ABSTRACT
Objective To investigate changes of serum silent information regulator 1 (Sirt1) and inflammatory cytokines in type 2 diabetes patients with different stages of diabetic nephropathy.To explore the relationship between serum Sirt1 level and inflammatory cytokines in type 2 diabetic patients with different urinary albumin excretion rates.Methods A total of 436 cases with type 2 diabetes were divided into three groups:normoalbuminuric [D1,n =168],microalbuminuric [D2;n =152],and macroalbuminuric [D3,n =116].Serum Sirt1,hypoxia-inducible factor-1α (HIF-1α),early growth response protein 1 (EGR1),insulin-like growth factors-Ⅰ (IGF-Ⅰ),and monocyte chemotactic protein-1 (MCP-1) were determined by enzyme-linked immunosorbent assay (ELISA).Results The levels of serum Sirt1 in type 2 diabetes patients were significantly lower than that in control group,and with the increase of urinary protein excretion rate,the levels of serum Sirt1 in group D1,D2 and D3 were decreased gradually (P ＜ 0.01).Compared to control,serum inflammatory cytokines (HIF-1α,EGR1,IGF-Ⅰ,and MCP-1) levels were significantly increased in type 2 diabetic patients and gradually increased in the patients of D1,D2 and D3 groups (P ＜0.01).Furthermore,Serum Sirt1 was negatively correlated with the levels of inflammatory cytokines.Age,duration,fasting blood glucose (FBG),fasting insulin (FINS),homeostasis model assessment insulin resistance index (HOMA-IR),glycosylated hemoglobin (HbA1c),low density lipoprotein (LDL),total cholesterol (TC),triglyceride (TG),serum creatinine (Scr),blood urea nitrogen (BUN),uric acid (UA),HIF-1α,EGR1,IGF-Ⅰ,and MCP-1 were positively correlated with Ln Koc of urinary albumin to creatinine ratio [Ln(ACR)] (P ＜ 0.05);and Sirt1 were negatively correlated with Ln(ACR)(P ＜ 0.01).HIF-1α,MCP-1,IGF-Ⅰ,duration,BUN,Sirt1,UA,LDL,and EGR1 were independent factors that significantly influenced Ln (ACR) (P ＜ 0.05).Conclusions Serum Sirt1 might be a new target for the treatment of diabetic nephropathy.Enhancing serum Sirt1 levels might have a role in delaying the progression of diabetic nephropathy.
ABSTRACT
Background:Hepatic veno-occlusive disease( HVOD) is a disease characterized by hepatomegaly,jaundice, ascites,weight gain and lack of effective treatment currently. Our prophase research showed that ligustrazine had therapeutic effect on Sedum aizoon induced HVOD in mice. Aims:To investigate the mechanism of therapeutic effect of ligustrazine on Sedum aizoon induced HVOD in mice. Methods:A total of 115 mice were randomly divided into 4 groups:mice in group A were intragastrically administrated with 30 mg·kg-1 ·d-1 Sedum aizoon to induce HVOD and served as model group;mice in group B were given 30 mg·kg-1 ·d-1 Sedum aizoon + 100 mg·kg-1 ·d-1 ligustrazine and served as low dose ligustrazine intervention group;mice in group C were given 30 mg·kg-1 ·d-1 Sedum aizoon + 200 mg·kg-1 ·d-1 ligustrazine and served as high dose ligustrazine intervention group;mice in group D were given 30 mg·kg-1 ·d-1 PBS and served as normal control group. After 30 days,all the mice were sacrificed. HE staining and Masson staining were performed for histological examination. The mRNA and protein expressions of tissue factor(TF),nuclear factor(NF)-κBp65 and early growth response factor( Egr)-1 in liver tissue were determined by RT-PCR and Western blotting, respectively. Results:HE staining and Masson staining histological examination showed that ligustrazine could obviously ameliorate the pathological injury of liver tissue in HVOD mice. Compared with group D,the mRNA and protein expressions of TF,NF-κBp65,Egr-1 were significantly increased in group A( P 0. 05). Conclusions:Ligustrazine has therapeutic effect on HVOD,the possible mechanism is that ligustrazine could interrupt the activation of coagulation system by reducing the expression of TF via down regulating the expressions of NF-κBp65 and Egr-1,especially in high dose ligustrazine group.
