ABSTRACT
<p><b>OBJECTIVE</b>RNA interference was used to silence geranylgeranyltransferase Ⅰ(GGTase-Ⅰ) in vitro and to study the effect of GGTase-Ⅰ on the migration and invasion of tongue squamous cancer cells.</p><p><b>METHODS</b>Three small interfering RNAs (siRNA) were designed according to the GGTase-Ⅰ sequence by Genebank and were transfected into tongue squamous cancer cells Cal-27 to knock down GGTase-Ⅰ expression. The tested cells were divided into three groups, as follows: the RNA-interfered groups (GGTase-Ⅰ siRNA1, GGTase-Ⅰ siRNA 2, GGTase-Ⅰ siRNA 3), a negative control group (disrupted by random sequence NC-siRNA), and a blank control group. GGTase-Ⅰ and RhoA gene expressions were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The optimum interference group was screened by qRT-PCR and Western blot and was assigned as the experimental group. Matrix metalloproteinase (MMP)-2 and MMP-9 protein expressions were examined by Western blot. GTP-RhoA expression of protein was examined by GST-pull down. The migration and invasion abilities were analyzed by wound healing assay and Transwell motility assay.</p><p><b>RESULTS</b>GGTase-Ⅰ mRNA and protein expression in Cal-27 decreased significantly after transfection of GGTase-I siRNA (P<0.05). No significant difference of RhoA gene expression was detected. MMP-2, MMP-9, and GTP-RhoA protein expressions decreased significantly (P<0.05). The migration and invasion abilities were inhibited (P<0.05).</p><p><b>CONCLUSIONS</b>To inhibit GGTase-Ⅰ expression, the migration and invasion abilities of tongue squamous cancer cells should also be inhibited. Further studies on GGTase-Ⅰ may provide novel effective molecular targets for tongue squamous cancer cells.</p>
ABSTRACT
BACKGROUND: Inflammatory response is one of potential cellular mechanisms by which hyperhomocysteinemia accelerates atherosclerosis. There are still many details need to be illuminated. TNFSF4 gene and interleukin (IL)-10 are two inflammatory factors related to atherosclerosis. But their correlation with hyperhomocysteinemia is seldom reported. OBJECTIVE: To investigate the effects of homocysteine (Hcy) on expression of TNFSF4 gene and IL-10 in the monocytes cultured in vitro. DESIGN, TIME AND SETTING: A randomized control experiment in vitro was conducted from July to December in 2007 in the Pathophysiology Laboratory of West China School of Preclinical and Forensic Medical Sciences of Sichuan University. MATERIALS: THP-1 monocytes were offered by the Pathophysiology Laboratory of West China School of Preclinical and Forensic Medical Sciences of Sichuan University); Hcy, dexamethasone (Dex) and vitamin D3 (Vit D3) were all purchased from Sigma. METHODS: Cultured THP-1 monocytes were induced to macrophages by 0.1 ?mol/L phorbol myristate acetate, and the differentiated THP-1 macrophages were incubated with 200 ?mol/L Hcy for 24, 48 and 72 hours respectively. Then cell supernatant and lysate were collected as condition medium. Any other differentiated THP-1 macrophages were incubated with a combination of Dex and Vit D3 for 6 days, and then the condition mediums were added to incubate together for 24 hours. Cells were divided into blank control group, 200 ?mol/L Hcy group, Dex+Vit D3 group, 24-hour Hcy+Dex+Vit D3 group, 48-hour Hcy+Dex+Vit D3 group, and 72-hour Hcy+ Dex+Vit D3 group. MAIN OUTCOME MEASURES: The expression level of TNFSF4 mRNA was determined by reverse transcription polymerase chain reaction. The expression level of IL-10 protein was determined by ELISA. RESULTS: Compared with blank control group, no significant difference of TNFSF4 mRNA expression was found in the 24-hour Hcy group, while the expression level was significantly higher in 48-hour and 72-hour Hcy groups. Compared with Dex+Vit D3 group, the IL-10 expression of all Hcy+Dex+Vit D3 groups was significantly lower than that in the Dex+Vit D3 group. CONCLUSION: Hcy may increase TNFSF4 mRNA expression and decrease IL-10 expression in THP-1 macrophages.
