Metabolism of Mequindox in Isolated Rat Liver Cells

Mequindox （MEQ）, 3-methyl-2-quinoxalinacetyl-l,4-dioxide, is widely used in Chinese veterinary medicine as an antimicrobial agent and feed additive. Its toxicity has been reported to be closely related to its metabolism. To understand the pathways underlying MEQ＇s metabolism more clearly, we studied its metabolism in isolated rat liver cells by using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap （LC-LTQ-Orbitrap） mass spectrometry. The structures of MEQ metabolites and their product ions were readily and reliably characterized on the basis of accurate MS2 spectra and known structure of MEQ. Eleven metabolites were detected in isolated rat liver cells, two of which were detected for the first time in vitro. The major metabolic pathways reported previously for in vitro metabolism of MEQ in rat microsomes were confirmed in this study, including N O group reduction, carbonyl reduction, and methyl monohydroxylation. In addition, we fotmd that acetyl hydroxylation was an important pathway of MEQ metabolism. The results also demonstrate that cellular systems more closely simulate in vivo conditions than do other in vitro systems such as microsomes. Taken together, these data contribute to our understanding of the in vivo metabolism of MEQ.

The role of liver sinusoidal endothelial cells in liver remodeling after injury

Liver transplantation is the optimal treatment for patients with end-stage liver disease,metabolic liver diseases,and hepatic malignancies that are not amenable to resection.Hepatic ischemia-reperfusion injury(IRI)is the main problem in liver transplantation and liver resection,leading to parenchymal cell injury and organ dysfunction.The damage of liver sinusoidal endothelial cells(LSECs)is a critical event in IRI.LSECs work as an important regulating factor of liver regeneration after partial hepatectomy.This review primarily describes the mechanisms of LSECs injury in IRI and explores the roles of LSECs in liver regeneration,and briefly introduces the protective strategies targeting LSECs damaged in IRI.

Effects of ethanol on liver sinusoidal endothelial cells-fenestrae of rats

Important advances have been made in research into the mechanism of alcoholic liver disease (ALD) over the past few years,but the role of liver sinusoidal endothelial cell (LSEC) in ALD has not been elucidated adequately. This study was undertaken to investigate the effect of ethanol on fenestrae of LSECs in rats. METHODS: A rat model of alcoholic liver disease was established by means of direct intragastric instillation of ethanol. Fifty-five rats of experimental (35 rats) and control (20) groups were sacrificed at the end of 4,8,12 weeks respectively, and also at the end of 12-week abstinence. After heart perfusion, the liver tissue was fixed and stained with hematoxylin and eosin for observation of serial changes of LSEC-fenestrae under a transmission electron microscope. RESULTS: Normal LESC was flat with a nucleus and organelles arranged regularly. The distal cytoplasm displayed as a lamina with many fenestrae, lacking the basement membrane(BM) underneath the endothelium. At the end of 4-week alcohol feeding, the number of fenestrae decreased at the distal cytoplasm in some LSECs, without the formation of the BM underneath the endothelium. At the end of 8 weeks, the number of fenestrae decreased significantly or even disappeared. The BM began to develop incompletely underneath the endothelium, while the active fibroblast appeared. At the end of 12 weeks, the number of fenestrae decreased more significantly and the complete BM could even be seen. But the changes were mostly limited in the single or adjoining sinus, and fibrosis was scarcely formed. At the end of 12-week abstinence, defenestration and formation of the endothelial BM lightened significantly. CONCLUSIONS:Defenestration and formation of the BM in LSECs develop gradually with the chronic stimulation of ethanol. Hepatic sinusoidal capillarization and fibrosis will be seen if their state is more serious. These early changes, i. e., limited and regional defenestration and capillarization may be the basis of alcoholic peri-fibrosis. This kind of he- patic fibrosis is reversible after removal of etiological factors.

Regulators of liver cancer stem cells

Hepatocellular carcinoma(HCC)is a leading cause of cancer deaths.It is often detected at a stage when there are few therapeutic options.Liver cancer stem cells(LCSCs)are highly tumorigenic and resistant to chemotherapy and radiation therapy.Their presence in HCC is a major reason why HCC is difficult to treat.The development of LCSCs is regulated by a variety of factors.This review summarizes recent advances on the factors that regulate the development of LCSCs.Due to the importance of LCSCs in the development of HCC,a better understanding of how LCSCs are regulated will help to improve the treatments for HCC patients.

