Objective To investigate the changes of expression of Fas-associated proteins named Fas-associated death domain protein(FADD)and death-associated protein(Daxx)in the ischemic penumbra following transient focal cerebra ischemia in rats.Methods ①Adult male Sprague-Dawley rats were randomly divided into the sham-operated group and the cerebral ischemia model group.Rats underwent right middle cerebral artery occlusion(MCAO)for 2 h and reperfusion for 1,3,6,12 and 24 h using an intraluminal suture technique.The expression of FADD and Daxx mRNA and protein were measured with methods of immunohistochemistry.Western blot and reverse transcription-polymerase chain reaction(RT- PCR)respectively were used in the ischemic penumbra of rats.②Double-label fluorescence confocal laser scanning microscopy(CLSM)was performed to monitor FADD and Daxx intracellular location before and after ischemia.Results RT-PCR,Immunohistochemistry,Western blot experiments indicated that a very low level of FADD mRNA and protein were detected in the cerebral cortex of sham rats.The expression level both of FADD mRNA and protein increased significantly at 3 h after reperfusion,peaked at 12 h,then declined markedly at 24 h in the ischemic penumbra of model rats.RT-PCR,Immunohistochemistry indicated that a relatively high level of Daxx mRNA was detected in the cerebral cotex of sham rats.The expression level of Daxx mRNA increased significantly at 3 h after reperfusion and persisted to 24 h at a high level,whose protein had a same change of expression level in the ischemic penumbra of model rats. Immunofluorescence double-staining laser scanning by CLSM showed that the immunoreactivity of FADD was located in cytoplasm,and the intracellular translocation of the immunoreactivity of Daxx from nucleus to cytoplasm was monitored by measuring the green fluorescence after ischemia.Conclusion The transient upregulation of FADD and the persistant high level of expression of Daxx may contribute to neuronal apoptosis following cerebral ischemia/reperfusion.

Limit test of flavones in diterpene ginkgolides meglumine injection materials by UV-Vis and HPLC-DAD method was studied in this essay. The HPLC-DAD method has lower LOD (about 1% of the UV-Vis), that is, the sensitivity is higher than UV-Vis method. Through the analysis of the kinds of flavonoids ingredients in the samples by LC-MS, the three compounds with highest contents are kaempferol, quercetin and isorhamnetin. Kaempferol, quercetin and isorhamnetin were chosen as reference compounds for HPLC analysis, and the HPLC separation analysis was carried on an Agilent Eclipse plus C18 column (4.6 mm x 250 mm, 5 μm) with methanol and water containing 0.4% phosphoric acid (50: 50) as mobile phase, and the flow rate was 1.0 mL x min(-1). The detection wavelength was set at 360 nm. This method has good specificity, precision and reproducibility. The LODs of quercetin, kaempferide and isorhamnetin were 27.6, 22.3, 29.5 μg x L(-1). The average recovery was 87.9% (RSD 3.3%), 91.7% (RSD 3.1%), 88.3 (RSD 1.3%) for quercetin, kaempferide and isorhamnetin, respectively. Based on the 10 batches of sample results and sensitivity of different HPLC, the content of total flavonoids ingredients of diterpene ginkgolides meglumine injection materials was limited no more than 2 x 10(-5). This method is simple, quick and has good maneuverability, and could be used to the limit test of flavonoids in the diterpene ginkgolides meglumine injection materials.
Chromatography, High Pressure Liquid ; methods ; Diterpenes ; analysis ; Drugs, Chinese Herbal ; analysis ; Flavones ; analysis ; Ginkgolides ; analysis ; Limit of Detection ; Mass Spectrometry ; methods

Objective To investigate the effect of brain-derived neurotrophic factor (BDNF) gene modified neural stem cells (NSCs)transplantation on cerebral ischemic injury in rats.Methods NSCs from newborn rat hippocampus were isolated,cultured in a medium containing fibroblast factor (FGF) in vitro. Their proliferation and differentiation were detected by immunohistochemistry.Virus vectors pLXSN-BDNF were built and BDNF were transfected into NSCs.Biological activity were detected to obtained engineering stem cells of BDNF protein with secretary activity.Middle cerebral artery occlusion (MCAO) models were made and transplanted with NSCs-BDNF by stereotaxic technique.The effect of transplantation on MCAO models was evaluated histologically and behaviorally.Results Statistical analysis showed that the behavioral performance of the animals improved after transplantation (Longa mark being 1.343?0.293 four weeks after stem cell transplantation).The number of hippocampal dentate gyrus neurons increased to 87.5%?6.6% , four weeks after stem cell transplantation on Nissle stained hippocampal sections,which was significantly different from that of controls.Positively BrdU stained neural stem cells revealed by immunohistochemistry in the cultured cells and in the transplanted brain sections,indicated that the engineering cells transplanted had survived in the host brain and began to proliferate.Conclusion Transplantation of BDNF genetically modified NSCs can effectively promote the recovery from cerebral ischemic injury.