ABSTRACT
Hyperhomocysteinemia, which is an independent risk factor for atherosclerosis, may cause aberrant methylation and dysregulation of gene expression, but the characteristics of the aberrant methylation and its key links involved in its pathogenic mechanisms are still poorly understood. The effect of hyperhomocysteine on DNA methylation in vascular smooth muscle cells, its characteristics and the underlying mechanism of Hcy-induced changing in DNA methylation patterns were investigated. Clinical relevant concentrations of homocysteine was added into the cultured vascular smooth muscle cells of the Homo sapien umbilical vein for 24 h. The level of SAM and SAH was detected by HPLC. The activity of SAH Hydrolase was detected by real-time quantitative reverse transcription-PCR and Western blotting analysis. The level and patterns of DNA methylation were measured by endogenous C-5 DNA methyltransferase(C-5 MT-ase) activity and capacity of genomic DNA to accept methyl groups and methylation-dependent restriction analysis. The results indicated that an increased Hcy concentration induced elevated SAH, declined SAM and the ratio of SAM/SAH, reduced expression of SAH Hydrolase, but increased activity of C-5MT-ase. The methylation status of gDNA analyzed by methyl-accepting capacity of gDNA uncovered a demethylation process in gDNA, or homocysteine-caused hypomethylation in gDNA.With different methylation-dependent restriction endonucleases, the aberrant demethylation was found to prefer C↓CGG sequences to CpG islands. The impacts of different dosage of Hcy showed that the varied detrimental effects of Hcy could be attributed to different concentrations via different mechanisms. In mild and moderate hyperhomocysteinemia, the Hcy may primarily influence the epigenetic regulation of gene expression through the interference of transferring methyl-group metabolism, while in more higher Hcy concentration, the notorious impacts may be more directly caused via oxidative stress, apoptosis, inflammation etc.
ABSTRACT
Objective To study the effect of matrine on ATP binding cassette transporter A1 (ABCA1) expression and cholesterol in monocyte derived foam cells. Methods The lipid peroxide in cells was measured by TBARS method; The foam cells were examined by oil red O stain. The accumulation of cholesterol in monocyte was measured by fluorescence spectrophotometric method;ABCA1 mRNA and its protein level were determined by RT-PCR and Western blot, respectively. Results In the homonocytes incubated with PMA and low density lipoprotein (LDL), the intracellular accumulated total cholesterol (TC), free cholesterol (FC), cholesteryl ester (CE) and lipid peroxide increased obviously, and a huge amount of foam cells were found by oil red O stain. This was accompanied by a reduction in the membrane content of ABCA1. matrine alter ABCA1 mRNA abundance, indicating that increased ABCA1 transcription, enhanced mRNA level.Conclusion The mechanism of anti-artherosclerosis by matrine may be related to reduce cholesterol and to increase the level of ABCA1 expression.
ABSTRACT
OBJECTIVE: To explore the effects of Xuezhikang Capsules (ZXKC) and probucol on blood lipids, vascular endothelial functions and redox status in patients with coronary heart disease. METHODS: One hundred and twelve patients with coronary heart disease were randomly divided into XZKC-treated group and probucol-treated group, 56 in each. Before and after 8-week treatment, the blood levels of total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-C), nitric oxide (NO), endothelin-1 (ET-1), reduced glutathione (GSH) and oxidized glutathione (GSSG) were all measured in both groups. The GSH/GSSG redox potential (Eh) was calculated according to the Nernst equation. RESULTS: In the XZKC-treated group, the blood levels of TC, LDL-C and TG were significantly decreased after 8-week treatment as compared with those before treatment. The blood levels of TC and LDL-C were also significantly decreased in the probucol-treated group as compared with those before treatment. In the XZKC-treated group, the blood levels of ET-1 and GSSG and the GSH/GSSG Eh after treatment were all significantly lower than those before treatment, whereas the blood levels of GSH and NO, the NO/ET-1 ratio, and the GSH/GSSG ratio after treatment were all significantly higher than those before treatment. CONCLUSION: The XZKC or probucol treatment can yield a significant decrease in blood lipids in patients with coronary heart disease. Furthermore, XZKC exerts effective protection on vascular endothelial function, and can make GSH/GSSG redox status shift towards deoxidation.
ABSTRACT
In order to unveil the anti-angiogenic mechanism of triptolide, we investigated the effects of triptolide on the proliferation, migration, angiogenesis and u-PA expression of cultured HUVECs. MTT assay found that triptolide could inhibit the proliferation of HUVECs, and three-dimensional culture system revealed that triptolide could curb the migration of HUVECs. The chick embryo chorioallantoic membrane (CAM) test showed that triptolide could inhibit angiogenesis. Real time quantitive RT-PCR showed the expression of u-PA of HUVECs was down-regulated by triptolide. Therefore, triptolide may inhibit the proliferation and migration of endothelial cell by way of reducing the expression of u-PA. These findings may conduce to the elucidation of the anti-angiogenic mechanism of triptolide.
