Effect of siRNA-mediated Smad3 silence on proliferation and apoptosis in activated hepatic stellate cells

AIM: To investigate the effects of siRNA-mediated Smad3 silence on proliferation and apoptosis in activated hepatic stellate cells (HSCs).METHODS: HSCs-T6 cells were divided into 3 groups: blank group, negative control group and siRNA-Smad3 transfection group. The siRNA-Smad3 was transfected into HSCs-T6 cells. At different time points after transfection, cell proliferation was measured by CCK-8, cell apoptosis was detected by flow cytometry, and protein levels of P53 and Bcl-2 were determined by immunocytochemistry.RESULTS: HSCs proliferation was significantly inhibited at the time points of 24 h, 48 h and 72 h after transfection. Meanwhile, the apoptosis of HSCs was significantly increased in siRNA-Smad3 transfection group (P<0.01). Compared to the control cells, the protein expression of P53 was significantly increased while Bcl-2 protein was significantly decreased 48 h after transfection in siRNA-Smad3 transfection group (P<0.01).CONCLUSION: The siRNA-mediated Smad3 silence significantly inhibits HSCs proliferation and induces apoptosis by up-regulating the P53 expression and down-regulating the Bcl-2 expression in HSCs.     

Effects of Arg-Gly-Asp-Ser tetrapeptide on proliferation and apoptosis of hepatic stellate cells in vitro

AIM:To investigate the effects of Arg-Gly-Asp-Ser (RGDS) tetrapeptide on proliferation, apoptosis and caspase 3 expression in FN-stimulated HSCs in vitro. METHODS:[³H]-thymidine incorporation, Annexin-V/Propidium Iodide double-labeled flow cytometry(FCM), TUNEL, scanning electron microscope and transmission electron microscopy were employed to estimate the influence of RGDS on proliferation and apoptosis of HSCs. The adhesion rates were observed by toluidine blue colorimetric assay. The expression of caspase-3 protein was detected by FCM. RESULTS:①Compared with control and FN groups, RGDS tetrapeptide at concentrations of 25 mg·L^-1, 50mg·L^-1 and 100 mg·L^-1 inhibited the proliferation of HSCs (P<0.01), and the inhibition rates of 100 mg·L^-1 at 12 h, 24 h and 48 h were 62.73%, 74.23%, 80.22%, respectively.②RGDS tetrapeptide induced the HSC apoptosis in dose-dependent and time-dependent manners(P<0.01). Observed with scanning electron microscope, the cell bodies and cellular processes of HSCs exposed to RGDS tetrapeptide were seen to be diminished. Microvilli on the cell surface decreased, became short even disappeared. Observed with transmission electron microscopy, the chromatins condensed, shrunk and aggregated along inside of nuclear membrane to exist in the form of ball, petal and crescent. Sometimes, apoptotic bodies formed. ③After exposure of HSCs to RGDS tetrapeptide for 2 h, the inhibition rates of adhesion were 8.82%, 29.41% and 45.59%, respectively, but that of RGES group was only 4.41%, P<0.01. ④ The expression of caspase 3 was obviously higher in RGDS tetrapeptide group than that in FN group, RGES tetrapeptide. CONCLUSION: These results suggest that RGDS tetrapeptide may inhibit proliferation and induce apoptosis of HSCs in both dose- dependent and time- dependent manners in vitro, which may be related to the abrogation of cell adhesion and caspase 3.     

Effects of adenovirus-mediated shRNA targeting PTEN on proliferation and apoptosis of activated hepatic stellate cells in vitro

