Effect of Beclin 1 RNA interference on Vit K3 injured SMMC-7721 cells

AIM: To observe the effect of Beclin 1 silencing by RNA interference (RNAi) technique to the injury of SMMC-7721 hepatoma cells by vitamin K3 (Vit K3).METHODS: The recombinant plasmid Psilencer 3.1-siRNA-Beclin 1 was transfected into SMMC-7721 hepatoma cells by eukaryotic cell transfection technique. Plasmid vector and cell culture medium were used as negative and control, respectively. The cells were collected 48 h later to extract cell RNA and total protein and to detect Beclin 1 gene expression by RT-PCR and Western blotting. 40 μmol/L Vit K3 was used to treate the Beclin 1-siRNA cells, Hoechst33342 staining was used for the determination of the percentage of cell apoptosis.RESULTS: Compared with the control group, the synthetic siRNA of Beclin 1 significantly decreased the levels of Beclin 1 mRNA and protein expressions. Beclin 1 mRNA was up-regulated in 40 μmol/L Vit K3 treated SMMC-7721 hepatoma cells, the percentage of apoptosis cells increased (P﹤0.01). In beclin 1-siRNA cells, Beclin 1 mRNA was down-regulated obviously, the percentage of apoptosis cells increased significantly compared with the 40 μmol/L Vit K3 group (P﹤0.01).CONCLUSION: The transfection of SMMC-7721 hepatoma cells by Psilencer3.1-siRNA-Beclin 1 effectively inhibits the expressions of Beclin 1 mRNA and protein, inhibits the activation of Beclin 1 dependent autophagic signaling pathway, and aggravates the apoptosis induced by Vit K3.     

Effects of 2-deoxy-D-glucose and oxaliplatin on proliferation and apoptosis of human hepatoma cell line SMMC-7721

AIM: To investigate the effects of 2-deoxy-D-glucose (2-DG) or oxaliplatin (L-OHP) alone and the combination of both on the proliferation and apoptosis of hepatoma cell line SMMC-7721 in vitro. METHODS: Human hepatoma cell line SMMC-7721 was treated with 2-DG or L-OHP alone, or both. The inhibitory effect on the proliferation of SMMC-7721 cells was estimated by MTT method. The q value, which represents synergistic effect, was determined. Apoptotic rate and cell cycle were assayed by flow cytometry. The activity of caspase-3 was detected by a caspase-3 activity assay kit. RESULTS: 2-DG or L-OHP at different concentrations inhibited the growth of SMMC-7721 cells obviously and the inhibitory effect on SMMC-7721 cell growth strongly depended on the exposure time and dose. When the 2 drugs worked together, the inhibitory effect was improved (P<0.05). 2-DG induced cell apoptosis and arrested the cells at G₂/M phase. When combined with L-OHP,the 2 drugs induced more severe apoptosis and arrested the cells at S and G₂/M phase. Meanwhile, the activity of caspase-3 increased when the 2 drugs used together. CONCLUSION: 2-DG inhibits the growth of hepatoma cell line SMMC-7721. The combination of 2-DG and L-OHP improves the ability of L-OHP to attack the tumor cells. The mechanism might be related to increasing the activity of caspase-3.     

Antitumor activity of Paecilomyces tenuipes polysaccharide and its mechanism in vitro

AIM:To investigate the antitumor activity and mechanism of Paecilomyces tenuipes polysaccharide(PTPS).METHODS:PTPS-I was obtained by water extraction and alcohol precipitation, and purified by DEAE-cellulose and Sephadex G-100 chromatography. Human erythroleukemia cell line K562, laryngocarcinoma cell line Hep2 and hepatic carcinoma cell line SMMC-7721were co-cultured with PTPS-I or the conditioned medium which prepared with PTPS-I-stimulated human mononuclear cells (PTPS-I-MNC-CM), and the proliferation of tumor cells was determined. The cell counting kit-8 (CCK-8) was used to determine the proliferation of MNCs. The FQ-RT-PCR was applied to investigate the expression of TNF-α and IL-6 mRNA in MNCs. RESULTS:PTPS-I-MNC-CM inhibited the proliferation of K562, Hep2 and SMMC-7721 cells in vitro (P<0.01). Cytotoxicity of PTPS-I against K562, Hep2 and SMMC-7721 cells was not observed (P<0.01). PTPS-I stimulated the proliferation of MNCs (P<0.01) and significantly enhanced the expression of TNF-α and IL-6 mRNA in MNCs (P<0.05, P<0.01). CONCLUSION:The results suggest that PTPS-I is an immunomodulator and its antitumoral activity is through the immunomodulatory mechanism rather than the direct cytotoxicity against tumor cells.     