Subject(s)
Animals , Chick Embryo , Humans , Angiogenesis Inhibitors , Pharmacology , Cells, Cultured , Chorioallantoic Membrane , Diterpenes , Pharmacology , Down-Regulation , Endothelial Cells , Cell Biology , Metabolism , Epoxy Compounds , Pharmacology , Phenanthrenes , Pharmacology , RNA, Messenger , Genetics , Umbilical Veins , Cell Biology , Metabolism , Urokinase-Type Plasminogen Activator , GeneticsABSTRACT
<p><b>BACKGROUND</b>To construct the Bifidobacterium Infantis/CD tumor targeting gene therapy system.</p><p><b>METHODS</b>PCR expanded CD gene and pGEX-1LambdaT plasmid was transferred into E.Coli. JM109. with TSS solution. Then a dual restriction enzyme (EcoR I and BamH I) digestion was carried out to cut CD gene and pGEX-1LambdaT plasmid. Two segments (4.9 kb and 1.3 kb) were selected and extracted from the gel. T4 DNA Ligase was added to these two segments' mixture. Finally, reconstruction fragment was transferred into Bifidobacterium Infantis by electroporation. The plasmid was extracted from the positive clone selected and testified by an agarose eletrophoresis after enzymed.</p><p><b>RESULTS</b>A positive transferred Bifidobacterium Infantis with 6.2 kb long reconstruction plasmid fragment with CD gene was established which was in accordance with theoretical calculation.</p><p><b>CONCLUSIONS</b>The Bifidobacterium Infantis/CD tumor targeting gene therapy system could be successfully constructed.</p>
ABSTRACT
Objective To investigate the effects of homocysteine (Hcy) on methylation status and mRNA expression of TNF superfamily member 4 (TNFSF4) gene in THP-1 macrophages. Methods Cultured THP-1 monocytes were induced to macrophages by 0.1 ?mol/L PMA treatment for 72 h, then the differentiated THP-1 macrophages were incubated with homocysteine (50-1 000 ?mol/L) for 24, 48 or 72 h. The status of methylation of TNFSF4 gene in THP-1 macrophages was analyzed by methylation specific polymerase chain reaction (MS-PCR).The expression level of TNFSF4 mRNA was determined by RT-PCR. Results The MS-PCR results showed that the unmethylated bands gradually became stronger, and the methylated bands gradually became weaker along with the prolongation of treatment time and the increased Hcy concentrations. The 72-hour treatment with Hcy induced a complete demethylation in the promotor region of TNFSF4 gene, where left the only unmethylated bands. Meanwhile, the TNFSF4 mRNA expression was also increased in a dose-dependent manner. Conclusion Hcy may contribute to atherogenesis by inducing demethylation in the promotion region of the TNFSF4 gene and increasing TNFSF4 expression in THP-1 macrophages.
ABSTRACT
AIM: This study is to detail its possible mechanisms that homocysteine (Hcy) induces injury of cultured endothelial cells.METHODS: Hcy in sequential concentrations was added into the cultured human umbilial vein endothelial cells for 24 hours in serum-free medium. The lipid peroxidation, release of LDH, cell total protein content, cell apoptosis and necrosis were assessed. RESULTS: Hcy increased the apoptosis of endothelial cells.In high Hcy concentration the cells also showed obvious injurious and necrotic morphological changes. Lipid peroxidation increased, with LDH releasing up and cell total protein content down, and they showed a positive dose-effect relationship with the Hcy concentration. All the above effects of Hcy was strengthened by low density lipoprotein (LDL) which may suggest synergetic effects of Hcy and LDL.CONCLUSION: Our results indicate that Hcy has a strong oxidizing effect, which may be one of its major mechanism for injury of EC.
ABSTRACT
AIM: To investigate the changes of the redox status and the antioxidative capability in the tissue of malignant tumors. METHODS: The carcinoma tissues collected from 42 patients with primary cancer in digestive tract (13 cases of esophageal cancer, 14 cases of gastric cancer and 15 cases of colorectal cancer),the corresponding paratumor mucosa tissues were taken as the control samples. The content of oxidized and reduced glutathion (GSSG and GSH), oxidized and reduced coenzyme II (NADP+ and NADPH) were measured, the GSH/GSSG, NADPH/NADP+ ratios, and the GSH/GSSG, NADPH/NADP+ redox potentials were calculated according to Nernst formula. RESULTS: The levels of GSH and NADPH in cancer tissues were significantly higher than those in corresponding paratumor tissues (P0.05). CONCLUSIONS: The significant increase in GSH and NADPH contents in cancer tissues indicates a notable enhancement of its antioxidative capability compared with the corresponding paratumor tissues. Based on this changes, the redox potential in the cancer tissues has only slightly reductive shift, which may suggest an apparent oxidative stress existed in the cancer tissues.
