Hepatocyte Nuclei Research Articles

The proliferation of atypical hepatocytes and CDT1 expression in noncancerous tissue are associated with the postoperative recurrence of hepatocellular carcinoma

Recently, we reported that extent of proliferation of atypical hepatocytes (atypical hepatocytes) was most important histological risk factor for development of hepatocellular carcinoma (HCC) from chronic hepatitis C or liver cirrhosis. Here, we aimed to clarify whether the atypical hepatocytes in noncancerous sections is also involved in postoperative recurrence. Furthermore, we investigated significant genes involved in the atypical hepatocytes. Association between the extent of atypical hepatocytes in noncancerous tissue and postoperative recurrence was validated in 356 patients with HCC. Next, we identified putative signature genes involved in extent of atypical hepatocytes. First, atypical hepatocytes or hepatocytes other than the atypical hepatocyte in noncancerous sections of 4 HCC patients were selectively collected by laser capture microdissection (LCM). Second, the gene expression profiles of the selected hepatocyte populations were compared using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher SCIENTIFIC, Waltham, MA, USA) analysis. Finally, we validated the mRNA expression of the extracted genes in noncancerous frozen liver tissue from 62 patients with HCC by RT-qPCR to identify the signature genes involved in both the extent of atypical hepatocytes and postoperative recurrence. Furthermore, the extent of atypical hepatocytes and CDT1 expression in noncancerous sections from 8 patients with HCC were also validated by selectively collecting samples using LCM. The extent of atypical hepatocytes was associated with postoperative recurrence. Of the genes that showed significant differences in expression levels between two populations, the expression of the chromatin licensing and DNA replication factor 1 (CDT1) gene was most strongly associated with the extent of atypical hepatocytes and was also associated with postoperative recurrence. Furthermore, CDT1-positive cells that exhibited stronger expression resembled those morphologically considered to be atypical hepatocytes. CDT1 and Ki-67 were colocalized in the nuclei of both hepatocytes and cancer cells. The hepatocytes in noncancerous livers were not uniform in each hepatocyte population, suggesting that the accumulation of genetic abnormalities was variable. We found that the strong degree of atypical hepatocytes and high CDT1 mRNA expression represent a high carcinogenic state of the liver. Thus, we consider the evaluation of degree of these could support the personalized medicine.

Microcystin-LR-induced nuclear translocation of cGAS promotes mutagenesis in human hepatocytes by impeding homologous recombination repair

Microcystin-LR (MC-LR) has been recognized as a typical hepatotoxic cyclic peptides produced by cyanobacteria. Nowadays, due to the frequent occurrence of cyanobacterial blooms, the underlying hepatotoxic mechanism of MC-LR has become the focus of attention. In our present work, the mutagenic effect of MC-LR on human normal hepatic (HL-7702) cells regulated by cGAS was mainly studied. Here, we showed that exposure to MC-LR for 1–4 days could activate the cGAS-STING signaling pathway and then trigger immune response in HL-7702 cells. Notably, relative to the treatment with 1 μM MC-LR for 1–3 days, it was observed that when HL-7702 cells were exposed to 1 μM MC-LR for 4 days, the mutation frequency at the Hprt locus was remarkably increased. In addition, cGAS in HL-7702 cells was also found to complete the nuclear translocation after 4-day exposure. Moreover, co-immunoprecipitation and homologous recombination (HR)-directed DSB repair assay were applied to show that homologous recombination repair was inhibited after 4-day exposure. However, the intervention of the nuclear translocation of cGAS by transfecting BLK overexpression plasmid restored homologous recombination repair and reduced the mutation frequency at the Hprt locus in HL-7702 cells exposed to MC-LR. Our study unveiled the distinct roles of cGAS in the cytoplasm and nucleus of human hepatocytes as well as potential mutagenic mechanism under the early and late stage of exposure to MC-LR, and provided a novel insight into the prevention and control measures about the hazards of cGAS-targeted MC-LR.

