1.Rosiglitazone Inhibitory Effect on Mesangial Cell Proliferation and Extracellular Matrix Expression Induced by Angiotensin Ⅱ
Journal of Applied Clinical Pediatrics 2006;0(23):-
ObjectiveTo investigate the inhibitory effect of peroxisome proliferator-activated receptor-?(PPAR?) agonist on mesangial cell(MC) proliferation and extracellular matrix expression induced by angiotensin Ⅱ(Ang Ⅱ).MethodsThe incorporation of 3H-thymidine(3H-TdR) and cell count were used as the measurement of MC proliferation.MC cell-cycle was analyzed by flow cytometry.Mouse primary MC was treated with various concentration of Ang Ⅱ(1,10,100 nmol/L) in the presence or Absence of N-acytosistin(NAC) or rosiglitazone.Transforming growth factor-?1(TGF-?1),plasminogen activator inhibitor-1(PAI-1),and fibronectin(FN) mRNA expression were determined by real time-PCR.Reactive oxygen species(ROS) production was measured by 2,7-dichlorofluorescein diacetate(DCFDA) fluorescence.Results1.One hundred nmol/L Ang Ⅱ increased 3H-TdR incorporation and cell number by 2.14 and 2.32 fold,respectively.Ang Ⅱ-induced MC proliferation was inhibited by PPAR? agonist rosiglitazone with dose-dependent manner in mouse MC.2.One hundred nmol/L Ang Ⅱ stimulation for 24 h induced 48% MC processed to S and G2/M phase.Rosiglitazone significantly blocked Ang Ⅱ increased cell number in S and G2/M phase.3.Rosiglitazone reduced Ang Ⅱ-induced TGF-?1,PAI-1,and FN mRNA expression with dose-dependent manner.4.One hundred nmol/L Ang Ⅱ stimulation for 60 min increased ROS production by 3.85 folds.Rosiglitazone significantly inhibited Ang Ⅱ-induced ROS production.Ten ?mol/L rosiglitazone almost completely blocked Ang Ⅱ-induced ROS production.ConclusionPPAR? agonist rosiglitazone could block Ang Ⅱ-induced MC proliferation and extracellular matrix expression via inhibition of ROS production.
3.Effects of sodium arsenite on hypermethylation, transcription and expression of O6-methylguanine-DNA methyltransferase gene in HaCaT cells
Chinese Journal of Endemiology 2011;30(3):273-278
Objective To investigate the DNA methylation feature and DNA methylation regulation to its transcription and expression of O6-methylguanine-DNA methyltransferase gene (MGMT) in NaAsO2-treated HaCaT cells. Methods HaCaT cells were treated 72 hours at intervals and repeatedly by 3.13, 6.25,12.50, and 25.00 μmol/L NaAsO2, MGMT gene promoter region was amplified in the transcription initiation site - 329 - + 93 region by bisulfate-sequencing polymerase chain reaction (BSP), the mRNA transcription and the protein expression of MGMT was detected by real-time quantitative PCR and Western blotting. NaAsO2-untreated HaCaT cell was set as a blank control, and human epidermal squamous carcinoma cell strain A431 was set as a positive control. Results Among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, the positive rates of the DNA methylation of promoter region in MGMT gene were 0.63%(l/160), 6.25% (10/160), 10.63%( 17/160) and 18.75% (30/160), respectively, and methylated CpG sites were mainly located in - 249--146 region relative to transcription start site. There was no DNA methylation in the blank control. There were significant differences between the blank control and the NaAsO2-treated cells (x2 = 76.687, P< 0.05). Average levels of MGMT mRNA were 1.518 31 ± 0.180 54, 1.425 22 ± 0.180 39, 1.014 54 ± 0.096 79 and 0.887 72 ± 0.020 00, respectively among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, compared with the blank control cells(1.198 29 ± 0.159 97), there were significant differences(F = 37.359, P < 0.05). Average levels of MGMT protein were 1.174 47 ± 0.064 75, 0.848 83 ± 0.057 01, 0.471 63 ± 0.023 34 and 0.240 34 ± 0.014 43, respectively among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, compared with the blank control cells (1.066 19 ± 0.061 24), there were significant differences(F = 20.687, P < 0.05). Conclusions Arsenic can cause CpC island hypermethylation in the promoter region of MGMT gene, which results in inhibited MGMT mRNA transcription and protein expression. It might be one of the important mechanisms of arsenic-induced skin lesion.
