Over-expression of P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, represents one of the major mechanisms that contribute to multidrug resistance (MDR) in cancer cells. This study examined the effects of troglitazone, a ligand of peroxisome proliferator-activated receptor gamma (PPARgamma), on P-gp-mediated MDR in SGC7901/VCR cells (a vincristine-resistant human gastric cancer cell line). The expression of P-gp was detected by RT-PCR and Western blotting, respectively. The SGC7901/VCR cells were treated with 0.1 mg/L vincristine (VCR) alone or in combination with 1, 5, 10 mumol/L troglitazone for 24 h. PPARgamma was measured by electrophoretic mobility shift assay (EMSA). The intracellular concentration of Rhodamine123 (Rh123, a fluorescent P-gp substrate) was assayed to evaluate the activity of P-gp. The cell cycle and apoptosis were measured by flow cytometry. The results showed that the P-gp was increasingly expressed in SGC7901, BGC823 and SGC7901/VCR cells in turn, suggesting that MDR in the SGC7901/VCR cells was mediated by the increased expression of P-gp. In the SGC7901/VCR cells, the expression level of total PPARgamma was increased, however, the protein level and activity of PPARgamma in the nuclei of cells decreased significantly. Troglitazone elevated the PPARgamma activity in SGC7901/VCR cells in a dose-dependent manner. Troglitazone decreased the P-gp expression and markedly enhanced the accumulation of Rh123 in SGC7901/VCR cells in a dose-dependent manner. We also found that troglitazone significantly increased the percentage of SGC7901/VCR cells in the G(2)/M phase and decreased the cell percentage in G(1) and S phase in a dose-dependent manner. Troglitazone significantly increased the apoptotic rate of SGC7901/VCR cells treated by VCR or ADR in a dose-dependent manner. It was concluded that P-gp-overexpressed SGC7901/VCR cells have minor endogenous PPARgamma activity. Elevation of the PPARgamma activity by troglitazone can reverse P-gp-mediated MDR via down-regulating the expression and activity of P-gp in SGC7901/VCR cells. It was suggested that troglitazone can dramatically enhance the sensitivity of P-gp-mediated MDR cancer cells to chemotherapeutic agents.

Objective To investigate the therapeutic effect of frozen-thawed murine islets which were transplanted into diabetic rats after cultured with hyperbaric oxygenated rotary cell culture system (HORCCS). Methods The purified rat islets were divided into two groups: A. In vitro experiment groups (IvEG) : The rat islets in each subgroup were cultured in HORCCS or common medium for 30 days, then evaluated for the intracellular DNA and insulin contents of islets, and the viability and insulin secreting level of islets. B. Islet transplantation experimental groups (TxEG) : The frozen-thawed islets were cultured in HORCCS or common medium for 7 days, and then transplanted into the recipients. We observed the blood glucose level (BGL) and insulin secreting level in the recipients as well as the uhrastructure change of islets in TxEG. Results The viability and insulin secreting level of islets cultured with HORCCS at 14th day were much higher than those cultured with common medium (P <0.05). The blood glucose level in recipients transplanted with islets cultured with HORCCS recovered to normal value at the 2nd week and lasted for 8 weeks. All these recipients maintained the normal glucose tolerance curve. Electronic microscopy found microchannel outlets on the surface of the frozen-thawed islets cultured with HORCCS. Conclusions Frozen-thawed islets cultured with HORCCS could establish nutrient transmission microchannels, which were not only capable of oxygen and nutrients transmission, but also improving cryopreservation solution to diffuse inside the islet cells evenly and uniformly. So this method not only lessens islet damage from cryopreservation, but also improves the effect of transplantation.

Objective To explore the effects of adriamycin-loaded immuno-nanoparticles on multidrug resistance (MDR) of hepatoma cell line SMMC-7721/ADM. Methods The cytotoxicity of the adriamycin-loaded immuno-nanoparticles on the bepatoma cell line SMMC-7721/ADM in vitro and the tumor cell-binding ability of adriamycin-loaded immuno-nanoparticles were detected. Results The effect of the cytotoxicity of adriamycin-loaded immuno-nanoparticles on the hepatoma cell line SMMC-7721/ADM was significantly better than that of adriamycin-loaded nanoparticles. Adriamycin-loaded immuno-nanoparticles had the specific binding ability with the hepatoma cell line SMMC-7721/ADM. Conclusions Adriamycin-loaded immuno-nanoparticles can overcome the MDR of the tumor in vitro. The mechanism may be that immuno-nanoparticles could adhere to the tumor cell membrane, and the release of the loaded adriamycin creates a high local concentration in the extracellular medium. The increased concentration gradient improves the diffusion of adriamycin from the extracellular medium to the intracellular medium.

