Effects of silymarin on homocysteine-induced apoptosis in human umbilical vein endothelial cells

AIM:To investigate the effect of silymarin on homocysteine-induced cell viability and apoptosis in human umbilical vein endothelial cells (HUVECs). METHODS:Cell viability was analyzed by using MTT and LDH assay. Apoptotic cells were detected by using DNA fragmentation and flow cytometric analysis. The level of intracellular reactive oxygen species (ROS) and the potential of mitochondrial membrane were determined by flow cytometric assay. The activity of caspase-3, -6 and -9 were measured with microplate spectrofluorometer. Protein levels were examined by Western blotting. RESULTS:Treatment of cultured HUVECs with HCY for 48 h induced a significant decrease in cell viability, and the percentage of apoptosis increased to 76.8%. The level of intracellular ROS and activity of caspase-3, -6 and -9 enhanced, and the red/green ratios of mitochondrial membrane decreased. However, simultaneous treatment with silymarin exhibited cytoprotective effects, reduced formation of the DNA ladder, prevented the levels of Bax and Bcl-2 proteins and the accumulation of ROS as well as caspase-3, -6 and -9 activation, reconverted the potential of mitochondrial membrane, and the percentage of apoptosis/necrosis was significantly decreased to 12.7% in a dose-dependent manner. CONCLUSION:These results demonstrate that silymarin has the protective capacity to antagonize HCY-induced apoptosis in HUVECs. The antiapoptotic action of silymarin may be partially dependent on an anti-oxidative stress effects, inhibition of caspases activity, and maintenance of mitochondria function.     

Function of Shh gene in the development of embryos

Mammals have three genes with homology to the hh gene. These comprise Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Shh has been shown to play a crucial role in embryogenesis and the development of ectoderm. Shh has the great relationship with the forming all the system. Shh-Patched-Smoothened signal pathway will lead the embryogenesis in the normal way. Otherwise, the abnormal embryogenesis, malformation, and tumor will be happened.     

Effects of Chinese yellow wine on homocysteine-induced dysfunction in rat endothelial progenitor cells

AIM: To investigate whether Chinese yellow wine has influences on homocysteine (Hcy)-induced dysfunction in rat endothelial progenitor cells (EPCs). METHODS: Rat bone marrow was extracted to harvest mononuclear cells (MNCs) by density gradient centrifugation. The MNCs were plated on fibronectin-coated culture dishes, and were induced into EPCs by EGM-2 complete medium supplemented with cell growth factor. The adherent cells were collected 7 d later for all studies. EPCs were characterized as adherent cells double positive for DiI-ac-LDL uptaking and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. The viability, migration, apoptosis and in vitro vasculogenic activity of the EPCs were determined by MTT assay, Transwell chamber assay, apoptosis kit and in vitro vasculogenesis kit, respectively. RESULTS: Compared with control group, the viability, migration and in vitro vasculogenic capacity of the EPCs in Hcy group were significantly decreased (P<0.01). Compared with Hcy group, yellow wine group and red wine group both significantly improved the viability, migration and in vitro vasculogenic capacity of Hcy-induced EPCs (P<0.01). Compared with control group, yellow wine group and red wine group both significantly improved the above-mentioned functions of EPCs (P<0.05). However, no significant difference of apoptosis in all groups was observed. CONCLUSION: Hcy may result in dysfuction of EPCs. Treatment with yellow wine improves Hcy-induced EPC functions.     

Expression of zinc-finger transcription factor Snail 1 in the kidney of diabetic rats

AIM: To explore the expression of Snail 1 in renal tissues of diabetic rats, and to investigate its contribution to the progression of diabetic nephropathy. METHODS: Streptozotocin-induced diabetic rats were randomly divided into 2, 4, 8, 12, 16, 20, 24 weeks groups and 16 week A, 20 week A and 24 week A groups. A groups were treated with insulin to control blood glucose to normal level from the 13th week. Control groups were set up in age-matched time points. Blood glucose, 24 h urine protein, serum creatinine (Scr) and kidney index of rats were measured. Periodic acid-silver (PAS) staining was used to observe the renal pathological changes. The mRNA and protein expressions of Snail 1 and FN in renal cortex were detected by RT-PCR and immunohistochemical staining, respectively. Western blotting was employed to detect the expression of Snail 1 protein in the renal cortex. RESULTS: The levels of blood glucose, Scr, kidney weight index were increased remarkably in diabetic rats as compared with those in control groups (P<0.05, P<0.01), and decreased remarkably in the insulin-treated rats as compared with those in the diabetic rats (P<0.05, P<0.01). The Snail 1 protein was not detected by immunohistochemical staining in normal renal tissues. However, strongly positive staining was observed in renal tubules of diabetic rats. A time-dependent loss of Snail 1 expression was detected in the kidney in insulin-treated rats. The Snail 1 protein and mRNA of Snail 1 and FN were significantly up-regulated in the diabetic rats as compared with those in controls (P<0.01), while down-regulated in the insulin-treated diabetic rats (P<0.01). A close positive relationship existed between the mRNA expression of Snail 1 and FN (r=0.800, P<0.01). The level of Snail 1 protein expression was positively correlated with blood glucose, urine protein, Scr, kidney index (r=0.877, 0.694, 0.522, 0.875, P<0.01).CONCLUSION: These findings suggest that Snail 1 gene and protein expression are up-regulated in the kidney of rats with diabetes and may be involved in the pathogenesis of diabetic nephropathy.     

