Temporal Integrative Omics Reveals an Increase in Nondegradative Ubiquitylation during Primary Hepatocyte Dedifferentiation

Primary hepatocytes(PHCs)are widely used in various fields,but the progressive deterioration of liverspecific features in vitro significantly limits their application.While the transcriptional regulation and whole cell proteome(WCP)of PHCs have been extensively studied,only a small number of studies have addressed the role of posttranslational modifications in this process.To elucidate the underlying mechanisms that induce dedifferentiation,we carried out parallel quantifications of the transcriptome,WCP,ubiquitinome,and phosphoproteome of rat PHCs after 0,6,12,24,and 48 h of in vitro culture.Our data constitute a detailed proteomic analysis of dedifferentiated PHCs including 2196 proteins,2056 ubiquitinated sites,and 4932 phosphorylated peptides.We revealed a low correlation between the transcriptome and WCP during dedifferentiation.A combined analysis of the ubiquitinome with the corresponding WCP indicated that the dedifferentiation of PHCs led to an increase in nondegradative K27 ubiquitination.Functional analysis of the altered phosphoproteins suggested a significant enrichment in ferroptosis.In all,404 proteins with both ubiquitination and phosphorylation were identified to be involved in critical metabolic events.Furthermore,Ptbph Hnqjd,Hnrnpu,and Srrm2 were identified as hub genes.Taken together,our data provide new insights into proteome dynamics during PHC dedifferentiation and potential targets to inhibit the dedifferentiation process.

Forkhead box protein O1(FoxO1)regulates lipids metabolism and cell proliferation mediated by insulin and PI3K-Akt-mTOR pathway in goose primary hepatocytes

In order to explore the role of forkhead box protein O1(FoxO1)in the lipid metabolism and cell proliferation,goose primary hepatocytes were isolated and incubated with insulin or PI3K-Akt-mTOR pathway dual inhibitor NVPBEZ235,and then transfected with FoxO1 interference plasmid.The related parameters of lipid metabolism and cell proliferation were measured.The results firstly showed that FoxO1 interference increased the intracellular TG and lipids concentration(P<0.05);and increased the proliferative index(PI),cell DNA synthesis,protein expression of Cyclin D1 in goose primary hepatocytes(P<0.05).Secondly,the co-treatment of insulin and FoxO1 interference increased the mRNA level and protein content of Cyclin D1(P<0.05);however,there was no significant difference between the insulin treatment and the co-treatment of insulin and miR-FoxO1 interference in the intracellular TG and lipids concentration and PI(P>0.05).Lastly,the decrease of intracellular TG and lipids concentration and PI induced by NVP-BEZ235 was up-regulated by FoxO1 interference significantly(P<0.05).In summary,FoxO1 could regulate the lipids metabolism and cell proliferation mediated by PI3K-Akt-mTOR signaling pathway in goose primary hepatocytes.Further investigations are required to highlight the potential role of FoxO1 in the lipid metabolism and cell proliferation mediated by insulin in goose primary hepatocyte.

Dexamethasone potentiates the insulin-induced Srebp-1c expression in primary rat hepatocytes

The impacts of dexamethasone(Dex)and thyroid hormone T3 on the insulin-stimulated Srebp-1c expression were studied in primary rat hepatocytes. Primary hepatocytes from Sprague-Dawley rats were isolated, cultured and treated with insulin in the presence or absence of the indicated reagents over time. The mRNA levels of indicated genes were determined using real-time PCR. Insulin treatment induced the Srebp-1c expression and suppressed the Pck1 expression in a time-dependent manner. Dex treatment alone reduced the Srebp-1c expression, whereas potentiated the insulin-induced its expression, which reached to a level that was higher than the insulin alone group. On the other hand, insulin treatment completely suppressed the Dex-induced Pck1 expression in the same cells. T3 treatment did not affect the expressions of Srebp-1c and Pck1 alone or in the presence of absence of insulin or Dex. Interestingly, insulin treatment induced the Rxrg m RNA expression level in the absence or presence of T0901317, a specific agonist for the liver X receptor. Dex and insulin mutually affect each other's ability to regulate the expression levels of hepatic genes involved in glucose and fatty acid metabolism. Insulin induced Rxrg expression in primary hepatocytes, which may contribute to the induction of Srebp-1c expression in the same cells.