Effect of abnormal expression of miR-141 on malignant biological behaviors of human hepatocarcinoma cells

AIM: To investigate the expression of microRNA-141 (miR-141) in human hepatocellular carcinoma (HCC) cell line SMMC-7721 and normal hepatocyte line HL-7702, and to analyze the effect of abnormal expression of miR-141 on the malignant biological behaviors of human hepatocarcinoma cells. METHODS: The RNA from SMMC-7721 cells and HL-7702 cells was extracted. SYBR Green real-time PCR was performed to detect the expression of miR-141. Synthetic miR-141 mimic and its negative control were transfected into the SMMC-7721 cells, and miR-141 inhibitor and its negative control were transfected into the HL-7702 cells by the method of Lipofectamine. After transfection, MTS assay and BrdU-ELISA were employed to evaluate the effect of miR-141 on the cell proliferation. Flow cytometry was used to detect cell cycle and apoptosis. The changes of migration ability were investigated by Transwell invasion assay. RESULTS: The expression of miR-141 in the SMMC-7721 cells was significantly lower than that in the HL-7702 cells (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of the SMMC-7721 cells transfected with 25 nmol/L miR-141 mimic was significantly inhibited in a time-dependent manner (P < 0.05). The percentages of G₁ phase cells and early apoptotic rate were significantly increased when miR-141 was up-regulated, but the migration ability was inhibited (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of HL-7702 cells transfected with 50 nmol/L miR-141 inhibitor was significantly increased in a time-dependent manner (P < 0.05). When miR-141 was down-regulated, the percentages of G₁ phase cells and early apoptotic rate were significantly decreased, but the migration ability was enhanced (P < 0.05). CONCLUSION: miR-141 is down-regulated in human hepatocarcinoma cell line. Up-regulation of miR-141 will not only inhibit cell proliferation and migration ability, but also affect the cell cycle and apoptosis of SMMC-7721 cells. miR-141 may function as a tumor suppressor gene during HCC development.     

In vitro growth inhibition effects of rhHGF/cHGF on SMMC-7721 human HCC cell line

AIM:To examine the effects of recombinant human hepatocyte growth factor(rhHGF) and native calf HGF(cHGF) on SMMC-7721 human hepatocellular carcinoma(HCC) cell line. METHODS:Human HCC cell line culture, photometric assay, and flow cytometric assay were used in this study .RESULTS:A similar type of dose-dependent cell growth inhibition effect on SMMC-7721 human HCC cells by rhHGF(5-20 μg/L) as well as by cHGF(25-100 mg/L) had been found, with the maximal effect at the highest concentration used. Approximately over 50% of the cells treated with rhHGF(5 μg/L, 10 μg/L, 20 μg/L) accumulated in the quiescent G₀/G₁ phase of the cell cycle over incubation periods for 3 d. CONCLUSION:The growth of SMMC-7721 human HCC cells was strongly inhibited by both rhHGF and cHGF. This might be because the cells exposed to HGF became arrested in the G₀/G₁ phase.     