Single-cell transcriptomics shows dose-dependent disruption of hepatic zonation by TCDD in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) dose-dependently induces the development of hepatic fat accumulation and inflammation with fibrosis in mice initially in the portal region. Conversely, differential gene and protein expression is first detected in the central region. To further investigate cell-specific and spatially resolved dose-dependent changes in gene expression elicited by TCDD, single-nuclei RNA sequencing and spatial transcriptomics were used for livers of male mice gavaged with TCDD every 4 days for 28 days. The proportion of 11 cell (sub)types across 131 613 nuclei dose-dependently changed with 68% of all portal and central hepatocyte nuclei in control mice being overtaken by macrophages following TCDD treatment. We identified 368 (portal fibroblasts) to 1339 (macrophages) differentially expressed genes. Spatial analyses revealed initial loss of portal identity that eventually spanned the entire liver lobule with increasing dose. Induction of R-spondin 3 (Rspo3) and pericentral Apc, suggested dysregulation of the Wnt/β-catenin signaling cascade in zonally resolved steatosis. Collectively, the integrated results suggest disruption of zonation contributes to the pattern of TCDD-elicited NAFLD pathologies.

Morphofunctional State and Circadian Rhythms of the Liver of Female Rats under the Influence of Chronic Alcohol Intoxication and Constant Lighting

A separate and combined effect of constant illumination and chronic alcohol intoxication (CAI) on diurnal dynamics of micromorphometric parameters of hepatocytes in female Wistar rats and p53, Ki-67, PER2, BMAL1, and ADH5 expression in these cells were studied. The increase in apoptotic activity and proliferation in all animals under the action of chronodestructors is shown. All experimental animals showed a decrease in BMAL1 expression and increase in PER2 expression; ADH5 is overexpressed under the influence of ethanol. Circadian rhythms (CRs) of BMAL1, PER2, p53, and Ki-67 expression persist in all groups, except combined action of chronodestructors, and ADH5 CRs persist in all groups—thus, these rhythms in females are quite stable. CRs of the hepatocyte nuclei area are preserved in all the studied groups, although they undergo a significant shift. At the same time, the CRs of the hepatocyte area are destroyed under the action of light, both independently and in combination with CAI, and the CR of the nuclear-cytoplasmic ratio (NCR) is destroyed by exposure to CAI. It can be assumed that CRs of the hepatocyte area are significantly affected by dark deprivation and NCR rhythm is sensitive to ethanol consumption, while the stability of studied genes’ expression rhythms at separate influences of studied chronodestructors is maintained by yet unknown adaptation mechanisms. It is necessary to note that, according to our previous studies of male rats, rat females show significantly greater stability of the studied CRs.

Interleukin-33 mediates both immune-related and non-immune-related inhibitory effects against hepatitis B virus

Chronic infection by hepatitis B virus (HBV) is associated with high risks of liver fibrosis, cirrhosis and hepatocellular carcinoma. HBV covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocyte serves as transcription template. Neither natural resolution of acute infection nor current treatment options for chronic infection are believed to cause cccDNA clearance. We previously showed that injection of IL-33-expressing plasmid facilitated clearance of intrahepatic HBV DNA in a mouse model of HBV persistence. In this work, HBV-targeting therapeutic effects of IL-33 were further explored. Murine IL-33 delivered by recombinant adeno-associated virus (AAV-mIL-33) induced clearance of both serum HBV markers and intrahepatic HBV DNA in two mouse models of HBV persistence based on replicon plasmid and recombinant cccDNA (rcccDNA) respectively. Clearance was associated with serum ALT elevations and liver infiltrations by CD4+ and CD8+ T cells, indicating IL-33-induced cellular immune responses against HBV-harboring cells. Adoptive transfer of splenocytes from AAV-mIL-33-cured mice was indeed sufficient to engender similar clearance in recipient mice. In vitro, intracellular, instead of extracellular, IL-33 was mainly responsible for repressing viral transcription, protein production and genome replication in Huh7 cells transfected with HBV replicon or rcccDNA. IL-33 was shown to be recruited onto rcccDNA minichromosome accompanied by loss of transcriptional activation epigenetic marks. Finally, transfection of IL-33 into HBV-infected HepG2/NTCP cells resulted in reduced transcription, antigen expression and genome replication, suggesting repression of canonical cccDNA. These data demonstrated diverse inhibitory effects on HBV and HBV-infected cells mediated by IL-33, and suggest IL-33 as an interesting therapeutic candidate.

