: Correlation of surface activity-cytotoxicity and hepatocyte apoptosis mechanism study

Saponin is an active component of many phytomedicine, which has extensive pharmacology effects. Meanwhile, it is reported that cytotoxicity, especially hemolysis and hepatotoxicity, in pentacyclic triterpenoid saponin (PTS) hindered their further development and application. Surface activity, a unique physical property of saponins, is believed to be related to membrane toxicity. However, the correlation between the surface activity and cytotoxicity of saponins is still unexplained. In this paper, our aim was to explore the relationship between surface activity-cytotoxicity of pulchinenosides and the hepatotoxicity mechanism of PTS in vitro. The surface activity of different saponins was investigated by contact angle, surface free energy (SFE), and oil/water partition coefficient (log Papp). In the cytotoxicity study, the hemolysis and hepatotoxicity activity of different saponins was compared by HD50 of erythrocyte and MTT, flow cytometry and LDH assay in LO2 cells respectively. And in the hepatotoxicity mechanism study, western blot was used for observing the expression of proteins related to apoptosis and exploring the liver injury mechanism of PTS. The results suggested that the influences of surface activity on hepatocytes and erythrocytes were different, indicating that the correlation of surface activity-cytotoxicity could provide more information for development of PTS. And the result of hepatotoxicity mechanism study of saponins suggested that endogenous and exogenous apoptotic pathways could be the potential targets of PTS, which could not only provide basis for clinical monitoring and treatment of the toxicity in saponins, but also provide more reference for the clinical application of PTS and phytomedicine containing PTS.

Discovery of new chalone adamantyl arotinoids having RXRα-modulating and anticancer activities

In the present study, a new series of chalcone adamantly arotinoids (chalcone AdArs) derived from RAR antagonist MX781, are synthesized, characterized, and evaluated for the biological activities in vitro. The studies of antiproliferative activity and RXRα-binding affinity of target compounds result in the discovery of a lead candidate (WA15), which is a good RXRα binder (Kd = 2.89 × 10−6 M) with potent antiproliferative activity against human cancer cell lines (IC50 ≈ 10 μM) and low toxic to normal LO2 and MRC-5 cells (IC50 > 50 μM). Different from MX781, WA15 eliminates RARα antagonist activity but inhibits 9-cis-RA-induced RXRα transactivation activity in a dose-dependent manner. Compound WA15 is found to be a good apoptosis inducer in various cancer cells and promotes cell apoptosis in an RXRα-independent manner. Besides, WA15 shows the induction of proteasome-dependent RXRα degradation which might enhance the WA15-induced apoptosis. Finally, the immunoblotting indicates that WA15 can inhibit the TNFα-induced IKK activation and IκBα degradation, suggesting that the anticancer activity of WA15 might be related to the inhibition of IKK/NF-κB signal pathway.

Hesperidin alleviates insulin resistance by improving HG-induced oxidative stress and mitochondrial dysfunction by restoring miR-149

BackgroundHesperidin, a natural flavanone, has been proven to have multiple protective effects in diabetic rats, such as antioxidant, anti-inflammatory and anti-apoptotic effects. However, the molecular mechanisms underlying the effects of hesperidin are not well elucidated.MethodsLO2 cells were stimulated with high glucose (HG, 33 mM) for 24 h to establish a model of oxidative stress. Then, cell viability was determined using the MTT assay. The antioxidant activities, including the reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels, mitochondrial membrane potential (MMP) and adenosine-triphosphate (ATP) production, were measured with the corresponding kits. The levels of gene expression, protein expression and methylation were detected using qRT-PCR, western blotting and methylation-specific PCR (MSP) assays, respectively.ResultsCompared to the NG treatment, hesperidin treatment increased the viability and improved the oxidative stress, mitochondrial dysfunction and insulin resistance of HG-treated LO2 cells, and these effects were correlated with heightened SOD and GPx activities, increased MMP level and ATP generation, reduced MDA, ROS and glucose levels, and activated GSK3β/AKT and inactivated IRS1 signals. Mechanistically, hesperidin treatment enhanced the miR-149 expression level by reducing its promoter methylation by inhibiting DNMT1. Importantly, knockdown of miR-149 obviously abolished the biological roles of hesperidin.ConclusionsOur findings demonstrated that hesperidin treatment ameliorated HG-induced insulin resistance by reducing oxidative stress and mitochondrial dysfunction partly by suppressing DNMT1-mediated miR-149 silencing.