ABSTRACT
Background Flicker light can induce myopia,but its mechanism remains unclear.As one of immediate early genes,early growth response-1 (Egr-1) gene can generate rapid response to visual stimulation,however,its effect on the formation and development of myopia is below understood.Objective This study was to investigate the dynamic expression of Egr-1 gene in retinas of flicker light-induced eyes (FL) and compare the results with form deprived eyes (FD).Methods One hundred and fifty 28-day-old C57BL/6J mice were randomly assigned to the normal control group,FD group and FL group.The right eyes of mice were occluded with a semitransparent hemispherical thin plastic shell for 2 weeks in the FD group,and the right eyes of mice were stimulated by 2 Hz flicker light for 2 weeks in the FL group,and then the mice were fed in the normal light environment for 1 week.The refractive state and axial length of the model eyes were measured by murine-specific eccentric infrared photorefraction and A-scan ultrasonography before modeling and 1 hour,I day,1 week,2 weeks after modeling as well as 1 week after termination,respectively.The mice were sacrificed in above-mentioned time points to isolate the retinas.The expressions and location of Egr-1 protein and mRNA in the retinas were detected by Western blot,and reverse transcription PCR (RT-PCR) and immunochemistry.The expressions of Egr-1 markers,neuron and protein kinase C (PKC)-α,in the retinas were assayed by using immunofluorescence.The care and use of the animals followed the administration regulations for experimental animals of Jiangsu Province.Results Two weeks after modeling,the refraction of the FL group was (0.32±0.14) D,which was significantly lower than (-0.66±0.43)D in the FD group (t=6.78,P=0.00).One hour after modeling,The expression levels of Egr-1 mRNA in mouse retinas were 0.626±0.044 and 0.695±0.058 in the FD group and FL group,which were significantly declined in comparison with 1.009±0.089 of the normal group (t=14.81,P=0.01;t=9.15,P=0.03).In 2 weeks after modeling,the expression levels of Egr-1 mRNA were still lower in the FD group and F:L group compared with the normal group (all at P＜0.05).However,the expression levels were significantly elevated in the FD group and FL group compared with the normal group (t=4.13,P=0.01;t=4.26,P=0.01) at 1 week after termination.Western blot showed a dynamic decrease in the expressions of Egr-1 protein with lapse of time in the FD group and FL group with the lowest expressing level in the second week after modeling.In I week after termination of modeling,the expressing level was raised in the FD group or the FL group,but it was still lower than that ir the normal group (t =6.32,P=0.00;t =5.45,P=0.01).Egr-1 protein was mainly expressed in the retinal ganglion cell (RGC) layer,inner nuclear layer and photoreceptor layer in the normal mice,and the expression intensity was obviously weaker in the FD mice and FL mice 2 weeks after modeling.Htowever,the expression was enhanced in 1 week after termination of modeling.Neuron and PKC-α were strongly expressed in the RGCs and bipolar cells in the normal mice.Conclusions The eyes show a myopic trend after induce of flicker light in B6 mice.The expression level of Egr-1 gene in the retina down-regulates with the reduce of refraction in FL eyes,and its dynamic expressing change is consistent between the FD eyes and FL eyes.
ABSTRACT
AIM:To observe the effects of Egr-1 gene transfection on the expression of tumor necrosis factor-α( TNF-α) and intercellular adhesion molecule-1 ( ICAM-1) , and to investigate the role of Egr-1 in the pathogenesis of dia-betic nephropathy .METHODS:The diabetic mouse model was established .Ten mice were randomly selected as the dia-betic group .The remaining 40 mice were injected with empty plasmid , Egr-1 expression plasmid or Egr-1 siRNA plasmid via the tail vein once a week.The normal control group was also set up .The animals were sacrificed at the end of the 4th week.The renal tissues were harvested .The expressions of Egr-1, TNF-αand ICAM-1 were detected by immunohisto-chemistry and Western blot .The pathological changes were observed under electron microscope .RESULTS: In diabetic mouse kidney, the expression of Egr-1, TNF-αand ICAM-1 was increased, and irregular thickening of glomerular basement membrane , mesangial expansion and fusion of foot were observed .The change trend was more significant in Egr-1 gene transfection group , and these changes in siRNA plasmid transfection group were obviously reduced compared with diabetes group.CONCLUSION:Egr-1 up-regulates the expression of TNF-αand ICAM-1, and induces mesangial cell proliferation and mesangial extracellular matrix accumulation , which is probably one of the mechanisms of accelerating glomerulosclerosis .