Heavy Metal Bioaccumulation and Histopathological Studies of Fish Tissues from Ose River, Ondo State, Nigeria

The exposure of the aquatic ecosystem to heavy metal from both natural and anthropogenic activities are on the rise and have harmful health effects on all aquatic organisms and human. This study assessed the concentrations of heavy metals (Fe, Pb, Cd, Cr, As and Zn) in surface water, sediments and fish tissues of Ose River and examined the histopathological changes of the tissues of two fish species (Mormyrus rume and Clarias garienpinus). Surface water, sediments and fish samples were collected and procured from Ose River in Ondo State, Nigeria from February to April 2021. Heavy metal digestions were done in the laboratory using HNO3/HCl while the concentrations were determined using Atomic Absorption Spectrometer. Histopathological alterations in the fish tissues were also assessed. Bioaccumulation factor (BAF), ecological risk index and potential ecological risk level were used to assess the investigated metal concentrations. In the surface water, the concentration of Cd (0.010–0.030 mg/l) and As (0.010–0.05 mg/l) exceeded the WHO limits of 0.01mg/l respectively while Fe, Pb, Cr and Zn were within the limits. In the sediments, all metals exceeded the limits except Pb and As. The concentrations of Pb (0.30±0.000–4.12±0.030 mg/l), Zn (9.970±0.010–30.77±0.023 mg/l), Cd (0.026±0.002–0.331±0.004 mg/l) and Cr (ND-0.331±0.004 mg/l) in the fish tissues were seen to be higher than the permissible limits of 0.5 mg/l, 5 mg/l, 0.005 mgl and 0.05-0.15 mg/l respectively. The BAF showed no probability. The alterations observed in the kidneys of the fish were cholestasis, necrosis, loss of glomeruli structure and tubular necrosis. While the gills showed congestion of the central veins and interlamellae hyperplasia caused by the presence of a monogenean parasite. The livers however revealed loss of hepatocytes nuclei, necrosis in hepatocytes and lymphocytic infiltrate. The study showed that the study site was polluted with heavy metals and may pose serious health issues to fishers, those using the water and people feeding on fish from the river. This study therefore suggests good indicators for bio-monitoring of heavy metals in Ose River. Seeing that the site was polluted with heavy metals hence, a more comprehensive assessment of the river is recommended.

Induced arthritis, hepatocytes cytotoxicity and non-steroidal anti-inflammatory drugs (NSAIDs) – experimental study