This study was aimed to establish a quality evaluation method, quantitative analysis of multi-components with a single-marker (QAMS) to determine the contents of five saponins in Yao-Bi-Tong (YBT) capsules. Ginseno-side Rg1 was used as the internal standard; the relative correction factor (RCF) of notoginsenoside R1, ginsenoside Re, ginsenoside Rb1 and ginsenoside Rd were calculated and evaluated. The contents of 5 saponins were determined by the external standard method and QAMS, respectively. Rationality, feasibility and repeatability of the QAMS method were verified by comparing the results obtained from two different methods. The results showed that RCFs of notoginsenoside R1, ginsenoside Re and ginsenoside Rb1 to ginsenoside Rg1 against YBT capsules were 0.999, 1.228, 0.990 and 1.094, respectively, indicating good reproducibility. These results of two methods had no significant dif-ference. It was concluded that the QAMS method can be accurately, rapidly and reasonably used as a new quality assessment model for ginsenosides in YBT capsules.

This study was aimed to screen main active site to reduce blood lipid from Xin-Mai (XM) capsule and establish HPLC fingerprint of the site, in order to study the correlativity between active site and relevant fractions of its herbs. Solvent extraction was used to separate XM capsule into different polar fractions. Intraperitoneal injection of 75% egg-yolk emulsion was used to establish mice hyperlipidemia models. And the active site was screened. Chromatographic fingerprints of the site and relevant fractions of its herbs were configured by HPLC analysis. The
retention time of peaks was utilized as index to evaluate the correlativity. The results showed that lipid-lowering effect of ethyl acetate extract and garlic essential oil was significant (P<0.01). Fingerprint of the active site in XM capsule was established with 28 fingerprint peaks and the assignment results of 27 peaks were indicated. It was concluded that the active sites to reduce blood lipid of XM capsule were ethyl acetate extract and garlic essential oil. The established fingerprint method can effectively determine the correlativity between the active site and its relevant fractions, which contributed to pharmacodynamic material foundation and quality standard.

To observe the expression of N - cadherin and fibronectin in retinal pigment epithelium ( RPE) cells in vitro under high glucose conditions, furthermore, to explore the effects of high glucose on epithelial -mesenchymal transition (EMT) in RPE cells.
●METHODS: Human RPE (hRPE) cells were cultured in vitro. Containing a final concentration of 60mmol/ L glucose was used for high glucose treatment. The cells were divided into normal glucose group (5. 5mmol/ L, NG) and high glucose group (24, 48 and 72h) respectively. The expression of N - cadherin and fibronectin in hRPE cells were evaluated by immunofluorescence and real -time PCR.
●RESULTS:RPE cells became disorganized and swollen over time under high glucose conditions, especially in 72h subgroup. lmmunohistochemical analysis revealed that the expression of N - cadherin in RPE cells under high glucose conditions was decreased compared with that in the control group, while the expression of fibronectin was increased. Real - time PCR results showed that the expression of N - cadherin mRNA in high glucose group was decreased at 24h compared with that in the control group, and declined markedly at 72h ( F = 12. 252, P =0. 000). There were no significant differences between the control group and the high glucose group at 24h, while the differences between the control group and the high glucose group (48 and 72h) were significant respectively (P < 0. 05 ). Meanwhile, the expression of fibronectin mRNA in RPE cells was increased in high glucose group at 24h, and reached the peak at 72h (F = 50. 543, P = 0. 000). There were no significant differences between the control group and the high glucose group at 24h. Compared with the control group, the expression of fibronectin mRNA in hRPE cells was increased significantly in high glucose group (48 and 72h) respectively (P= 0. 000, P= 0. 000).
●CONCLUSlON: The expression of epithelium marker N-cadherin is down - regulated under high glucose conditions in hRPE cells in vitro. Meanwhile, the expression of mesenchymal maker fibronectin is induced and appeared to EMT changes. Results of this study will enrich our growing understanding in proliferative diabetic retinopathy and hopefully lead to novel insights for the pathogenesis and therapeutic treatments.