BACKGROUND: Inflammatory and proliferating effect after mechanical injury of vascular wall is the major cause of restenosis. Nuclear factor-kappa B (NF-κB) in the NF-κB/Rel family is expressed in a variety of cell types and activates a series of target genes, which are related to the pathophysioiogy of vascular wall.OBJECTIVE: To investigate the effect of antisense and decoy NF-κB oligonucleotides on vascular smooth muscle cell (VSMS) proliferation in vitro and neointimal proliferation and monocyte chemotactic protein-1 (MCP-1) in the balloon-injured carotid artery of rats.DESTGN: Randomized controlled animal trial.SETTTNG: Department of Cardiology, Ruijin Hospital, Medical College, Shanghai Jiaotong University.MATERTALS: Totally 126 male Sprague-Dawley (SD) rats, aged 3 months, weighing 350 to 380 g, were involved in this study. Synthesis of primer and oligonucleotide: they were synthesized and designed by Shanghai Bioengineering Co. Ltd according to literatures and international internet cDNA library.METHODS: This study was carried out in the Laboratory of Cell Biology, Medical College, Shanghai Jiao Tong University and Cardiovascular Laboratory, Ruijin Hospital Affiliated to Shanghai Jiaotong University from May 2001 to March 2003.Rat aortic smooth muscle cells were isolated from May 2001 to March 2003. Rat thoracic aorta vascular smooth muscle cells were cultured by primary-explant method. And the third to fifth generations of VSMCs were involved in the experiment. Proliferating cell nuclear antigen (PCNA) NF-κB p65 protein synthesis in proliferating smooth muscle cells were detected. SD rat carotid artery underwent balloon injury. The involved 126 rats were randomly divided into 7 groups with 18in each group: normal group: normal group (the procedure was the same as other group except for balloon injury), sense group, antisense group, decoy group, scramble group, antisense plus decoy group, model group. Each group includes 6time points (6 hours, and 1,3,5,7,14 days, n =3). Then, the effect of antisense and decoy NF-κB oligonucleotides on intimai proliferation and MCP-1 and NF-κB p65 and extracellular signal regulated kinase(ERK2) expression in the balloon-injured carotid artery of rats were detected.MAIN OUTCOME MEASURES: ①Effect of oligonucleotide of NF-κB p65 on VSMCs proliferation; ② NF-κB p65 gene expression and protein synthesis; ③ Patho-morphological change after carotid balloon-injury. ④ Vascular MCP-1 mRNA Expression in balloon-injured rat carotid artery; ⑤ MCP-1 immunoreactivity in the injured arterial wall detected by immunohistochemistry; ⑥ NF-κBp65 and ERK2 protein synthesis after balloon-injury detected by Western blot in injured rat carotid arteries.RESULTS: ①PCNA protein synthesis increased in proliferating smooth muscle cells. ②NF-κB p65 gene expression was found in the cytoplasm and nucleus of proliferating smooth musclecells by in situ hybridization and NF-κB p 65 protein level increased in proliferating smooth muscle cells by flow cytometry. NF-κB p65 gene expression in antisense group decreased 53.66% compared with in sense group; it decreased 57.35% in decoy group compared with in scramble group. There were all statistical differences(P＜0.05).③ PCNA expression were inhibited in proliferating smooth musclecells by antisense and decoy oligonucleotides. Compared with positive control group, PCNA protein expression in antisense group and decoy group decreased 45.12% and 45.05%,respectively. ④ In model group, sense group and scramble group, vessel intimal area, medial area and intimal area/medial area increased at the 5th day after balloon-injury and reached the maximum at the 7th day after injury. The intimal area/medial area was significantly decreased in the antisense group and decoy group. The effect of antisense plus decoy oligonucleotides was more obvious than that of antisense group and decoy group alone but there were not significant differences among three groups. ⑤ Reverse transcription-polymerase chain reaction showed that MCP-1 mRNA expression was significantly increased 6 hours after balloon-injury, but not evident after 1 day. It was increased at the 3th, 5th and 7th days continuously, but decreased at the 14th day. MCP-1 mRNA expression was decreased at each time point in antisense group, decoy group, antisense plus decoy group (P＜0.05). ⑥Western blot analysis showed that NF-κB p65 was weakly expressed at 6 hours after vascular balloon-injury, increased significantly at 1 day, reached the peak at 7 days and weakened at 14 days, while ERK2 protein was weakly expressed, a little increased at 1 day, reached the peak at 7 days and weakened at 14 days. Treatment of antisense group, decoy group and antisense plus decoy group inhibited protein synthesis more significantly than those of model group, sense group and scramble group (P＜0.05).CONCLUSTON: NF-κB expression increases in proliferating smooth muscle cells. NF-κB modulates genes expression and protein synthesis of MCP-1 and ERK2. Cellular proliferation in vessel wall dynamically changes after balloon angioplasty injury. Antisense and decoy oligonucleotide of NF-κB by local lipofectamine transfer inhibit the expression of regulated target gene.

