Objective Improving the existed HPLC methods in order to detect the levels of global DNA methylation rapidly, stably and conveniently. Methods The HP1100 high performance liquid chrom-atographic (HPLC) system was used in this study. The analytic column was Macherey-Nagel (MN) EC 250/4.6 Nucleosil 100-5SA (250mm x 4. 6mm, 5u,m) cation exchange chromatographic column. We used 60mM acetic acid + 15% acetonitrile as mobile phase (adjust to pH =4. 6 by NaOH). The flow rate was 1. 0 ml/min, detective UV wavelength was set to 276 nm and column temperature was set to 28℃. The injection volume was 50μl. The global DNA methylation was expressed as 5mdC/(dC +5mdC) x 100%. Results Under these conditions, we can isolate dC and 5mdC completely in ten minutes. The level of leukocyte global DNA methylation in healthy people is (4. 389 ±0. 0159) %. Conclusions This method can determine the levels of global DNA methylation rapidly, and it can be widely applied in some laboratories.

Objective To study the effects of keto-valine-calcium and keto-isoleucine-calcium on(human mesangial cells)HMCs. Method HMCs were stimulated by keto-valine-calcium and keto-isoleucine-calcium. The AT1R and TGF-β1 were detected. MTr method was used to measure the proliferation of HMCs, and cell cycle was studied by flow cytometry. Results The expression of AT1R and TGF-β1was increased in the experiment groups compared with negative and DMSO control groups. Cell cycle G1 attest and cell apoptosis were observed in the experiment groups. Conclusions 10mM keto-valine-calcium and keto-isoleueine-calcium have multiple effects on HMCs in vitro, which not only increased the expression of AT1R,but also the expression of TGF-β1.Furthermore,keto-valine-calcium and keto-isoleucine-calcium can induce cell cycle G1 arrest and cell apoptosis.

BACKGROUND: It has confirmed that angiotensin converting enzyme inhibitor benazepril can delay fibrosis of varied organs. However, whether benazepril has inhabited effect on peritoneal fibrosis in the process of peritoneal dialysis is poorly understood. OBJECTIVE: It is assumed that benazepril could inhabit peritoneal fibrosis of peritoneum with peritoneal dialysis, in addition, to compare the effect to other mehods. METHODS: All rats were randomly and evenly divided into 4 groups. There was no intervention in the control group; saline solution, and 20 mL 42.5 g/L Dianeal solution, was injected into rats in the saline solution and peritoneal dialysis groups; in the combination group, 20 mL 42.5 g/L Dianeal solution was injected combined with oral taken benazepril 20 mg/(kg·d). The intraperitoneal injection performed once a day, for 4 successive weeks. The ultrafiltration function was performed 4 weeks later. Meantime, Paraffin sections were cut and stained by Van Gieson to measure peritoneal thickness. RESULTS AND CONCLUSION: Two rats in the peritoneal dialysis group and 1 rat in the combination group were dead. The remained 37 rats were included in the final analysis. Compared to the control and saline solution groups, the ultrafiltration volume of the peritoneal dialysis and combination groups were obviously decreased (P_(all)< 0.05), especially notably decreased in the combination group (P< 0.05). Compared to the control group end saline solution groups, the peritoneal thickness was significantly elevated in the combination group, but not as much as in the peritoneal dialysis group (P < 0.05). In the long-term peritoneal dialysis rats, administration of benazepril can effectively protect the ultrofiltration function of peritoneum and delay the progression of peritoneal fibrosis.

It had been proved by many evidences that several heat shock proteins (HSPs) expression is up-regulated in tissue-derived primary lung cancer, and HSPs may play important roles in development, in resistant to drugs and in prognosis of lung cancer. However, there have not still systemic research on which HSPs,especially HSP70 can be or not thought as a new biological target in the therapy to lung cancer. In order to address the expression and roles of heat shock protein 70 (HSP70) in lung adenocarcinoma, immunoblotting was performed to detect the expression of HSP70 in tissue specimens from lung adenocarcinoma which were diagnosed unambiguously by branch fibromicroscopy and were excised. It showed that in the normal lung tissues, the expression of HSP70 was less then that in cancer tissues. After down-regulation of HSP70 protein by HSP70 anti-sense oligonucleotides in A549 cell line, MTT assay showed that the proliferation of A549 cells was inhibited remarkably after the treatment with HSP70 antisense oligonucleotides and Act D. There had significant differential in HSP70 antisense treatment group followed by Act D treatment and Act D treatment group. Results of Hoechst33258 staining revealed that HSP70 antisense oligonucleotides could promote Act D-mediated apoptosis in A549 cells with a higher percentage of apoptotic cells (26.91?3.73)% than that of Act D-treated group (16.83?3.41)% (P

