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.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.
5.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.
8.Transcription and expression of excision repair cross complementing 1 in endemic arsenism caused by coal-burning
Yun, XIAO ; Ai-hua, ZHANG ; Xiao-xin, HUANG
Chinese Journal of Endemiology 2011;30(1):4-8
Objective To study the transcription and expression of excision repair cross complementing 1(ERCC1) in the peripheral blood and the skin tissue in coal-burning borne endemic arsenism, and to explore the role of arsenism in its pathogenic or carcinogenesis mechanism. Methods According to "Endemic arsenism diagnostic criteria" (WS/T 211-2001), 110 arsenism patients were chosen as case group in Xingren county,Guizhou province and they were divided into 3 groups according to their hnir arsenic: < 2(31 cases),2 ~< 4(31 cases),≥4 mg/kg(48 cases), respectively. Another 36 healthy residents about 13 km away from the endemic area were chosen as healthy control group. Under the principle of informed consent, hair samples were collected for arsenic analysis by Ag-DDC and blood samples were collected to determine mRNA expression levels of ERCCI by real-time fluorescence quantitative PCR. At the same time, skin tissue samples were collected from the voluntary surgical treatment of 62 patients with endemic arsenism as case group which were divided into 3 groups according to their hair arsenic: < 2(16 cases), 2 ~< 4(20 cases) and ≥4 mg/kg(26 cases), respectively, and these patients were also divided into general pathological changes (32 cases), precancerous (19 cases) and cancerous groups( 11cases), respectively, according to their skin pathologic diagnosis of skin lesions. Another 13 cases pathologically normal without skin cancer surgery from a certain hospital were chosen as control group. Skin samples were collected to detect the ERCC1 protein by immunohistochemical method. Results The mRNA levels of ERCC1 were 0.7156(0.2158 ~ 1.2405),0.5772(0.0843 ~ 1.1234) and 0.5490(0.1895 ~ 0.8431 ), respectively, among < 2, 2 ~< 4and ≥4 mg/kg groups, which were lower than the mRNA levels of ERCC1 in the control group[1.5128(1.0000 ~2.1295)], and the difference was statistically significant(all P < 0.05). The expression rate of ERCC1 protein were 87.5%, 80.0% and 77.0%, respectively, among < 2, 2 ~< 4 and ≥4 mg/kg groups. The expression rate of ERCC1 protein in 2 ~< 4 and ≥4 mg/kg groups were lower than the rate in the control group(100.0%), and the difference was statistically significant (all P < 0.05). The expression rate of ERCC1 protein were 84.4%, 79.0%and 72.8%, respectively, among general pathological changes, precancerous and cancerous groups compared with the control group( 100.0% ), and the difference was statistically significant(all P < 0.05). Conclusions Arsenic from coal-burning can lead to abnormal ERCC1 gene transcription and protein expression, which may inhibit DNA repair through influencing the removal of damaged DNA and promoting the incidence of arsenism development and even skin carcinogenesis.
9.Relationship between glutathione S-transferase GSTO 1 Glu155 △Glu genetic polymorphism and arsenic poisoning caused by coal-burning
Bing, LIANG ; Ai-hua, ZHANG ; Xue-xin, DONG
Chinese Journal of Endemiology 2012;31(1):20-23
ObjectiveTo investigate glutathione S-transferase GSTO 1 Glu155△Glu genetic polymorphism and risks of arsenic poisoning caused by coal-burning in Guizhou.Methods GSTO1 Glu155 △Glu gene polymorphism was analyzed by polymerase chain reaction-with confronting two-pair primers among one hundred and thirty arsenic poisoning patients and one hundred and thirty healthy controls.The results were verified by DNA sequencing.The association between different genotypes and arsenic poisoning was analyzed by unconditional Logistic regression model.ResultsThe results of Glu/Glu and Glu/△Glu genotype detected by this method were consistent with those of DNA sequencing.The frequencies of GSTO1 Glu/Glu genotype and Glu/△Glu genotype were 94.85%(92/97) and 5.15%(5/97) in the patients,99.15%(117/118) and 0.85%(1/118) in the controls,respectively.The difference was statistically significant(x2 =3.896,P < 0.05).△Glu/△Glu genotype was not found in both patients and controls.After age and sex adjusting,GSTO1 Glu155 △Glu polymorphism was found to be a risk factor of arsenic poisoning [odds ratio (OR) =1.85,95% confidence interval (CI):1.39 - 17.48].ConclusionsThe study finds that GSTO1 Glu 155 △ Glu polymorphism is associated with risk of arsenic poisoning.The relationship between them should be further studied through increasing sample size.
10.Expression of DNA methyltransferase 1 mRNA in patients of endemic arsenism and its clinical significances
Xue-li, PAN ; Ai-hua, ZHANG ; Xiao-xin, HUANG
Chinese Journal of Endemiology 2010;29(1):13-17
Objective To investigate the transcription and expression of DNA methyltransferase 1 (DNMT1) mRNA in endemic arsenism patients by burning coal usage,to probe its effects on the development and carcinogenesis of arsenism. Methods In 2008,68 arsenism patients(including 24 mild cases,28 moderate cases and 16 severe cases) were selected in the areas with endemic arsenism according to Standarding of Diagnosis for Endemic Arsenism from Xingren county,Guizhou province. Among the subjects,40 cases were diagnosed by pathological methods,and they were divided into general pathological changes(20),precancerous(14) and cancerous group(6). Tweleve kilometer away from the endemic arsenism area,23 controls were selected in Daguoduo village (non-arsenism exposure). Under the principle of informed consent,blood samples were collected from individuals. The mRNA expression of DNMTI was detected by real-time quantitative reverse transcription polymerase chain reaction(FQ-PCR). At the same time,skin tissue samples were collected from the voluntary surgical treatment patients with endemic arsenism (total 61 cases,including 34 general pathological changes cases,21 precancerous cases and 6 cancerous cases) and from the control(15 cases). DNMT1 protein was detected by immunohistochemical method.Results Average level of DNMT1 mRNA were 0.221 83±0.595 09,0.246 11±0.509 79 and 0.389 27±0.411 33 respectively among mild,moderate and severe arsenism group. DNMT1 mRNA level of mild and moderate group were obviously lower than the control group(0.695 95±0.463 98,all P < 0.01). The mRNA average level of DNMT1 were 0.320 64±0.547 46,0.313 09±0.529 13 and 0.159 07±0.342 56 individually among general pathological changes,precancerous and cancerous group,which were obviously lower than the control group(0.695 95±0.463 98,all P < 0.05). The expression rates of DNMT1 protein in skin were 88.24%(30/34),100%(21/21) and 100% (6/6) among general pathological changes,precancerous and cancerous group were higher than the control group [0(0/15),all P < 0.01],and the extent of expression gradually increased with the aggravation of skin damage(r,= 0.740,P < 0.01). Conclusions DNMT1 participated in the development of the arsenism. High expression of its protein was an early event during the process of the arsenism. DNMT1 may be the new target markers for early diagnosis and treatment of arsenism.