OBJECTIVES: To investigate the mechanism of comorbidity between non-alcoholic fatty liver disease (NAFLD) and atherosclerosis (AS) based on metabolomics and network pharmacology. METHODS: Six ApoE^－/－ mice were fed with a high-fat diet for 16 weeks as a comorbid model of NAFLD and AS (model group). Normal diet was given to 6 wildtype C57BL/6J mice (control group). Serum samples were taken from both groups for a non-targeted metabolomics assay to identify differential metabolites. Network pharmacology was applied to explore the possible mechanistic effects of differential metabolites on AS and NAFLD. An in vitro comorbid cell model was constructed using NCTC1469 cells and RAW264.7 macrophage. Cellular lipid accumulation, cell viability, morphology and function of mitochondria were detected with oil red O staining, CCK-8 assay, transmission electron microscopy and JC-1 staining, respectively. RESULTS: A total of 85 differential metabolites associated with comorbidity of NAFLD and AS were identified. The top 20 differential metabolites were subjected to network pharmacology analysis, which showed that the core targets of differential metabolites related to AS and NAFLD were STAT3, EGFR, MAPK14, PPARG, NFKB1, PTGS2, ESR1, PPARA, PTPN1 and SCD. The Kyoto Encyclopedia of Genes and Genomes showed the top 10 signaling pathways were PPAR signaling pathway, AGE-RAGE signaling pathway in diabetic complications, alcoholic liver disease, prolactin signaling pathway, insulin resistance, TNF signaling pathway, hepatitis B, the relax in signaling pathway, IL-17 signaling pathway and NAFLD. Experimental validation showed that lipid metabolism-related genes PPARG, PPARA, PTPN1, and SCD were significantly changed in hepatocyte models, and steatotic hepatocytes affected the expression of macrophage inflammation-related genes STAT3, NFKB1 and PTGS2; steatotic hepatocytes promoted the formation of foam cells and exacerbated the accumulation of lipids in foam cells; the disrupted morphology, impaired function, and increased reactive oxygen species production were observed in steatotic hepatocyte mitochondria, while the formation of foam cells aggravated mitochondrial damage. CONCLUSIONS Abnormal lipid metabolism and inflammatory response are distinctive features of comorbid AS and NAFLD. Hepatocyte steatosis causes mitochondrial damage, which leads to mitochondrial dysfunction, increased reactive oxygen species and activation of macrophage inflammatory response, resulting in the acceleration of AS development.
Animals ; Mice ; Non-alcoholic Fatty Liver Disease/metabolism* ; Cyclooxygenase 2/metabolism* ; PPAR gamma/metabolism* ; Reactive Oxygen Species/metabolism* ; Mice, Inbred C57BL ; Hepatocytes ; Macrophages/metabolism* ; Liver

OBJECTIVE: To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout. METHODS: The Sidt2 knockout (Sidt2^-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed. RESULTS: Sidt2^-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2^-/- HL7702 cells. CONCLUSION Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.
Humans ; Lysosome-Associated Membrane Glycoproteins/metabolism* ; Autophagy ; Apoptosis ; Hepatocytes ; Lysosomes/metabolism* ; Chloroquine/pharmacology* ; Nucleotide Transport Proteins/metabolism*

The liver has a complex cellular composition and a remarkable regenerative capacity. The primary cell types in the liver are two parenchymal cell populations, hepatocytes and cholangiocytes, that perform most of the functions of the liver and that are helped through interactions with non-parenchymal cell types comprising stellate cells, endothelia and various hemopoietic cell populations. The regulation of the cells in the liver is mediated by an insoluble complex of proteins and carbohydrates, the extracellular matrix, working synergistically with soluble paracrine and systemic signals. In recent years, with the rapid development of genetic sequencing technologies, research on the liver's cellular composition and its regulatory mechanisms during various conditions has been extensively explored. Meanwhile breakthroughs in strategies for cell transplantation are enabling a future in which there can be a rescue of patients with end-stage liver diseases, offering potential solutions to the chronic shortage of livers and alternatives to liver transplantation. This review will focus on the cellular mechanisms of liver homeostasis and how to select ideal sources of cells to be transplanted to achieve liver regeneration and repair. Recent advances are summarized for promoting the treatment of end-stage liver diseases by forms of cell transplantation that now include grafting strategies.
Humans ; Liver/surgery* ; Hepatocytes/transplantation* ; Stem Cells/metabolism* ; Liver Diseases/surgery*

