HSC-T6 Hepatic Stellate Cells Research Articles

Peach Kernel Extracts Inhibit Lipopolysaccharide-Induced Activation of HSC-T6 Hepatic Stellate Cells.

There is an important role for hepatic stellate cells (HSCs) in liver fibrosis. As it stands, many traditional Chinese medicine formulations can effectively improve liver fibrosis, whether it is clinically used or in animal studies; however, the efficacy and mechanism of the main formulations remain unclear, including the peach kernel, which contains numerous phytochemicals with a wide range of biological activities. The purpose of this study was to investigate peach kernel's anti-liver fibrosis effects. In this study, peach kernel extracts inhibited lipopolysaccharide (LPS) activation in HSC-T6 cells and the expression of α-smooth muscle actin and connective tissue growth factor induced by LPS in HSC-T6 cells. Furthermore, peach kernel extracts inhibited signal transducers involving protein kinase B and mitogen-activated protein kinase, which regulate downstream genes associated with inflammation. As a result, peach kernel extracts inhibited inflammatory responses and subsequently inhibited LPS-induced transformation of activated HSC-T6 cells.

In vitro study on the role and mechanism of interferon α-1b in regulating the inhibition of protein kinase Cε and Cα on fibrosis of hepatic stellate cells

Objective To investigate the regulation of interferon α-1b (IFNα-1b) on protein kinase Ce (PKCe) and protein kinase Cα (PKCα) which inhibit the fibrosis of hepatic stellate cells (HSC), and to explore its mechanism. Methods HSC-T6 cells were treated with different levels of IFNα-1b (100, 200, 400, 800 and 1 000 U/mL) and the proliferation of HSC-T6 cells was analyzed by methyl thiazol tetrazolium (MTT) assay. Changes of hydroxyproline level were analyzed. The expressions of PKCe and PKCα were detected by immunofluorescence staining. PKCe, PKCα, β-catenin and Survivin mRNA levels were detected by RT-PCR. PKCe, PKCα, β-catenin and Survivin protein levels were detected by Western blot. Variance analysis was conducted by using one-way ANOVA approach. Results The inhibition rates of 100, 200, 400, 800 and 1 000 U/mL IFNα-1b treatment after 24 hours of administration were (15.85±1.05)%, (36.59±1.03)%, (45.12±1.05)%, (50.00±1.01)% and (62.20±1.02)%, respectively, with statistically significant differences among groups (F=27.478, P<0.01). The 48h inhibition rates were (20.87±1.09)%, (43.96±1.08)%, (53.85±1.08)%, (64.84±1.06)% and (74.72±1.07)%, respectively, with statistically significant differences among groups (F=25.321, P<0.01). half maximal inhibitory concentration at 48 h was 343.47 U/mL. The levels of hydroxyproline in 100, 200 and 400 U/mL IFNα-1b groups were (7.48±0.28), (6.26±0.17) and (3.86±0.20) μg/mL, respectively, which were lower than that in control group (8.47±0.32) μg/mL. The differences were all statistically significant (t=4.033, 10.564 and 21.160, respective, all P<0.05). The fluorescence intensities of PKCe in 100, 200 and 400 U/mL IFNα-1b groups were all lower than that of control group. The differences were statistically significant (t=1.984, 2.457 and 7.771, respectively, all P<0.05). The fluorescence intensities of PKCα were also significantly lower than that of control group (t=9.232, 15.921 and 22.222, respectively, all P<0.01). With the increase of IFNα-1b level, the levels of HSC-T6 PKCe, PKCα, β-catenin and survivin were significantly lower than those of control group (t=7.020, 24.562, 45.701 and 14.241, respectively, all P<0.01). With the increase of IFNα-1b, the levels of HSC-T6 PKCe, PKCα, β-catenin and survivin were significantly lower than those of control group (t=9.564, 4.409, 10.036 and 6.794, respectively, all P<0.01). Conclusions IFNα-1b can down-regulate the expression of collagen in hepatic stellate cells in a dose-dependent manner, reduce the expressions of PKCe, PKCα, β-catenin and Survivin, and inhibit the proliferation of HSC-T6 hepatic stellate cells. Key words: IFNα-1b; hepatic stellate cells; proliferation; molecular mechanism

Lipopolysaccharides induce Smad2 phosphorylation through PI3K/Akt and MAPK cascades in HSC-T6 hepatic stellate cells