Taking guizhi fuling capsule (GZFL) for instance, a new method about reference Chinese medicine preparation which was used as standard substance for the quality evaluation of complex Chinese medicine preparation by the fingerprint of reference preparation instead of standard fingerprint was proposed. It could eliminate the errors from different instruments, chromatographic columns and solve the problem of similarity matching in the absence of standard fingerprint. The qualification of reference GZFL was evaluated according to the quality control method of GZFL from Chinese Pharmacopoeia. Then multiple batches of GZFL were estimated, taking fingerprint of reference preparation and standard fingerprint as references, respectively, at different instruments and chromatographic columns. Finally, the packaging and expiration date for reference GZFL were confirmed according to the results of stability investigation. The results indicated that the fingerprint of reference GZFL could be used to assess the quality of GZFL better than standard fingerprint. The data of accelerated stability and long-term stability test demonstrated that reference GZFL was stable in the conditions of double blister package. Therefore, reference GZFL can be used as standard substance in quality control of GZFL.
Capsules ; Drug Stability ; Drugs, Chinese Herbal ; chemistry ; standards ; Reference Standards

Objective:To observe the effect of herbal cake-partitioned moxibustion on the expressions of mitogen-activated protein kinase (MEK1/2) and extracellular regulatory protein kinase (ERK1/2) in gastric tissues of rats with spleen deficiency syndrome, and to explore the possible mechanisms of herbal cake-partitioned moxibustion in treating spleen deficiency syndrome. Methods:Sixty Sprague-Dawley (SD) rats were randomly divided into a blank control group (group A), a model group (group B), a ranitidine group (group C), and a herbal cake-partitioned moxibustion group (group D) by random digit, 15 rats in each group. Rat models of spleen deficiency syndrome were made by intragastric administration of 4℃ 200% concentrated Da Huang (Radix et Rhizoma Rhei). After successful modeling, the rats in group C were treated with 25 mg/(kg·bw) ranitidine by intragastric adminstration and rats in group D were treated with herbal cake-partitioned moxibustion at Zusanli (ST 36) and Zhongwan (CV 12), for 8 d. Excepted for rats in group A, all the other rats were treated with indomethacin at 5 mg/(kg·bw) at 8:00 a.m. on the second day after finishing all the intervention and sacrificed 7 h later to isolate the stomach. Histopathological changes of the gastric tissues were observed under light microscope after hematoxylin-eosin (HE) staining. The protein expressions of MEK1/2 and ERK1/2 in the gastric tissues were detected by immunohistochemistry. Results:After intervention, the gastric mucosal injury in group B was significantly severer than that in group A, with large breakage and ablating; the damage of gastric mucosa was decreased in group C compared with group B; the gastric mucosal surface remained relatively complete, and the status of breakage and ablating was significantly improved. After intervention, compared with group A, the protein expressions of MEK1/2 and ERK1/2 in gastric tissues of the other groups were significantly higher (P<0.01). Compared with group B, the protein expressions of MEK1/2 and ERK1/2 in group C and D were significantly higher (allP<0.01). Compared with group C, the protein expressions of MEK1/2 and ERK1/2 in group D were significantly higher (P<0.01). Conclusion: Herbal cake-partitioned moxibustion promotes the repair of gastric mucosa in rats with spleen deficiency syndrome, via improving protein expressions of MEK1/2 and ERK1/2 in gastric tissues, as well as activating MEK/ERK signaling pathway.

BACKGROUNDPolypharmacy and potentially inappropriate medications (PIMs) are prominent prescribing issues in elderly patients. This study was to investigate the different prevalence of PIM use in elderly inpatients between 65-79 years of age and 80 years or older, who were discharged from Geriatric Department in West China Hospital.

METHODSA large-scale cohort of 1796 inpatients aged 65 years or over was recruited. Respectively, 618 patients were 65-79 years and 1178 patients were 80 years or older. Updated 2012 Beers Criteria by the American Geriatric Society was applied to assess the use of PIM among the investigated samples.

RESULTSA review of the prescribed medications identified 686 patients aged 80 years or older consumed at least one PIM giving a rate of 58.2%. Conversely, 268 (43.4%) patients aged 65-79 years consumed at least one PIM (χ2 = 40.18, P < 0.001). Patients aged 80 years or older had higher hospitalization expenses, length of stay, co-morbidities, medical prescription, and mortality than patients aged 65-79 years (all with P < 0.001). Patients aged 80 years or older were prescribed with more benzodiazepines, drugs with strong anticholinergic properties, megestrol, antipsychotics, theophylline, and aspirin. In multiple regression analysis, PIM use was significantly associated with female gender, age, number of diagnostic disease, and number of prescribed medication.

CONCLUSIONSThe finding from this study revealed that inpatients aged 80 years or older encountered more PIM use than those aged 65-79 years. Anticholinergic properties, megestrol, antipsychotics, theophylline, and aspirin are medications that often prescribed to inpatients aged 80 years or older. Doctors should carefully choose drugs for the elderly, especially the elderly aged 80 years or older.