In vitro culture of isolated primary hepatocytes and stem cell-derived hepatocyte-like cells for liver regeneration

Various liver diseases result in terminal hepatic failure, and liver transplantation, cell transplantation and artificial liver support systems are emerging as effective therapies for severe hepatic disease. However, all of these treatments are limited by organ or cell resources, so developing a sufficient number of functional hepatocytes for liver regeneration is a priority. Liver regeneration is a complex process regulated by growth factors （GFs）, cytokines, transcription factors （TFs）, hormones, oxidative stress products, metabolic net- works, and microRNA. It is well-known that the function of isolated primary hepatocytes is hard to maintain; when cultured in vitro, these cells readily undergo dedifferentia- tion, causing them to lose hepatocyte function. For this mason, most studies focus on inducing stem cells, such as embryonic stem cells （ESCs）, induced pluripotent stem cells （iPSCs）, hepatic progenitor cells （HPCs）, and mesenchymal stem cells （MSCs）, to differentiate into hepatocyte-like cells （HLCs） in vitro. In this review, we mainly focus on the nature of the liver regeneration process and discuss how to main- tain and enhance in vitro hepatic function of isolated primary hepatocytes or stem cell-derived HLCs for liver regenera- tion. In this way, hepatocytes or HLCs may be applied for clinical use for the treatment of terminal liver diseases and may prolong the survival time of patients in the near future.

Effect of Diallyl Trisulfide on Induction of UDS by Mutagenic Drugs in Primary Rat Hepatocytes

Mosl of anticancer drugs are mutagenic. A possible exeeption is diallyl .trisulfide(DAT ), a component of garlic. It is an antimutagenic anticunccr chemical although it ismainly uscd as antibiotic. Its modifying effeci on induction of UDS by mutagenicmitomycin C (MMC), cyclophosphamide (CP) and cis-diamine dichloroplatin (DDP) was invcstigiltcd with the UDS assay in the primary cultures of Wistar rat hepatocytes (hpc)using the autoradiographic technique. Resultsshowed that 1.0-4.0 nmol/ml of DAT didnot inducc UDS and that MMC, CP and DDP resulted in a significant induction ofdosc-dependent UDS. DAT enhanced induction of UDS by these drugs. A dose-effectrclationship was observed betwecn dose of DAT and enhancement of induction of UDS.Howcvcr, thc mcchanism of the enhancement is not clear.

Creating rat hepatocyte organoid as an in vitro model for drug testing

BACKGROUND Liver organoids have recently been applied as models for liver disease and drug screening,especially when combined with liver-on-a-chip technologies.Compared to hepatocyte-like cells,primary hepatocytes have high functionality but cannot maintain their function when cultured in vitro.Mesenchymal stem cells(MSCs)enhance hepatocyte function and maintain hepatocyte metabolism when co-cultured with hepatocytes.MSCs can help induced pluripotent stem cells to generate an organoid structure via the MSC-based traction force triggered by extracellular matrix(ECM)proteins.In this study,primary hepatocytes were cocultured with MSCs on a liver-derived ECM to generate liver organoids within a short duration.AIM To create hepatocyte organoids by co-culturing primary hepatocytes with MSCs on a porcine liver extracellular matrix(PLECM)gel.METHODS Perfusion and enzymatic hydrolysis were used to form the PLECM gel.Rat hepatocytes and human MSCs were mixed and plated on pre-solidified PLECM gel in a 48-well plate for 48 h to generate organoids.Generated organoids were evaluated through hematoxylin and eosin,periodic acid-Schiff,immunohistological,and immunofluorescence staining,and quantitative PCR for alb,CYP450 gene markers,and urea cycle genes.Culture medium was collected to detect albumin(ALB)and urea production on days 2,4,6,8,14,and 20.RESULTS The whole porcine liver was perfused and enzymatically hydrolyzed to form a PLECM gel.The structural components and basement membrane composition of the ECM,such as collagen type I,collagen type IV,fibronectin,and laminin,were demonstrated to be retained.Through interaction of human MSCs with the liverderived ECM,primary hepatocytes and human MSCs assembled together into a 3D construction and generated primary hepatocyte organoids for 48 h.The mRNAs of the gene alb,the CYP450 gene markers cyp1a1,cyp1a2,and cyp3a2 as well as urea cycle genes arg-1,asl,ass-1,cps-1,nags were highly expressed in hepatocyte organoids.Long-term survival of the primary hepatocyte organoids,as well as stable functionality,was demonstrated via ALB and urea production in vitro.CONCLUSION Our new method of creating primary hepatocyte organoids by co-culturing hepatocytes with MSCs on liver-derived ECM hydrogels could be used to develop models for liver disease and for drug screening.