This study aimed to histologically evaluate the pharmacological activity of meloxicam, COX-2 inhibitor, on liver function in arthritic rats. Meloxicam is a non-steroidal anti-inflammatory drug (NSAID), COX-2 inhibitor, generally prescribed in the area of rheumatology. Thus, 18 Wistar rats were divided into 3 groups with 6 animals each: G1 (Negative Control), G2 (Arthritis induced with Zymosan [Zy]), G3 (Arthritis induced with Zymosan [Zy] and treated with meloxicam). The G3 treatment started on the 21st day after induction. The animals were sacrificed, by overdose of Ketamine/Xylazine, seven, 14 and 21 days after the beginning of the meloxicam treatment. Liver samples were collected from the animals, fixed in Millonig buffer containing 10% formaldehyde and processed for conventional histological analysis. G1 animals showed absence of inflammation and degeneration of hepatocytes. In the G2 group, a slight cytoplasmic alteration was observed at seven and 14 days. At 21 days, intense cytoplasmic and nuclear disorganization was detected, accompanied by cell necrosis. In G3, large spacing between sinusoid capillaries was detected. In some 14-day-old animals, amyloidosis was observed, with the presence of a large number of vacuolated hepatocytes involved in an amorphous mass of necrotic cells. Most of the hepatocyte nuclei had compacted chromatin, evidencing cellular degeneration. Meloxican, used as an anti-inflammatory in the synovial region, showed that, in addition to its beneficial effects for the treatment of rheumatological diseases, it is capable of, when administered orally, promoting a high level of liver damage.

HEPATO-PATHOLOGICAL STUDY OF THE REHABILITATIVE POTENTIALS OF JAMBUL (Syzygium cumini) PLANT EXTRACT IN LEAD INTOXICATED MICE

The ameliorative prospective of Jambul (Syzygium cumini) fruit pulp extract was studied against Pb-based hepato-histological and micrometric changes in albino laboratory mice (Mus musculus). There were 3 experimental groups: (i) Control group (untreated) (ii) Pb group (15 days 50ppm Pb ions from Lead acetate in drinking water (iii) Pb+Jm group (Pb ions treatment as in Pb group followed by 0.2ml/per day jambul fruit pulp extract through gavage and plain water for the next 5 days). Animals were euthanized on the 21st day to recover liver for histopathological and micrometric studies.
 The misalignment of the hepatic cord with narrowed and chocked sinusoidal spaces and some lesions in hepatic tissue were seen as persistent histopathological signs of lead exposure. Micrometric observations indicate that the mean cross-sectional area of the hepatocytes in the Pb group (483.5µ2) increased significantly than the control group (420.1µ2), similarly, the mean cross-sectional area of the hepatocytes nuclei in the Pb group was also significantly higher (76.69µ2) than the control (62.37µ2). These pathological and micrometric signs were convincingly reversed in Pb+Jm, additionally, hepatoblasts progenitor cells were frequently observed, indicating the rehabilitative and regenerative potential of jambul fruit pulp extract. Results indicate that jambul fruit pulp extract possesses precious hepato-protective and regenerative potentials against lead exposure-related hepato-histopathology. The findings of the present study indicate the nutraceutical role of jambul fruit pulp extract for potential human consumption.

Hepatitis B core-related antigen: Are we near a treatment endpoint?

Different serological and virological markers in chronic hepatitis B patients guide staging of viral infection, and initiation and response to therapy. Due to the persistence of intrahepatic covalently closed circular DNA (cccDNA) in the hepatocyte nucleus, hepatitis B is not curable. Even after undetectable hepatitis B virus DNA levels, the persistence of hepatitis B surface antigen and novel markers such as hepatitis B core-related antigen (HBcrAg) indicate the persistence of intrahepatic cccDNA. In this study, HBcrAg levels at baseline and after 24 and 48 wk of antiviral therapy predicted hepatitis B e antigen seroconversion. Due to the poor sensitivity of assays and detectable levels in HBsAg-negative patients, the long-term utility of HBcrAg needs future research.

Drug Discovery Study Aimed at a Functional Cure for HBV.