Efficient generation of functional hepatocytelike cells from mouse liver progenitor cells via indirect co-culture with immortalized human hepatic stellate cells

BACKGROUND： Differentiation of liver progenitor cells（LPCs） to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system.METHODS： Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line（HSCLi） we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS： Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, lowdensity lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity.CONCLUSIONS： Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.

Discussion on Serum Technique and Cryopreservation Technique in the Culture of Liver Sinusoidal Endothelial Cells

Hepatic sinusoidal endothelial cells are a kind of highly differentiated cells in hepatic sinusoids, which play an important role in the occurrence and development of liver fibrosis. Hepatic sinusoidal endothelial cells are often used in the study of liver fibrosis. In the process of culturing liver sinusoidal endothelial cells, the treatment of serum for culture and cryopreservation of cells are relatively complicated and error-prone. If the technical details of serum treatment and cell cryopreservation are not handled properly, it will have a more obvious effect on the effect of hepatic sinusoidal endothelial cell culture. We have gained experience in the theoretical study of liver fibrosis and the operation of cell culture experiments, and this paper summarized the details of liver sinusoidal endothelial cell culture such as the problems of serum preservation, thawing, precipitates and heat inactivation, as well as methods and precautions for cell cryopreservation.

A NEW METHOD OF ISOLATING KUPFFER CELLS FROM BIOPSY TISSUE OF RAT LIVER

The isolation of a high yield and purity of Kupffer cells has been reported in detail.1 This paper reports into the research about isolation Kupffer cells from biopsy tissue of liver. This method includes 5 important steps: (1) take fresh liver tissue, and mince with scissors. (2) spin at low speed to wash off red blood cells. (3) digest in collagenase for suitable time. (4) isolate Kupffer cells on a percoll density gradient. (5) cell charaterization was observed by N.S.E stain and peroxidatic activity with lumino-meter measurement and phagocytosis with latex beads.2.3

Investigating the cytotoxic effect of ibuprofen concentration in liver cancer cells(HepG2)and normal fibroblast(AGO)

Objective:Although many studies have reported that nonsteroidal anti-inflammatory drugs can have anticancer effects,the results are still challenging.The aim of this research is to Mechanism and effect of anti-inflammatory drugs in cancer treatment.Methods:In this laboratory study,cell lines were randomly divided into control group(no exposure to ibuprofen and groups exposed to ibuprofen concentrations of 10,1,0.1,and 0.001 mg/mL.The cytotoxic effect of ibuprofen was measured at 24 and 72 hours using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT).Data were compared between groups using a one-way variance test.Results:The results showed that the viability rate of HepG2 cancer cells at concentrations of 1 and 10 mg/mL decreased significantly compared to the control group in 24 hours(P<0.0001).Also,the viability rate at concentrations of 1,10,0.1,and 0.001 mg/mL decreased significantly compared to the control group in 72 h(P<0.0001).Only the concentration of 10 mg/mL ibuprofen decreased the viability of normal cells compared to the control(P<0.05).Conclusion:Overall,the results of this research showed that different concentrations of ibuprofen had a cytotoxic effect on liver cancer cells,and except for the concentration of 10 mg/mL,the other concentrations did not have a cytotoxic effect on normal cells.

A STUDY OF THE FORMATION OF LIVER METASTASIS AND ITS MECHANISM USING THE INTRASPLENIC INOCULATION OF CANCER CELLS

In order to establish an animal model with hepatic metastasis intrasplenic inoculation of carcinoma cells from murine uterine cervical carcinoma (U14) was employed. Results showed a high incidence of hepatic metastasis could be obtained through the intrasplenic inoculation of 1 × 106 carcinoma cells. Removal of the primary carcinoma through splenec-tomy at different intervals after intrasplenic inoculation proved that the hepatic metastatic mechanism was not due to mechanical pressure but occurred spontaneously. This experimental model provides a useful means for studying the mechanism and prevention of hepatic metastasis.