TWIST2 and the PPAR signaling pathway are important in the progression of nonalcoholic steatohepatitis

BackgroundTo investigate the roles of the transcription factors twist family bHLH transcription factor 1 (TWIST1), twist family bHLH transcription factor 2 (TWIST2), and peroxisome proliferator activated receptor gamma (PPARγ) in the progression of nonalcoholic steatohepatitis.MethodsThe protein levels of TWIST1, TWIST2 and PPARγ were determined in the serum of nonalcoholic fatty liver disease (NAFLD) patients and healthy controls by enzyme-linked immunosorbent assay (ELISA). An in vivo model for fatty liver was established by feeding C57BL/6 J mice a high-fat diet (HFD). An in vitro model of steatosis was established by treating LO-2 cells with oleic acid (OA). RNA sequencing was performed on untreated and OA-treated LO-2 cells followed by TWIST1, TWIST2 and PPARγ gene mRNA levels analysis, Gene Ontology (GO) enrichment and pathway analysis.ResultsThe TWIST2 serum protein levels decreased significantly in all fatty liver groups (P < 0.05), while TWIST1 varied. TWIST2 tended to be lower in mice fed an HFD and was significantly lower at 3 months. Similarly, in the in vitro model, the TWIST2 protein level was downregulated significantly at 48 and 72 h after OA treatment. RNA sequencing of LO-2 cells showed an approximately 2.3-fold decrease in TWIST2, with no obvious change in TWIST1 and PPARγ. The PPAR signaling pathway was enriched, with 4 genes upregulated in OA-treated cells (P = 0.0018). The interleukin (IL)-17 and tumor necrosis factor (TNF) signaling pathways were enriched in OA-treated cells.ConclusionsThe results provide evidence that the TWIST2 and PPAR signaling pathways are important in NAFLD and shed light on a potential mechanism of steatosis.

Roles of SET7/9 and LSD1 in the Pathogenesis of Arsenic-induced Hepatocyte Apoptosis.

Background and AimsMultiple regulatory mechanisms play an important role in arsenic-induced liver injury. To investigate whether histone H3 lysine 4 (H3K4) methyltransferase (SET7/9) and histone H3K4 demethyltransferase (LSD1/KDM1A) can regulate endoplasmic reticulum stress (ERS)-related apoptosis by modulating the changes of H3K4 methylations in liver cells treated with arsenic.MethodsApoptosis, proliferation and cell cycles were quantified by flow cytometry and real-time cell analyzer. The expression of ERS- and epigenetic-related proteins was detected by Western blot analysis. The antisense SET7/9 expression vector and the overexpressed LSD1 plasmid were used for transient transfection of LO2 cells. The effects of NaAsO2 on the methylation of H3 in the promoter regions of 78 kDa glucose-regulated protein, activating transcription factor 4 and C/EBP-homologous protein were evaluated by chromatin immunoprecipitation assay.ResultsThe protein expression of LSD1 (1.25±0.08 vs. 1.77±0.08, p=0.02) was markedly decreased by treatment with 100 µM NaAsO2, whereas the SET7/9 (0.68±0.05 vs. 1.10±0.13, p=0.002) expression level was notably increased, which resulted in increased H3K4me1/2 (0.93±0.64, 1.19±0.22 vs. 0.71±0.13, 0.84±0.13, p=0.03 and p=0.003). After silencing SET7/9 and overexpressing LSD1 by transfection, apoptosis rate (in percentage: 3.26±0.34 vs. 7.04±0.42, 4.80±0.32 vs. 7.52±0.38, p=0.004 and p=0.02) was significantly decreased and proliferation rate was notably increased, which is reversed after inhibiting LSD1 (in percentage: 9.31±0.40 vs. 7.52±0.38, p=0.03). Furthermore, the methylation levels of H3 in the promoter regions of GRP78 (20.80±2.40 vs. 11.75±2.47, 20.46±2.23 vs. 14.37±0.91, p=0.03 and p=0.01) and CHOP (48.67±4.04 vs. 16.67±7.02, 59.33±4.51 vs. 20.67±3.06, p=0.004 and p=0.001) were significantly increased in LO2 cells exposed to 100 µM NaAsO2 for 24 h.ConclusionsHistone methyltransferase SET7/9 and histone demethyltransferase LSD1 jointly regulate the changes of H3K4me1/me2 levels in arsenic-induced apoptosis. NaAsO2 induces apoptosis in LO2 cells by activating the ERS-mediated apoptotic signaling pathway, at least partially by enhancing the methylation of H3 on the promoter regions of ERS-associated genes, including GRP78 and CHOP.