Effects of marrow stromal cell-mediated microenvironment on human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the effects of marrow stromal HS-5 cells on hepatocellular carcinoma SMMC-7721 cells in the tumor microenvironment. METHODS: The effects of HS-5 cell-conditioned medium (HS-5-CM) on the proliferation, migration and invasion abilities of SMMC-7721 cells were detected by MTT, wound-healing and Transwell assays. After co-culture of SMMC-7721 cells with HS-5 cells in the Transwell chamber, the expression of chemokine CCL5 and its receptor CCR5 at mRNA and protein levels in SMMC-7721 cells was examined by quantitative real-time PCR (qRT-PCR), ELISA or Western blotting. Akt and p-Akt473 protein levels in SMMC-7721 cells treated with PI3K inhibitor LY294002 were observed by Western blotting. RESULTS: HS-5-CM promoted the proliferation, migration and invasion abilities of SMMC-7721 cells. The expression of CCL5 and CCR5 at mRNA and protein levels in SMMC-7721 cells was increased after co-cultured with HS-5 cells. PI3K inhibitor LY294002 inhibited the activation of PI3K-Akt signaling pathway and the secretion of CCL5 in SMMC-7721 cells after co-cultured with HS-5 cells. CONCLUSION: HS-5 cells significantly promote the proliferation, migration and invasion abilities of SMMC-7721 cells. Co-culture of SMMC-7721 cells with HS-5 cells activates PI3K-Akt signaling pathway to increase the secretion of CCL5 in SMMC-7721 cells.     

miR-15a-5p inhibits proliferation and migration of human hepatocellular carcinoma SMMC-7721 cells

AIM: To observe the influence of high expression of miR-15a-5p on the proliferation and migration of human hepatocellular carcinoma SMMC-7721 cells.METHODS: The miR-15a-5p oligonucleotide, which was reconstructed with additional restriction sites of EcoR Ⅰ and Hind Ⅲ, was chemically synthesized and confirmed by sequencing. The miR-15a-5p eukaryotic expression system was constructed by pcDNA6.2-GW/Em-GFP-pre-miR-15a-5p plasmid. The miR-15a-5p was transfected into the SMMC-7721 cells transiently by plasmid, and quantified by quantitative real-time PCR at the mRNA level. The cell viability was measured by CCK-8 assay, and the living cell counting was performed by the method of Trypan blue exclusion. The migration ability of the SMMC-7721 cells with high expression of miR-15a-5p was detected by wound healing test.RESULTS: The sequence of miR-15a-5p oligonucleotide 100% matched the designed sequence. Compared with control group, the miR-15a-5p expression was increased significantly (P<0.05). The viability, the living cell number and the migration ability of the SMMC-7721 cells were decreased in high expression of miR-15a-5p group with statistically significant difference (P<0.05).CONCLUSION: The abilities of proliferation and migration in human hepatocellular carcinoma SMMC-7721 cells are decreased by high expression of miR-15a-5p.     

Different inhibition of hepatocarcinoma cell growth by As2O3 in SMMC-7721 and BEL-7402 cell lines

AIM: To explore the different inhibitory effect of arsenic trioxide (As₂O₃) on hepatocarcinoma cell growth in SMMC-7721 and BEL-7402 cell lines and its mechanism. METHODS: The cell culture and trypan blue staining were used to study the inhibitory effect of arsenic trioxide on cell growth, and the glutathione (GSH) contents in hepatocarcinoma cells treated with arsenic trioxide were detected. RESULTS: Arsenic trioxide inhibited the growth of BEL-7402 cells in a time and dose-dependent manner. The inhibitory effect was significant at a lower dose of 0.50 μmol/L for 24 h, however, to SMMC-7721 cells, a higher dose of 2.00 μmol/L for 96 h was needed. The inhibitory rate of arsenic trioxide (0.25-2.00 μmol/L) on BEL-7402 cell growth was higher than that on SMMC-7721 cells. The content of GSH in SMMC-7721 cells was much higher than that in BEL-7402 cells . CONCLUSION: There was a significant difference in inhibition of hepatocarcinoma cell growth by arsenic trioxide between BEL-7402 and SMMC-7721 cell lines, the cause of which may be due to the difference in GSH content in BEL-7402 and SMMC-7721 cells.     