Rapamycin increased development of CD4^+ CD25~h ighFoxp3^+ T cells of peripheral blood in liver transplant recipients

Objective To investigate the possible influence of immunosuppressive therapy,including sirolimus ( SRL) and calcineurin inhibitors ( CNI,tacrolimus) ,on level of Treg in liver allo - graft recipients. Methods Forty - seven liver transplant recipients with stable liver function were assessed for at least 2 years,and divided into

Advances in cell sources of hepatocytes for bioartificial liver

BACKGROUND: Orthotopic liver transplantation (OLT) is the most effective therapy for liver failure. However, OLT is severely limited by the shortage of liver donors. Bioartificial liver (BAL) shows great potential as an alternative therapy for liver failure In recent years, progress has been made in BAL regarding genetically engineered cell lines, immortalized human hepatocytes, methods for preserving the phenotype of primary human hepatocytes, and other functional hepatocytes derived from stem cells. DATA SOURCES: A systematic search of PubMed and ISI Web of Science was performed to identify relevant studies in English language literature using the Key words such as liver failure bioartificial liver, hepatocyte, stem cells, differentiation, and immortalization. More than 200 articles related to the cell sources of hepatocyte in BAL were systematically reviewed. RESULTS: Methods for preserving the phenotype of primary human hepatocytes have been successfully developed. Many genetically engineered cell lines and immortalized human hepatocytes have also been established. Among these cell lines the incorporation of BAL with GS-HepG2 cells or alginate encapsulated HepG2 cells could prolong the survival time and improve pathophysiological parameters in an animal model of liver failure. The cBAL111 cells were evaluated using the AMC-BAL bioreactor, which could eliminate ammonia and lidocaine, and produce albumin. Importantly, BAL loading with HepLi-4 cells could significantly improve the blood biochemical parameters, and prolong the survival time in pigs with liver failure. Other functional hepatocytes differentiated from stem cells, such as human liver progenitor cells, have been successfully achieved. CONCLUSIONS: Aside from genetically modified liver cell lines and immortalized human hepatocytes, other functionalhepatocytes derived from stem cells show great potential as cell sources for BAL. BAL with safe and effective liver cells may be achieved for clinical liver failure in the near future.

Autophagy in liver diseases

Autophagy is the liver cell energy recycling system regulating a variety of homeostatic mechanisms.Damaged organelles,lipids and proteins are degraded in the lysosomes and their elements are re-used by the cell.Investigations on autophagy have led to the award of two Nobel Prizes and a health of important reports.In this review we describe the fundamental functions of autophagy in the liver including new data on the regulation of autophagy.Moreover we emphasize the fact that autophagy acts like a two edge sword in many occasions with the most prominent paradigm being its involvement in the initiation and progress of hepatocellular carcinoma.We also focused to the implication of autophagy and its specialized forms of lipophagy and mitophagy in the pathogenesis of various liver diseases.We analyzed autophagy not only in well studied diseases,like alcoholic and nonalcoholic fatty liver and liver fibrosis but also in viral hepatitis,biliary diseases,autoimmune hepatitis and rare diseases including inherited metabolic diseases and also acetaminophene hepatotoxicity.We also stressed the different consequences that activation or impairment of autophagy may have in hepatocytes as opposed to Kupffer cells,sinusoidal endothelial cells or hepatic stellate cells.Finally,we analyzed the limited clinical data compared to the extensive experimental evidence and the possible future therapeutic interventions based on autophagy manipulation.

Liver sinusoidal endothelial and biliary cell repopulation following irradiation and partial hepatectomy

AIM: To investigate whether irradiation (IR) and partial hepatectomy (PH) may prepare the host liver for nonparenchymal cell (NPC) transplantation.METHODS: Livers of dipeptidyl peptidase(DPP)-deficient rats were pre-conditioned with external beam IR (25 Gy) delivered to two-thirds of the right liver lobules followed by a one-third PH of the untreated lob-ule. DPP-positive liver cells (NPC preparations enriched for liver sinusoidal endothelial cells (LSECs) and hepatocytes) were transplanted via the spleen into the recipient livers. The extent and quality of donor cell engraftment and growth was studied over a long-term interval of 16 wk after transplantation.RESULTS: Host liver staining demonstrated 3 different repopulation types. Well def ined clusters of donor-derived hepatocytes with canalicular expression of DPP were detectable either adjacent to or in between large areas of donor cells (covering up to 90% of the section plane) co-expressing the endothelial marker platelet endothelial cell adhesion molecule. The third type consisted of formations of DPP-positive duct-like structures which co-localized with biliary epithelial CD49f.CONCLUSION: Liver IR and PH as a preconditioning stimulus enables multiple cell liver repopulation by donor hepatocytes, LSECs, and bile duct cells.

