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.     

Metanephric cell microenvironment induces embryonic stem cells to differentiate toward renal cells

AIM: To explore the effects of metanephric cell microenvironment on inducing embryonic stem cells (ESCs) to differentiate toward renal cells.METHODS: Embryoid bodies (EBs) of D3 mouse embryonic stem cells were prepared by hanging drop culture, and the EBs were co-cultured indirectly with metanephric cells derived from E12.5 d mouse embryo. The EBs cell with spontaneous differentiation was used as the control. The proteins of Pax2 and WT-1 were analyzed by immunofluorescence assay. The mRNA expression of Pax2, WT-1, Lim1, Sall1, Emx2, GDNF, Wnt4, BMP7, Nephl, Nephrin, KSP and CD24 genes was detected by RT- PCR.RESULTS: The genes related to kidney development were expressed in the EBs cells after co-culture on day 3, and the mRNA expression of Pax2, WT-1, Emx2, GDNF, Nephl, Nephrin, KSP and CD24 was stronger than those in control group. Pax2 positive cells were found on day 3 in the co-cultured EBs cells, and the positive cells increased on day 5 and day 7. WT-1 protein positive cells were found in the co-cultured EBs cells on day 5. No Pax2 or WT-1 positive cell was observed in control group.CONCLUSION: Metanephric cell microenvironment promotes ESCs differentiation toward renal cells.     

Homocysteine thiolactone damages cultured endothelial cells

AIM: To approach the mechanisms of homocysteine thiolactone (HTL)-induced damage in endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were incubated with HTL. The concentrations of soluble intercellular adhesion molecule (sICAM)-1 and TNF-α in the conditioned medium were measured by ELISA. The activity of NF-κB and the level of ROS were determined by fluorescence microscopy. Cell viability,activity of lactate dehydrogenase (LDH) and content of nitric oxide (NO) in the medium were also detected. RESULTS: Exposure of HUVECs to HTL at concentration of 1 mmol/L for 3 h potentiated the activity of NF-κB and increased the level of ROS. Incubation of HUVECs with HTL (1 mmol/ L for 24 h) markedly decreased the cell viability and NO content, and increased the level of LDH, sICAM-1 and TNF-α in the culture medium. Pretreatment with NAC, apocynin or PDTC markedly inhibited the increased activity of NF-κB and decreased the levels of ROS, TNF-α, sICAM-1, NO and LDH in a dose-dependent manner. CONCLUSION: The dysfunction of endothelial cells induced by homocysteine thiolactone in vitro may be related to the oxidative stress and the activation of NF-κB.     

Effect of growth arrest-specific protein 6 on H9c2 cell apoptosis induced by anoxia-reoxygenation via PI3K/Akt survival pathway

AIM: To investigate the effect of growth arrest-specific protein 6(Gas 6) on H9c2 cell apoptosis induced by anoxia-reoxygenation (A/R) and its possible relationship with PI3K/Akt pathway. METHODS: Cultured H9c2 cell line of cardiomyocytes was randomly divided into 4 groups: normal control group, anoxia-reoxygenation group (A/R), anoxia-reoxygenation+Gas6 group (A/R+Gas6) and anoxia/reoxygenation+Gas6+LY294002 group (A/R+Gas6+LY294002). The procedure of A/R was performed in cultured H9c2 cells by 3 h of anoxia and then 3 h of reoxygenation. The viability of the cells and the activity of caspase-3 were detected by automatic biochemistry analytic instrument. Cell apoptotic rates were evaluated by flow cytometry. The protein level of phosphorylated Akt(p-Akt) was determined by Western blotting. RESULTS: Compared with control group, the cell viability was significantly decreased, and caspase-3 activity, cell apoptotic rate and the protein level of p-Akt were increased in A/R group. Compared with A/R group, the caspase-3 activity and cell apoptotic rate reduced markedly, while the cell viability and the protein level of p-Akt were significantly increased in A/R+Gas6 group .The effect of Gas6 was inhibited by LY294002. CONCLUSION: Gas6 may protect the H9c2 cells from anoxia-reoxygenation-induced apoptosis. Its mechanism is possibly involved in the activation of PI3K/Akt survival pathway via increasing the phosphorylation of Akt protein.     