Hepatitis B virus (HBV) causes acute and, most importantly, chronic hepatitis B worldwide. Antiviral treatments have been developed to reduce viral loads but few patients with chronic hepatitis B (CHB) achieve a functional cure. The development of new therapeutic agents is desirable. Recently, many novel agents have been developed, including drugs targeting HBV-DNA and HBV-RNA. This review provides an overview of the developmental status of these drugs, especially direct acting antiviral agents (DAAs). Serological biomarkers of HBV infection are essential for predicting the clinical course of CHB. It is also important to determine the amount and activity of covalently closed circular DNA (cccDNA) in the nuclei of infected hepatocytes. Hepatitis B core-associated antigen (HBcrAg) is a new HBV marker that has an important role in reflecting cccDNA in CHB, because it is associated with hepatic cccDNA, as well as serum HBV DNA. The highly sensitive HBcrAg (iTACT-HBcrAg) assay could be a very sensitive HBV activation marker and an alternative to HBV DNA testing for monitoring reactivation. Many of the drugs currently in clinical trials have shown efficacy in reducing hepatitis B surface antigen (HBsAg) levels. Combination therapies with DAAs and boost immune response are also under development; finding the best combinations will be important for therapeutic development.

Histopathological changes resulting from the effect of nano-graphene oxide on the liver in laboratory rats

The current study included the use of (48) white male laboratory rats, where the rats were divided into six groups, with 8 rats in each group. The second group with a concentration of 30 mg/kg, the third group with a concentration of 40 mg/kg, the fourth group with a concentration of 50 mg/kg, and the fifth group with a concentration of 60 mg/kg of nano graphene oxide. The sixth group is the control group. The results of the statistical analysis of the study showed a significant decrease in the average body weight. On the last day of the experiment when comparing between the control group and the treated groups, and the results showed a significant decrease in the average liver weight when comparing between the control group and the treated groups. Central vein, hepatic sinusoidal dilatation, hepatocyte nucleus replication, hepatocyte rupture, and nucleolysis As well as thickening of the nuclei in some hepatocytes, infiltration of inflammatory cells, hemorrhage, and dissolution and necrosis of hepatocytes. The results of the over-study by transmission electron microscope also showed changes at the cellular level, represented by the rupture of the nucleus and cytoplasm, in addition to the dissolution of the nucleus.

Prevention of HBV Reactivation in Hemato-Oncologic Setting during COVID-19.

Onco-hematologic patients are highly susceptible to SARS-CoV-2 infection and, once infected, frequently develop COVID-19 due to the immunosuppression caused by tumor growth, chemotherapy and immunosuppressive therapy. In addition, COVID-19 has also been recognized as a further cause of HBV reactivation, since its treatment includes the administration of corticosteroids and some immunosuppressive drugs. Consequently, onco-hematologic patients should undergo SARS-CoV-2 vaccination and comply with the rules imposed by lockdowns or other forms of social distancing. Furthermore, onco-hematologic facilities should be adapted to new needs and provided with numerically adequate health personnel vaccinated against SARS-CoV-2 infection. Onco-hematologic patients, both HBsAg-positive and HBsAg-negative/HBcAb-positive, may develop HBV reactivation, made possible by the support of the covalently closed circular DNA (cccDNA) persisting in the hepatocytic nuclei of patients with an ongoing or past HBV infection. This occurrence must be prevented by administering high genetic barrier HBV nucleo(t)side analogues before and throughout the antineoplastic treatment, and then during a long-term post-treatment follow up. The prevention of HBV reactivation during the SARS-CoV-2 pandemic is the topic of this narrative review.

PreS/2-21-Guided siRNA Nanoparticles Target to Inhibit Hepatitis B Virus Infection and Replication.

A viable therapy is needed to overcome the deadlock of the incurable chronic hepatitis B (CHB). The prolonged existence of covalently closed circular DNA (cccDNA) and integrated HBV DNA in the nucleus of hepatocytes is the root cause of CHB. As a result, it is critical to successfully suppress HBV DNA replication and eliminate cccDNA. RNA interference has been proven in recent research to silence the expression of target genes and thereby decrease HBV replication. However, siRNA is susceptible to be degraded by RNA enzymes in vivo, making it difficult to deliver successfully and lacking of tissue targeting. To exploit the advantages of siRNA technology while also overcoming its limitations, we designed a new strategy and prepared biomimetic nanoparticles that were directed by PreS/2-21 peptides and precisely loaded HBV siRNA. Experiments on these nanoparticles in vitro and in vivo revealed that they are tiny, stable, safe and highly targetable, with high inhibitory effects on HBV DNA, pgRNA, cccDNA, HBeAg and HBsAg. PreS/2-21-directed nanoparticles loaded with HBV gene therapy drugs are expected to be promising for the treatment of CHB.