The mechanism of TiaoGanYiPi formula for treating chronic hepatitis B by network pharmacology and molecular docking verification

The Chinese herbal formula TiaoGanYiPi (TGYP) showed effective against chronic hepatitis B (CHB) caused by hepatitis B virus (HBV) infection. Hence, we aimed to clarify the mechanisms and potential targets between TGYP and CHB. The active compounds and related putative targets of TGYP, and disease targets of CHB were obtained from the public databases. The key targets between TGYP and CHB were identified through the network construction and module analysis. The expression of the key targets was detected in Gene Expression Omnibus (GEO) dataset and normal hepatocyte cell line LO2. We first obtained 11 key targets which were predominantly enriched in the Cancer, Cell cycle and HBV-related pathways. And the expression of the key targets was related to HBV infection and liver inflammation verified in GSE83148 database. Furthermore, the results of real-time quantitative PCR and CCK-8 assay indicated that TGYP could regulate the expression of key targets including CCNA2, ABL1, CDK4, CDKN1A, IGFR and MAP2K1, and promote proliferation of LO2 cells. In coclusion, we identified the active compounds and key targets btween TGYP and CHB, and found that the TGYP might exhibite curative effect on CHB via promoting hepatocyte proliferation and inhibiting the liver inflammatory processes.

Different Influences of trans Fatty Acids on the Phospholipase A2 and Arachidonic Acid Metabolic Pathway in Hepatocytes.

This study investigated the effects of 9t18:1 (representing I-TFAs), 9t16:1, and 11t18:1 (representing R-TFAs) and their mixtures on the normal human hepatocyte LO2 cell function, the possible mechanism of lipid metabolism by lipidomics, and the relationship between phospholipase A2 (PLA2) and the arachidonic acid (AA) metabolic pathway. Here, we found that the damaging effect of 9t18:1 on the LO2 cell function was significantly greater than those of 11t18:1 and 9t16:1 (p < 0.05), and the damaging effects of CHB and HSO were significantly greater than those of HHB and CM (p < 0.05). The lipidomic results showed that TFAs and TFA mixtures caused a significant change in the lipid profiles of LO2 cells, in which the TAG, PL, and OL contents increased significantly. Moreover, 9t18:1 regulated only the protein expression of cPLA2 but did not participate in the AA metabolic pathway, while 11t18:1 and 9t16:1 participated in the COX-2 and CYP450 pathways, respectively.

Novel carbazole-oxadiazoles as potential Staphylococcus aureus germicides

Staphylococcus aureus resistance poses nonnegligible threats to the livestock industry. In light of this, carbazole-oxadiazoles were designed and synthesized for treating S. aureus infection. Bioassay discovered that 3,6-dibromocarbazole derivative 13a had effective inhibitory activities to several Gram-positive bacteria, in particular to S. aureus, S. aureus ATCC 29213, MRSA and S. aureus ATCC 25923 (MICs = 0.6–4.6 nmol/mL), which was more active than norfloxacin (MICs = 6–40 nmol/mL). Subsequent studies showed that 3,6-dibromocarbazole derivative 13a acted rapidly on S. aureus ATCC 29213 and possessed no obvious tendency to induce bacterial resistance. Further evaluations indicated that 3,6-dibromocarbazole derivative 13a showed strong abilities to disrupt bacterial biofilm and interfere with DNA, which might be the power sources of antibacterial performances. Moreover, 3,6-dibromocarbazole derivative 13a also exhibited slight cell lethality toward Hek 293 T and LO2 cells and low hemolytic toxicity to red blood cells. The above results implied that the active molecule 13a could be studied in the future development of agricultural available antibiotics.

