To study the relation between drug release and the drug status within curcumin-loaded microsphere, SPG (shirasu porous glass) membrane emulsification was used to prepare the curcumin-PLGA (polylactic-co-glycolic acid) microspheres with three levels of drug loading respectively, and the in vitro release was studied with high-performance liquid chromatography (HPLC). The morphology of microspheres was observed with scanning electron microscopy (SEM), and the drug status was studied with X-ray diffraction (XRD), differential scanning calorimetry (DSC) and infrared analysis (IR). The drug loading of microspheres was (5.85 ± 0.21)%, (11.71 ± 0.39)%, (15.41 ± 0.40)%, respectively. No chemical connection was found between curcumin and PLGA. According to the results of XRD, curcumin dispersed in PLGA as amorphous form within the microspheres of the lowest drug loading, while (2.12 ± 0.64)% and (5.66 ± 0.07)% curcumin crystals was detected in the other two kinds of microspheres, respectively, indicating that the drug status was different within three kinds of microspheres. In the data analysis, we found that PLGA had a limited capacity of dissolving curcumin. When the drug loading exceeded the limit, the excess curcumin would exist in the form of crystals in microspheres independently. Meanwhile, this factor contributes to the difference in drug release behavior of the three groups of microspheres.
Calorimetry, Differential Scanning ; Curcumin ; chemistry ; Drug Liberation ; Lactic Acid ; Microscopy, Electron, Scanning ; Microspheres ; Polyglycolic Acid ; X-Ray Diffraction

Objective A HPLC method was established for determination of the content of ATST in the aqueous humor of rabbits. MethodsThe mobile phase was consisting of methanol-1％ triethylamine(57:43) and omeprazole (OMZ) as internal standard. The detection was carried out with an ultraviolet detector operated at 235nm. ResultsThere was linearity over the range of 2. 056～41.12 ug/ml in the humor aquosus, r=0.9997. The average recovery of ATST was 94.58 ％. Intra-day and in- ter-day RSD were less than 5 ％ and 10 ％ ( n = 5), respectively. Conclusion The method is reliable. It can be used for the study on the pharmacokinetics of ATST.

To study the relation between drug release and the drug status within curcumin-loaded microsphere, SPG (shirasu porous glass) membrane emulsification was used to prepare the curcumin-PLGA (polylactic-co-glycolic acid) microspheres with three levels of drug loading respectively, and the in vitro release was studied with high-performance liquid chromatography (HPLC). The morphology of microspheres was observed with scanning electron microscopy (SEM), and the drug status was studied with X-ray diffraction (XRD), differential scanning calorimetry (DSC) and infrared analysis (IR). The drug loading of microspheres was (5.85 ± 0.21)%, (11.71 ± 0.39)%, (15.41 ± 0.40)%, respectively. No chemical connection was found between curcumin and PLGA. According to the results of XRD, curcumin dispersed in PLGA as amorphous form within the microspheres of the lowest drug loading, while (2.12 ± 0.64)% and (5.66 ± 0.07)% curcumin crystals was detected in the other two kinds of microspheres, respectively, indicating that the drug status was different within three kinds of microspheres. In the data analysis, we found that PLGA had a limited capacity of dissolving curcumin. When the drug loading exceeded the limit, the excess curcumin would exist in the form of crystals in microspheres independently. Meanwhile, this factor contributes to the difference in drug release behavior of the three groups of microspheres.