Biological characteristics of side population cells sorted from hepatoma SMMC-7721 cell line

AIM: To study the biological characteristics of side population (SP) cells sorted from hepatoma SMMC-7721 cell line. METHODS: Fluorescence-activated cell sorter (FACS) was used to sort SP cells and non-SP (NSP) cells from SMMC-7721 cell line. The colony-formation ability and proliferation ability between SP cells and NSP cells were compared in terms of plate colony assay and growth curve. The migratory and invasive properties of SP cells and NSP cells were tested by Transwell method. The cell cycle and apoptosis were analyzed by flow cytometry. The oncogenicity of the cells was analyzed by nude mouse transplantation tumor experiment in vivo. RESULTS: The results of FACS analysis indicated that (9.2±0.2)% of the SMMC-7721 cells were SP cells. The proportion of G₀/G₁ phase of SP cells was higher, and the apoptotic rate was lower than those of NSP cells (P<0.05). The proliferation ability and colony-forming ability and migratory and invasive properties of SP cells were significantly higher than those of NSP cells (P<0.05). The nude mouse transplantation tumor experiment displayed that the oncogenicity of SP cells was higher than that of NSP cells. CONCLUSION: The SP cells sorted from SMMC-7721 cell line may enrich tumor stem cells.     

Effect of ginsenoside Rh2 on cell proliferation and cytoskeleton in hepatocarcinoma SMMC-7721 cells

AIM: To investigate the changes of cell growth and cytoskeleton in hepatocarcinoma SMMC-7721 cells treated with ginsenoside Rh₂.METHODS: Cell viability and apoptosis under the conditions of ginsenoside Rh₂ exposure at different concentrations were measured by MTT test and flow cytometry,respectively. The morphological changes of F-actin labeled with FITC-phalloidin were observed under confocal laser scanning microscope. The structures of nuclear matrix-intermediate fibre system were observed under transmission electron microscope (TEM).RESULTS: Rh₂ at 40 mg/L for 4 days inhibited the proliferation and induced apoptosis in SMMC-7721 cells more than those in control group, 10 mg/L Rh₂ group and 20 mg/L Rh₂ group. The F-actin in the cells treated with Rh₂ was well-distributed, lined up in order and the number of fibers increased, while those in the control cells were in disorder and punctiform. The results of whole mount TEM indicated that the intermediate fiber was plentiful, well-distributed and interweaved into a regular network in Rh₂ treated cells.CONCLUSION: Rh₂ effectively inhibits the cell proliferation, increases the cell apoptosis and induces the change of the cytoskeleton alignment in SMMC-7721 cells.     

Construction of siArid2 recombinant adenovirus and its effect on proliferation of hepatocarcinoma cells

AIM: To observe the effects of Arid2gene on cell proliferation and cell cycle by interference of endogenous Arid2 expression in hepatoma cells. METHODS: Three pairs of shRNA targeting Arid2gene were cloned into a shuttle vector to construct recombinant adenovirus plasmids. HEK293 cells were transfected with the recombinant adenovirus plasmids. After several rounds of the package and amplification, the high-titer adenoviruses AdsiArid2-1~3 were obtained. To verify the inhibitory effects of AdsiArid2 adenoviruses, Western blotting was used to detect the endogenous Arid2 protien expression in SMMC-7721 cells. Cell growth and cell cycle analysis were carried out by MTS assay and flow cytometry. RESULTS: High- titer recombinant adenovirus of siArid2 were successfully obtained, and named AdsiArid2-1~3, among which the AdsiArid2-3 had the best inhibitory effects. MTS assay showed that the absorbance values at 490 nm were increased at 72 h and 96 h after transduction compared with the mock and Adsicontrol groups. These data indicated that knockdown of Arid2 promoted the proliferation rate of SMMC-7721 cells(P<0.05). Moreover, the flow cytometry analysis revealed that the G1-phase distribution at 72 h in AdsiArid2 group was lower than that in mock group and Adsicontrol group. In contrast, the S-phase distribution in AdsiArid2 group was much higher than that in mock group and Adsicontrol group. CONCLUSION: The recombinant plasmids and recombinant adenovirus were successfully constructed. shRNA-mediated knockdown of Arid2 promotes the proliferation and the transition from G1 phase to S phase of hepatoma cells.     