Non-alcoholic fatty liver disease (NAFLD) is a metabolic-related disorder induced by multiple factors and mainly characterized by excessive fat buildup in hepatocytes. With the consumption of a Western-style diet and obesity prevalence in recent years, the incidence of NAFLD has gradually increased, becoming an increasingly serious public health problem. Bilirubin is a heme metabolite and a potent antioxidant. Studies have demonstrated that bilirubin levels have an inverse correlation with the incidence rate of NAFLD; however, which form of bilirubin plays the main protective role is still controversial. It is considered that the main protective mechanisms for NAFLD are bilirubin antioxidant properties, insulin resistance reduction, and mitochondrial function. This article summarizes the correlation, protective mechanism, and possible clinical application of NAFLD and bilirubin.
Humans ; Non-alcoholic Fatty Liver Disease/metabolism* ; Bilirubin ; Antioxidants ; Obesity/complications* ; Hepatocytes/metabolism* ; Liver/metabolism*

Hepatic parenchymal cells are a type of liver cells that performs important functions such as metabolism and detoxification. The contribution of hepatic parenchymal cells, bile duct cells, and hepatic stem/progenitor cells to new hepatic parenchymal cells in the process of liver injury repair has become a controversial issue due to their strong proliferation ability. Lineage tracing technology, which has emerged in the past decade as a new method for exploring the origin of cells, can trace specific type of cells and their daughter cells by labeling cells that express the specific gene and their progeny. The article reviews the current literature on the origin and contribution of hepatic parenchymal cells by this technique. About 98% of new hepatic parenchymal cells originate from the existing hepatic parenchymal cells during liver homeostasis and after acute injury. However, under conditions of severe liver injury, such as inhibition of hepatic parenchymal cell proliferation, bile duct cells (mainly liver stem/progenitor cells) become the predominant source of hepatic parenchymal cells, contributing a steady increased hepatocyte regeneration with the extension of time.
Hepatocytes/metabolism* ; Liver/metabolism* ; Bile Ducts ; Stem Cells ; Liver Regeneration/physiology* ; Cell Differentiation

Objective To study the effect and mechanism of blueberry on regulating the mitochondrial inner membrane protein mitofilin/Mic60 in an in vitro model of metabolic dysfunction-associated liver disease (MAFLD). Methods L02 human hepatocytes were induced by free fatty acids (FFA) to establish MAFLD cell model. A normal group, a model group, an 80 μg/mL blueberry treatment group, a Mic60 short hairpin RNA (Mic60 shRNA) transfection group, and Mic60 knockdown combined with an 80 μg/mL blueberry treatment group were established. The intracellular lipid deposition was observed by oil red O staining, and the effect of different concentrations of blueberry pulp on the survival rate of L02 cells treated with FFA was measured by MTT assay. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), superoxide dismutase (SOD) activity, glutathione (GSH) and malondialdehyde (MDA) contents were measured by visible spectrophotometry. The expression of reactive oxygen species (ROS) in hepatocytes was observed by fluorescence microscopy, and the mRNA and protein expression of Mic60 were detected by real-time quantitative PCR and Western blot analysis, respectively. Results After 24 hours of FFA stimulation, a large number of red lipid droplets in the cytoplasm of L02 cells was observed, and the survival rate of L02 cells treated with 80 μg/mL blueberry was higher. The results of ALT, AST, TG, TC, MDA and the fluorescence intensity of ROS in blueberry treated group were lower than those in model group, while the levels of SOD, GSH, Mic60 mRNA and protein in blueberry treated group were higher than those in model group. Conclusion Blueberry promotes the expression of Mic60, increases the levels of SOD and GSH in hepatocytes, and reduces the production of ROS, thus alleviating the injury of MAFLD hepatocytes and regulating the disorder of lipid metabolism.
Humans ; Blueberry Plants/chemistry* ; Hepatocytes/metabolism* ; Liver/metabolism* ; Liver Diseases/metabolism* ; Reactive Oxygen Species/metabolism* ; Superoxide Dismutase/metabolism* ; Superoxides/metabolism* ; Mitochondrial Membranes/metabolism* ; Mitochondrial Proteins/metabolism* ; Plant Extracts/pharmacology*