ABSTRACT
0.05). CONCLUSION:HDL of human plasma could attenuate or inhibit the decrease in BP induced by endotoxin and prolong the survival time.These results indicated that HDL has therapeutic and protective effect on rat with endotoxemia.Inhibition of TNF release might be one of mechanisms.
ABSTRACT
AIM: To investigate therapeutic effects of recombinant human growth hormone(rhGH) on rat sepsis and its possible mechanisms. METHODS: Mean arterial pressure (MAP), levels of plasma TNF?,IL-1? and endotoxin,leukocyte count and survival rate within 1 week were determined after E. coli injection among control group,sepsis group and sepsis+rhGH group. RESULTS: (1)rhGH diminished the decrease of MAP, reduced plasma endotoxin and TNF? levels and increased neutrophil ratio in total leukocytes in sepsis rat. rhGH increased survival rate within 1 week on sepsis rat. (2)No changes were found in IL-1? level among the three groups. CONCLUSION: rhGH showed desirable beneficial effects on rat sepsis, which may attribute to: improving circulatory function; maintaining intestinal mucosa barrier, attenuating bacteria/endotoxin translocation and inhibiting the production and release of TNF?.
ABSTRACT
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-? 5?10 5 U/L, IL-1? 2?10 5 U/L, INF-? 2?10 5 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-?, IL-1? and INF-?) and LPS significant activated iNOS and NO production which, in turn, induced oxidative reaction in endothelial cells.
ABSTRACT
AIM: In this research, the apoptosis mechanism in human umbilical vein endothelial cells (HUVEC) exposed to increasingly high Hcy concentrations was investigated by examining the intracellular signaling pathways. METHODS: HUVEC were cultured and pretreated with Hcy. Twenty-four h after Hcy treatment, cell apoptosis was detected by Annexin V and DNA ladder analysis. Caspase3 and c-IAP1/2 mRNA expression were examined using reverse transcription-polymerase chain reaction (RT-PCR) and protein expression detected by Western blotting. The activation of caspase 3 was measured with the specific substrate DEVD-AMC. RESULTS: Hcy (0.3 mmol/L) induced apoptosis of HUVEC. Caspase 3 mRNA and protein expressions increased and activation enhanced with the concentration of increasing Hcy, but c-IAP2 mRNA and protein expression decreased. CONCLUSION: Hcy induces apoptosis of HUVEC in vivo by activating caspase 3 and inhibiting protein c-IAP-2 mRNA and protein expression.
ABSTRACT
AIM:To evaluated weather homocysteine(Hcy) and low density lipoprotein(LDL) had co-effects in pathogenesis of atherosclerosis (As). METHOD: Thiobarbituric acid reactive substance (TBARS) and apoptotic cells were measured after endothelial cell(EC) being exposed commonly or separated to Hcy and LDL. RESULTS: TBARS content in Hcy +LDL group was 4.9～7.7 times of that in single Hcy or LDL group, even put together TBARS contents of single Hcy or LDL groups, the co-effect of Hcy+LDL still showed 3 times higher than the former. Furthermore, Hcy+LDL showed obvious apoptosization effect on EC. The lipid peroxidization and EC apoptosization effects of Hcy+LDL were inhibited by adding folic acid , L-arginine+folic acid or glutamate+glycine. CONCLUSION: Hcy+LDL have co-injury effects on EC which may lead to As. The pathogenic factors of these effects probably involve the thiol groups of Hcy and their injury on nitric oxide system of EC.ESULTS :TBARScontentinHcy +LDLgro
ABSTRACT
Aim To investigate the role of K562 cells conditioned media on redox state of human umbilical vein endothelial cells (HUVECs) and the role of buthionine sulfoxine(BSO), a selective inhibitor of ?-glutamylcysteine synthetase, on HUVECs cultured with K562 cells conditioned media. Methods Glutathione(GSH)、oxidized glutathione (GSSG)、NADP~+、NADPH concentration and the viability of HUVECs under various conditions were determinated. Results GSSG、GSH、NADP~+、NADPH concentration of HUVECs increased when HUVECs were cultured with K562 cells conditioned media. The inhibition of HUVECs growth by Bso enhanced when K562 cells conditioned media were used at the same time. Conclusion Under the effection of chronic myelogenous leukemia cells conditioned media, endothelial cells may be more sensitive to BSO.