Effect of Metformin on Lactate Metabolism in Normal Hepatocytes under High Glucose Stress in Vitro

Objective To study the effect of metformin on lactate metabolism in hepatocytes in vitro under high glucose stress. In vitro LO2 cells, liver cells were randomly divided into blank control group, 25 tendency/L glucose solution, 27 tendency/L glucose solution,29 tendency/L glucose solution, 31 tendency/L glucose solution, 33 tendency/L glucose solution,35 tendency/L glucose solution treatment group, the optimal concentration of 31 tendency after L, use 30 tendency for L metformin solution, and then divided into blank control group, the optimal concentration of glucose solution, normal liver cells + metformin solution normal liver cells. The optimal concentration of glucose solution normal liver cells + metformin solution respectively in the 12 h, 24 h,48 h on cell count plate to calculate the number of liver cells, and using lactic acid determination kit the optimal concentration of glucose solution + normal liver cells and normal liver cells + the optimal concentration of glucose solution + metformin solution respectively in the 12 h, 24 h, 48 h of cell cultures of lactic acid value. There was no significant change in the lactic acid concentration but significant increase in the number of surviving hepatocytes in the highglycemic control group compared with that in the high-glycemic control group without metformin. Metformin has no significant effect on lactic acid metabolism of hepatocytes under high glucose stress in vitro, and has a protective effect on hepatocytes under high glucose stress. Based on this,it is preliminarily believed that metformin is not the direct factor leading to diabetic lactic acidosis.

Paeoniflorin inhibits lipopolysaccharide-induced inflammation in LO2 cells by regulating RhoA/NLRP3 pathway

Inflammation is an essential component of liver diseases. Paeoniflorin (PF), a monoterpenoid component derived from peony root ( Paeonia lactiflora Pall.), has anti-inflammatory, immunoregulatory, and hepatoprotective activities. However, whether PF affects liver inflammation and its underlying mechanisms is unclear. In this study, we investigated the effects of PF on lipopolysaccharide (LPS)-induced inflammation in LO2 cells and the underlying molecular mechanism. LPS was used to induce inflammation. After PF pretreatment for 2 h, the cells were treated with PF and LPS. Cell counting kit-8 was used to measure cell viability. Tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were tested by Enzyme-linked immunosorbent assay. Western blot was used to evaluate TNF-α, Ras homolog family member A (RhoA), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and IL-1β proteins expression. In LPS-induced LO2 cells, PF reduced TNF-α and IL-6 inflammatory cytokine production in a dose-dependent manner. LPS-induced TNF-α expression was also suppressed by PF. In addition, PF significantly inhibited LPS-induced RhoA activation ( P = .0014). Finally, PF suppressed LPS-induced NLRP3 inflammasome activation by downregulating NLRP3, ASC, caspase-1, and IL-1β expression. These findings suggest that PF alleviates inflammation induced by LPS and further suggest the anti-inflammatory effect of PF may follow via reduced RhoA and NLRP3 inflammasome activity.

Astragaloside IV Alleviates Liver Inflammation, Oxidative Stress and Apoptosis to Protect Against Experimental Non-Alcoholic Fatty Liver Disease.

PurposeNon-alcoholic fatty liver disease (NAFLD) is the main form of chronic liver disease in the world. Astragaloside IV (ASIV) has been tested in experimental models of different diseases. The purpose of this study was to evaluate the effect and protective mechanism of ASIV on NAFLD.MethodsLipopolysaccharide (LPS)- and palmitate acid (PA)-induced RAW264.7 cells and LO2 cells were used as a NAFLD model. The mice NAFLD model was evaluated by hematoxylin-eosin staining (HE staining), and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Liver lipid metabolism was evaluated by triglyceride (TG) and total cholesterol (TC) kits and oil red O staining. Oxidative stress indicators were examined through biochemical methods. Inflammatory factors were explored through enzyme-linked immuno sorbent assay (ELISA), real-time quantitative PCR and oxidative stress indicator kits. The expression levels of 5-LO (5-lipoxygenase) and leukotriene A4 hydrolase (LTA4H) were checked by real-time quantitative PCR and Western blotting. Apoptosis was detected by Annexin V-FITC/PI cell apoptosis detection kit.ResultsOur results showed that in vivo ASIV significantly reduced liver tissue damage, and serum AST, ALT and serum TG levels in NAFLD mice. In vitro, ASIV reduced cell supernatant TG and TC content increased by PA treatment, and significantly decreased the accumulation of intracellular lipid droplets induced by PA treatment. Additionally, ASIV reduced reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and restored glutathione peroxidase (GSH-Px) levels in PA-treated LO2 cell supernatant. Furthermore, ASIV inhibited the production of proinflammatory cytokines (IL-6 and TNF-α) in RAW264.7 cells induced by LPS. We also found that ASIV downregulated the expression of 5-LO and LTB4 (leukotriene B4) in NAFLD mice. Moreover, ASIV restored apoptotic protein (Bax and Bcl-2) expression in PA-treated LO2 cells.ConclusionASIV may reduce liver steatosis, hepatocyte oxidative stress and apoptosis, and decrease liver inflammation, thereby attenuating the progression of NAFLD and thus might be of therapeutic interest.