The present study investigated the main components of fenugreek(Trigonella foenum-graecum L.) leaf flavonoids(FLFs) and their antioxidant activity. FLFs were prepared and enriched by solvent extraction, and the flavonoids were characterized by high-performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS). The protective effect of FLFs against H_2O_2-induced stress damage to L02 hepatocytes was also investigated. Firstly, the cell viability was measured by MTT assay. The oxidative stress injury model was induced by H_2O_2 in L02 cells. The release of lactate dehydrogenase(LDH), the content of reduced glutathione(GSH) and malondialdehyde(MDA), and the activities of superoxide dismutase(SOD) and catalase(CAT) were measured by assay kits. Hoechst fluorescence staining was performed to observe the cell apoptosis. The expression levels of c-Jun N-terminal kinase(JNK), extracellular signal-regulated kinase 1/2(ERK1/2), nuclear factor erythroid-2 related factor 2(Nrf2), heme oxygenase 1(HO-1), and their phosphorylated proteins were detected by Western blot. Based on the MS fragment ion information and data in databases, FLFs contained eight flavonoids with quercetin and kaempferol as the main aglycons. The cell viabi-lity assay revealed that as compared with the conditions in the H_2O_2 treatment group, 3.125-25 μg·mL~(-1) FLFs could increase the viability of L02 cells, reduce LDH release and MDA content in a dose-dependent manner, potentiate the activities of SOD, CAT, and GSH, decrease the phosphorylation of JNK and ERK1/2 proteins, and up-regulate the expression of Nrf2 and HO-1. The results of fluorescence staining showed that the nucleus of the H_2O_2 treatment group showed concentrated and dense strong blue fluorescence, while the blue fluorescence intensity of the FLFs group decreased significantly. FLFs showed a protective effect against H_2O_2-induced oxidative damage in L02 cells, and the underlying mechanism is associated with the enhancement of cell capability in clearing oxygen free radicals and the inhibition of apoptosis by the activation of the MAPKs/Nrf2/HO-1 signaling pathway. The antioxidant effect of fenugreek leaf is related to its rich flavonoids.
Antioxidants/pharmacology* ; Apoptosis ; Flavonoids/pharmacology* ; Hepatocytes/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Oxidative Stress ; Plant Leaves/metabolism* ; Superoxide Dismutase/metabolism* ; Tandem Mass Spectrometry ; Trigonella/metabolism*

Taking lipophagy as the breakthrough point, we explored the mechanism of Zexie Decoction(ZXD) in improving lipid metabolism in the hepatocyte model induced by palmitic acid(PA) and in the animal model induced by high-fat diet(HFD) on the basis of protein kinase B(Akt)/transcription factor EB(TFEB) signaling pathway. Co-localization was carried out for the microtubule-associated protein light chain 3(LC3) plasmid labeled with green fluorescent protein(GFP) and lipid droplets(LDs), and immunofluorescence co-localization for liver LC3 of HFD mice and perilipin 2(PLIN2). The results showed that ZXD up-regulated the expression of LC3, reduced lipid accumulation in hepatocytes, and increased the co-localization of LC3 and LDs, thereby activating lipo-phagy. Western blot results confirmed that ZXD increased autophagy-related protein LC3Ⅱ/LC3Ⅰ transformation ratio and lysosome-associated membrane protein 2(LAMP2) in vivo and in vitro and promoted the degradation of sequestosome-1(SQSTM1/p62)(P<0.05). The results above jointly explained that ZXD regulated lipophagy. Furthermore, ZXD activated TFEB expression(P<0.05) and reversed the PA-and HFD-induced decrease of TFEB nuclear localization in hepatocytes(P<0.05). Meanwhile, ZXD activated liver TFEB to up-regulate the expression of the targets Lamp2, Lc3 B, Bcl2, and Atg5(P<0.05). Additionally, ZXD down-regulated the protein level of p-Akt upstream of TFEB in vivo and in vitro. In conclusion, ZXD may promote lipophagy by regulating the Akt/TFEB pathway.
Animals ; Mice ; Autophagy/drug effects* ; Hepatocytes/metabolism* ; Microtubule-Associated Proteins/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction ; Drugs, Chinese Herbal/pharmacology*

