Regulation of EMT in lung cancer cells by interaction of Hsp90 and SIRT1 / 国际肿瘤学杂志

Objective:To investigate the interaction between heat shock protein 90 (Hsp90) and silent mating-type information regulation 2 homolog 1 (SIRT1) and evaluate its effect on epithelial-mesenchymal transition (EMT) of lung cancer A549 cells.Methods:EMT model was established by treating lung cancer A549 cells with 5 μg/L transforming growth factor-β1 (TGF-β1), which was used as TGF-β1 group, and the normal lung cancer A549 cells were used as control group. The interaction between Hsp90 and SIRT1 in lung cancer A549 cells was detected by immunocoprecipitation method. The expression of Hsp90 gene was silenced by RNA interference technique, and the cells were divided into TGF-β1 group, TGF-β1+ siRNA-Hsp90-neg group and TGF-β1+ siRNA-Hsp90 group. Transwell invasion assay was used to investigate the effect of the interaction of Hsp90 and SIRT1 on the invasion ability of lung cancer A549 cells. The expressions of Hsp90, SIRT1, E-cadherin and vimentin were detected by Western blotting. The effect of inhibiting Hsp90 expression on the stability of SIRT1 protein and EMT of lung cancer A549 cells was observed.Results:After 48 h induction with TGF-β1, EMT characteristics of lung cancer A549 cells were induced successfully. The relative expression levels of Hsp90 protein in the control group and TGF-β1 group were 0.45±0.05 and 1.31±0.06, respectively, the relative expression levels of SIRT1 protein were 0.29±0.04 and 0.95±0.08, respectively, and there were statistically signigicant differences ( t=10.98, P=0.018; t=7.39, P=0.028). The results of immunocoprecipitation showed that there was an interaction between Hsp90 and SIRT1 protein in lung cancer A549 cells. The relative expression levels of Hsp90 in the TGF-β1 group, TGF-β1+ siRNA-Hsp90-neg group and TGF-β1+ siRNA-Hsp90 group were 0.75±0.07, 0.63±0.06 and 0.23±0.05, respectively, and there was a statistically significant difference ( F=18.85, P=0.012). The relative expression levels of SIRT1 in the above three groups were 0.99±0.08, 0.97±0.12 and 0.35±0.05, respectively, and there was a statistically significant difference ( F=16.52, P=0.014). The expression levels of Hsp90 and SIRT1 in the TGF-β1+ siRNA-Hsp90 group were significantly lower than those in the TGF-β1 group ( P=0.019, P=0.016). The numbers of cells passing Matrigel in the above three groups were 378.13±27.70, 323.52±19.82 and 142.51±22.54, respectively, and there was a statistically significant difference ( F=27.35, P=0.022). The number of cells passing Matrigel in the TGF-β1+ siRNA-Hsp90 group was significantly less than that in the TGF-β1 group ( P=0.028). The relative expression levels of E-cadherin in the above three groups were 0.31±0.02, 0.34±0.04 and 0.63±0.05, respectively, and there was a statistically significant difference ( F=19.39, P=0.031). The relative expression levels of vimentin in the above three groups were 0.33±0.02, 0.27±0.05 and 0.09±0.03, respectively, and there was a statistically significant difference ( F=12.58, P=0.012). The expression level of E-cadherin in the TGF-β1+ siRNA-Hsp90 group was significantly higher than that in the TGF-β1 group ( P=0.017), while the expression level of vimentin was significantly lower than that in the TGF-β1 group ( P=0.023). Conclusion:Hsp90 interacts with SIRT1, and Hsp90 inhibition can lead to the decrease of SIRT1 protein level. Hsp90 may play a role of molecular chaperone to maintain the conformation stability of SIRT1, and the interaction between Hsp90 and SIRT1 may be one of the molecular mechanisms for the occurrence of EMT and the enhancement of invasion ability of lung cancer A549 cells.