AIM:To investigate the down-regulation of phosphatase and tensin homolog deleted on chromosome 10(PTEN) gene by adenovirus-mediated short hairpin RNA(shRNA) on proliferation and apoptosis of activated hepatic stellate cells(HSCs) in vitro and the related signaling transduction pathways. METHODS:The activated HSCs were cultured in vitro and transfected with recombinant adenovirus expressing shRNA targeting PTEN. The proliferation of HSCs was measured by MTT assay and the apoptosis was assessed by TUNEL and flow cytometry. Western blotting was used to detect the protein levels of PTEN, Bax, Bcl-2, Akt, p-Akt, ERK1/2 and p-ERK1/2 in HSCs, and real-time fluorescent quantitative PCR was applied to detect the mRNA expression of Akt and ERK1. RESULTS:The recombinant adenovirus expressing shRNA targeting PTEN was successfully transfected into activated HSCs in vitro, and significantly promoted the proliferation of HSCs in a time-dependent manner within a certain extent. The apoptotic rate of HSCs was significantly decreased 72 h after transfection(P<0.05). Meanwhile, reduced expression of Bax and elevated expression of Bcl-2 were induced 72 h after transfection(P<0.05). Furthermore, the expression of p-Akt and p-ERK1/2 were increased significantly(P<0.05), while no significant difference in the expression of Akt and ERK1 at mRNA and protein levels was observed(P>0.05). CONCLUSION:Down-regulation of PTEN by adenovirus-mediated shRNA dramatically promotes the proliferation of activated HSCs, and inhibits the apoptosis through Bcl-2/Bax pathway. In addition, the phosphorylation of Akt and ERK1/2 is increased, indicating that PI3K/Akt and ERK1/2 signal transduction pathways may play an important role in the regulation of proliferation and apoptosis of HSCs.     

Effect of fat-specific protein 27 on activation of rat hepatic stellate cells in vitro

AIM：To investigate the effects of fat-specific protein 27 (Fsp27) on the proliferation and activation of hepatic stellate cells (HSCs) in vitro. METHODS：HSCs were isolated from the liver of SD rats. The mRNA and protein expression of Fsp27 in primary HSCs and activated HSCs was detected by real-time fluorescence quantitative PCR, immunofluorescence staining and Western blotting. After 72 h of transfection with Fsp27-carrying lentivirus (pLV-Fsp27), the proliferation of HSCs was tested by CCK-8 assay, the protein expression of α-smooth muscle actin (α-SMA) in HSCs was detected by Western blotting, and the mRNA expression of fibrosis-related proteins, including matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinase 1 (TIMP-1) and transforming growth factor beta 1 (TGF-β1), was determined by real-time fluorescence quantitative PCR. RESULTS：Rat HSCs were successfully isolated and cultured. The difference of Fsp27 expression between primary HSCs and activated HSCs was significant (P<0.01). The proliferation and activation of HSCs was inhibited 72 h after pLV-Fsp27 transfection (P<0.05). Fsp27 enhanced the mRNA expression of MMP-2 and down-regulate the mRNA expression of TIMP-1 and TGF-β1 in activated HSCs (P<0.05). CONCLUSION：Fsp27 inhibits the proliferation and activation of HSCs and regulates the expression of fibrosis-related proteins. Fsp27 may play an important role in maintenance of the quiescent phenotype of HSCs.     

Inhibitory effects of FRNK on collagen synthesis in hepatic stellate cells

AIM:To investigate the effect of focal adhesion kinase-related non-kinase (FRNK) on collagen synthesis in hepatic stellate cells (HSCs).METHODS:HSCs were transfected with FRNK by cationic liposome method. The expressions of FRNK at the protein level in HSCs were detected by Western blotting analysis. HSCs collagen synthesis capability was examined by［3H］-Pro incorporation assay.RESULTS:The exposure of HSCs to FRNK led to the up-regulated expression of FRNK protein, and it was at 48 h after transfection that the FRNK protein content was the highest. Moreover, after FRNK was transfected successfully in HSCs, the total collagen synthesis and type I collagen synthesis were inhibited.CONCLUSION:After FRNK is transfected successfully in HSCs using Lipofectamine, the synthesis of total and type I collagen in HSCs is inhibited.     