Objective To evaluate the effect of curcumin on the proliferation of and apoptosis in HaCaT cells induced by tumor necrosis factor α (TNF-α).Methods HaCaT cells were cultured with the presence of different concentrations (0,1,5,10,25,50,100 ng/ml) of recombinant TNF-α,curcumin of 20 μmol/L,or the combination of recombinant TNF-α (25 ng/ml) and curcumin (20 μmol/L),for 24 hours followed by the determination of cell proliferation with methyl thiazolyl tetrazolium (MTT) assay.Western blot was conducted to measure the protein expression of proliferating cell nuclear antigen (PCNA) and Notch-1 in HaCaT cells treated with recombinant TNF-α (25 ng/ml) and curcumin (20 μ mol/L) alone or in combination for 24 hours.Flow cytometry using annexin-V/propidium iodine (PI) was performed to assess the early apoptosis in HaCaT cells incubated with recombinant TNF-α of 25 ng/ml and curcumin of 20 μmol/L alone or in combination for 12 hours.Statistical analysis was carried out with one-way analysis of variance.Results Recombinant TNF-α promoted the proliferation of HaCaT cells in a dose-dependent manner,with the maximum proliferation activity observed in HaCaT cells treated with TNF-α of 25 ng/ml,while curcumin of 20 μmol/L effectively inhibited the proliferation of HaCaT cells induced by TNF-α of 25 ng/ml (P ＜ 0.01).TNF-α of 25 ng/ml had no obvious effect on cell apoptosis,while curcumin of 20 μ mol/L markedly induced the apoptosis in HaCaT cells,and there was a synergy between TNF-α of 25 ng/ml and curcumin of 20 μmol/L in the induction of apoptosis in HaCaT cells,with the apoptosis rate being 2.3％,3.4％,11.6％ and 16.8％ respectively in untreated cells,cells treated with TNF-α,curcumin,and the combination of TNF-α and curcumin,respectively.Conclusions Curcumin could enhance the inductive effect of TNF-α on the apoptosis in,but suppress the promotive effect of TNF-α on the proliferation of,HaCaT cells.

Objective To estimate the effect of glycyrrhetinic acid on epidermal growth factor (EGF)-induced proliferation of HaCaT cells,and to investigate its possible mechanism.Methods Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the proliferation of HaCaT cells treated with different concentrations of EGF (0,1,5,10,25,50,100 μg/L) and glycyrrhetinic acid (0,0.1,1.0,10,25,50,100μmol/L) alone,or the combination of 25 μg/L EGF with 25 μ mol/L glycyrrhetinic acid or 10 μ mol/L U0126 (an inhibitor of MEK1/2).Western blot was carried out to measure the protein expression of proliferating cell nuclear antigen (PCNA),Notch-1,ERK 1/2 and phosphorylated ERK 1/2 in HaCaT cells treated with 25 μg/L EGF,10 μmol/L U0126,25μmol/L glycyrrhetinic acid alone or in combination.Data were statistically analyzed by using t test,analysis of variance and correlation analysis with SPSS 17.0 software.Results EGF of 0-100 μg/L promoted the proliferation of HaCaT cells in a dose-dependent manner (r =0.798,P ＜ 0.05),and there was a linear correlation between the effect and concentration within the concentration range 0-50 μg/L (r =0.859,P ＜ 0.05).However,glycyrrhetinic acid of 10-100 μmol/L inhibited the proliferation of HaCaT cells in a dose-dependent manner (r =-0.945,P ＜0.01),and 10 μmol/L glycyrrhetinic acid could suppress the EGF (25 μg/L)-induced proliferation and phosphorylation of ERK1/2 in HaCaT cells.Also,both 25 μmol/L glycyrrhetinic acid and 10 μmol/L U0126 could attenuate the increase in PCNA and Notch-1 expression in HaCaT cells induced by 25 μg/L EGF.Conclusion Glycyrrhetinic acid can inhibit the EGF-induced proliferation of HaCaT cells,likely by suppressing the activation of ERK1/2 signaling pathway.