Effect of fluoride on the expression of 5-methylcytosine in blood, liver, kidney and brain of rats / 中华地方病学杂志

Objective To investigate the effects of fluoride exposure dose and exposure time on the expression of 5-methylcytosine (5-mC) in blood,liver,kidney and brain of rats;and to understand whether there is a difference in the effects of fluoride on DNA methylation levels in different tissues.Methods Eighty three-week old SPF male Wistar rats were randomly divided into four groups according to body weight [(82.34 ± 10.60) g],with 20 rats in each group.The rats of control group drank distilled water and the fluoride group's drank distilled water containing 25,50 and 100 mg/L of F ion,respectively.Rats were sacrificed after fed for 1 month and 3 months (n =10),and peripheral blood and tissue samples were collected.The incidence of dental fluorosis was observed in rats.Bone and urine fluoride content was detected by ion selective electrode method.The content of 5-mC in blood,liver,kidney and brain was detected by enzyme-linked immunosorbent assay (ELISA).The independent and interactive effects of fluoride exposure dose and exposure time on 5-mC in rat peripheral blood and different tissues were analyzed by factorial design anova.Results After feeding for 1 month and 3 months,all rats in the fluoride group had dental fluorosis with different severities,while none dental fluorosis was found in the control groups.Fluoride exposure dose and exposure time had a main effect on bone fluoride contents [1 month:(324.985 + 127.094),(846.148 ± 331.861),(1 886.601 + 250.140),(2 420.971 + 135.883) mg/kg;3 months:(417.591 ± 88.324),(1 582.243 ± 347.975),(2 163.519 ± 614.932),(2 755.434 ± 265.370)mg/kg;F =96.692,13.077,P ＜ 0.01],respectively,but there was no interaction effect (F =2.013,P ＞ 0.05);fluoride exposure dose had a main effect on urinary fluoride contents (F =62.358,P ＜ 0.01),the exposure time had no effect on it (F =0.862,P ＞ 0.05),and there was no interaction effect (F =0.081,P ＞ 0.05).Fluoride exposure dose had a main effect on the 5-mC content in the blood (F =8.446,P ＜ 0.01),the exposure time had no effect on it (F =0.095,P ＞0.05),and there had an interaction effect (F =4.676,P ＜ 0.01).Fluoride exposure dose and exposure time had a main effect on the 5-mC content in the liver,respectively (F =4.737,7.064,P ＜ 0.01 or ＜ 0.05),and an interaction effect was exist (F =8.302,P ＜ 0.01).Fluoride exposure time had a main effect on the 5-mC content in the kidney (F =6.340,P ＜ 0.05),the exposure dose had no effect on it (F =0.140,P ＞ 0.05),and there was no interaction effect (F =1.269,P ＞ 0.05).Fluoride exposure dose and exposure time had no effect on 5-mC content in the brain (F =0.633,2.065,P ＞ 0.05).Conclusion Fluoride exposure dose and exposure time have the different effect on the levels of 5-mC in blood,liver,kidney and brain,suggesting that there may be differences in the effects of fluoride on DNA methylation levels in different tissues.

Effects of fluoride on oxidative damage of protein in rat plasma / 中华地方病学杂志

Objective To investigate the effects of fluoride on protein oxidative damage in rat plasma by measuring oxidative stress levels,advanced glycation end products (AGEs) and advanced oxidation protein products (AOPP).Methods Eighty SPF male 3-week-old Wistar rats weighing (82.34 ± 10.60) g were randomly divided into 4 groups,20 rats in each group.The control group drank distilled water,and the fluoride groups drank distilled water with fluoride concentrations of 25,50 and 100 mg/L,respectively.Rats were allowed to eat and drink freely,and they were sacrificed at 1 month and 3 month,respectively,and samples such as urine,femur and peripheral blood were collected for experiments.Fluoride contents in urine and bone were detected by ion selective electrode method,the superoxide dismutase (SOD) activity was detected by hydroxylamine method,malondialdehyde (MDA) content was detected by thiobarbituric acid (TBA) method,and AGEs and AOPP contents were detected by enzyme linked immunosorbent assay (ELISA).Results For 1 month and 3 months,compared urinary fluoride contents (mg/L:2.088 + 0.638,9.170 ± 2.865,20.094 ± 8.186,54.866 ± 2.866;2.202 ± 1.282,9.112 ± 2.364,21.854 ±8.325,52.513 ± 16.211),and bone fluoride contents (mg/kg:324.985 ± 127.094,846.148 ± 331.861,1 886.601 ±250.140,2 420.971 ± 135.883;417.591 ± 88.324,1 582.243 ± 347.975,2 163.519 ± 614.932,2 755.434 ±265.370) in control group and fluoride concentrations of 25,50 and 100 mg/L groups,the differences were statistically significant (F =88.379,29.225;87.440,33.998,P ＜ 0.05).For 1 month and 3 months,compared SOD activity (U/ml:32.469 ± 5.674,35.931 ± 2.262,36.746 ± 3.994,38.042 ± 4.632;31.027 ± 4.147,30.777 ±4.791,34.148 ± 1.755,36.585 ± 2.860) and AGEs contents (μg/L:26.977 ± 5.285,33.303 ± 6.226,28.021 ±5.946,34.117 ± 6.706;35.681 ± 3.802,33.651 ± 7.214,28.114 ± 4.660,24.330 ± 3.581) in control group and fluoride concentrations of 25,50 and 100 mg/L groups,the differences were statistically significant (F =2.896,5.780;3.565,10.195,P ＜ 0.05).By factorial design anova,there was an interaction between the exposure concentration and exposure time of fluorine and the content of AGEs (F =8.957,P ＜ 0.01).Conclusion Excessive fluoride can affect urinary,bone fluoride contents,SOD activity,AGEs content,suggesting that excessive fluoride may regulate protein expression through direct and indirect oxidative damage pathways,which leading to fluorosis.