Effect of IGFBP-rP1 on hepatic stellate cells and the activity of nuclear factor-κB

AIM:To identify the effect of insulin-like growth factor binding protein-related protein 1(IGFBP-rP1) on the activation of hepatic stellate cells (HSC) and the synthesis of extracellular matrix (ECM), and to investigate the possible pathway of signal transduction. METHODS:Rat HSC-T6 was cultured in vitro and established respectively the control group (incubated with PBS) and the groups treated with different concentrations of IGFBP-rP1. Cell-coated dishes were attained, and the single cell suspension was prepared after 24 h, then the expression of alpha-smooth muscle actin (α-SMA), collagen I and fibronectin (FN) were detected by immunocytochemical staining. The DNA binding activity of NF-κB p65 was detected by flow cytometry. RESULTS:The results of immunocytochemical staining showed that the positive stainings of α-SMA, collagen I and FN in the groups of IGFBP-rP1 were significantly higher than those in control group, and a dose-dependent relationship in a certain range was observed. Flow cytometry analysis showed that the percentage of NF-κB p65 positive cells in the groups of IGFBP-rP1 markedly increased compared with control group. CONCLUSION:HSC is activated by IGFBP-rP1 and the level of HSC activation gradually enhances in a certain dose range of IGFBP-rP1. The synthesis of both collagen I and FN is increased by IGFBP-rP1. IGFBP-rP1 enhances the DNA binding activity of NF-κB p65 in HSC.     

Inhibitory effect of sorafenib on collagen synthesis in human hepatic stellate cells

AIM: To investigate the effects of sorafenib on collagen synthesis in human hepatic stellate cells (HSCs). METHODS: HSC cell line LX-2 was used in vitro in this study. -proline incorporation assay was performed to measure the collagen synthesis. Immunocytochemistry was applied to detect type I collagen and real-time PCR was used to determine the mRNA expression of collagen α1 (I). RESULTS: Stimulation with platelet-derived growth factor (PDGF) induced the increase in type I collagen synthesis, while treatment with sorafenib (10.0 μmol/L) for 24 h markedly decreased the collagen synthesis. Sorafenib resulted in dose-dependent and time-dependent decrease in collagen synthesis in LX-2 cells in the absence or presence of PDGF by -proline incorporation assay. The inhibition rates were 22.69%, 37.52% and 71.74%, respectively, when LX-2 cells was treated with sorafenib at 10.0 μmol/L for 12 h, 24 h and 48 h. Sorafenib dose-dependently blocked the mRNA expression of collagen α1 (I) in LX-2 cells stimulated with PDGF. Sorafenib at the concentrations of 2.5 μmol/L, 5.0 μmol/L and 10.0 μmol/L down-regulated the mRNA expression of collagen α1 (I) in LX-2 cells by 58.66%, 67.06% and 81.64%, respectively. CONCLUSION: Sorafenib inhibits the collagen synthesis and blocks the expression of type I collagen at mRNA and protein levels in vitro in LX-2 cells. Therefore, sorafenib may be a potential therapeutic agent in the treatment of liver fibrosis.     

Effect of plumbagin on levels of Nox4/ROS and α-SMA in human hepatic stellate cells

AIM: To observe the effect of plumbagin on the mRNA and protein expression of nicotinamide adenine dinucleotidephosphate oxidase 4 (Nox4), reactive oxygen species (ROS) level and protein expression of α-smooth muscle actin (α-SMA) in the HSC-LX2 cells stimulated with transforming growth factor β1 (TGF-β1) in vitro. METHODS: HSC-LX2 cells were cultured in vitro and divided into blank group, model group, high-, medium- and low-dose (2, 1.5 and 1 μmol/L) plumbagin groups. After incubated with each drug for 72 h, the mRNA expression of Nox4 was detected by RT-PCR. ROS levels were tested by in situ loading probe method. The protein contents of Nox4 and α-SMA were measured by Western blot. RESULTS: Compared with model group, after treated with plumbagin for 72 h, the mRNA expression of Nox4, ROS level and α-SMA protein were significantly decreased in high-and medium-dose plumbagin groups (P<0.01). CONCLUSION: Plumbagin inhibits the activation of HSC-LX2 cells via decreasing the expression of Nox4, thus decreasing ROS levels.     