4.Study on the expression of Dickkopf-3 protein in diabetic retinopathy
Shu-Yan, LI ; Lei, ZHANG ; Ai-Hua, ZHANG
International Eye Science 2016;16(10):1891-1893
AIM:To observe the effects of Dickkopf-3 ( Dkk-3 ) in diabetic retinopathy ( DR ) circulating blood in patients with the expression level, the Dkk - 3 development changes in the diabetic retinopathy of significance in the diagnosis of early DR.
● METHODS: Eighty - five type 2 diabetic patients, included the non - proliferative diabetic retinopathy ( NPDR ) 23 patients, proliferative diabetic retinopathy, proliferative DR ( PDR ) in patients with 30 and non-diabetic retinopathy ( NDR ) with 32 cases. The same period of healthy physical examination was selected as control group ( 80 cases ) . Serum samples were collected, and the relative expression level of Dkk-3 was detected by enzyme - linked immunosorbent assay ( ELlSA) double antibody sandwich assay. The statistical differences were compared between groups.
●RESULTS: The plasma level of Dkk - 3 ( 430. 16 ± 198. 11pg/mL) in DR patients was significantly lower than that in healthy control group (627. 48±294. 45 pg/mL; P<0. 05 ) and NDR patients ( 601. 99 ± 194. 16 pg/mL; P<0. 05). While there was no significant difference in Dkk-3 level between NDR and healthy control group ( P =0. 729). The level of PDR in patients with Dkk-3 (396. 38± 185. 59 pg/mL) was lower than that of NPDR (538. 82 ± 187. 20 pg/mL;P=0. 002).
●CONCLUSION:The decrease of Dkk-3 level may be related to the occurrence and development of diabetic retinopathy, and there is a significant correlation with PDR. Circulating blood Dkk - 3 protein in diabetic retinopathy has a certain differential efficacy, it is likely to become diabetic retinopathy patients peripheral blood test indicators.
5.The effect of NaAsO2 on the binding of methyl CpG binding protein 2, DNA methyltransferase 1 and histone deacetylase 1 to the promoter of MGMT gene in HaCaT cells
Bo, ZHANG ; Xue-li, PAN ; Ai-hua, ZHANG
Chinese Journal of Endemiology 2013;(1):16-20
Objective To investigate the effect of NaAsO2 on the binding levels of methyl CpG binding protein 2(MeCP2),DNA methyltransferase 1 (DNMT1) and histone deacetylase 1(HDAC1) to the hypermethylation promoter region of MGMT gene in HaCaT cells,in order to provide a basis to deepen the interpretation of the role of arsenic poisoning mechanism.Methods HaCaT cells were treated repeatedly and interval with different concentrations of NaAsO2(3.13,6.25,12.50,25.00 μnol/L,respectively) for 72 h.Untreated HaCaT was used as blank control group and human epidermal squamous carcinoma cell line(A431 cells) as positive control group.The binding levels to the two transcription regulatory regions(ChIP1,ChIP2) and to the coding region(ChIP3) of MGMT 8ene were detected by chromatin immuno-precipitation combined with quantitative PCR.Results The differences of binding levels of MeCP2,DNMT1 and HDAC1 to ChIP1 and ChIP2 in each group were significant (F=7.387,84.634,78.442 and 19.263,69.649,26.546,all P < 0.05).The binding levels of MeCP2,DNMT1 and HDAC1 to ChIP1 and ChIP2 in each NaAsO2 exposed group[3.13 μmol/L NaAsO2 exposed group:(136.00 ±16.97)%,(145.00 ± 2.83)%,(88.50 ± 19.09)% and (106.50 ± 37.48)%,(112.34 ± 8.73)%,(59.71 ± 8.49)%;6.25 μmol/L NaAsO2 exposed group:(130.00 ± 42.43)%,(154.50 ± 4.95)%,(101.00 ± 1.27)% and (88.50 ±3.54)%,(134.32 ± 2.82)%,(102.75 ± 19.91)% ; 12.50 μmol/L NaAsO2 exposed group:(141.50 ± 23.33)%,(161.50 ± 7.78)%,(125.00 ± 11.31)% and (119.50 ± 24.75)%,(171.59 ± 3.54)%,(167.61 ± 10.61)%; 