Inhibitory effect of 5-fluorouracil on ultra-microstructure and insulin secretion of pancreatic β cells

AIM: To investigate the molecular mechanisms of β cell dysfunction induced by 5-fluorouracil (5-FU) in islet β cell line (NIT-1 cells). METHODS: The NIT-1 cells were treated with different concentrations of 5-FU. The content of insulin in the culture medium was determined by radioimmunoassay. Cell apoptosis was observed by flow cytometry with annexin V/PI staining. The ultra-microstructural changes of NIT-1 cells were observed under transmission electron microscope. The expression of pancreatic and duodenal homeobox protein 1(PDX-1) at mRNA and protein levels in NIT-1 cells was examined by RT-PCR and Western blotting, respectively. RESULTS: Exposed to the low glucose concentration (5.6 mmol/L), insulin secretion in NIT-1 cells was not significantly decreased following a 24 h treatment with 5.0 to 40.0 mg/L 5-FU (P>0.05). On the contrary, the high glucose (16.7 mmol/L)-stimulated insulin secretion in NIT-1 cells was inhibited by 5.0 to 40.0 mg/L of 5-FU in a dose-dependent manner after 24 h of incubation (P<0.01). The apoptosis rate of NIT-1 cells was significantly increased as compared to those in the control levels(P<0.05). The structural changes of mitochondria were the main apoptotic changes under transmission electron microscope. Significant down-regulation of PDX-1 expression at mRNA and protein levels was observed in NIT-1 cells treated with 5-FU at the concentration of 10.0 mg/L to 40.0 mg/L(P<0.05).CONCLUSION: 5-FU inhibits the insulin secretion in islet β cell induced by high glucose. A relative deficiency in insulin secretion following 5-FU treatment is related to the changes of β cell ultra-microstructure and the reduction of β cell numbers, by which an increase in apoptosis of pancreatic β cells is induced. Down-regulation of PDX-1 expression may play a pivotal role in increasing the apoptosis of pancreatic β cells induced by 5-FU in high-glucose condition.     

Effects of PAR-2 agonist peptide on proliferation and cytosolic calcium level in hepatoma cells

AIM: To investigate the effects of PAR-2 agonist peptide on the proliferation and cytosolic calcium concentration (［Ca2+］c) in human hepatoma cells HepG2. METHODS: Human hepatoma cell line HepG2 was cultured. The cells were treated with PAR-2 agonist peptide SLIGKV-NH2 and the reverse PAR-2 agonist peptide VKGILS-NH2, respectively. The ［Ca2+］c of hepatoma cells were measured by microfluorimetric techniques based on calcium indicator fura-2/AM. The influences on proliferation of hepatoma cells were determined by MTT method. The changes of cell cycle were evaluated by flow cytometry, and the changes of cyclin D1 mRNA expression were detected by RT-PCR. RESULTS: After treated with 50 μmol/L SLIGKV-NH2, a rapid rise of ［Ca2+］c in HepG2 cells was induced (P<0.01), percent S phase, G2/M phase and proliferation index (PI) of HepG2 cells were elevated (P<0.01), and cyclin D1 mRNA expression was significantly upregulated (P<0.01). The proliferation rates of HepG2 cells treated with 1-50 μmol/L SLIGKV-NH2 were significantly increased, and the effect was in a dose-dependent manner (P<0.01 or P<0.05). No statistical significance of the difference between VKGILS-NH2 and control group was observed (P>0.05). CONCLUSION: PAR-2 agonist peptide induces the rise of ［Ca2+］c in HepG2 cells, upregulates the expression of cyclin D1 mRNA, accelerates the progress of cell cycle, promotes the synthesis of DNA and the proliferation of hepatoma cells via activating PAR-2 in vitro.     

