Objective To evaluate the role of phosphoinositide 3 kinase/protein kinase B/glycogen synthase kinase 3β (PI3K/Akt/GSK-3β) signaling pathway in mitigation of ischemia-reperfusion (I/R) injury by diazoxide postconditioning in isolated rat hearts.Methods Pathogen-free Sprague-Dawley rats were used in the study.Thirty hearts were excised and passively perfused in a Langendorff apparatus with oxygenated K-H solution at 37 ℃.The hearts were randomly divided into 5 groups (n =6 each) using a random number table:control group (group C),I/R group,diazoxide postconditioning group (group DZ),PI3K inhibitor LY294002 group (group LY),and diazoxide postconditioning + LY294002 group (group DZ + LY).In group C,the hearts were continuously perfused with K-H solution for 70 min.In group I/R,the hearts were perfused with cardioplegic solution 4 ℃ ST-Thomas 10 ml/kg,the perfusion pump was then stopped to induce global ischemia,and 40 min later the hearts were perfused with K-H solution for 30 min.In DZ group,5 min of retrograde perfusion with diazoxide 50μmol/L was performed through the aorta starting from the onset of reperfusion.In LY group,5 min of retrograde perfusion with LY294002 15 μnol/L was performed through the aorta starting from the onset of reperfusion.In LY + DZ group,5 min of retrograde perfusion with LY294002 15 μmol/L was performed through the aorta starting from the onset of reperfusion,followed by 5 min of retrograde perfusion with diazoxide 50 μmol/L.At 20 min of stabilization (T1) and 30 min of reperfusion (T2),heart rate (HR),coronary flow (CF),left ventricular developed pressure (LVDP),left ventricular developed pressure (LVEDP) and ± dp/dtmax were measured.The expression of total Akt (t-Akt) and total GSK-3β (t-GSK-3β) and phosphorylation of Akt and GSK-3β in myocardial tissues were determined by Western blot.Results Compared with C group,HR,LVDP and ± dp/dtmax were significantly decreased,and LVEDP was increased at T2 in the other four groups,CF was decreased in I/R,LY and DZ + LY groups,and the phosphorylation of Akt and GSK-3β in myocardial tissues was increased in DZ group.Compared with I/R group,HR,CF,LVDP and ± dp/dtmax were significantly increased,and LVEDP was decreased at T2,and the phosphorylation of Akt and GSK-3β in myocardial tissues was increased in DZ group,and no significant changes were found in the phosphorylation of Akt and GSK-3 β in LY and DZ + LY groups.Compared with DZ group,HR,CF,LVDP and ± dp/dtmax were significantly decreased,LVEDP was increased,and the phosphorylation of Akt and GSK-3β in myocardial tissues was decreased in LY and DZ + LY groups.Conclusion PI3K/Akt/GSK-3β signaling pathway is involved in the mechanism by which diazoxide postconditioning mitigates I/R injury in isolated rat hearts.

OBJECTIVETo determine the interaction between miR-21 and DNA methylation in different breast cancer cells.

METHODSFluorescence tagged miR-21 inhibitor and its negative control (NC) were transient transfected into MCF-7 and MDA-MB-231 cell, the transfection efficiency was observed using fluorescence microscopy, and the miR-21 expression level and genome DNA methylation status before and after transfection were assessed by real-time PCR and bisulfite-qMSP respectively. To investigate the regulation effect of DNA methylation on miR-21, cells were treated with 5-AZA (2.5 µmol/L) for 72 h, with dimethyl sulfoxide (DMSO) treatment as its negative control (NC), and the expression level of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and AKT(also known as Protein Kinase B), two downstream genes of miR-21 were detected by Western blot.

RESULTSThe expression of miR-21 in MCF-7 cell was significantly knocked down (P < 0.01) by miR-21 inhibitor, with the genome DNA methylation level (P < 0.05) and all the three Dnmts: Dnmt1, Dnmt3a, and Dnmt3b unregulated. In contrast, the miR-21 expression in MDA-MB-231 cell was elevated ( P < 0.01) by miR-21 inhibitor, meanwhile, down- regulated of genome DNA methylation (P < 0.05) and Dnmt3b expression, upregulation of Dnmt3a were also observed. In addition, treated with 5-AZA resulted in significant increases of miR-21 expression in both MCF-7 and MDA-MB-231 cells (P < 0.01), with the protein level of PTEN increased in MCF-7 cell, which was further involved in the downregulation of AKT.