AimsEndotoxemia and its pro-fibrogenic signaling play a significant role in the development of hepatic fibrosis. This study investigated whether lipopolysaccharide (LPS) directly activate cultured HSC-T6 hepatic stellate cells (HSCs) through triggering Smad-dependent pro-fibrogenic signaling pathway. Main methodsDirect cell counting and assays for cell proliferation and migration were used to measure the effects of LPS on HSC behaviors. Quantitative PCR, Western blot, and gelatin zymography were used to quantify the molecular effects of LPS on expression of HSC activation markers and signaling activity. Key findingsLong-term exposure to LPS exhibited moderately stimulatory effect on HSC cell growth. A wound-healing cell migration assay showed that LPS suppressed HSC-T6 cell migration. qPCR and Western blotting detection indicated that LPS treatment induced upregulation of type I and IV collagens, α-smooth muscle actin (α-SMA), and matrix metalloproteinase-9 (MMP-9). Gelatin zymography confirmed that LPS elevated MMP-9, but not MMP-2 gelatinolytic activity. Moreover, LPS immediately stimulated Akt, EKR1/2, JNK, p38 MAPK, and Smad2 hyperphosphorylation, supporting that LPS directly triggers pro-fibrogenic Smad signaling cascade without TGF-β1 stimulation. Kinase blockade experiments demonstrated the involvement of PI3K/Akt, JNK, p38 MAPK, but not ERK1/2 signaling activation in the LPS-elicited Smad2 phosphorylation as well as the overexpression of type I collagen and α-SMA in HSC-T6 cells. SignificanceThese findings demonstrate that LPS exerts pro-fibrogenic effect through activation and transformation of HSCs. The tissue-remodeling effect of LPS may be attributable to its ability to activate non-canonical Smad pathway through PI3K/Akt and MAPK signaling cascades.

Ceanothane- and lupane-type triterpene esters from the roots of Hovenia dulcis and their antiproliferative activity on HSC-T6 cells

Three ceanothane-type and three lupane-type triterpenoids, as well as three known compounds, were isolated from the roots of Hovenia dulcis (Rhamnaceae), based on LC–MS dereplication. The previously undescribed compounds were determined to be 27-O-protocatechuoyl-3-dehydroxyisoceanothanolic acid, 27-O-protocatechuoyl-3-dehydroxycolubrinic acid, 27-O-protocatechuoyl-3-dehydroxyepicolubrinic acid, 27-O-protocatechuoylbetulinic acid, 27-O-p-hydroxybenzoylbetulinic acid, and 27-O-syringoylbetulinic acid by 1D and 2D NMR spectroscopic and HR mass spectrometric data analysis. The isolates were examined for their antiproliferative activity in HSC-T6 hepatic stellate cells; compounds 1, 2, 3, and 6 showed IC50 values in the range of 15–50 μM.

Catechin-Bound Ceanothane-Type Triterpenoid Derivatives from the Roots of Zizyphus jujuba.

Three unprecedented ceanothane-type triterpenoids, ent-epicatechinoceanothic acid A (1), ent-epicatechinoceanothic acid B (2), and epicatechino-3-deoxyceanothetric acid A (3), containing C-C bond linkages with catechin moieties, were isolated from the roots of Zizyphus jujuba. Their chemical structures, including absolute configurations, were established by spectroscopic analysis and calculation of their ECD spectra. A possible biogenetic pathway for C-C bond formation between the catechin and the triterpenoid moieties is presented. Compound 1 was evaluated for its antiproliferative activity on HSC-T6 hepatic stellate cells.

Inhibitory effects of Phyllanthus emblica L. on hepatic steatosis and liver fibrosis in vitro

Phyllanthus emblica L. exerts multiple biological functions, and its fruit possesses hepatoprotective benefits. However, the underlying mechanisms of these activities remain unclear. This study aimed to investigate the potential impact of P. emblica on improving nonalcoholic fatty liver disease (NAFLD). Free fatty acid mixture-induced HepG2 cells and leptin-induced HSC-T6 hepatic stellate cells were used to mimic nonalcoholic steatohepatitis and hepatic fibrosis, respectively. Ellagic acid (EA) was identified as a major compound in the water extract of P. emblica fruits (WEPL). WEPL significantly decreased fat accumulation and ROS production by altering lipogenesis-related gene expression and stimulating AMP-activated protein kinase (AMPK) signalling in treated HepG2 cells. WEPL also inhibited hepatic fibrosis in HSC-T6 cells and induced mitochondrial apoptosis. Importantly, EA was shown to inhibit cellular steatosis and liver fibrosis in vitro. In conclusion, WEPL reduces lipogenesis in hepatocytes and fibrotic cells, suggesting that WEPL has the potential to improve NAFLD.