Proliferation of human hepatoma cells are regulated by the intracrine and autocrine loop of the macrophage colony-stimulating factor system

AIM: To study the expression and characterization of intracellular macrophage colony-stimulating factor (M-CSF) in human hepatoma cell line, SMMC 7721 cell, and to explore the mechanism by which M-CSF regulates the proliferation of human hepatoma cells. METHODS: The immunohistochemical staining, flow cytometry, antisense technique and Western blotting were used to study the effects and mechanisms of intracellular M-CSF on the proliferation of human hepatoma cells. RESULTS: SMMC 7721 cells highly expressed M-CSF and its receptor. The localization of positive reactions was mainly in cytoplasma and nucleus in SMMC 7721 cells. In cytoplasma and nucleus, one isoforms of M-CSF was found with the molecular weight (MW) of 20 kD, while one type of M-CSFR was discovered with MW of 120 kD. Immunoprecipitation assay showed that these ligands existed in binding with its receptor. Monoclonal antibody (McAb) against M-CSF and antisense oligodeoxynucleotides (ASODN) blocking M-CSF expression inhibited the proliferation of SMMC 7721 cells. McAb and ASODN regulated the expression of cyclin D1/E and p16. Simultaneous administration of both McAb and ASODN inhibited the proliferation of SMMC 7721 cells and modulated the expression of cyclins at greater degrees. CONCLUSION: Our results suggest that an autocrine and an intracrine loop of M-CSF/M-CSFR are present in SMMC 7721 cells.     

Expression of eEF1A2 in hepatocellular carcinoma

AIM: To study the expression of eukaryotic elongation factor 1A2 (eEF1A2) in the hepatocellular carcinoma (HCC) tissues and the effects of eEF1A2 over-expression on the biological behaviors of the HCC cells. ME-THODS: The expression of eEF1A2 at mRNA and protein levels in the HCC tissues and matched liver tissues from 62 HCC patients, and 20 normal liver tissues were detected by the methods of real-time PCR and immunohistochemical staining, respectively. The mRNA and protein expression of eEF1A2 in the HCC cells was also determined by real-time PCR and Western blot, respectively. The lentivirus containing eEF1A2 gene was constructed, and was used to infect the HCC cells with low eEF1A2 expression. The expression of eEF1A2 at mRNA and protein levels in the infected cells was detected by real-time PCR and Western blot, respectively. The cell activity, cell cycle and mRNA expression of albumin were measured by MTT assay, DNA ploid analysis and real-time PCR, respectively.RESULTS: The mRNA expression levels and protein expression positive rates of eEF1A2 in the 62 cases of HCC tissues, were significantly higher than those of 62 matched liver tissues and 20 normal liver tissues (P<0.01). eEF1A2 mRNA and protein were highly expressed in SMMC-7721 cells and BEL-7402 cells, and expressed in SK-HEP-1 cells at low level. The expression of eEF1A2 at mRNA and protein levels in the SK-HEP-1 cells was significantly enhanced by infection of GV287-eEF1A2 expression lentivirus.Compared with negative control group (transfected with negative control lentivirus), the cell activity in eEF1A2 over-expression group (transfected with GV287-eEF1A2 expression lentivirus) was significantly enhanced, the mRNA expression of albumin was remarkably reduced, and the cells in G₀/G₁ phase were significantly decreased with increased percentage of the cells in S and G₂/M phases.CONCLUSION: eEF1A2 is selectively over-expressed in human HCC cancer tissues. eEF1A2 might be a putative oncoprotein in HCC. eEF1A2 over-expression has noticeable effects on the HCC cell proliferation enhancement, differentiation inhibition, and cell cycle acceleration through the G₀/G₁ phase to S phase and G₂/M phases.     

