Self-Protection against Triptolide-Induced Toxicity in Human Hepatic Cells via Nrf2-ARE-NQO1 Pathway

Objective: To find the signaling pathway of triptolide(TP)-induced liver injury and to reveal whether NF-E2-related factor 2(Nrf2) plays an important role in cellular self-protection. Methods: The L-02 and HepG2 cells were cultured and treated with various concentrations of TP. The cell viability was observed, and the cell medium was collected for detecting the aspartate aminotransferase(ALT), alanine aminotransferase(AST), lactate dehydrogenase(LDH), superoxide dismutase(SOD) and L-glutathione production(GSH) levels. Nrf2 and its downstream target NAD(P)H: quinine oxidoreductase 1(NQO1) and heme oxygenase-1(HO-1) expression, the nuclear translocation of Nrf2, and the binding ability of Nrf2 and antioxidant response element(ARE) were also identified. Meanwhile,shRNA was used to silence Nrf2 in L-02 cells to find out whether Nrf2 plays a protective role. Results: The viability of the L-02 and HepG2 cells treated with TP decreased in a doseand time-dependent manner, and TP(20–80 μg/mL) markedly induced the release of ALT, AST and LDH(P<0.05 or P<0.01), reduced the levels of SOD and GSH(P<0.01), and increased the intracellular reactive oxygen species. Meanwhile, TP augmented the Nrf2 expression in L-02 and HepG2 cells(P<0.05 or P<0.01), induced Nrf2 nuclear translocation, increased the Nrf2 ARE binding activity, and increased HO-1 and NQO1 expressions. Nrf2 knockdown revealed a more severe toxic effect of TP(P<0.05 or P<0.01). Conclusions: Human hepatic cells treated with TP induced oxidative stress, and led to cytotoxicity. Self-protection against TP-induced toxicity in human hepatic cells might be via Nrf2-ARE-NQO1 transcriptional pathway.

Herbal compound 861 regulates mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells

AIM： To identify the role of herbal compound 861 （Cpd 861） in the regulation of mRNA expression of collagen synthesis- and degradation-related genes in human hepatic stellate cells （HSCs）. METHODS： mRNA levels of collagen types I and III, matrix metalloproteinase 1 （MMP-1）, matrix metalloproteinase 2 （MMP-2）, membrane type-1 matrix metalloproteinase （MT1-MMP）, tissue inhibitor of metalloproteinase 1 （TIMP-1）, and transforming growth factor β1 （TGF-β1） in cultured-activated HSCs treated with Cpd 861 or interferon-γ, （IFN-γ,） were determined by real-time PCR. RESULTS： Both Cpd 861 and IFN-γ reduced the mRNA levels of collagen type Ⅲ, MMP-2 and TGF-β1. Moreover, Cpd 861 significantly enhanced the MMP-1 mRNA levels while down-regulated the TIMP-1 mRNA expression, increasing the ratio of MMP-1 to TIMP-1 to （6.3 ＋ 0.3）- fold compared to the control group. CONCLUSION： The anti-fibrosis function of Cpd 861 may be mediated by both decreased interstitial collagen sythesis by inhibiting the transcription of collagen type Ⅲ and TGF-β1 and increased degradation of these collagens by up-regulating MMP-1 and down-regulating TIMP-1 mRNA levels.

Mesenchymal stem cells from the human umbilical cord ameliorate fulminant hepatic failure and increase survival in mice

BACKGROUND：Cell therapy has been promising for various diseases.We investigated whether transplantation of human umbilical cord mesenchymal stem cells（h UCMSCs）has any therapeutic effects on D-galactosamine/lipopolysaccharide（Gal N/LPS）-induced fulminant hepatic failure in mice.METHODS：h UCMSCs isolated from human umbilical cord were cultured and transplanted via the tail vein into severe combined immune deficiency mice with Gal N/LPS-induced fulminant hepatic failure.After transplantation,the localization and differentiation of h UCMSCs in the injured livers were investigated by immunohistochemical and genetic analy- ses. The recovery of the injured livers was evaluated histologi- cally. The survival rate of experimental animals was analyzed by the Kaplan-Meier method and log-rank test. RESULTS： hUCMSCs expressed high levels of CD29, CD73, CD13, CD105 and CD90, but did not express CD31, CD79b, CD133, CD34, and CD45. Cultured hUCMSCs displayed adip- ogenic and osteogenic differentiation potential. Hematoxylin and eosin staining revealed that transplantation of hUCMSCs reduced hepatic necrosis and promoted liver regeneration. Transplantation of hUCMSCs prolonged the survival rate of mice with fulminant hepatic failure. Polymerase chain reaction for human alu sequences showed the presence of human cells in mouse livers. Positive staining for human albumin, human alpha-fetoprotein and human cytokeratin 18 suggested the for- mation of hUCMSCs-derived hepatocyte-like cells in vivo.CONCLUSIONS： hUCMSC was a potential candidate for stem cell based therapies. After transplantation, hUCMSCs partially repaired hepatic damage induced by GalN/LPS in mice. hUC- MSCs engrafted into the injured liver and differentiated into hepatocyte-like cells.

Trichloroethylene Induces Biphasic Concentration-dependent Changes in Cell Proliferation and the Expression of SET-Associated Proteins in Human Hepatic L-02 Cells

Trichloroethylene （TCE） is a major pollutant that affects both occupational and general environments. The liver is an important target organ of TCEE. Substantial efforts and remarkable progress into understanding TCE cytotoxicity have been made in cultured liver cells. However, the molecular mechanisms by which TCE induces hepatotoxicity are not well understood. SET （also known as protein phosphatase 2A inhibitor, 12PP2A, or template-activating factor-I, TAF-D is a nuclear protein that regulates histone modification, gene transcription, DNA replication, nucleosome assembly,

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.

KN-93,a specific inhibitor of CaMKⅡ inhibits human hepatic stellate cell proliferation in vitro

AIM： To investigate the effects of KN-93, a CaMKⅡ selective inhibitor on cell proliferation and the expression of p53 or p21 protein in human hepatic stellate cells. METHODS： Human hepatic stellate cells （LX-2） were incubated with various concentrations （0-50 μmol/L） of KN-93 or its inactive derivative, KN-92. Cell proliferation was measured by CCK-8 assay, and the expression of two cell cycle regulators, p53 and p21, was determined by SDS-PAGE and Western blotting. RESULTS： KN-93 （5-50 μmol/L） decreased the proliferation of human hepatic stellate cells in a dosedependent manner from 81.76% （81.76% ± 2.58% vs 96.63% ± 2.69%, P 〈 0.05） to 27.15% （27.15% ± 2.86% vs 96.59% ± 2.44%, P 〈 0.01） after 24 h treatment. Incubation of 10 μmol/L KN-93 induced the cell growth reduction in a time-dependent manner from 78.27% at 8 h to 11.48% at 48 h. However, KN-92, an inactive derivative of KN-93, did not inhibit cell proliferation effectively. Moreover, analysis of cell cycle regulator expression revealed that KN-93 rather than KN-92 reduced the expression of p53 and p21. CONCLUSION： KN-93 has potent inhibitory effect on proliferation of LX-2 cells by modulating the expression of two special cell cycle regulators, p53 and p21.