The effect of fluoride on osteoclast in bone tissue of rats and its mechanism / 中华地方病学杂志

Objective To investigate the effect of fluoride on osteoclast in bone tissue of rats and its mechanism.Methods Twenty specific pathogen free male Wistar rats aged 3 weeks were randomly divided into two groups by weight (each group has 10).The rats of control group drink distilled water and treatment group drink distilled water containing 100 mg/L fluoride.The rats were fed for 3 month.The dental fluorosis in rats was observed.The ion selective electrode method was used to measure bone fluoride accumulation.The pathological changes of bone tissue in rats were observed under light microscope.The osteoclast was identified by tartrateresistant acid phosphatase (TRAP) staining.The calcineurin (CaN) activity of serum was measured by detection of free phosphate with malachite green.The bicinchoninic acid (BCA) method was used to detect total protein concentration of serum.The colorimetry method was used to detect calcium and malondialdehyde (MDA) levels in serum.The enzyme linked immunosorbent assay (ELISA) method was used to detect calmodulin (CaM) content.Results By the end of the experiment,none dental fluorosis was detected in control group,all rats in fluoride group had dental fluorosis.The bone fluoride content of rats in fluoride group [(4 460.671 ± 418.548) mg/kg] was about 7.6 times higher than that in control group [(582.534 ± 58.342) mg/kg,t =-29.020,P ＜ 0.01].Compared with the control group,the bone tissue of rats in fluoride group showed thicker bone trabecular,sclerotin fusion and incomplete mineralization.Positive signal intensity of TRAP staining of bone tissue in fluoride group was significantly higher than that in control group.The number of osteoclast formation in fluoride group [10 (5-12)] was significantly higher than that in control group [3 (2-4);U =92.5,P ＜ 0.01].CaN activity in serum of rats in fluoride group [(3.334 ± 0.654) nmol/mg prot] was significantly higher than that in control group [(1.289 ± 0.361) nmol/mg prot;t =-6.346,P ＜ 0.01].The Ca and CaM content of serum in rats were not significantly different between the two groups.However MDA content in fluoride group [(7.703 ± 2.954) μmol/L] was significantly higher than that in control group [(3.958 ± 1.965) μmol/L,t =-2.968,P ＜ 0.05].Conclusion Excessive fluoride may increase osteoclast formation in bone tissue of rats,and the mechanism might be fluoride stimulated CaN activity through oxidative stress pathway.

Clinical significance of monitoring leptin level in adolescent patients with polycystic ovary syndrome / 中国综合临床

Objective To discuss the relationship between leptin level and polycystic ovary syndrome (PCOS) in adolescent patients with polycystic ovary syndrome, and to explore the classification diagnosis method of adolescent PCOS and indicator for clinical monitoring of obese patients. Methods All enrolled adolescent individuals were assigned into four groups: 30 normal adolescent individuals in the control group, 30 simple adolescent obese individuals in the simple obesity group,27 obese adolescent PCOS patients in the obese PCOS group and 14 nonobese adolescent PCOS patients in the nonobese PCOS group. The fasting serum samples were prepared for leptin level measurement and analysis, Results The serum leptin level of in the control group, the simple obesity group, the obese PCOS group and the nonobese PCOS group were ( 19.44 ± 6. 63 ) μg/L vs.(23.09 ±7. 39) μg/L, (42. 99 ±9. 83) μg/L and (31, 92 ±7, 02) μg/L,respectively. Leptin in the obese PCOS group was significantly higher than that in the control group and the simple obese group (t = 2. 903 and 2. 714 respectively,Ps ＜ 0. 05 ). Conclusion Monitoring the serum level of leptin can not only aid the classification of adolescent PCOS patients and guide the treatment, but also can serve as a indicator for therapeutic monitoring of obese adolescent PCOS.