灵芝提取物对人肝癌SMMC─7721细胞DNA合成的影响

本文以人肝癌ＳＭＭＣ－７７２１细胞为材料，对水溶性灵芝提取物（ＥＧＬ）抗癌作用机理进行了研究。结果表明，ＥＧＬ能明显抑制ＳＭＭＣ－７７２１细胞的ＤＮＡ合成：ＥＧＬ在５０ｕｇ／ｍｌ到３００ｕｇ／ｍｌ范围表现出明显的剂量效应；ＥＧＬ最小有效作用剂量为１００ｕｇ／ｍｌ，最大有效作用剂量为３００ｕｇ／ｍｌ。结果提示ＥＧＬ的抗癌机理主要是通过抑制癌细胞ＤＮＡ合成而起作用的。     

Effect of catalpol on activity of osteoblasts/osteoclasts and osteoblast ERα/β mRNA expression in osteoblast-osteoclast co-culture system

AIM: To investigate the effect of catalpol on the activity of osteoblasts (OB) and osteoclasts (OC), and OB estrogen receptor (ER) α/β mRNA expression in the OB-OC co-culture system. METHODS: OB and OC were isolated from the SD rats of 1 and 5 days old. In the OB-OC co-culture system, different concentrations of catalpol including low dosage (0.05, 0.1, 0.5 and 1 mg/L), middle dosage (2, 5 and 10 mg/L), and high dosage (20, 50 and 100 mg/L) were added into the culture medium to detect the changes of OB proliferation by MTT assay. The catalpol at maximal dosage was added to OB section to detect the alkaline phosphatase (ALP) activity of OB by pNPP method. The mRNA expression of ERα/β in the OB treated with catalpol in the co-culture system was detected by RT-PCR. The catalpol at maximal dosage was added to OC group to detect the activity of OC by microscopy and tartrate-resistantacid phosphatase (TRAP) activity detection. RESULTS: In 0.05~2 mg/L catalpol groups, the proliferation of OB was significantly increased as compared with control group in the co-culture system, and it reached the maximum value when catalpol was at 0.05 mg/L, while in 5~100 mg/L catalpol groups, the proliferation of OB was not increased. The ALP activity of OB in 0.05 mg/L catalpol group was higher than that in control group. The catalpal at 0.05 mg/L promoted the mRNA expression of ERβ in OB in the co-culture system, but did not increase the mRNA expression of ERα as compared with control group. Catalpol at 0.05 mg/L obviously inhibited the bone resorption and the TRAP activity in OC. CONCLUSION: Catalpol stimulates the proliferation and activity of OB, inhibits the bone resorption and activity of OC, and increases the mRNA expression of ERβ in OB in the OB-OC co-culture system, suggesting that high mRNA expression of ERβ may be the regulatory pathway of catalpol in response to bone metabolism.     

Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

The cytotoxicity of nitric oxide induced by inflammatory cytokine in combination with LPS in endothelial cells

AIM: To explore the mechanism underlying inducible nitric oxide (NO) caused injury of endothelial cells during inflammation. METHODS:The activity of iso-enzymes of NO synthase (NOS), NO level and iNOS expression were examined using NADPH method, Griess reaction and RT-PCR, respectively. Furthermore, the lactate dehydrogenase (LDH) release rate, malondialdehyde (MDA) content were also measured. RESULTS:Co-administration of cytokines (TNF-α 5×10⁵ U/L, IL-1β 2×10⁵ U/L, INF-γ 2×10⁵ U/L) and LPS (10 mg/L) caused an obvious increase in NOS activity, NO levels (about two-fold) and a significant injury of the cells. At the same time, a significant increase in iNOS mRNA was also detected. Wheareas, treatment of the cells separately with cytokines or LPS for 24 h had no significant effect on NOS activity and NO level in cell lysates, however, it caused a significant increase in LDH release and MDA content. Also, the effect of cytokines and LPS on cell viability was concentration-and time-dependent. L-NMMA, a inhibitor of NOS, can suppress inducible NO production and protect cells against NO induced injury. CONCLUSION:Co-administration of cytokines (TNF-α, IL-1β and INF-γ) and LPS significant activated iNOS and NO production which, in turn, induced oxidative reaction in endothelial cells.