Objective To explore the effect of probiotics on patients with severe acute pancreatitis.Methods Randomized controlled trials of probiotics in patients with severe acute pancreatitis were searched in Cochrane Library(2010),Medline(1966 to September 2010),China Academic Literature Full-text Database,Chinese Science and Technology Journal Database and WanFang Database between 1966 and September 2010.Evaluation of literature quality and data extraction were independently completed by two researchers.Data were analyzed with RevMan4.3 software.Results A total of 313 papers had been searched.After excluded duplicated and low-level paper,six articles were included in this study.The result showed that the infection rate(OR=0.88,95％CI[0.56,1.38])and mortality rate(OR=1.44,95％CI[0.78,2.64])of patients with severe acute pancreatitis in intervention group were not lower than control group,however the length of stay in hospital was shorter in intervention group than control group(WMD=-4.69,95％ CI[-7.73,-1.65]).There was no significant difference between intervention group and control group in systemic inflammatory response syndrome(OR =1.33,95 ％ CI[0.63,2.83]),multiple organ failure (OR=1.50,95 ％ CI[0.89,2.52])and transfering to surgery department for further treatment(OR=0.92,95％CI[0.56,1.51]).Conclusion So far the studies indicated that probiotics could not lower the infection rate and mortality rate in patients with severe acute pancreatitis.Large sample and highquality randomized controlled trials are needed for further evaluation.

OBJECTIVE: To investigate the protective effect of epigallocatechin-3 -gallate (EGCG) on apoptosis of cerebellar granule neurons (CGNs) induced by acrylamide (ACR). METHODS: CGNs were cultured with the addition of 5 mmol/L ACR for 24 hours to set up a cell injury model. Prior to ACR treatment, CGNs were treated with different concentrations of EGCG (0, 5, 10, 25, 50, 100 μmol/L) for 48 hours. Neuronal viability was measured with metylthiazdyltetrazolium (MTT). The activity of SOD and the content of MDA were assayed. Hoechst33342 staining was employed to observe morphological changes of the cell nucleus. Reverse transcription-polymerase chain reaction (RT-PCR) was used to measure expression of bcl- 2 mRNA and bax mRNA. RESULTS: At the concentrations of 10, 25 or 50 μmol/L, EGCG played a protective role against ACRinduced CGN injury. Compared with ACR injured group (no EGCG), EGCG improved the cell viability, enhanced SOD activity, decreased the level of MDA as well as the cell apoptosis ratio (P<0.05). Bcl-2 mRNA expression was increased and bax mRNA expression was reduced (P<0.05). 25 μmol/L EGCG had the largest effect. However, 100 μmol/L EGCG did not have a significantly protective effect. CONCLUSION EGCG at appropriate concentration has protective effect against the CGNs on apoptosis induced by ACR.
Acrylamide ; toxicity ; Animals ; Apoptosis ; drug effects ; Catechin ; analogs & derivatives ; pharmacology ; Cells, Cultured ; Cerebellum ; cytology ; drug effects ; Cytoplasmic Granules ; Female ; Male ; Neurons ; cytology ; drug effects ; Neuroprotective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley

The aim of this study is to explore the tissue distribution of PEGylated puerarin in acute myocardial ischemia model rats. Healthy male SD rats were randomly divided into two groups (30 each). Both were given PEGylated puerarin at a dose of 488 mg x kg(-1). After 5 min of medication, one group was normal rats, another group with acute myocardial ischemia was established by peritoneal injection of 50 mg x kg(-1) isoprenaline. After administration, the animals were executed at 30, 60, 90, 120, 150 and 180 min, then heart, liver, spleen, lung, kidney were extracted. The content of puerarin in organ tissue was determined by HPLC. The results showed that the AUC of tissue distribution of PEGylated puerarin in normal rats was liver > kidney > heart ≈ spleen > lung > brain. While the AUC of tissue distribution of PEGylated puerarin in acute myocardial ischemia model rats was liver ≈ heart > kidney > lung ≈ spleen > brain. AUC(heart) of PEGylated puerarin in acute myocardial ischemia model rats was 1.7 times than that of the normal rats, and there was significant difference (P < 0.05). Thus, PEGylated puerarin had a good heart-targeting property in early myocardial infarction area, drugs could accumulate in the ischemic myocardium. It provided important information for further study and clinic use of PEGylated puerarin.