Rational selection of virologic and immunological biomarkers and therapeutic endpoints in the clinical trials of new drugs treating chronic hepatitis B

Hepatitis B virus (HBV) infection remains to be the major cause of chronic liver diseases in China. Since the nucleos(t)ide analogues and pegylated interferon-alpha do not directly target the covalently closed circular DNA (cccDNA) in the nuclei of HBV-infected hepatocytes, those standard-of-care medications cannot efficiently cure the infected hepatocytes and rarely achieve the functional cure of chronic hepatitis B (CHB). Therefore, new antiviral drugs targeting distinct steps of HBV replication and immunotherapeutics reinvigorating antiviral immune responses are urgently needed for the functional cure of CHB. Based on the extensive discussion of the biological and clinical significance of new virologic biomarkers and distinct mechanism of drug candidates currently in clinical development, we propose that the selection of virologic and immunological biomarkers for evaluation of therapeutic efficacy as well as setting the therapeutic endpoints in the clinical trials should be based on the mode of action of investigational drugs. In addition, due to the complexity of CHB pathogenesis, selection of specific subpopulation of CHB patients for the clinical trials of drugs with a specific mode of action should also be considered.

The effect of stiffness and impurities on bubble nucleation in polyacrylamide hydrogels

Tissue mimicking hydrogels can help us understand how viscoelastic properties (stiffness, elastic modulus, etc.) of biological tissues impact bubble nucleation. Adding impurities to hydrogels introduces inhomogeneities and increases their similarity to biological tissues. In this study, we evaluated the effect of stiffness and impurities on bubble nucleation in polyacrylamide (PA) hydrogels. Bubble nuclei were evaluated in 17.5%, 20%, and 22.5% v/v PA hydrogels, after which 0.25% w/v cholesterol crystals (maximum dimension = 0.6 mm) were embedded in the gels (n = 3 each). A 1.5 MHz focused ultrasound transducer was used to induce cavitation using 10-ms pulses with pressures ranging up to p+ = 89 MPa, and p− = 26 MPa and −6 dB focal dimensions of 9.4 × 1.2 mm (p−). Image analysis from high-speed photography showed bubble nucleation increases with increasing peak negative pressure and decreasing hydrogel stiffness. Adding cholesterol crystals largely decreases the acoustic cavitation threshold from p− = 19 MPa for 17.5% v/v hydrogels with no added impurities to p− = 12 MPa for the same concentration hydrogel with added cholesterol crystals. This suggests that hydrophobic cholesterol crystals weaken the gel or trap bubble nuclei, thus lowering the cavitation threshold. Future work includes investigating bubble nuclei in rat hepatocytes. [Work supported by NSF CAREER 1943937 and PSU Riess Fellowship.]

Morphology of Acute Liver Failure During Treatment With a Combination of Drug Therapy and Transplanted Cultured Hepatocytes.

