Objective · To investigate the expression of the regulator of calcineurin 1 (RCAN1) and calcineurin A (CnA) in tissues of in-stent restenosis after intervention of arteriosclerosis obliterans (ASO), and to explore the relationship between their expression levels and the occurance of in-stent restenosis. Methods · Superficial femoral arterial tissues were collected from 15 ASO patients undergoing lower extremity amputation for in-stent restenosis in Department of Vascular Surgery, The First Affiliated Hospital of Chongqing Medical University from September 2013 to June 2016. H-E staining and Masson staining were performed on the stenosis tissues, as well as on the proximal and distal tissues, and the morphological changes of these tissues were observed under optical microscope. Western blotting was used to detect the protein levels of RCAN1, CnA and proliferating cell nuclear antigen (PCNA). The distribution of RCAN1 and CnA proteins was observed by immunohistochemistry and immunofluorescence methods. In addition, co-immunoprecipitation was used to validate the protein-protein interaction between RCAN1 and CnA in vascular tissues. Results · The expression of RCAN1 in the distal tissues was significantly elevated compared with the proximal tissues and the stenosis tissues (P<0.05). The expression of RCAN1 in the proximal tissues was higher than that in the stenosis tissues (P <0.05). The expression of CnA and PCNA in the stenosis tissues was significantly elevated compared with the proximal tissues and the distal tissues (P<0.05). Immunohistochemistry and immunofluorescence analyses showed that RCAN1 and CN proteins were mainly expressed in the cytoplasm of vascular smooth muscle cells. Co-immunoprecipitation analysis showed there is protein-protein interaction between RCAN1 and CnA in arterial tissues. Conclusion · The low expression of RCAN1 and the high expression of CnA are probably related to the occurrence of in-stent restenosis.

0.05).The median survival time was 32 weeks in group A compared to 27 weeks in group B(P

Objective To evaluate the protecting effects of matrine on chemotherapy related hepatic lesion and its possible mechanism.Methods The positive rate and severity of hepatic lesion were compared between pa- tients being treated with or without matrine during chemotherapy processes.Furthermore,the difference of liver pro- tecting effect of this Chinese medicine between hepatitis B virus(HBV)infection chemotherapy patients and disinfec- tion patients were also analyzed.Results Both the rate of hepatic lesion and level of ALT in matrine treated group were much lower than those in untreated group in chemotherapy patients.The rate of hepatic lesion and level of ALT in HBV infection patients were higher than those in HBV disinfection patients in untreated group,while the signifi- cant difference of these two parameters between HBV infection patients and disinfection patients were disappeared in matrine treated group.Conclusion Matrine has hepatic protecting effect in chemotherapy related liver lesion.

Objective To study the effect of aquaporin 1 on intestinal capillary endothelial barrier in rats with experimental acute necrotizing pancreatitis (ANP).Methods In this study,160 male Sprague-Dawley rats were randomly divided into five groups:Control group ( n =32),ANP group (n =32),NS group,Dexamethasone group,and Acetazolamide group.Eight rats in each group were sacrificed at 3,6,12 and 18 h after induction of experimental models.Volume of ascites and levels of serum amylases were deternined at each time point.Pathological changes in intestine tissues were observed under electron microscope after HE staining.Capillary permeeabilities in intestine tissues were detected by Evans blue (EB) extravasation experiment.The mRNA and protein expressions of AQP1 in intestine tissue were determined by real-time PCR and Western blotting,respectively.Results Serum amylase level in ANP group was significantly higher than that in control group.Amylase level in dexamethasone group was lower than that in ANP group,and amylase level in acetazolamide group was higher than that in ANP group at 12 h (P ＜0.05 ) ; The concentration of EB in intestine tissues at each time point in ANP group was significantly higher than those in control group,and EB in dexamethasone group was lower than those in ANP group at 6,12 and 18 h.EB in acetazolamide group was higher than that in ANP group at 3 h ( P ＜ 0.05 ) ; The mRNA expression of AQPI in ANP group was significantly lower than that in control group.The expression of AQP1 in dexamethasone group was higher than those in ANP group at 6,12 and 18 h,and the expression of AQP1 in acetazolamide group was lower than that in ANP group at 3,6,12 h in intestine tissue ( P ＜ 0.05 ).Protein expression of AQPI in tissues in ANP group was significantly lower than that in control group.The expression of AQP1 in dexamethasone group increased more than that in ANP group at 3,6,12 h,and the expression of AQP1 in acetazolamide group was lower than that in ANP group at 3 h,6 h ( P ＜ 0.05 ).Conclusions The expression of AQP1 is down-regulated in intestine tissue in rats with acute necrotizing pancreatitis,and AQP1 could play an important role in the pathogenesis of capillary endothelial barrier dysfunction.