CONCLUSIONThe regulation effects of DNA methylation by transient transfection of miR-21 in MCF-7 and MDA-MB-231 cells are almost opposite, whilst the expression of miR-21 in two cell lines were all upregulated by decreased DNA methylation level and our results may provide some experimental evidences for the future development of rational therapy for different breast cancer.

Azacitidine ; Breast Neoplasms ; genetics ; Cell Line, Tumor ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Humans ; MCF-7 Cells ; MicroRNAs ; genetics ; PTEN Phosphohydrolase ; metabolism ; Promoter Regions, Genetic ; Proto-Oncogene Proteins c-akt ; metabolism ; Real-Time Polymerase Chain Reaction ; Up-Regulation

Adolescent ; Adult ; Cyclophosphamide ; adverse effects ; Female ; Femur Head Necrosis ; etiology ; Humans ; Immunosuppressive Agents ; adverse effects ; Male ; Middle Aged ; Paraquat ; poisoning ; Young Adult

AIM:To investigate the effects of estrogen on the expression of matrix metalloproteinases-2(MMP-2), tissue inhibitor of metalloproteinases-2(TIMP-2) and transforming growth factor-β1(TGF-β1) in cultured human corneal stromal cells.METHODS: Inflammatory environments of human corneal stromal cells were simulated by using 1.5ng/mL IL-1β.The cells were then treated with or without different concentrations of estrogen(0, 1×10-4, 1×10-6, 1×10-8, 1×10-10mol/L estradiol)in vitro.Cell viability was evaluated by MTT.Expression levels of MMP-2, TIMP-2 and TGF-β1 proteins were measured by enzyme-linked immunosorbent assay(ELISA).RESULTS:Estrogen did not affect the viability of human corneal stromal cells.Compared with the control group, expression levels of MMP-2 and TGF-β1 proteins in E2 treatment group significantly decreased after being treated with estrogen, while the expression level of TIMP-2 significantly increased.CONCLUSION: Estrogen could, to some extent, down-regulate the expression of MMP-2 and TGF-β1 and up-regulate the expression of TIMP-2, which might contribute to protecting human cornea.

Aim To discuss whether specific mitochon-drial ATP-sensitive potassium channel opener diazoxide ( DZ ) postconditioning activates RISK signaling path-way to protect isolated rat hearts against ischemica reperfusion injury ( IRI ) . Methods Langendorff de-vice was used to establish rat in vitro model of myocar-dial ischemia reperfusion. SD rats were randomly di-vided into normal group ( NOR ) , control group ( CON ) , diazoxide after treatment group ( DZ ) , and LY294002 antagonistic nitrogen Triazine group ( DZ +LY) , with 8 cases in each. The following was com-pared:①whether heart function of each group changed at the end of equilibration and reperfusion; ② at the end of myocardial perfusion and separation, protein was extracted, and protein kinase B ( PKB / Akt ) , P70S6 kinase (P70S6K), endothelial nitric oxide syn-thase ( eNOS) phosphorylation level of expression were analysed by Western blot. Results ① Indicators of changes in heart function: for DZ group at the end of reperfusion , HR , CF , LVDP , LVEDP , +d p/d tmax and -dp/dtmax were significantly better than those in CON group and DZ + LY group ( P <0.01 ) , but worse than those in NOR group ( P <0.01 ); there was no statistical difference in cardioac function at the end of equilibration. ② For DZ group at the end of reperfu-sion Akt, P70S6K, eNOS phosphorylation level of ex-pression were significantly higher than those in NOR group, CON group, and DZ + LY group (P<0.01). There was no difference in expression level of ERK1/2 phosphorylation ( P >0.05 ) . Conclusion Diazoxide postconditioning through the activation of RISK signa-ling pathway can protect isolated rat hearts against is-chemia reperfusion injury.