Primary human hepatocytes (PHH) are the gold standard of in vitro human liver model for drug screening. However, a problem of culturing PHH in vitro is the rapid decline of cytochrome P450 (CYP450) activity, which plays an important role in drug metabolism. In this study, thermo-responsive culture dishes were used to explore the conditions for murine embryonic 3T3-J2 fibroblasts to form cell sheet. Based on the cell sheet engineering technology, a three-dimensional (3D) "sandwich" co-culture system of 3T3-J2 cell sheet/PHH/collagen gel was constructed. The tissue structure and protein expression of the model section were observed by hematoxylin eosin staining and immunofluorescence staining respectively. Phenacetin and bupropion were used as substrates to determine the activity of CYP450. The contents of albumin and urea in the system were determined by enzyme linked immunosorbent assay (ELISA). The results showed that the complete 3T3-J2 cell sheet could be obtained when the cell seeding density was 1.5×10⁶ /dish (35 mm dish) and the incubation time at low temperature was 60 min. Through cell sheet stacking, a 3D in vitro liver model was developed. Compared with the two-dimensional (2D) model, in the 3D model, the cell-cell and cell-matrix connections were tighter, the activities of cytochrome P450 CYP1A2 and cytochrome P450 CYP2B6 were significantly increased, and the secretion levels of albumin and urea were increased. These indexes could be maintained stably for 21 d. Therefore, cell sheet stacking is helpful to improve the level of liver function of 3D liver model. This model is expected to be used to predict the metabolism of low-clearance drugs in preclinical, which is of great significance for drug evaluation and other studies.
Albumins/metabolism* ; Animals ; Cytochrome P-450 Enzyme System/metabolism* ; Hepatocytes/metabolism* ; Humans ; Liver ; Mice ; Urea/metabolism*

OBJECTIVE: To explore the changes in Yes-associated protein (YAP) activity at the early stage of nonalcoholic steatohepatitis (NASH) and the spatiotemporal relationship between YAP and ductular reaction (DR). METHODS: Male C57BL/6J mouse models of NASH were established by feeding with a methionine- and choline-deficient (MCD) diet or a thioacetamide (TAA) diet for 12 weeks. At different time points during the feeding, liver histology of the mice was observed with HE and Masson trichrome staining. The mRNA expressions of YAP and its target genes (Ctgf, Cyr61, Acta2) were determined by qPCR, and the total protein expression level of YAP was measured with immunoblotting. The expression and distribution of YAP and the markers of DR (K19 and Sox9) were observed with immunohistochemical staining. RESULTS: At the early stage of NASH induced by MCD diet (1 to 4 weeks), the mRNA expression of YAP and its target genes and the total protein expression of YAP increased significantly (P < 0.01). The number of YAP-positive hepatocytes reached the peak level of 90.8 (cells per ×400 field of view) at week 2 and then decreased to 30.8 at week 4 (P < 0.001); YAP-positive ductular cells appeared near the portal area, where DR began to occur. From 8 to 12 weeks, numerous K19/Sox9-positive DR cells were observed in the hepatic lobules around the central vein (P < 0.01), while only a few YAP-positive hepatocytes were present in the liver tissue (P > 0.05), and the number of YAP-positive ductular cells gradually increased with time (P < 0.001). At the early stage of NASH induced by TAA diet (3 days to 2 weeks), the mRNA expression of YAP and its target genes and the total protein expression of YAP increased significantly (P < 0.05), and the number of YAP-positive hepatocytes reached the peak of 69.2 at week 2 and then decreased to 55.2 at week 4 (P < 0.001); YAP-positive ductular cells first appeared at the initial location of DR near the central vein. From 6 to 12 weeks, numerous K19/Sox9-positive DR cells occurred in the hepatic lobules around the central vein (P < 0.01). While the number of YAP-positive hepatocytes decreased (P < 0.001), the number of YAP-positive ductular cells continued to increase (P < 0.001). CONCLUSION During the development of NASH, YAP activation occurs earlier than DR but they are spatiotemporally correlated. YAP activation in hepatocytes may participate in DR by promoting hepatocyte dedifferentiation.
Animals ; Choline ; Disease Models, Animal ; Hepatocytes ; Liver/metabolism* ; Male ; Methionine/metabolism* ; Mice ; Mice, Inbred C57BL ; Non-alcoholic Fatty Liver Disease/metabolism* ; RNA, Messenger/metabolism* ; Thioacetamide/metabolism* ; YAP-Signaling Proteins