Objective To investigate the related factors of serum carnitine deficiency in critical ill patients, and the influence of its deficiency on the length of hospital stay. Methods A prospective study was conducted. Critical ill patients with acute physiology and chronic health evaluationⅡ(APACHEⅡ)score>12 admitted to Department of Critical Care Medicine of the First Affiliated Hospital of Sun Yat-sen University from March 2013 to September 2013 were enrolled. Serum carnitine concentration and indexes of organ function were determined,and the tolerance of enteral nutrition within 5 days,the length of hospital stay,the length of intensive care unit(ICU)stay,and the hospital mortality were recorded. The relationship between serum carnitine and indexes mentioned above was analyzed. Results Thirty critically ill patients were enrolled. Serum carnitine concentration was very low in all critically ill patients,i.e. (8.92±5.05)μmol/L(normal reference value at 43.5 μmol/L)at hospital admission. Serum carnitine concentration in patients with APACHEⅡscore>23(7 cases)was significantly lower than that in those with APACHEⅡscore 12-23(23 cases,μmol/L:5.33±1.72 vs. 10.02±5.24,t=2.300,P=0.001). Serum carnitine concentration in patients with serum total bilirubin(TBil)>19μmol/L(9 cases)was significantly lower than that in those with TBil≤19μmol/L(21 cases,μmol/L:5.54±2.70 vs. 9.84±5.08,t=2.750,P=0.014). Serum carnitine concentration was negatively correlated with the APACHEⅡscore and the TBil(r=-0.387,P=0.035;r=-0.346,P=0.048). During the 5-day observation period,enteral feeding amount〔(5 134±1 173)mL〕was positively correlated with serum carnitine concentration(r=0.430,P=0.022). In 30 critical patients,the incidence of abdominal distension was 40.0%(12/30),and the serum carnitine concentration of patients with abdominal distension was lower compared with that of patients without abdominal distension(μmol/L:7.83±4.98 vs. 9.12±5.35,t=0.707,P=0.383). The incidence of diarrhea was 26.7%(8/30),and the serum carnitine concentration of diarrhea patients was lower compared with that of patients without diarrhea(μmol/L:8.27±5.78 vs. 9.73±4.78,t=0.607,P=0.576). The mean length of hospital stay was(34.72±16.66)days. The serum carnitine concentrations in patients with hospital stay≥45 days (8 cases)were lower compared with those in those<45 days(22 cases,μmol/L:5.71±3.23 vs. 9.95±5.26,t=1.627,P=0.020). No correlation was found between serum carnitine concentrations and the hospital stay(r=-0.165, P=0.385). The length of ICU stay was(18.60±10.72)days. Serum carnitine concentration in patients with the length of ICU stay>7 days(27 cases)was slightly lower than that in those with the length of ICU stay≤7 days (3 cases,μmol/L:8.44±5.00 vs. 13.24±3.65,t=1.610,P=0.119). No correlation was found between serum carnitine concentrations and the length of ICU stay(r=-0.019,P= 0.293). In-hospital mortality was 26.67%(8/30). No significant difference in serum carnitine concentrations was found between the death group and the survival group(μmol/L:12.24±6.52 vs. 7.72±3.91,t=-1.846,P=0.098). No correlation was found between serum carnitine concentrations and in-hospital mortality(r=0.340,P=0.066). Conclusions Carnitine deficiency is significant in critically ill patients,and it is correlated with disease severity and serum TBil. The total amount of lenteral feeding was lower,and hospital stay was prolonged in critically ill patients with low serum carnitine level.

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Objective The objective of this study was to investigate the effect of acidic conditions on epithelial mesenchymal transition(EMT) of HepG2 cells.Methods HepG2 cells were cultured under acidic and alkaline conditions to observe the difference of cell morphology.Wound healing assays were performed to detect the migration ability of the two groups.Matrigel invasion assays were used to detect the invasive ability of the cells.The expression of EMT-related genes at mRNA level was detected by RT-PCR.The expression level of EMT-related gene protein was detected by Western blot.Results The morphology of HepG2 cells was changed from epithelium to interstitial type under acid conditions.The migration ability of HepG2 cells under acidic condition was stronger than that of alkaline conditions.The number of HepG2 cells crossing Matrigel was higher than that of alkaline condition.The mRNA expression of Vimentin,Slug,Snail and Zeb1 related to EMT and the protein expression of Vimentin and MMP9 related to EMT in HepG2 cells were significantly higher than those under alkaline condition.Conclusion The acid conditions can induce the occurrence of EMT in HepG2 cells.