We analyzed liver tissue morphology dynamics in experimental animals with acute liver failure during treatment with a combination of transplanted cultured embryonic hepatocytes and the hepatoprotective drug Erbisol. We studied 30 white outbred male rats weighing 250 to 350 g, divided into 6 groups with 5 animals in each group. Acute liver damage was induced by a hepatotoxic model of acute liver failure for which carbon tetrachloride was applied (200 μg CCl4/100 g body weight with toxin-to-oil volume ratio of 1:1). To obtain cultured embryonic hepatocytes, we used an enzymatic-mechanical process that caused only minimal cell damage. This method is known to improve the output of viable cultured embryonic hepatocytes, improve the morphological-functional properties of the hepatocytes, and reduce the process time during procurement, and thereby reduce the overall time from procurement to the subsequent culturing of the obtained cells. Transplant of cultured embryonic hepatocytes was performed intrape-ritoneally at a dose of 50 million (0.5 × 108) cells. Morphological studies were performed on day 7 and day 21 of the experiment. There were significant morphological changes in livers from animals with acute liver failure, caused by widespread necrosis and an elevated inflammatory response. Treatment with injections of cultured embryonic hepatocytes induced regeneration of the liver parenchyma cells and reduced the inflammatory response, both of which were further reduced in rats that received combined treatment of transplant with Erbisol. Combined application of cultured embryonic hepatocytes and Erbisol potentiated the effects of both treatments, which produced intensive proliferation of hepatocytes, hypertrophy and polyploidization of hepatocyte nuclei, and an early restoration of liver structure and organ mass.

Влияние гипобарической барокамерной гипоксии на состояние гепатоцитов крыс с неалкогольной жировой дистрофией печени

The problem of non-alcoholic fatty liver disease (NAFLD) has acquired particular relevance due to its widespread prevalence among the population throughout the world. The mechanisms of fat accumulation in hepatocytes are not fully understood. A certain role in this process is assigned to insulin resistance, which entails hyperproduction of triglycerides in the liver. The effect of hypoxia on hepatocytes has a multi-loop character. The relevance of the work is dictated by the lack of knowledge about the features of the course of fatty degeneration of the liver in conditions of hypobaric hypoxia. The article reflects the results of the influence of hypobaric hypoxia on the structural organization of the liver of rats suffering from. To study the features of the influence of hypobaric hypoxia on the structural organization of the liver of rats suffering from NAFLD. Materials and methods. The work was performed on 180 outbred male rats weighing 150–250 g. Before the start of the experiment, the animals were divided into 2 groups — the control group, where the animals were kept on a modified Ackerman diet, and the main group, in which animals on an identical diet were simultaneously exposed to hypobaric hypoxia (6000 m a. s. l., exposure time 6 hours, duration of the experiment 35 and 70 days). After the expiration of the experiments, the animals were decapitated, a post-mortem examination and material sampling were performed. Finished histological preparations were studied under a microscope with morphometry. The analysis of the obtained results was carried out using the statistical package of application programs SPSS 16.0 for Windows. A p value of <0.05 was considered statistically significant at a 95% confidence level. Results. It was found that in rats on a modified diet according to Ackerman, by the 35th day of the experiment, persistent changes are formed in hepatocytes that are characteristic of non-alcoholic fatty degeneration. The development of non-alcoholic fatty degeneration of the liver in the experiment leads to a significant increase in the area of hepatocytes (by 70% on the 35th day and by 85% on the 70th day), while the volume of the nuclei undergoes an increase of 50% only by the 70th day of the experiment. Hypobaric hypoxia in the pressure chamber reduces the phenomena of cell infiltration, which is expressed by the absence of a pronounced increase in the area of hepatocytes, but the volume of the nucleus increases by 41%. Conclusion. Non-alcoholic fatty degeneration of the liver under conditions of hypobaric hypoxia occurs with an increase in the volume of the hepatocyte nucleus and minor changes in its area, which requires further studies of pathogenetic mechanisms.

G-Quadruplex in hepatitis B virus

Over 257 million people worldwide are chronically infected with Hepatitis B virus (HBV), making HBV the leading cause of hepatocellular carcinoma and liver failure which subsequently results in ∼800,000 deaths annually. Chronic HBV infection is established in part through the virus’ highly stable covalently closed circular DNA (cccDNA) persisting in the nuclei of infected hepatocytes. The cccDNA serves as the template for transcription of all HBV mRNA transcripts. None of the currently approved treatments for HBV target the source of persistent infection — the cccDNA.