Objective To explore the simplified method by examining the levels of luteinizing hormone(LH),follicle-stimulating hormone(FSH),estradid(E2) and stosterone(T) at different times in the stimutation test of luteinizing hormone-releasing hormone(LHRH).Methods Sixty patients with precocious puberty accepted LHRH stimutation test.The levels of E2,T,FSH,LH before injection and after 30,60,90 minutes were compaired.The levels of stimutation test of LHRH examined with ACS:180 chemiluminesence.Results There were 39 patients in the group of central precocious puberty(CPP).The levels of FSH and LH in CPP group significantly increased after LHRH 30 minutes injection.The ratio of LH/FSH was higher than 1.The peak level of LH was higher than 12 IU/L.There were 21 patients in group of peripheral precocious puberty(PPP).Compared with the results before injection,the levels of LH were similar to the results of 30,60 and 90 minutes after LHRH injection.Compared with the result before injection,the levels of E2 and T were similar to the result of 60 minutes after LHRH injection.The peak levels of LH and FSH in two groups all focused in 30,60 minutes.Conclusions LHRH stimutation test is mainly based on the peak level of LH and the ratio of LH/FSH,the test can be simplified to examine the levels the of LH and FSH before the test and 30,60 minutes after injection as a basis for the clinical diagnosis.

Objective To evaluate the effect of hypoxic-ischemic time on reduction of hypoxic-ischemic brain injury by sevoflurane postconditioning in neonatal rats.Methods Two hundred and ten 7-day-old Sprague-Dawley rats (105 male,105 female),weighing 13-17 g,were randomly divided into 7groups (n=30 each) using a random number table:sham operation group (group Sham),hypoxia-ischemia group (group HI),and sevoflurane postconditioning at different hypoxic-ischemic time point groups (P0,P3,P6,P 12 and P24 groups).Immediately after ligation of the left common carotid artery,and at 3,6,12 and 24 h after ligation,the rats inhaled the mixed gas containing 2％ sevoflurane for 30 min in P0,P3,P6,P13 and P24 groups,respectively.The fatality was recorded within 7 days after establishment of the model.At 7 days after establishment of the model,the rats were sacrificed,the brains were removed,and the right and left cerebral hemispheres were weighed separately,and the left/right cerebral hemisphere weight ratio was calculated.The hippocampal CA1 region and posterior cingulate gyrus were isolated,and the ratio of density of normal neurons in the left to the right was calculated.Results Compared with group Sham,the left cerebral hemisphere weight,left/right cerebral hemisphere weight ratio,and ratio of density of normal neurons were significantly decreased,and the fatality rate was increased in the other six groups (P＜0.05).Compared with group HI,the left cerebral hemisphere weight,left/right cerebral hemisphere weight ratio,and ratio of density of normal ncurons were significantly increased in P0,P3 and P6 groups (P＜0.05),and no significant change was found in the parameters mentioned above in P12 and P24 groups (P＞0.05).Compared with group P6,the left cerebral hemisphere weight,left/right cerebral hemisphere weight ratio,and ratio of density of normal neurons were significantly increased in P0 and P3 groups (P＜ 0.05).There was no significant difference in the parameters mentioned above between group P0 and group P3 (P＞0.05).Conclusion Sevoflurane postconditioning performed within 6 h of hypoxia-ischemia can reduce hypoxic-ischemic brain injury,and it provides no cerebral protection if exceeding 12 h.