ABSTRACT
Objective To construct a recombinant baculovirus dual expression vector containing NIS gene under the control of human telomerase reverse transcriptase (hTERT) promoter and plasminogen kringle 5 (K5) gene driven by early growth response 1 (Egr1) promoter,and to explore the feasibility of targeting both tumor and tumor vessel with combination of radioiodide and antiangiogenic therapy.Methods The hTERT-NIS gene and Egr1-K5 gene fragments were subcloned into baculovirus vector,then packaged and amplified in the sf9 cells to obtain recombinant baculovirus Bac-hTERT-NIS-Egr1-K5.Bac-CMV-NISEgr1-K5,Bac-hTERT-0-Egr1-K5 and Bac-hTERT-NIS-Egr1-0 were constructed as controls.The expression of NIS and K5 genes in human cervix cancers cells (HeLa) was examined by Western blot and quantitative real-time PCR.Functional NIS activity was confirmed by the uptake of 125I,the inhibition of NaClO4 and the cytotoxicity of 131I.The apoptotic effect of 131I-inducedK5 on human umbilical veins endothelial cells (HUVEC)was analyzed by an apoptosis assay using flow cytometry.Statistical analysis was performed using the analysis of variance.Results The recombinant baculovirus Bac-hTERT-NIS-Egr1-K5 was successfully constructed.The NIS gene under the control of hTERT promoter was specifically expressed in HeLa cells.The baculovirusinfected HeLa cells showed a significant increase of 125I uptake,which was significantly inhibited by NaClO4(F199.296,P＜0.05).Furthermore,a notable decreased cell survival rate (38.3％) was found after 131I treatment.The expression of K5 gene induced by 131I was elevated in a dose or time dependent manner and resulted in obvious inhibition with cell survival rate of 30.8％ in baculovirus-infected HUVEC cells,which was significantly higher than that in the control groups (11.2％ and 10.9％ respectively,F=19.926,45.409;both P＜0.05).Conclusions A recombinant baculovirus dual expression vector containing the NIS and K5 genes has been successfully constructed.This study suggests the feasibility of a synergistic strategy of NISbased raidoiodide therapy and K5-based antiangiogenic therapy in vitro,and make it possible to perform in vivo study in the near future.
ABSTRACT
Sex-determining region Y box 18 (Sox18/SOX18) gene is an important regulator of vascular development playing a role in endothelial cell specification or differentiation, angiogenesis and atherogenesis. The aim of this study was to perform comprehensive functional characterization of the human SOX18 promoter, including determination of transcription start point (tsp) and identification of control elements involved in the regulation of SOX18 gene expression, with an emphasis on angiogenesis-related transcription factors. Analyses were performed in HeLa cells, representing a tumor cell line, and in EA.hy926 cells used as an endothelial model system. We have determined unique tsp of SOX18 gene, located 172 nucleotides upstream from ATG codon. Further, we have shown that SOX18 promoter region, -726 to -89 bp relative to tsp, contains positive cis-regulatory element(s) that stimulates SOX18 promoter activity, while region -89 to + 166 represents the minimal promoter. Within this region we have recognized the presence of essential element(s), positioned from -89 to +29, which harbors cluster of three putative early growth response 1 (EGR1) binding sites. By in vitro binding assays and functional analyses we have shown that these three putative binding sites are functionally relevant and sufficient for EGR1-induced SOX18 transcription. Mutations of these binding sites significantly impaired activity of the SOX18 promoter, particularly in EA.hy926 cells, indicating the importance of these regulatory elements for SOX18 promoter activity in endothelial setting. By data presented in this study, we have established SOX18 as a novel target gene regulated by EGR1 transcription factor, thus providing the first functional link between two transcription factors previously shown to be involved in the control of angiogenesis.
Subject(s)
Humans , Early Growth Response Protein 1/genetics , Electrophoretic Mobility Shift Assay , Endothelium/metabolism , Gene Expression Regulation , HeLa Cells , Mutagenesis, Site-Directed , Neovascularization, Physiologic/genetics , Promoter Regions, Genetic , Protein Binding/genetics , SOXF Transcription Factors/genetics , Transcription Initiation Site , Transcriptional ActivationABSTRACT
The early growth response-1 gene (egr-1) encodes a zinc-finger transcription factor Egr-1 and is rapidly inducible by a variety of extracellular stimuli. Anisomycin (ANX), a protein synthesis inhibitor, stimulates mitogen-activated protein kinase (MAPK) pathways and thereby causes a rapid induction of immediate-early response genes. We found that anisomycin treatment of U87MG glioma cells resulted in a marked, time-dependent increase in levels of Egr-1 protein. The results of Northern blot analysis and reporter gene assay of egr-1 gene promoter (Pegr-1) activity indicate that the ANX- induced increase in Egr-1 occurs at the transcriptional level. Deletion of the serum response element (SRE) in the 5'-flanking region of egr-1 gene abolished ANX-induced Pegr-1 activity. ANX induced the phosphorylation of the ERK1/2, JNK, and p38 MAPKs in a time-dependent manner and also induced transactivation of Gal4-Elk-1, suggesting that Elk-1 is involved in SRE-mediated egr-1 transcription. Transient transfection of dominant-negative constructs of MAPK pathways blocked ANX-induced Pegr-1 activity. Furthermore, pretreatment with specific MAPK pathway inhibitors, including the MEK inhibitor U0126, the JNK inhibitor SP600125, and the p38 kinase inhibitor SB202190, completely inhibited ANX-inducible expression of Egr-1. Taken together, these results suggest that all three MAPK pathways play a crucial role in ANX-induced transcriptional activation of Pegr-1 through SRE-mediated transactivation of Elk