Effects of maxadilan on proliferation of human induced pluripotent stem cells

AIM: To investigate the promoting effect of maxadilan, which specifically activate the type I receptor for pituitary adenylate cyclase-activating polypeptide (PACAP), on the proliferation of human induced pluripotent stem cells (IPSCs). METHODS: PACAP type I (PAC1) receptor in IPSCs was detected by RT-PCR and Western blotting. maxadilan at various concentrations was added to the medium of IPSCs as experimental groups. The medium in control group was without maxadilan treatment. The effect of maxadilan on theproliferation of IPSCs was measured with Cell Counting Kit-8 (CCK-8). The changes of cell cycle caused by maxadilan in IPSCs were analyzed by flow cytometry. The analysis of karyotype was carried out in IPSCs treated with maxadilan. Proteins and gene expression levels of both Nanog and OCT4 in IPSCs treated with maxadilan were detected by real-time quantitative polymerase chain reaction (real-time-qPCR) and immunofluorescence. The gene expression levels of Nestin and PAX6 in both IPSCs treated with maxadilan and cells of embryonic body, which was birthed from IPSCs with maxadilan treatment, were detected by real-time qPCR. The ability of IPSCs treated with maxadilan differentiating into 3 embryonic layers was evaluated by analyzing the component of embryo using RT-PCR. RESULTS: The PAC1 receptor in IPSCs was identified by RT-PCR and Western blotting. Viability of the IPSCs with 100 nmol/L maxadilan treatment was increased by 16% compared with control group. The differences with statistical significance were found in the cell viability between 100 nmol/L maxadilan treatment group and control group (P<0.05). The average values of proliferation index (PI) in IPSCs with 100 nmol/L maxadilan treatment for 3 h, 6 h and 9 h were 47.23%, 59.70% and 55.67%,respectively, while that in control group was 37.00%. The differences with statistical significance were found in PI between 100 nmol/L maxadilan treatment for 3 h group, 6 h group, 9 h group and control group (P<0.05). Normal karyotype and unchanged pluripotent state in IPSCs treated with maxadilan were observed. Compared with control group, the gene expression levels of Nestin and PAX6 were not significantly different in both IPSCs and the cells of embryonic body birthed from IPSCs with maxadilan treatment. The ability of differentiation into 3 embryonic layers in IPSCs treated with 100 nmol/L maxadilan was found. CONCLUSION: PAC1 receptor presents in IPSCs. Maxadilan promotes the proliferation of IPSCs but does not affect their pluripotent state and karyotype.     

Inhibitory effect of 1,25-(OH)2D3 on proliferation of airway smooth muscle cells from passively-sensitized patients

AIM: To investigate the effects of 1,25-dihydroxyvitamin D₃ on the proliferation of passively-sensitized human airway smooth muscle cells (HASMCs), and to explore its potential role in asthmatic airway remodeling.METHODS: HASMCs were passively sensitized with 10% serum from asthmatic patients.1,25-(OH)₂D₃ was used as the interventor.The effect of 1,25-(OH)₂D₃ on the cell proliferation and its optimal concentration were determined by MTT colorimetric assay.The cell cycle analysis was performed by flow cytometry.The expression of proliferating cell nuclear antigen (PCNA) was measured by the method of immunocytochemical staining.RESULTS: 1,25-(OH)₂D₃ at the concentrations of 10^-9-10^-7 mol/L markedly inhibited the cell proliferation and the maximum effect was observed at the concentration of 10^-7 mol/L.This concentration of 1,25-(OH)₂D₃ markedly suppressed the PCNA-positive rate and hampered the G₁/S transition in HASMCs passively-sensitized by asthmatic serum.CONCLUSION: 1,25-(OH)₂D₃ has direct inhibitory effects on the proliferation of passively-sensitized HASMCs in vitro, which may be concerned with the beneficial role of 1,25-(OH)₂D₃ on the prevention and therapy of asthmatic airway remodeling.     