Hepatoprotective Effects of Folium syringae Extracts Against Ethanolinduced Acute Liver Injury

The aim of the study was to investigate the hepatoprotective effects of Folium syringae(FS) extracts against ethanolinduced acute liver injury. Mice and primary hepatocytes were pretreated with FS extracts at different dosages before ethanol administration. Transaminases, glutathione S-transferase A1 level and hepatic biochemical indices(malondialdehyde, superoxide dismutase, glutathione and glutathione peroxidase) were determined. Pretreatment with FS extracts significantly inhibited the damage caused by ethanol and the hepatoprotective effects of FS were almost similar to Silymarin that was used to treat alcoholic liver injury. GSTA1 contents in all the FS extract-treated groups were significantly different from those in the ethanol-induced acute liver injury model group(p<0.01), and similar trends were observed in transaminases and hepatic indices level both in vitro and in vivo. The results showed that FS extracts had hepatoprotective effects against ethanol-induced injury. Those effects might be related to the enhancement of antioxidant capacity of liver cells, and FS extracts could reduce the release of liver GSTA1, which contributed to improve liver detoxification.

Altered integrity of hepatocyte tight junctions in rats with triptolide-induced cholestasis

Triptolide(TP),an active component of Tripterygium wilfordii Hook.f.(TWHF),has been widely used for centuries as a traditional Chinese medicine.However,the clinical application of TP has been restricted due to multitarget toxicity,such as hepatotoxicity.In this study,28 days of oral TP administration(100,200,or 400μg·kg^(-1)·d^(-1))induced the occurrence of cholestasis in female Wistar rats,as evidenced by increased serum levels ofγ-glutamyl transpeptidase(γ-GGT),alkaline phosphatase(ALP)and hepatic total bile acids(TBAs).In addition,the heptocyte polarity associated with the strcture of tight junctions(TJs)was disrupted in both rats and sandwich-cultured primary hepatocytes.Immunoblotting revealed decreased expression of the TJ-associated proteins occludin,claudin^(-1),and zonula occludens protein(ZO^(-1)),and downregulated m RNA levels of these TJs was also detected by real-time PCR.An immunofluorescence analysis showed abnormal subcellular localization of occludin,claudin^(-1) and ZO^(-1),which was also confirmed by transmission electron microscopy.Moreover,the concentration of FITC-dextran,a marker of paracellular penetration,was found to increase rapidly in bile increased rapidly(within 6 minutes)after treatment with TP,which indicated the functional impairment of TJs.Taken together,these results suggest that the administration of TP for 28 consecutive days to rats could induce cholestatic injury in the liver,and the increased paracellular permeability might play an important role in these pathological changes.

Detecting cell-secreted growth factors in microfluidic devices using bead-based biosensors

Microfluidic systems provide an interesting alternative to standard macroscale cell cultures due to the decrease in the number of cells and reagents as well as the improved physiology of cells confined to small volumes.However,the tools available for cell-secreted molecules inside microfluidic devices remain limited.In this paper,we describe an integrated microsystem composed of a microfluidic device and a fluorescent microbead-based assay for the detection of the hepatocyte growth factor(HGF)and the transforming growth factor(TGF)-β1 secreted by primary hepatocytes.This microfluidic system is designed to separate a cell culture chamber from sensing chambers using a permeable hydrogel barrier.Cell-secreted HGF and TGF-β1 diffuse through the hydrogel barrier into adjacent sensing channels and are detected using fluorescent microbead-based sensors.The specificity of sensing microbeads is defined by the choice of antibodies;therefore,our microfluidic culture system and sensing microbeads may be applied to a variety of cells and cell-secreted factors.