Objective To explore the possible role of neuropilin-1 (NRP-1) and extracellular regulated protein kinases (ERK) in the mechanisms underlying the promotive effect of vascular endothelial growth factor (VEGF) on the proliferation of HaCaT cells.Methods HaCaT cells were cultured in vitro and transfected with a NRP-1 expression plasmid EX-O0008-M02.Reverse transcription (RT)-PCR and Western blot were performed to detect the mRNA and protein expression of NRP-1 in HaCaT cells respectively before and after the transfection.Some HaCaT cells were divided into three groups to be transfected with liposome (liposome control group),control plasmid pReceiver-M02 (plasmid control group),and objective plasmid EX-O0008-M02 (objective plasmid group),respectively,and each of the three groups was classified into several subgroups to be treated with phosphate buffer solution (PBS),U0126 (MEK1/2 inhibitor) and VEGF alone or in combination.After additional culture,methyl thiazolyl tetrazolium (MTT) assay was performed to determine the proliferative activity of HaCaT cells,Western blot to quantify the expression of total and phosphorylated ERK1/2 as well as proliferating cell nuclear antigen (PCNA) protein in HaCaT cells.Intergroup differences were assessed by one-way analysis of variance (ANOVA),and multiple comparisons and correction were done by using the least significant difference (LSD) test.Results RT-PCR and Western blot analysis confirmed that the transfection with NRP-1 effectively promoted the mRNA and protein expression of NRP-1 in HaCaT cells.A significant increase was observed in cellular proliferative activity (absorbence value at 570 nm) in the objective plasmid group compared with the liposome control group and plasmid control group (0.88 ± 0.14 vs.0.63 ± 0.07 and 0.62 ± 0.13,F =8.755,P ＜ 0.05),also in the VEGF-stimulated objective plasmid group compared with the VEGF-stimulated plasmid control group (1.14 ± 0.18 vs.0.88 ± 0.10,F =4.591,P ＜ 0.05).The U0126 pretreatment markedly suppressed the VEGF-induced proliferation of A375 cells in the objective plasmid group (0.50 ± 0.13 vs.1.14 ± 0.18,F =42.106,P ＜ 0.01).As Western blot analysis suggested,the objective plasmid significantly enhanced the VEGF-induced increase in ERK1/2 phosphorylation degree and PCNA expression intensity in HaCaT cells compared with the control plasmid,but the enhancing effect of objective plasmid was effectively inhibited by U0126.Conclusion The activation of ERK1/2 signaling pathway may play a key role in the NRP-1 protein-mediated promotive effect of VEGF on the proliferation of HaCaT cells.

This article was aimed to study the quality control of pineapple leaf in order to further develop the appli-cations of pineapple leave as medicinal resources. The tissue structure of pineapple leaf was observed by tissue biop-sy assay. Thin-layer chromatography (TLC) was employed on the qualitative identification of ananasate in the leave. The p-coumaric acid and ananasate were detected with high-performance liquid chromatography (HPLC) assay in the content determination. The results showed that pineapple leaf can be divided into the upper and lower leaf epidermis with stoma under microscope observation. Ananasate of the leave can be indentified clearly by TLC. HPLC method can accurately detect the content of p-coumaric acid and ananasate of pineapple leave. There are different contents of p-coumaric acid and ananasate of pineapple leave in different origins. In Y unnan province, the contents of p-coumaric acid and ananasate of pineapple leave are the highest; while the contents of p-coumaric acid and ananasate in Guangxi province are the lowest. It was concluded that ananasate can be used in the quality identification. The p-coumaric acid and ananasate can be used in the quantitative determination in the better control of the quality of pineapple leaf.

Objective To analyze the clinical features,methods of treatment and prognosis of primary neurogenic tumors of mediastinum in patients taking surgical intervention.Methods A database was maintained retrospectively of all patients undergoing surgery for tumor and pathologically diagnosed with primary neurogenic tumors of mediastinum,managed in the Department of Thoracic Surgery,Zhongshan Hospital,Fudan University,Shanghai between Jan.,2008 and Dec.,2014.This work analyzed retrospectively the information about clinical and imaging features,surgical techniques and outcome extracted from medical records.Results Among the 131 cases,78 cases (59.5％) were males,53 cases (40.5％) were females;72 cases were diagnosed incidentally (55.0％),while the other 59 cases (45.0 ％) suffered from different symptoms.The posterior mediastinum was the most principal location with 61 cases in the left and 69 cases in the right,and 1 case remained in the anterior mediastinum.Total 98 cases (74.8％) underwent surgeries via video-assisted thoracic surgery (VATS),5 cases (3.8％) took VATS surgery with small incision,and 28 cases (21.4％) experienced open thoracotomy,with no mortality during perioperative period.Gross total resection was obtained in 130 patients (99.2％).The remaining patient underwent a palliative resection for malignant schwannomas.Of the patients,98 cases had benign schwannomas (74.8％),24 cases had gangliocytomas (18.3％),2 cases had malignant schwannomas (1.5％),2 cases had neurofibromas (1.5％),2 cases had paragangliomas (1.5％),2 cases hadprimitive neurotodermal tumor (PNET) (1.5％) and 1 case had neuroblastomas (0.8％).All patients were followed up from 12 to 95 months with an average of 53 months.A patient with PNET died of tumor metastasis,a patient with malignant schwannomas died after palliative ectomy,and 2 cases died of other reasons.The rest survived until Jan.,2016 with tumor free.Conclusions Nearly no specific clinical symptoms occur in neurogenic tumors of mediastinum.Most of neurogenic tumors of mediastinum are benign with optimistic prognosis after surgical treatment.While malignant neurogenic tumorsusually come with poor prognosis,which places special emphasis on early diagnose together with surgical treatment.