25.00μmol/L NaAsO2 exposed group:(134.50 ± 43.13)%,(472.50+ 50.20)%,(383.50 ± 30.41)% and (180.09 ±12.73)%,(348.50 ± 27.58)%,(158.45 ± 12.02)%] were higher than that in the blank control group[(51.50 ±9.19)%,(82.00 ± 12.73)%,(25.03 ± 2.91)% and (37.02 ± 4.24)%,(91.56 ± 26.16)%,(19.09 ± 2.90)%,all P < 0.05].The differences of binding levels of MeCP2 to ChIP3 in each group were not significant(F =1.670,P >0.05),but the differences of binding levels of DNMT1 and HDAC1 to ChIP3 were significant (F =4.404,9.863,all P < 0.05),and only the binding levels in the 25.00 μmol/L NaAsO2 exposed group [(615.85 ± 29.63)%,(306.09 ± 59.40)%] were higher than that in the blank control group[(99.70 ± 12.02)%,(92.45 ± 48.79)%,all P < 0.05].Conclusions MeCP2 can bind to the methylated MGMT gene transcriptional regulatory regions which are induced by arsenic and leads to histone deacetylation by the recruitment of DNMT1 and HDAC1 and,meanwhile,DNMT1 can bind to the coding region of MGMT gene to recruit HDAC1 in a methyl DNA binding protein(MBD) independence manner and media MGMT gene silencing through the chromatin remodeling way,which might be the early molecular events of arsenic poisoning.
10.The Effects of Exercise- and Food Intake Restriction-induced Weight Loss on the Expression of Ghrelin in Plasma and Stomach of Obese Rats
Wenqian LIU ; Jiangang ZHANG ; Lan XIE ; Hua AI
Chinese Journal of Sports Medicine 2010;(2):184-187,216
Objective To clarify the effects of exercise- and food intake restriction-induced weight reduction on expression of ghrelin in plasma and stomach of obese rats,and to explore the role of ghrelin during weight loss. Methods 100 weaned Sprague-Dawley(SD) rats were randomly divided into control group(fed with standard chow,n= 20) and model group (two-bottle-feeding method,fed with standard chow and high-fat diet simultaneously, n=80). After 20 weeks feeding, Lee' s index of control was used as the reference of diet-induced obesity identification, and then diet-induced obese rats were randomly divided into four groups:obese control group,swimming group,food intake restriction group and swimming+food intake restriction group, eight rats in each group. Rats in swimming group underwent long-term(40 min,6 days/week for 5 weeks)swimming exercise. Calorie in food intake restriction group was restricted 1/3 of normal kcal for 3 weeks,and subsequently restricted 1/3 for 2 weeks. Swimming+food restriction group had the same intervention as swimming and food restriction group. 8 normal diet rats were also chosen as controls. After five weeks of treatments,peripheral fat mass of testis and kidney,ghrelin proteins in plasma and stomach,and ghrelin mRNA in stomach were measured. Results Gastric ghrelin protein and mRNA were significantly lower in obese control group than those in normal control group (P<0.05). However, plasma ghrelin did not decrease markedly in obese control rats(P>0.05). Compared with obese control group,weight and fat mass in all weight loss groups declined notablely(P<0.05);in swimming and swimming+food restriction groups,the expression of ghrelin protein or mRNA in plasma or stomach increased remarkably( P<0.05); in food restriction group, the level of ghrelin mRNA raised significantly (P<0.05), while ghrelin protein in plasma and stomach had not change. Conclusion The level of ghrelin increased in exercise induced weight loss,while showed no significant change in food intake restriction group, suggested that ghrelin might be involved in the process of weight loss induced by exercise.