Proliferative effect of PDGF and anti-proliferative activity of AMPK on vascular smooth muscle cells

AIM: To investigate the proliferative effect of platelet-derived growth factor (PDGF) and anti-proliferative activity of AMP-activated protein kinase (AMPK) on vascular smooth muscle cells (VSMCs). METHODS: The proliferation of VSMCs cultured with PDGF and activation of AMPK were observed. VSMCs were divided in 4 groups: control group; PDGF group; 5-aminoimidazole-4 -carboxamide-1-β-D-riboside (AICAR) group and AICAR+PDGF group. The time course of AMPK activation was determined. The protein level of mTOR was also measured. RESULTS: Compared with control group, the proliferative rate in PDGF group was significantly increased. The growth of VSMCs was inhibited in a time-dependent manner and the activity of p-mTOR was significantly decreased in AICAR group. Compared with control group, the expression of p-AMPK in PDGF group was significantly decreased, and that in AICAR group and AICAR+PDGF group was significantly increased. The expression of p-AMPK in AICAR+PDGF group was higher than that in PDGF group. The activity of p-mTOR in PDGF group was significantly higher than that in control group, while that of AICAR group and AICAR+PDGF group was significantly decreased. The expression of p-mTOR in AICAR+PDGF group was lower than that in PDGF group. CONCLUSION: Stimulation of VSMCs with PDGF promotes the cell proliferation, which can be inhibited by AICAR. The proliferation of VSMCs activated by AMPK is probably correlated with the down-regulation of mTOR expression.     

Construction of a lentiviral RNA interference vector targeting rat myocardin and its effect on vascular smooth muscle cell differentiation

AIM: To construct a lentiviral RNA interference(RNAi)vector targeting rat myocardin mRNA and to investigate its effect on the differentiation of vascular smooth muscle cells(VSMCs).METHODS: Three pairs of dsDNA targeting rat myocardin mRNA were designed, synthesized and cloned into lentiviral vector pGCSIL-GFP to generate pGCSIL-GFP-shMyocd lentvirus. A Flag-tagged myocardin-overexpression vector pEGFP-N1-Myocd was constructed with pEGFP-N1/X124G. After these two vectors were cotransfected into 293T cells, the flag protein was assessed by Western blotting to analyze the knockdown effect of pGCSIL-GFP-shMyocd. The expression of myocardin and SM22α was also detected by RT-PCR and Western blotting after the pGCSIL-GFP-shMyocd viruses were transfected into primary cultured rat aortal VSMCs.RESULTS: The rat myocardin lentviral RNAi vector pGCSIL-GFP-shMyocd and myocardin-overexpression vector pEGFP-N1-Myocd were successfully constructed. After these two kinds of vectors were cotransfected into 293T cells,the No.1 interfering vector displayed the highest inhibitory effect on flag expression.After the No.1 lentvirus at the titer of 1×10¹² TU/L was transfected into VSMCs, the myocardin and SM22α expression was significantly attenuated. CONCLUSION: The lentiviral pGCSIL-GFP-shMyocd RNAi vector is successfully constructed, which is useful for further study regarding the molecular mechanism of the phenotypic switching in VSMCs under special pathological conditions such as atherosclerosis. Inhibition of myocardin expression in VSMCs leads to the decrease in the expression of differentiation marker, and implies a crucial role of myocardin in VSMCs differentiation.     

PI3K/Akt regulates endoplasmic reticulum stress-mediated GRP78 induction

AIM: To investigate the effect of PI3K/Akt pathway on endoplasmic reticulum (ER) stress-mediated glucose-regulated protein 78 (GRP78) induction in human embryonic kidney 293 cells (HEK293) cells.METHODS: PI3K inhibitor LY294002, dominant negative kinase-dead mutant vector for HA-Akt (K179M) and Akt1 siRNAs were used to block the PI3K/Akt pathway under ER stress. Constitutively active expression vectors for Akt (myr-HA-Akt) were used to up-regulate Akt activity under ER stress. The effects of PI3K/Akt on ER stress-mediated GRP78 induction in HEK293 cells were determined by Western blotting and RT-PCR. RESULTS: GRP78 induction was inhibited by LY294002, Akt1 (K179M) and Akt1 siRNA, but was increased by myr-Akt1 in dithiothreitol-and thapsigargin-treated HEK293 cells. However, both myr-Akt2/3 and Akt2/3 siRNA had no effect on GRP78 induction in HEK293 cells under ER stress. Furthermore, the PI3K/Akt pathway didnt regulated GRP78mRNA induction but increased GRP78 protein stability.CONCLUSION: PI3K/Akt promotes GRP78 accumulation through increasing the stability of GRP78 protein in HEK293 cells under ER stress.