Objective To investigate the molecular mechanism of miR-139-5p targeting transforming growth factor-β1 (TGF-β1) in the regulation of epithelial mesenchymal transition (EMT),thus inhibiting invasion and metastasis of hepatoma cells.Methods Bioinformatics methods were used to determine whether miR-139-5p was the best binding miRNA of TGF-β1.Correlation between the TGF-β1 expression as detected by immunohistochemistry and Western blot,and the miR-139-5p level by qRT-PCR in 56 hepatoma tissues and 20 normal tissues,respectively,was analyzed.The relationship between the miR-139-5p level as detected by qRT-PCR,and TGF-β1,E-cadherin and Vimentin by Western blot in the high and low metastatic hepatoma cell lines were investigated.In recombinant cell lines,whether miR-139-5p could bind to the 3'UTR site of TGF-β1 was evaluated,and the effect on invasive ability after modulating miR-139-5p level was also tested by the transwell method.Results A total of 20 miRNAs were found to be able to bind with TGF-β1 by bioinformatics methods and among these mRNAs,miR-139-5p was the best target miRNA with the highest specificity and strongest stability to bind TGF-β1.The positive expression rates of TGF-β1 in hepatoma tissues and adjacent normal liver tissues were 80.4％ (45/56) and 15.0％ (3/20),respectively,(P ＜0.05).There were significant differences on the expressions of TGF-β1,E-cadherin and Vimentin among the different metastatic cell lines (all P ＜ 0.05).After miR-139-5p was transfected into hepatoma cells,miR-139-5p could bind to the 3'UTR site of TGF-β1,resulting in downregulating TGF-β1 expression.When compared to the other three groups,the cell line with a high expression of miR-139-5p had a significantly lower count of invasive cells (53 ± 4/high magnification field) (P ＜ 0.05).Conclusion miRNA139-5p could specifically bind to the 3'UTR site of TGF-β1 and regulate the EMT signaling pathway,thus suppressing invasion and metastasis of hepatoma cells.

Objective To investigate the clinical features and management of bile duct injury caused by laparoscopic cholecysteetomy by using harmonic scalpel (UHS-LC), and its prevention. Methods The clinical data of 1863 UHS-LC cases from April 2003 to February 2008 were retrospectively analyzed. There were 11 patients suffering from UHS-LC related iatrogenic bile duct injury including intraoperatively immediate recognized injuries in 9 cases, and postoperatively found injuries in 2 cases. For those patients in which bile duct injury was found during the UHS-LC procedure, the patient was converted to open surgery, the injury was repaired accordingly by end-to-end bile duet anastomosis or Roux-en-Y procedure. For the injuries found postoperatively (all two cases were of CBD perforation), CBD was sutured by second stage. Results All the 11 patients recovered well and no biliary stricture occurred during the follow up of 1-5 years. Conclusions While UHS-LC is suitable for most cases of choleeystectomy, it causes significant porcentage of bile duct injury (0. 6% ) in hands of unskillful surgeons. It is important to be on alert against iatrugenic bile duct injuries.