Effects of angiotensin II and losartan on collagen synthesis in rat hepatic stellate cells

AIM: To investigate the effects of angiotensin II (Ang II) and AT_1a receptor antagonist (losartan) collagen synthesis in rat hepatic stellate cells (HSCs). METHODS: ① Rat HSCs were isolated, cultured and identified. ② Rat HSCs were incubated in the medium with different concentrations of AngII or losartan, then the quantity of collagen was examined by -proline release assay. RESULTS: ① The yield of HSCs was 2×10⁷-3×10⁷/per rat, their viability and purity was more than 95% and 90%, respectively. ② The yield of collagen in HSCs significantly got a rise in a concentration-dependent manner when HSCs were incubated with AngII (10^－6mol/L-10^－10 mol/L) (P<0.05). While HSCs were influenced by the antagonist of AT_1a (10^－6 mol/L-10^－9 mol/L), the quantity of collagen dropped greatly (P<0.05). CONCLUSIONS: Ang II stimulates HSCs to produce more collagen. Losartan inhibits the cell to synthesize collagen via AT_1a receptor (P<0.05). The results indicate that Ang II may play an important role in the development of hepatic fibrosis, and using AT_1a antagonist may offer a new strategy to prevent hepatic fibrosis.     

Effects of chloroquine on autophagy and collagen metabolism in activated hepatic stellate cells in vitro

AIM: To explore the effects of chloroquine (CQ) on collagen Ⅰand collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS: Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h. The cells were divided into 5 groups as follows:control group, TGF-β1 group, TGF-β1+CQ (15 μmol/L) group, TGF-β1+CQ (30 μmol/L) group and TGF-β1 + CQ (60 μmol/L) group. Western blot was used to determine the expression of LC3-Ⅱ/LC3-I, P62 and α-SMA in activated HSC-T6 cells. The expression of collagen I and collagen Ⅲ was detected by immunocytochemical staining, Western blot and RT-qPCR. Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS: The ratio of LC3-Ⅱ/LC3-Ⅰ and P62 expression were increased after CQ intervention. Moreover, they were significantly higher in the TGF-β1+CQ groups than those in TGF-β1 group (P<0.01). The expression of collagen I and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1+CQ groups as compared with TGF-β1 group (P<0.01), and it was markedly increased among TGF-β1+CQ groups in a dose-dependent manner. The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1+CQ groups as compared with TGF-β1 group (P<0.05).CONCLUSION: Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen I and collagen Ⅲ in a dose-dependent way, probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.     

Levels of histone modifications in activated primarily cultured rat hepatic stellate cells

AIM: To investigate the changes of histone modifications during the activation of primarily cultured rat hepatic stellate cells (HSCs) and the relationship between histone modification patterns and α-smooth muscle actin (α-SMA) expression, and to explore the roles of histone modifications in the activation of HSCs. METHODS: The rat HSCs were isolated by in situ perfusion of collagenase combined with density gradient centrifugation, cultured in vitro and identified by immunofluorescence staining. The morphological features of the cells were observed under inverted microscope. The changes of desmin and α-SMA during the activation of HSCs were detected by immunofluorescence staining and Western blotting. The levels of histone 3 lysine 4 dimethylation (H3K4me2), histone 3 lysine 9 dimethylation (H3K9me2), histone 3 lysine 9 acetylation (acH3K9) and histone 4 lysine 12 acetylation (acH4K12) in quiescent HSCs and activated HSCs were determined by Western blotting.RESULTS: The morphology of HSCs shifted from a quiescent phenotype to highly activated myofibroblast during the culture. Immunofluorescence staining and Western blotting showed that the expression levels of α-SMA and desmin were increased over time and reached maximum at 15 d. According to the results of cell morphology and immunofluorescence staining, the cells cultured for 24 h and 15 d were quiescent and activated HSCs, respectively. Compared with quiescent HSCs, there were higher H3K4me2 and lower H3K9me2, acH3K9 and acH4K12 modification levels in activated HSCs (P<0.01). CONCLUSION: Histone modifications show anomalous expression during the activation of primarily cultured rat HSCs. Histone modifications may contribute to the transdifferentiation of HSCs and the development of hepatic fibrosis.     