Study on genetic instability of nm23-H1 gene and expression of hMLH1,hMSH2 in sporadic gallbladder carcinoma

AIM: To examine the MSI and LOH of locus D17S396 and their influence on the expression of nm23-H1 in gallbladder tumors,and to examine the protein expression of hMLH1/hMSH2,which may provide experimental evidence for the tumor occurrence and metastasis.METHODS: Techniques such as DNA extraction,CR-SSCP,ordinary silver stain were used to study MSI and LOH of locus D17S396.Envision IHC was used to assess the expression of nm23-H1 and hMLH1/hMSH2.RESULTS: ① The frequency of heredity instability of gallbladder carcinoma was 42.55%.The frequency of LOH in liver and lymph node metastasis cases and in stage Nevin IV and V was significantly higher than that without metastasis and stage I,II and III.However,the frequency of MSI showed contrary correlation with some clinicopathologic characteristics.② The expression of nm23-H1 was 46.81%.The case with lymph node metastasis and Nevin stage IV and V showed significantly lower expression than that without lymph node metastasis and stage I,II and III.③ The expressions of hMLH1 and hMSH2 were 51.06% and 42.55% respectively.hMLH1 in lymph node and liver metastasis cases and in stage Nevin IV and V were significantly lower than that without metastasis and in stage I,II and III.④ Positive frequency of hMLH1 in MSI positive group was higher than that in MSI negative group.The positive frequency of nm23-H1 and hMSH2 protein in LOH positive group was lower than that in negative group.CONCLUSION: The heredity instability of nm23-H1 gene may be implicated pathogenesis and progression of gallbladder carcinoma.Both MSI and LOH of nm23-H1 control the development of gallbladder carcinoma independently in different paths.Abnormal expression of hMLH1/hMSH2 may be a molecule marker in early stage of gallbladder carcinoma.     

Effects of p27kip1 gene on DNA and protein synthesis in esophageal carcinoma cells

AIM: To investigate the effects of p27kip1 gene on DNA replication and protein synthesis in esophageal carcinoma cells. METHODS: Recombinant adenovirus Ad-p27kip1 was constructed and transfected into esophageal carcinoma cell EC-9706, and its effects on DNA replication, protein synthesis and the growth of esophageal carcinoma cells were explored by means of cell growth count, [3H]-TdR, [3H]-Leucine incorporation method and flow cytometry. RESULTS: The growth of esophageal carcinoma cells was inhibited obviously. The radioactive intensity of -TdR and -Leucine in Ad-p27kip1 group decreased significantly than that in control group after EC-9706 transfection (P<0.01), while the multiplication of esophageal carcinoma cell was inhibited obviously. CONCLUSION: Ad-p27kip1 inhibited the multiplication of the esophageal carcinoma cells, which may be related to its suppressive function for DNA replication and protein synthesis.     

Effects of folic acid and vitamin B12 on human umbilical vein endothelial cells against homocysteine-induced apoptosis

AIM: To investigate the effect of folic acid and vitamin B₁₂ on homocysteine (Hcy)-induced apoptosis of human umbilical vein endothelial cells (HUVECs) through mammalian sterile 20-like kinase 1 (MST1). ME-THODS: HUVECs were cultured in the absence (control group), or presence of 100 μmol/L Hcy alone (Hcy group) or 100 μmol/L Hcy plus 30 μmol/L folic acid and vitamin B₁₂ (intervention group) for 72 h. The effect of Hcy on the apoptosis of HUVECs was analyzed by flow cytometry. The transfection efficiency of DNA methyltransferase 1 (DNMT1)-overexpressing adenovirus was observed under fluorescence inverted microscope. The mRNA and the protein levels of DNMT1 and MST1 were determined by RT-qPCR and Western blot. The DNA methylation level of MST1 promoter was detected by methylation-specific PCR. RESULTS: Compared with control group, the apoptotic rate (P<0.01) and the expression of MST1 at mRNA (P<0.01) and protein (P<0.05) levels in the HUVECs were significantly increased, while the mRNA levels of DNMT1 was decreased in Hcy group (P<0.01). In addition, folic acid and vitamin B₁₂ treatment significantly inhibited Hcy-mediated apoptosis of HUVECs (P<0.01), increase in MST1 mRNA level (P<0.01) and decrease in DNMT1 mRNA level (P<0.01). Meantime, the mRNA level of MST1 was positively correlated with the apoptotic rate of the HUVECs (r=0.943 9, P<0.001). The expression of DNMT1 at mRNA and protein levels was significantly increased after the transfection of DNMT1-overexpressing adenovirus into HUVECs (P<0.01), and a large amount of green fluorescent protein expression was observed. Meanwhile, the DNA methylation level of MST1 promoter was increased (P<0.01), while the protein level of MST1 was decreased (P<0.01).CONCLUSION: Up-regulation of MST1 promotes Hcy-induced apoptosis of HUVECs, while folic acid and vitamin B₁₂ exert an anti-apoptosis effect, which might be regulated by hypermethylation of MST1 promoter region.