Study on rational daily administration frequency of Fufang Biejia Ruangan tablet based on integrated serum pharmacologic and pharmacokinetic model

To observe in vitro the effect of rat drug serum on the proliferation of HSC-T6 hepatic stellate cells in the pharmacokinetic model for determining peoniflorin in Fufang Biejia Ruangan tablet, in order to discover the rational daily administration frequency of Fufang Biejia Ruangan tablet. Fufang Biejia Ruangan tablet was orally administered to rats with different daily administration frequency. Their blood was collected from veins behind eye sockets at different time points before the administration and after the first administration, in order to determine the concentration of peoniflorin in blood plasma and the effect of rat drug serums on the proliferation of HSC-T6. A comprehensive analysis was made on the relationship between pharmacodynamics and pharmacokinetics to determine the rational daily administration frequency of Fufang Biejia Ruangan tablet. The results showed a good correlation between the inhibitory effect of Fufang Biejia Ruangan tablet-contained serum on HSC-T6 and the concentration of peoniflorin in blood. The two-time administration group showed higher pharmacologic and pharmacokinetic AUCs than one-time administration and three-time administration groups. In conclusion, Fufang Biejia Ruangan table is recommended to be taken twice a day for treating liver fibrosis in chronic hepatitis.

Sesquiterpene lactones from the roots of Lindera strychnifolia

Phytochemical investigation of the roots of Lindera strychnifolia resulted in the isolation of seven sesquiterpene lactones named linderolide G-M (1-7), together with twelve known sesquiterpenes. The structures and relative configurations of 1-7 were determined through spectroscopic observations, and the absolute configurations were defined by CD analysis. Linderolide G (1) and lindestrene (16) showed cytotoxicity against HSC-T6 hepatic stellate cells.

HGF regulates the activation of TGF‐β1 in rat hepatocytes and hepatic stellate cells

Hepatocyte growth factor (HGF) ameliorates experimental liver fibrosis through many mechanisms, including degradation of accumulated collagen and decreased expression of fibrotic genes. Investigating an upstream mechanism in which HGF could decrease many fibrotic effectors, we asked whether HGF regulates activation of the fibrotic cytokine transforming growth factor-beta 1 (TGF-β1). Specifically, we tested whether HGF decreases the levels of active TGF-β1, and whether such decrease depends on the predominantly hepatocyte-secreted protease plasmin, and whether it depends on the TGF-β1 activator thrombospondin-1 (TSP-1). With hepatocyte monocultures, we found HGF-induced hepatocyte proliferation did increase total levels of plasmin, while decreasing gene expression of fibrotic markers (PAI-1, TGF-β1, and TIMP-2). With in vitro models of fibrotic liver (HSC-T6 hepatic stellate cells, or co-cultures of HSC-T6 and hepatocytes), we found high levels of fibrosis-associated proteins such as TSP-1, active TGF-β1, and Collagen I. HGF treatment on these fibrotic cultures stimulated plasmin levels; increased TSP-1 protein cleavage; and decreased the levels of active TGF-β1 and Collagen I. When plasmin was blocked by the inhibitor aprotinin, HGF could no longer decrease TGF-β1 activation and Collagen I. Meanwhile, the TSP-1-specific peptide inhibitor, LSKL, reduced TGF-β1 to the same level as in the HGF-treated cultures; combining LSKL and HGF treatments caused no further decrease, suggesting that HGF affects the TSP-1 dependent pathway of TGF-β1 activation. Therefore, HGF can decrease TGF-β1 activation and TGF-β1-dependent fibrotic markers, by stimulating hepatocytes to produce plasmin, and by antagonizing TSP-1-dependent activation of TGF-β1.