The role of Physalis angulata as potential anti-type 2 diabetic agent

Background: Medicinal plants play important roles in the management of several diseases including diabetes. Objective: The aim of this study is to investigate the mechanism of action of Physalis angulata (PA) for its anti-diabetic activity using an in vitro model. Materials and Methods: Alpha-amylase inhibition was investigated through dinitrosalicylic acid assay. Glucose uptake was determined using LO-2 cell model. Radical scavenging activity was performed through 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS+) assay. Nitric oxide (NO) production was measured using the Griess reaction. Cell viability was examined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Results: The result showed that PA extract was able to inhibit enzyme alpha-amylase activity up to (56.6% ± 4.7%) at the concentration of 200 μg/ml. Moreover, PA possessed glucose adsorption and glucose uptake capacity up to (2.2 ± 0.18) mM glucose/g extract and (156% ± 10.1%), respectively. In addition, PA extract scavenged (52.6% ± 3.5%) DPPH and (59.7% ± 2.6%) ABTS + radicals and reduced NO production to (34.2% ± 3.8%) from RAW264.7 cells without any cytotoxic effects. Conclusion: PA could be suggested as pharmaceutical ingredient for the development of anti-diabetic products.

Landscape of Chimeric RNAs in Non-Cancerous Cells.

Gene fusions and their products (RNA and protein) have been traditionally recognized as unique features of cancer cells and are used as ideal biomarkers and drug targets for multiple cancer types. However, recent studies have demonstrated that chimeric RNAs generated by intergenic alternative splicing can also be found in normal cells and tissues. In this study, we aim to identify chimeric RNAs in different non-neoplastic cell lines and investigate the landscape and expression of these novel candidate chimeric RNAs. To do so, we used HEK-293T, HUVEC, and LO2 cell lines as models, performed paired-end RNA sequencing, and conducted analyses for chimeric RNA profiles. Several filtering criteria were applied, and the landscape of chimeric RNAs was characterized at multiple levels and from various angles. Further, we experimentally validated 17 chimeric RNAs from different classifications. Finally, we examined a number of validated chimeric RNAs in different cancer and non-cancer cells, including blood from healthy donors, and demonstrated their ubiquitous expression pattern.

Effects of chitinase-3-like protein 1 on brain death-induced hepatocyte apoptosis via PAR2-JNK-caspase-3

Hepatocyte apoptosis is a crucial factor affecting liver quality in brain-dead donors. The identification of key molecular proteins involved in brain-death (BD)-induced hepatocyte apoptosis may help determine an effective method for improving the quality of livers from brain-dead donors. In this study, we used in vivo and in vitro models to investigate the role of chitinase-3-like protein 1 (CHI3L1) in promoting liver cell apoptosis after BD. Chitin was used to inhibit CHI3L1 in a rat model of BD. Macrophage polarization of THP-1 cells and hypoxia/reoxygenation (H/R) of LO-2 cells were used to mimic BD-induced cell stress in liver. We found that CHI3L1 played a vital role in promoting liver cell apoptosis. Six hours after BD, CHI3L1 expression was significantly upregulated in liver macrophages and was associated with BD-induced M1 polarization of these cells. In liver cells cultured under H/R conditions, recombinant CHI3L1 activated the protease-activated receptor 2 (PAR2)/c-June N-terminal kinase (JNK) apoptotic pathway and aggravated apoptosis. Compared with the control group, chitin particles inhibited the expression of CHI3L1 in the liver of brain dead rats, thereby reducing activation of the hepatocyte surface receptor, PAR2, and its downstream JNK/caspase-3 signaling pathway, ultimately reducing hepatocyte apoptosis. In conclusion, our results indicate that CHI3L1 relies on a PAR2/JNK-mediated mechanism to promote BD-induced hepatocyte apoptosis.