Salvianolic acid A (Sal A) is one of the most effective compounds isolated from the root of Salvia miltiorrhiza. Up to now, several studies regarding the pharmacokinetic profiles of Sal A have been reported, however there is no such study reported in monkeys, the species which is more similar to human. The aim of this study is to develop a LC-MS method for the determination of Sal A in monkey plasma and apply it to the pharmacokinetic studies of monkeys. After single intravenous administration of Sal A, the plasma concentration-time curves were observed and the main pharmacokinetic parameters were calculated. The plasma concentration at 2 min (C2 (min)) values were (28.343 ± 6.426), (45.679 ± 12.301) and (113.293 ± 24.360) mg x L(-1) for Rhesus monkeys treated with Sal A at 2.5, 5 and 10 mg x kg(-1). The area under the concentration-time curve (AUC(0-∞)) values were (3.316 ± 0.871), (5.754 ± 2.150) and (13.761 ± 2.825) μg x L(-1) x h, respectively. Furthermore, this method was improved and applied to the simultaneous determination of Sal A, Sal B and Sal C, which provided useful information for preclinical studies and clinical trials of Sal A, Sal B and Sal C.
Administration, Intravenous ; Animals ; Caffeic Acids ; pharmacokinetics ; Chromatography, Liquid ; Drugs, Chinese Herbal ; pharmacokinetics ; Lactates ; pharmacokinetics ; Macaca mulatta ; Mass Spectrometry ; Plant Roots ; chemistry ; Salvia miltiorrhiza ; chemistry

Objective To demonstrate the relationship between Cdc20 mutation and the promotion of colon cancer via Cdc20loxp/+ APCmin/+ villin-cre+/-compound mutant mice.Methods Cdc20loxp/+ APCmin/+ villin-cre+/-compound mutant mice and APCmin/+ mutant mice were generated by mice mating strategy.The colon tumors of two group mice were compared by phenotypic analysis and histology analysis.Results Phenotypic analysis showed that the number of tumors in Cdc20loxp/+ APCmin/+ villin-cre+/-compound mutant mice group and APCmin/+ mutant mice group was 1.2±0.5 and 1.6±0.5, respectively (t =0.215, P =0.588), and the maximum diameter of tumors was (2.7±0.3) cm and (2.5±0.2) cm, respectively (t =0.568, P =0.575).Pathologic type of Cdc20loxp/+ APCmin/+ villin-cre+/-compound mutant mice was adenocarcinoma, while that of APCmin/+ mice was tubular adenoma.Conclusion Cdc20 carrying a null allele can accelerate the promotion of colon cancer in APCmin/+ mice without influence on the tumor number and size.

Salvianolic acid A (Sal A) is one of the most effective compounds isolated from the root of Salvia miltiorrhiza. Up to now, several studies regarding the pharmacokinetic profiles of Sal A have been reported, however there is no such study reported in monkeys, the species which is more similar to human. The aim of this study is to develop a LC-MS method for the determination of Sal A in monkey plasma and apply it to the pharmacokinetic studies of monkeys. After single intravenous administration of Sal A, the plasma concentration-time curves were observed and the main pharmacokinetic parameters were calculated. The plasma concentration at 2 min (C2 (min)) values were (28.343 ± 6.426), (45.679 ± 12.301) and (113.293 ± 24.360) mg x L(-1) for Rhesus monkeys treated with Sal A at 2.5, 5 and 10 mg x kg(-1). The area under the concentration-time curve (AUC(0-∞)) values were (3.316 ± 0.871), (5.754 ± 2.150) and (13.761 ± 2.825) μg x L(-1) x h, respectively. Furthermore, this method was improved and applied to the simultaneous determination of Sal A, Sal B and Sal C, which provided useful information for preclinical studies and clinical trials of Sal A, Sal B and Sal C.

Objective To figure out and verify infiltration related miRNAs in bladder urothelial carcinoma (BUC). Methods Fresh tissues (20 samples,12 were infiltrative BUC samples,8 were non-infiltrative BUC samples) were collected in liquid nitrogen.The total RNA was extracted by using Trizol reagents.RNA quality control; miRNA microarray hybridization; data analysis.Another 22 samples were collected in fresh (15 were infiltrative BUC samples,7 were non-infiltrative BUC samples) for verifying purpose.4 types of bladder cancer cell lines were used for the study.BUC cell strain; total RNA was extracted by Trizol reagents; RNA quality control; RT-PCR and analysis of the data. Results ①In infiltrative BUC group,compared with non-infiltrative BUC group,there were 7 differentially expressed miRNAs:hsa-miR29c,hsa-miR-200a,hsa-miR-378,hsa-miR-429,hsa-miR-200c and hsa-miR-141 were up-regulated; hsamiR-451 was down-regulated.②In collected samples,the result of RT-PCR was consistent with miRNA array.③In bladder cancer cell lines,only the results of T24 were consistent with miRNA array. Conclusion Infiltration of BUC might relate with different expression of miRNAs.