Vasonatrin peptide ameliorates hepatic fibrosis via NPR/cGMP/PKG signal transduction pathway

Objective To investigate the effects and signal transduction pathway of vasonatrin peptide(VNP),a novel man-made natriuretic peptide,on hepatic fibrosis.Methods Mice were injec ted with carbon tetrachloride(CCl4)for 12 weeks,with or without VNP treatment in the last 6 weeks.HE staining and Sirius red staining were performed to evaluate the status of hepatic fibrosis.In vitro after treatment of VNP,and DNA and collagen synthesis of cultured HSC-T6 hepatic stellate cells were assessed by[3H]-thymidine and[3H]proline incorporation,respectively.The signaling pathway involved was identified by radioimmunoassay to detect the levels of intracellular cGMP,and by mimicking experiments using 8-br-cGMP(a membrane-permeable cGMP analog).Blocking experi ments were performed using HS-142-1,an antagonist of guanylyl cyclase-coupled natriuretic peptide receptor(NPR),or KT-5823,the cGMP-dependent protein kinase(PKG)inhibitor.Results VNP markedly alleviated CCl4-induced liver fibrosis in mice.In vitro,HSC-T6 cells demonstrated a dosedependent reduction of DNA and collagen synthesis in the presence of VNP.In addition,VNP significantly increased intracellular levels of cGMP.The effects of VNP were mimicked by 8-br-cGMP,but they were inhibited by HS-142-1,or KT-5823.Conclusion VNP ameliorated liver fibrosis by inhibiting collagen production from hepatic stellate cells via guanylyl cyclase-coupled NPR/cGMP/PKG signal pathway,indicating that VNP might be a new effective agent in the treatment of liver fibrosis. Key words: Natriuretic peptides; Hepatic fibrosis; Carbon tetrachloride

Antifibrotic Constituents from Garcinia mangostana

From the CHCl3-soluble fraction of the fruits of Garcinia mangostana (Clusiaceae), six xanthone derivatives, alpha-mangostin (1), gamma-mangostin (2), gartanin (3), deoxygartanin (4), 1-isomangstanin (5) and garcinone E (6), were isolated. All these compounds significantly inhibited HSC-T6 viability as assessed by employing HSC-T6 hepatic stellate cells as an in vitro assay system. Among them, compounds 1 and 2, the most potent and major constituents of G. mangostana, inhibited HSC-T6 viability in dose- and time-dependent manners. In addition, compounds 1 and 2 significantly reduced collagen content, a pathological characteristic of liver fibrosis. Taken together, G. mangostana and its constituents might be beneficial for the treatment of liver fibrosis.

Antifibrotic activity of coumarins from Cnidium monnieri fruits in HSC-T6 hepatic stellate cells

The CHCl(3) fraction of Cnidium monnieri fruits significantly inhibited the proliferation of hepatic stellate cells in an in-vitro assay system employing HSC-T6 hepatic stellate cell lines. Activity-guided fractionation of the CHCl(3) fraction of C. monnieri led to the isolation of ten coumarins: osthol (1), meranzin (2), auraptenol (3), meranzin hydrate (4), 7-hydroxy-8-methoxy coumarin (5), imperatorin (6), xanthotoxol (7), xanthotoxin (8), bergapten (9) and isopimpinellin (10). Of these, compounds 1 and 6 significantly inhibited proliferation of HSCs in a time- and concentration-dependent manner. In addition, compounds 1 and 6 significantly reduced collagen content in HSC-T6 cells.

Protecting effects of vasonatrin peptide against carbon tetrachloride-induced liver fibrosis

In order to investigate the effects of vasonatrin peptide (VNP), a novel man-made natriuretic peptide, on liver fibrosis, mice received carbon tetrachloride (CCl 4) injection for 12 weeks and with or without VNP treatment during the last 6 weeks. Hematoxylin–eosin (HE) staining and Sirius red staining were performed to evaluate the status of liver fibrosis. After treatment of VNP, DNA and collagen synthesis of cultured HSC-T6 hepatic stellate cells were assessed by [ 3H]-thymidine and [ 3H]-proline incorporation, respectively. Additionally, involved signaling pathway was identified by radioimmunoassay to detect the levels of intracellular cGMP and by mimicking experiments using 8-br-cGMP (a membrane-permeable cGMP analog). Also, blocking experiments were performed using HS-142-1, an antagonist of guanylyl cyclase-coupled natriuretic peptide receptor (NPR), or KT-5823, the cGMP-dependent protein kinase (PKG) inhibitor. As a result , VNP markedly alleviated CCl 4-induced liver fibrosis in mice. In vitro, HSC-T6 cells demonstrated a dose-dependent reduction of DNA and collagen synthesis in the presence VNP. In addition, VNP significantly increased the intracellular levels of cGMP. These effects of VNP were mimicked by 8-br-cGMP, although inhibited by HS-142-1 or KT-5823. Taken together, VNP ameliorates liver fibrosis by inhibiting collagen production from hepatic stellate cells via guanylyl cyclase-coupled NPR/cGMP/PKG pathway, indicating that VNP might be a new effective reagent in the treatment of liver fibrosis.