Neuropilin-1: A feasible link between liver pathologies and COVID-19

The severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)pandemic has a tremendous impact on the health of millions of people worldwide.Unfortunately,those suffering from previous pathological conditions are more vulnerable and tend to develop more severe disease upon infection with the new SARS-CoV-2.This coronavirus interacts with the angiotensin-converting enzyme 2 receptor to invade the cells.Recently,another receptor,neuropilin-1(NRP-1),has been reported to amplify the viral infection.Interestingly,NRP-1 is expressed in nonparenchymal liver cells and is related to and upregulated in a wide variety of liver-related pathologies.It has been observed that SARS-CoV-2 infection promotes liver injury through several pathways that may be influenced by the previous pathological status of the patient and liver expression of NRP-1.Moreover,coronavirus disease 2019 causes an inflammatory cascade called cytokine storm in patients with severe disease.This cytokine storm may influence liver sinusoidal-cell phenotype,facilitating viral invasion.In this review,the shreds of evidence linking NRP-1 with liver pathologies such as hepatocellular carcinoma,liver fibrosis,nonalcoholic fatty liver disease and inflammatory disorders are discussed in the context of SARS-CoV-2 infection.In addition,the involvement of the infection-related cytokine storm in NRP-1 overexpression and the subsequent increased risk of SARS-CoV-2 infection are also analyzed.This review aims to shed some light on the involvement of liver NRP-1 during SARSCoV-2 infection and emphasizes the possible involvement this receptor with the observed liver damage.

Hepatitis E virus-related acute liver failure associated with pure red cell aplasia

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Liver regeneration using decellularized splenic scaffold: a novel approach in tissue engineering

BACKGROUND： The potential application of decellularized liver scaffold for liver regeneration is limited by severe shortage of donor organs. Attempt of using heterograft scaffold is accompanied with high risks of zoonosis and immunological rejection. We proposed that the spleen, which procured more extensively than the liver, could be an ideal source of decellularized scaffold for liver regeneration. METHODS： After harvested from donor rat, the spleen was processed by 12-hour freezing/thawing ×2 cycles, then circulation perfusion of 0.02% trypsin and 3% Triton X-100 sequentially through the splenic artery for 32 hours in total to prepare decellularized scaffold. The structure and component characteristics of the scaffold were determined by hematoxylin and eosin and immumohistochemical staining, scanning electron microscope, DNA detection, porosity measurement, biocompatibility and cytocompatibility test. Recellularization of scaffold by 5×106 bone marrow mesenchymal stem cells（BMSCs） was carried out to preliminarily evaluate the feasibility of liver regeneration by BMSCs reseeding and differentiation in decellularized splenic scaffold.RESULTS： After decellularization, a translucent scaffold, which retained the gross shape of the spleen, was generated. Histological evaluation and residual DNA quantitation revealed the remaining of extracellular matrix without nucleus and cytoplasm residue. Immunohistochemical study proved the existence of collagens I, IV, fibronectin, laminin and elastin in decellularized splenic scaffold, which showed a similarity with decellularized liver. A scanning electron microscope presented the remaining three-dimensional porous structure of extracellular matrix and small blood vessels. The poros-ity of scaffold, aperture of 45.36±4.87 μm and pore rate of 80.14%±2.99% was suitable for cell engraftment. Subcutaneous implantation of decellularized scaffold presented good histocompatibility, and recellularization of the splenic scaffold demonstrated that BMSCs could locate and survive in the decellularized matrix. CONCLUSION： Considering the more extensive organ source and satisfying biocompatibility, the present study indicated that the three-dimensional decellularized splenic scaffold might have considerable potential for liver regeneration when combined with BMSCs reseeding and differentiation.