Objective To evaluate the nephrotoxicity induced by radiographic contrast media with different osmolality in rats with hypercholesterolemia, and to explore the protective effect of pentoxifylline. Methods Forty-eight healthy SD male rats were randomly divided into normal dietary group (NN, n =8) and high cholesterol supplemented dietary group (H, 4% cholesterol and 1% cholic acid, n=40). At the end of 8th week, the rats with high cholesterol diet were randomly divided into five subgroups (n=8, respectively): high cholesterol diet group(HN), high cholesterol plus iso-osmolar contrast media (iodixanol, IOCM) group (HI), high cholesterol plus low-osmolar contrast media (iohexle, LOCM) group (HL), high cholesterol diet plus high-osmolar contrast media (diatrizoate, HOCM) group (HH) and high cholesterol plus HOCM plus pentoxifylline group (HHP). Forty-eight hours after contrast media injection, the rats were executed and blood samples were prepared to determine total cholesterol, triglyceride, serum creatinine, creatinine clearance(Ccr), fractional excretion of sodium and potassium(FeNa, FeK), and angtension Ⅱ (Ang Ⅱ) levels. The renal injury was assessed by HE staining and TUNEL staining, respectively. The expression of NF-κB protein in the renal tissue was detected by using immunohistocbemical method. Results An increase of cholesterol was observed in all the rats with high cholesterol diet. Scr, FeNa%, FeK% and Ang Ⅱ levels of rats in HH group were obviously higher than those in HL and HI groups respectively. Ccr in HH group [(0.11±0.02) ml·min-1·(100 g)-1] was significantly lower than that in HHP group [(0.43±0.03) ml·min-1·(100 g)-1], HL group [(0.25±0.02) ml·min-1·(100 g)-1] or HI group [(0.27±0.03) ml·min-1,(100 g)-1] (P<0.05). TUNEL staining showed that the pewentage of apoptotic cells in HH group [(89.60±6.40)% ] was higher than that of the other groups [NN (2.40±0.77)%, HN (5.60±1.08)%, HHP (8.91±1.44)%, HL (63.34±11.97)% and HI (61.50±9.40)%]. Immunohistochemistry staining showed that the average gray value of NF-κB positive cells in HH group decreased (P<0.05). There were no significant differences of all indices between HL and HI groups (P0.05). Conclusions Contrast media can cause kidney injuries in the rats with hypercholesterolemia. PTX can protect the renal tissue from nephrotoxicity induced by HOCM in hypercholesterolemia.

Objective To detect EPPB41L3 methylation frequency difference between esophageal squamous cell carcinoma (ESCC) tissues and the normal tissues and between ESCC patients′plasma and healthy volenteers′plasma, and to analyze the correlation with clinicopathological parameters. Methods We collected esophageal squamous cell carcinoma tissues (n = 42 patients) and adjacent surrounding normal tissues (n = 42 patients), and plasma from 42 patients with ESCC and from 50 healthy individuals. We used methylation specific PCR (MSP) combined with agarose gel electrophoresis to detect the methylation status of the EPB41L3. We used the SPSS 13.0 software for statistical analysis by χ2 test and Fisher′s exact test. Results EPB41L3 frequency of methylation was significantly higher in tumor tissues than in the adjacent tissues (59.5% vs. 4.8%), the difference was statistically significant (χ2 = 28.873, P < 0.001). For plasma, EPB41L3 methylation frequency was 31.0%in cancer patients, while was not detectable in the healthy volunteers. Methylation of EPB41L3 in tissues was more frequently found in patients with tumor size of ≥ 5 cm or T3 than in patients with tumor size of < 5 cm or T1-2. Conclusions The methylation frequency of EPB41L3 is higher in ESCC tissues than in control normal tissues, and higer in plasma from ESCC patients than that from the healthy volunteers. EPB41L3 methylation is more frequently found in patients with more advanced disease.