The effects of combined RNA interference (RNAi) of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) genes on telomerase activity in a bladder cancer cell line (BIU-87 cells) were investigated by using gene chip technology in vitro with an attempt to evaluate the role of RNAi in the gene therapy of bladder transitional cell cancer (BTCC). Three TR-specific double-stranded small interfering RNAs (siRNAs) and three TERT-specific double-stranded siRNAs were designed to target different regions of TR and TERT mRNA. The phTR-siRNA, phTERT-siRNA, and the combination of both plasmids phTR+phTERT-siRNA were transfected into BIU-87 cells. The expression of hTR and hTERT mRNA was detected by quantitative fluorescent reverse transcription-polymerase chain reaction, and a telomeric repeat amplification protocol was applied to detect telomerase activity. Growth inhibition of BIU-87 cells was measured by MTT assay. Gene chip analysis was performed to evaluate the effects of the combined RNAi of hTR+hTERT genes on telomerase activity and growth of BIU-87 cells in vitro. The results showed that the expression of hTERT and hTR mRNA was inhibited by pRNAT-hTERT-III, pRNAT-hTR-III, and pRNAT-hTR-III+hTERT-III in BIU-87 cells. The inhibition efficiency of pRNAT-hTERT-III, pRNAT-hTR-III, pRNAT-hTERT-III+pRNAT-hTR-III was 67% for TERT mRNA, 41% for TR mRNA, 57% for TR mRNA and 70% for TERT mRNA in BIU-87 cells respectively. The growth of BIU-87 cells was inhibited and telomerase activity was considerably decreased, especially in the cells treated with combined RNAi-hTR and -hTERT. Gene chip analysis revealed that 21 genes were down-regulated (ATM, BAX, BCL2, BCL2L1, BIRC5, CD44, CTNNB1, E2F1, JUN, MCAM, MTA1, MYC, NFKB1, NFKBIA, NME4, PNN, PNN, SERPINE1, THBS1, TNFRSF1A, and UCC1). The results indicated that hTR-siRNA and hTERT-siRNA, especially their combination, siRNA hTR+hTERT, specifically and effectively suppressed the expression of both hTR and hTERT mRNA and telomerase activity. Molecular biological mechanism by which combined siRNA-TR and -TERT inhibited telomerase activity and growth of BIU-87 cells in vitro may involve the down-regulation of the 21 genes.

The rate of corneal graft rejection is still high for high-risk keratoplasty although immune suppression drug is routinely used.The role of traditional Chinese medicine in corneal transplantation is concerned gradually.Heijingpaichitang on the prevention and treatment of rats with high-risk corneal allograft rejection needs further study.Objective This study was to investigate the inhibitory effect of heijingpaichitang on high-risk corneal transplantation immune rejection in rats.Methods Sixteen female SD rats were used as the donors and 32female Wistar rats were served as recipients.The high-risk corneal trasplantation models were established by corneal suture in 32 Wistar rats,and then homogeneity variant SD-Wistar corneal transplantation was performed.The recipients were randomized into model control group,cyclosporinc A (CsA)group,heijingpaichitang group and CsA +heijingpaichitang group.CsA,heijingpaichitang and CsA + heijingpaichitang was orally administered 4 days after operation once per day for 15 days,and normal saline solution was used at the same way in the model control group.Ocular anterior segment reaction was examined under the slit lamp and corneal opacification,edema and neovasculation were scored based on Larkin' s criteria.Rejection index of the corneal graft was recorded and the graft survival time was calculated.The pathological examination of the corneal graft was carried out in all rats,and the inflammatory cells in the corneas and CD4+ cells in the periphery blood were assayed using flow cytometry.The use of the animals complied with ARVO Statement.Results Corneal graft rejection occurred in 10 days after operation in the model control group,12-13 days in the CsA group and heijingpaichitang group and 22 days in the CsA +heijingpaichitang group.Compared with model control group,the scores of the corneal opacification,corneal edema and neovascularization were significantly lower in the CsA group,heijingpaichitang group and CsA+heijingpaichitang group (P＜0.05),and all the scores were declined in the CsA+ heijingpaichitang group compared with CsA group and heijingpaichitang group(P＜0.01),but no significant differences were seen in the scores between the CsA group and heijingpaichitang group(P＞0.05).The mean survival time of grafts was (10.38 ±1.69)days in the model control group,(22.50 ± 3.07) days in the CsA + heijingpaichitang group,with the significant difference (t =-9.790,P =0.000).The pathological examination of graft showed that the lymphocytes and new blood vessels were less in the CsA+heijingpaichitang group compared with CsA group and heijingpaichitang group 15 days after operation.Flow cytometry verified that the number of lymphocytes in graft,CD4+cells and CD4+/CD8+ in periphery blood were significantly lower in the heijingpaichitang group,CsA group and CsA+heijingpaichitang group compared with model control group (P＜0.05).Conclusions Heijingpaichitang can inhibit immune rejection to certain extent in high-risk corneal transplantation rat and has a similar effect to 0.1％ CsA.Heijingpaichitang and 0.1％ CsA have a synergistic effect.