Differential Proteomics in Malignant and Normal Liver Cell Lines

Objective： To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods： Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 （Immobilized Mental Affinity Capture） and WCX2 （Weak Cation Exchange） chips on the SELDI-TOF-MS ProteinChip reader. Results： Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion： Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

Simultaneous recovery of dual pathways for ammonia metabolism do not improve further detoxification of ammonia in HepG2 cells

BACKGROUND:Key enzyme deficiency in the dual-pathway of ammonia metabolism leads to low detoxification capacity of HepG2 cells.Previously,we established a HepG2/AFhGS cell line with overexpression of human glutamine synthetase(hGS) in pathway 1 and a HepG2/(hArgI+hOTC)4 cell line with overexpression of human arginase I(hArgI) and human ornithine transcarbamylase(hOTC) in pathway 2.The present study aimed to investigate whether simultaneous recovery of the two pathways contributes to the further improvement of ammonia detoxification in HepG2 cells.METHODS:We adopted a recombinant retrovirus carrying the hGS gene to infect HepG2/(hArgI+hOTC)4 cells and selected a new recombinant HepG2 cell line.The capacities of ammonia tolerance and detoxification in cells were detected by biochemical methods.Cell cycle PCR chip was used to assess the changes of gene expression.RESULTS:Introducing hGS into HepG2/(hArgI+hOTC)4 cells did not lead to hGS overexpression,but inhibited hArgI expression.The levels of synthetic glutamine and urea in HepG2/(hArgI+hOTC+AFhGS)1 cells were significantly lower than those in HepG2/(hArgI+hOTC)4 cells when cultured in the medium with 10 and 15 mmol/L glutamate(Glu) and with 60 and 180 mmol/L NH 4 Cl,respectively.In addition,the comparison of different cell growth showed that HepG2/AFhGS cells significantly lagged behind the other cells by the 5th and 7th day,indicating that introduction of hGS impedes HepG2 cell proliferation.Analysis of the mechanism suggested that the decreased expression of BCL2 played an important role.CONCLUSIONS:This study demonstrated that the recovery of two ammonia metabolic pathways in HepG2 cells is not helpful in increasing ammonia metabolism.The reinforcement of the pathway of urea metabolism is more important and valuable in improving the ammonia metabolism capacity in HepG2 cells.

QUANTITATIVE STUDIES ON THE DNA CONTENT AND MORPHOMETRIC FEATURES OF LIVER CELL DYSPLASIA

The DNA content and morphometric features of hepatocellular carcinoma (HCC) and liver cell dysplasia (LCD), including nuclear area, nuclear perimeter, nuclear maximum diameter and nuclear circle diameter, were quantitatively determined by means of image analysis technology. The results showed that in comparison with normal hepatocytes, LCD had a markedly increased DNA content and nuclear morphometric parameters, but the values were lower than those for HCC. LCD showed a slight increase in nuclear atypia represented by the nuclear irregular index, which was also less than HCC. The findings indicate that LCD may be a precaneerous lesion of HCC, to the cells in an abnormal proliferative state.

Effect of Qishen decoction on dedifferentiation of sinusoidal endothelial cells by autophagy

Objective:To observe the effect of Qishen decoction on dedifferentiation and autophagy of liver sinusoid endothelial cells(LSEC);Methods:LSEC were randomly divided into the control group,model group,Qishen Decoction low,medium,high dose group,and inhibitor group.The model was induced by 100μg/ml oxidized low-density lipoprotein(oxLDL)for 24 hours,and the corresponding drugs or medicated serum were given for intervention.The expression levels of VEGFR2 and ET1 were detected by RT-qPCR and immunofluorescence staining,the ultrastructure of LSEC was detected by transmission electron microscopy,the content of NO was detected by ELISA,the expression levels of autophagy related proteins(LC3BI,LC3BⅡand p62)and endothelial function related proteins(eNOS and p-eNOS)were detected by western blot;Results:The results of transmission electron microscopy showed that Qishen decoction medicated serum could increase the number of fenestra and autophagy in LSEC cells,and inhibit the formation of basement membrane under endothelium.Compared with the model group,Qishen decoction medicated serum could significantly up-regulate the expression level of VEGFR2 mRNA and protein in LSEC,down regulate the expression level of ET1 mRNA and protein,the difference was statistically significant(P<0.05).In addition,Qishen decoction medicated serum could significantly increase the expression of LC3BII,p-eNOS,eNOS protein and the ratio of LC3BII/LC3BI,p-eNOS/eNOS,and reduce the expression of LC3BI and p62 protein in LSEC,which is statistically significant compared with the model group(P<0.05).Conclusion:Qishen decoction can inhibit the dedifferentiation of LSEC by promoting the autophagy level of LSEC,and then play an anti-fibrosis role.