Effects of using distal protection device on neuroendocrine in patients with acute myocardial infarction / 中华急诊医学杂志

Objective To study changes of neuroendocrine in patients with ST segment elevation acute myocardial infarction (STEMI) after using distal protection device (GuardWire PlusTM). Method Seventy patients with STEMI received percutaneous coronary intervention (PCI) in Municipal Hospital Qingdao, during September 2004 to December 2006. They were randomdy (random numbs) enrolled in this prospective and control study. All the patients were divided into 2 groups: the distal protection device group (GW) and the non-distal protection device group (NGW).The inclusion criteria were:onset within 6 hours, chest pain more than 30 minutes without response to nitroglycerin, two or more adjacent ST segnents elevated over 0.2 mv,the proximal or middle diameter of infarction artery over 3 mm, and the increased plasma creatine kinase. The exclusion criteria were fluctuation in hemodynamics, severe heart failure, arteriopathy of left main coronary artery, mechanical complications of acute myocardial infarction and multi-vessel disease scheduled for coronary artery bypass. The plasma levels of endothelin(ET) , plasma renin activity (PRA),aldosterone (ALD),angiotensin Ⅱ (Ang Ⅱ), norepinephrine (NE) and epinephrine (E) were measured on the day of operation and on the 1st,2nd,3rd and 5th day after operation, respectively. The t-test was used to compare those neuroendocrine elements between two groups. Results There were no differences in plasma levels of all the neuroendocrine elements between two groups before operation. Compared with the NGW group, the levels of neuroendocrine elements in the plasma rapidly decreased in the GW group at 1 d after the operation ( P ＜ 0.05). Conclusions In patients with ST segment elevation acute myocardial infarction, the distal protection device can decrease the changes in neuroendocrine.

Study on neuroendocrine change in patients with ST segment elevation in acute myocardial infarction using thrombectomy / 介入放射学杂志

Objective To study neuroendocrine change and clinical value of percutaneous thrombectomy system(GuardWire PlusTM)in treatment of patients with ST-elevation acute myocardial infarction(STEMI). Methods 72 patients with STEMI underwent percutaneous coronary intervention(PCI)were divided into A group(38 patients)with direct stent placement after thrombectomy and B group(34 patients) with primary PCI. The plasma levels of ET, PRA, ALD, AngⅡ, NE, E were measured on the day of operation and the first, second, third and fifth days after PCI. Left ventricular ejection fraction(LVEF) was measured by echocardiography at one week and three months after PCI. Results The stents were successfully implanted in two groups. All the neuroendocrine factors have no difference between the two groups before operation. The first and second day after PCI, the levels of ET, PRA, ALD, AngⅡand E were significantly lower in A gronp than those in B group(P 0.05). Conclusions Deteriorated neuroendocrine changes are significantly improved with thrombectomy, providing potential benefits on heart function.

Clinical observation of distal protection device during primary angioplasty in acute myocardial infarction / 中国介入心脏病学杂志

Objective To evaluate the safety and efficacy of distal protection device(GuardWire PlusTM) during high risk PCI in patients with acute myocardial infarction(AMI).Methods Seventy-two patients with AMI admitted from September 2004 to May 2006 who received PCI were categotized into the GuardWire PlusTM group(GW group,n=38) and the conventional guidewire group(NGW group,n=34) according to the device used.The basic clinical characteristics,angiographic results,degree ST of resolution and changes in serum CK-MB and cTnI levels were compared.LVEF was measured by echocardiography at discharge and again at 3 months after PCI.Results All the distal protection deveices were applied successfully in the GW group.A greater percentage of patients in the GW group had post procedural ST-segment resolution ≥50% compared with the NGW group(68.4% vs 41.2%,P