Effects of MT1-MMP on collagen metabolism regulated by FRNK in hepatic stellate cells

AIM: To investigate the effect of FAK-related non-kinase (FRNK) on the expression of membrane-type matrix metalloproteinase-1 (MT1-MMP) in hepatic stellate cells (HSC). METHODS: FRNK were transfected into HSCs by cationic liposome method. The protein levels of FRNK in HSC were assayed by Western blotting. The levels of MT1-MMP were determined by RT-PCR for mRNA and by Western blotting for protein, respectively. RESULTS: The up-regulated expression of FRNK protein was observed and it was at 48 h after transfection that the FRNK protein content was the highest (P<0.05). The expressions of MT1-MMP mRNA and protein were also up-regulated by the transfection of FRNK, and it was at 48 h after transfection that the MT1-MMP protein content was significantly increased. CONCLUSION: The mRNA and protein of FRNK were over-expressed in HSC transfected with the gene of FRNK. The inhibitory effect of FRNK on the collagen synthesis in HSC may be through the up-regulation of MT1-MMP.     

Effect of antifibrotic herb serum on rat hepatic stellate cell proliferation and collagen synthesis

AIM: To investigate the effect of Chinese herbs, Ganxianfang(GXF), on rat hepatic stellate cells (HSC) proliferation and collagen synthesis. METHODS: Two types of herb serum, portal venous serum and circumferential venous serum, were prepared from rats infused intragastrically with 16, 8, 4 times adult dose of GXF decoction. HSC isolated from rat liver were processed with the above sera in vitro. Then we mensurated the radioactivity of HSC admixed with [[³H]H]proline and [[³H]H]thymine to judge the effect on proliferation and collagen synthesis of HSC. RESULTS: Both two types of serum collected 0.5, 1, 2 h after intragastrical infusion inhibited HSC proliferation (P＜0.05), and the serum collected 1 h after intragastrical infusion had the strongest effect (P＜0.05). Portal serum decreasea collagen synthesis (P＜0.05), but circumferential serum had no effect (P＞0.05). CONCLUSION: Inhibition of HSC proliferation and decrease of collagen synthesis may contribute to the GXF antifibrotic action.     

Regulatory effects of JNK transduction on interleukin-1β-induc ed proliferation in rat hepatic stellate cells

AIM:To study the action of JNK in stimulat ing proliferation in rat hepatic stellate cells (HSCs) induced by interleukin-1β.METHODS:Activation of JNK was detected with Western blotting,w hile the proliferation of HSCs induced by interleukin-1β and the effect of JNK specific blockade SP600125 were measured with cell counting kit-8.RESULTS:Interleukin-1β up-regulated proliferation in rat HSCs,which was obviously inhibited by SP600125,compared with control group (without SP600125 treatment) (1.560±0.110),the decrease was significant (1.427±0.113,P<0.05).Interleukin-1β activated JNK signaling pathway in a time-de pendent manner.At the six time points (0,5,15,30,60 and 120 min),the JNK a ctivities were 0.982±0.299,1.501±0.720,2.133±0.882,3.360±0.452,2.181±0.789,1.385±0.368,respectively.CONCLUSION:Interleukin-1β stimulates proliferation in rat HSCs ,and JNK signaling pathway is involved in this process.     

Effects of c-myb antisense RNA on the proliferation and collagen Ⅰ gene expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA on the proriferation and collagen Ⅰ gene expression in cultured hepatic stellate cells(HSC) in rats.METHODS:The c-myb antisense gene recombinant retroviral vector(pDOR-myb) was constructed, and then was transfected into retroviral package cell line PA 317 by means of N-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium methyl-sulfate(DOTAP) liposomal transfection reagent. The pseudoviruses produced from the resistant PA317 cells selected with G418 were collected, with which HSCs isolated from rat liver were infected. The cell proliferation was measured by MTT method, c-myb, α₁-Ⅰ collagen mRNA expression and c-myb protein in HSCs were detected with semi-quantitative RT-PCR and western-blot, respectively.RESULTS:HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and α₁-Ⅰ collagen mRNA expression were repressed significantly.CONCLUSIONS:c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and α₁-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.