Metabolically Improved Stem Cell Derived Hepatocyte-Like Cells Support HBV Life Cycle and Are a Promising Tool for HBV Studies and Antiviral Drug Screenings

More than 300 million people worldwide are diagnosed with a chronic hepatitis B virus (HBV) infection. Nucleos(t)ide viral polymerase inhibitors are available on the market and can efficiently treat patients with chronic HBV. However, life-long treatment is needed as covalently closed circular DNA (cccDNA) persists in the hepatocyte nucleus. Hence, there is a high demand for novel therapeutics that can eliminate cccDNA from the hepatocyte nucleus and cure chronically infected HBV patients. The gold standard for in vitro HBV studies is primary human hepatocytes (PHHs). However, alternatives are needed due to donor organ shortage and high batch-to-batch variability. Therefore, human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are being explored as an in vitro HBV infection model. We recently generated hPSC lines that overexpress three transcription factors (HC3x) and that, upon differentiation in a high amino-acid supplemented maturation medium, generate a more mature hepatocyte progeny (HC3x-AA-HLCs). Here, we demonstrate that HBV can efficiently infect these HC3x-AA-HLCs, as was shown by the presence of HBV core (HBc) and surface antigens. A clear increasing release of HBV surface and e antigens was detected, indicating the formation of functional cccDNA. Moreover, back-titration of culture supernatant of HBV-infected HC3x-AA-HLCs on HepG2-NTCP cells revealed the production of novel infectious HBV particles. Additionally, an increasing number of HBc-positive HC3x-AA-HLCs over time suggests viral spreading is occurring. Finally, the HC3x-AA-HLC model was validated for use in antiviral drug studies using the nucleoside reverse-transcriptase inhibitor, lamivudine, and the HBV entry inhibitor, Myrcludex B.

Pdcd4 promotes lipid deposition by attenuating PPARα-mediated fatty acid oxidation in hepatocytes

BackgroundNonalcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation in hepatocytes. The involvement of programmed cell death 4 (Pdcd4) in inflammation and metabolic diseases has been widely reported. However, the precise regulatory role of Pdcd4 in hepatocytic lipid metabolism and NAFLD is not well known. Research design and methodsWe established a high-fat diet–induced NAFLD (HFD-NAFLD) rat model and a free fatty acids (FFAs)-treated cell model, and analyzed the expression and distribution of PDCD4. The lentivirus for Pdcd4 knockout and the vector for Pdcd4 overexpression were used to alter Pdcd4 expression in BRL 3A cells. Thereafter, lipid accumulation, FA metabolic gene expression, and peroxisome proliferator-activated receptor alpha (Pparα)-dependent peroxisomal β-oxidation-related gene expression, especially that of the critical transcription factors and enzymes acyl-CoA oxidases 1–3 (Acox1-3), were detected both at the mRNA and protein levels. ResultsPDCD4 expression increased and it was mainly distributed in hepatocyte nuclei of the HFD-NAFLD rats. as well as the FFAs-treated CBRH-7919 and BRL 3A cell lines. Pdcd4 knockout significantly suppressed FFAs-induced lipid accumulation, and Pdcd4 overexpression accelerated FFAs-induced lipid accumulation in hepatocytes. Mechanistically, Pdcd4 negatively regulated the expression Pparα and Acox1-3. In addition, rescue experiments confirmed that Pparα knockdown could attenuate the expression of Acox1-3 in Pdcd4 knockout cells, which ultimately restored lipid deposition to normal levels. PPARα expression decreased in the liver of the HFD-NAFLD rats. The enrichment of PDCD4 in hepatocyte nuclei correlated with lower PPARα expression after FFAs treatment in vitro. ConclusionOur results indicate that the abundance of PDCD4 under high-fat conditions facilitates hepatocellular lipid accumulation by decreasing PPARα-dependent FA peroxisomal β-oxidation.