Objective To investigate the role of PI3K/Akt signaling in the regulation of epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) in peritoneal dialysis in vitro and in vivo.Methods The level of Phosphorylated serine/threonine kinase Akt and the expression of EMT associated gene and protein,including ZO-1,Vimentin and FN,were measured in mice EMT model.In vitro study,phosphorylation level and nuclear translocation of Akt,ZO-1 and Vimentin expression induced by TGF-β1 in human peritoneal mesothelial cells (HPMCs) were also observed.Moreover,HPMCs were pre-treated by one of PI3K/Akt inhibitor,LY294002,or transfected with dominant-negative Akt plasmid to inhibit PI3K/Akt signaling,then analyzed its effect on Zo-1 and Vimentin expression induced by TGF-β1.Results Compared with the control,thickened parietal peritoneum and remarkable decrease in mRNA and protein of the epithelial marker ZO-1,and notable increased in the expression of mesenchymal markers Vimentin and FN were observed in PD mice (all P ＜ 0.01).Moreover,the phosphorylation of Akt also significantly increased under above condition (P ＜ 0.01).In vitro study,with the stimulation of TGF-β1,the expression of Zo-l was down-regulated,while the expression of Vimentin increased (all P ＜ 0.01).In addition,TGF-β1 remarkably increased pAkt in HPMCs (all P ＜ 0.01) in dose-dependent.However,LY294002 and DN-Akt dramatically inhibited the vimentin expression in HPMCs induced by TGF-β1 after inhibition of pAkt.On the other hand,the expression of ZO-1 also was restored (P ＜ 0.01).Conclusion PI3K/Akt signaling is involved in EMT of peritoneal mesothelial cells in peritoneal dialysis,and may be a new target for the prevention and treatment of peritoneal fibrosis during PD.

Objective To investigate the role of microRNA-129-5p (miR-129-5p) in the regulation of epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) isolated from peritoneal dialysate effluents and TGF-β1 induced HPMCs line.Methods The isolated cells were cultured from peritoneal dialysate effluents overnight of 10 patients just started PD and 12 patients with PD over 6 months.Taqman PCR assay was used to determine the expression of miR-129-5p in the HPMCs.Moreover,the expression of miR-129-5p in HPMCs induced by 5 μg/L TGF-β1 for 0-72 h was also detected by Taqman PCR.HPMCs were pre-transfected with miR-129-5p precursor (pre-mir-129-5p) to overexpress miR-129-5p,then incubated with TGF-β1 for 48 h,and the expression of EMT associated gene and protein was detected by real-time PCR,Western blotting and immunofluorescence,respectively.Furthermore,the effect of TGF-β1 on the expression of Smad interacting protein-1 (SIP1) and the regulation of pre-miR-129-5p on the SIP1 expression also were investigated.Results MiR-129-5p expression significantly down-regulated in the HPMCs isolated from PD patients over 6 months than from PD start patients(P ＜ 0.01).Similarly,TGF-β1 remarkably decreased miR-129-5p in HPMCs lines on time-dependent manner (P ＜ 0.01).Pre-mir-129-5p dramatically restored the expression of epithelial marker E-cadherin,while inhibited the expression of Vimentin,a mesenchymal marker,in HPMCs induced by TGF-β1 (all P ＜ 0.01).In addition,TGF-β1 increased SIP1 expression in HPMCs time dependently,while the high level of SIP1 protein was obviously repressed after transfected of pre-miR-129-5p (P ＜ 0.01),but there was no obvious change of its mRNA expression.Conclusion MiR-129-5p modulates EMT formation of HPMCs in PD process,possibly by posttranscriptional inhibition of SIP1.Targeting miR-129-5p/SIP1 may provide a new approach for the prevention and treatment of peritoneal fibrosis during PD.

Objective To investigate the role of oxidative stress in the epithelial-to-mesenchymal transdifferentiation (EMT) of peritoneal mesothelial cells in rat model of peritoneal fibrosis and the effect of probucol on peritoneal fibrosis. Methods The rat model of peritoneal fibrosis was induced by 4.25% high glucose peritoneal dialysis fluid (PDF). The rats were randomly divided into 4 groups:the control group, the saline group, the peritoneal fibrosis group, and the probucol group. A 4 hour peritoneal equilibration test (PET) was performed 4 weeks later. The peritoneal function and net ultrafiltration (UF) volume were determined. The level of malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in peritoneal tissue were examined. The histology of peritoneal membrane was evaluated by light microscopy. E-cadherin and α-smooth muscle actin (α-SMA) protein expression was evaluated by immunohistochemical method and Western blot.Results The mesothelial cells were detached from peritoneal membrane in peritoneal firbosis rats. Comparing with the control rats, the thickness of visceral peritoneum, the level of MDA, and the-SMA protein expression were increased while the net ultrafiltration volume, the level of GSH-Px and E-cadherin protein expression were decreased in peritoneal firbosis rats. All these changes were reversed in the rats treated with probucol.Conclusion Oxidative stress plays an important role in transdifferentiation of peritoneal mesothelial cell in the peritoneal fibrosis rats. Probucol can improve structure and function of peritoneum, and partially reverse the EMT by reducing the oxidative stress.