Objective To evaluate the effectiveness offour in onenutrition management mode in the treatment of type 1 diabetes mellitus (T1DM) in children. Methods Ninety children diagnosed with T1DM in Tianjin Children's Hospital were selected during March 2011 to June 2013. Segmented balanced random method was used to generate a random number timer, and the children were divided into intervention group and control group (n=45) by random letters. The patients in the intervention group were treated withFour in one nutrition management. Those in the control group were treated with traditional nutrition education method. Observation period was one year. The change of food choices of the parents was assessed before and after the intervention. Nutritional treatment compliance of children,fasting plasma glucose (FPG),2 hours postpransial glucose (2 hPG),and hemoglobin A1c (HbA1c) levels and complications, and so on were also evaluated. Results One year after intervention, the percentages of parents who chose low glycemic index food (41/45), vegetables and fruits consumed every day (40/45), often ate soy products(41/45), did not drunk carbonated drinks (43/45) of treatment group were significantly higher than those of control group (24/45, 20/45, 19/45, 26/45, P all<0.001);diet treatment compliance of children in treatment group was significantly better than that of the control group (P=0.000 2);FPG, 2 hPG, HbA1c of two groups of children decreased significantly than before. FPG[(8.11 ± 2.82) mmol/L], 2 hPG [(12.82 ± 1.05) mmol/L], HbA1c [(6.10±0.93)%] of treatment group were significantly lower than those of the control group [(11.71± 2.75) mmol/L, (13.77±1.49) mmol/L, (9.02±0.93)%;t=6.13, 1.66, 14.89;P<0.001 for all comparisons]. After treatment, 14 cases of treatment group had acute ketoacidosis and hypoglycemia , while 39 cases of control group had these complications (P=0.000 00). Readmission occurred in 11 cases of treatment group and 33 of the control group (P=0.000 00). Conclusions Four in onenutrition management mode was effective in the management of children with T1DM.

Objective: To evaluate the diagnostic performance of conventional ultrasound, compression elastography (CE) and acoustic radiation force impulse imaging (ARFI) in diff erential diagnosis of benign and malignant breast tumors. Methods: A total of 98 patients with liver lesions were included in the study. The images of conventional ultrasound, CE and the values of virtual touch tissue quantiifcation (VTQ) of breast lesions were obtained. hTe diagnostic performance of conventional ultrasound, CE and ARFI were assessed by using pathology as the gold standard, and then evaluate the diagnosis effciency of these three approaches in differential diagnosing benign and malignant breast tumors. Results: The specificity, sensitivity and accuracy in the diagnosis of malignant breast tumors for conventional ultrasound were 80.0%, 81.1% and 81.7%, respectively, whereas for CE elastic score were 85.7%, 86.7% and 86.3%, respectively. With a cutoff value of 3.71 for the SR, the sensitivity, speciifcity, accuracy in diagnosis of malignant breast tumors were 97.1%, 83.3% and 88.4%, respectively. With a cutoff value of 3.78 m/s for VTQ, the sensitivity, speciifcity, accuracy in diagnosis of malignant breast tumors were 94.3%, 91.7% and 92.6%, respectively. The difference in diagnosis efficiency among ARFI, CE and conventional ultrasound in differential diagnosis of benign and malignant breast tumors was signiifcant (P<0.05). Conclusion: Conventional ultrasound, CE and ARFI are all useful for the differential diagnosis of benign and malignant breast tumors. But the diagnosis effciency of ARFI is superior to CE and conventional ultrasound. The three approaches can help each other in differential diagnosis of benign and malignant breast tumors.

Objective To observe the value of contrast-enhanced ultrasonography (CEUS) in quantitatively evaluating the evolution of the blood supply of rabbit liver VX2 tumor models in early stage. Methods Eighteen liver VX2 tumor models of rabbits were divided into 3 groups randomly (each n=6). CEUS was performed on the 10th, 15th and 20th day group after establishemnt, resepectively. Time intensity curve (TIC) was plotted, the rabbits' liver tumor and surrounding normal liver tissue were resected for immuno-histochemistry and microvessel density (MVD) count. Results CEUS showed that the rabbit liver VX2 tumor present a typical malignant tumor imaging fast-in and fast-out in early stage. TIC showed that the time to peak (TTP) of hepatic tumor is shorter than that of surrounding normal liver tissue in all three groups (P＜0.01), no statistical difference of tumor's TTP was detected among three groups (P>0.05). Absolute enhanced intensity (AEI) and MVD reduced in turn of 10th, 15th and 20th day group. MVD of tumor was higher in 10thday group than surrounding normal liver tissue (P＜0.01), while was equal in 15th day group (P>0.05) and lower in 20th day group (P＜0.01). AEI of liver tumor had positive correlation with MVD (r=0.83, P＜0.05). Conclusion AEI of TIC can be used to quantitatively assess the evolution of the blood supply in rabbit liver VX2 tumor models. The blood supply in early stage of rabbit liver VX2 tumor models shows a gradual descent process from abundant blood supply to medium blood supply and then low blood supply.