MTOR regulates PRMT1 expression and mitochondrial mass through STAT1 phosphorylation in hepatic cell

BackgroundFasting changes mitochondrial function, and mTOR acts as a major regulator of mitochondrial energy production ensuring the survival under reduced supply of nutrition. This study assessed the role of protein arginine methyltransferase 1 (PRMT1), which regulates mitochondrial function, in the context of fasting. MethodsThe effect of fasting on mTOR signaling and mTOR-regulated mitochondrial mass was assessed in LO2 cells (in vitro) and C57BL/6J mice (in vivo). Biochemical parameters of fasting were determined in blood samples of mice. PRMT1 expression was investigated by transfecting LO2 cells with an expression vector. Gene expression was determined by real-time quantitative PCR, protein interaction by chromatin immunoprecipitation, protein expression by Western blotting and immunofluorescence microscopy, and the mitochondrial mass by MitoTracker staining. ResultsAfter 48 h of fasting, mTOR and PRMT1 expression, as well as mitochondrial mass, were significantly reduced in LO2 cells, and in liver tissue sections. Fasting downregulated the expression of miR-21 and upregulated the expression of its target phosphatase and tensin homolog (PTEN), which was responsible for reduced mTOR expression. Inhibition of mTOR reduced phosphorylation of STAT1, and thereby PRMT1 expression in LO2 cells. Low PRMT1 down-regulated the expression of peroxisome proliferator-activated receptor (PPAR)-γ and thereby decreased mitochondrial mass. Supplementation of insulin contracted the effect of fasting on all mentioned parameters. ConclusionsFasting downregulates miR-21 and increases its target PTEN, thereby inhibiting mTOR signaling, p-STAT1, PRMT1, and mitochondrial mass. These findings highlight the role of mTOR and PRMT1 in the regulation of cellular energy availability.

Prenatal Exposure to Retrorsine Induces Developmental Toxicity and Hepatotoxicity of Fetal Rats in a Sex-Dependent Manner: The Role of Pregnane X Receptor Activation.

Pyrrolizidine alkaloids (PAs) are a type of natural phytotoxin that contaminate food and feed and become an environmental health risk to humans and livestock. PAs exert toxicity that requires metabolic activation by cytochrome P450 (CYP) 3A, and case reports showed that fetuses are quite susceptible to PAs toxicity. The aim of this study was to explore the characteristics of developmental toxicity and fetal hepatotoxicity induced by retrorsine (RTS, a typcial toxic PA) and the underlying mechanism. Pregnant Wistar rats were intragastrically administered with 20 mg/(kg·day) RTS from gestation day (GD) 9 to 20. Results showed that prenatal RTS exposure lowered fetal bodyweights, reduced hepatocyte numbers, and potentiated hepatic apoptosis in fetuses, particularly females. Simutaneously, RTS increased CYP3A expression and pregnane X receptor (PXR) activation in female fetal liver. We further confirmed that RTS was a PXR agonist in LO2 and HepG2 cell lines. Furthermore, agonism or antagonism of androgen receptor (AR) either induced or blocked RTS-mediated PXR activation, respectively. As a PXR agonist, RTS toxicity was exacerbated in female fetus due to the increased CYP3A induction and self-metabolism, while the inhibitory effect of AR on PXR activation reduced the susceptibility of male fetus to RTS. Our findings indicated that PXR may be a potential therapeutic target for PA toxicity.

Three Novel Dietary Phenolic Compounds from Pickled Raphanus Sativus L. Inhibit Lipid Accumulation in Obese Mice by Modulating the Gut Microbiota Composition.

Although pickled radish is widely consumed worldwide, few studies have investigated the nutritional benefits of bioactive compounds extracted from pickled radish. In this study, the authors investigate the relationship among dietary phenolic compounds, lipid accumulation, and gut microbiota. Three phenolic compounds 2,6-dihydroxyacetophenone (DHAP), 4-hydroxyphenethyl alcohol (4-HPEA), and 4-hydroxybenzaldehyde (HBA) are extracted from pickled radish. LO2 cells treated with free fatty acid are first used to explore the impact of the above three compounds at different doses on reducing lipid levels. The effects of the three compounds on obesity and the gut microbiota are further investigated in high-fat diet (HFD)-induced KM mice. Results show that three compounds inhibited the lipid accumulation in LO2 cells. The results of animal experiments reveal that three compounds prevented body weight gain and significantly decreased serum lipid levels. Treatment with DHAP, HPEA, and HBA reversed gut microbiome dysbiosis in HFD-induced mice. The three phenolic compounds increase Odoribacter, and decrease Helicobacter and Mucispirillum. Notably, DHAP and HBA reduce the HFD-induced increase in the Firmicutes/Bacteroidetes ratio. These data suggest that phenolic compounds extracted from pickled radish possess excellent lipid-lowering capacity, providing a theoretical basis for further analysis of the nutritional value of pickled radish.