To make a preliminary study on the mechanism of Coptidis Rhizoma(CR) and Rehmanniae Radix(RR) before and after the combined administration in treating type II diabetes mellitus. The type I diabetes animal model in rats was established by fat emulsion and intraperitoneal injection with streptozotocin, in order to compare the hpyerglycemic and hypolipidemic effects of CR, RR and their combined administration of different ratio. The urinary metabolic profiling in rats of Coptidis Rhizoma and Rehmanniae Radix before and after the combined administration was analyzed by using the gas chromatography-mass spectrometry. The differences among groups in metabolome were analyzed by the principal component analysis (PCA). The biochemical index results indicated that both CR and RR before and after the combined administration could lower high blood glucose, hypertriglyceride and high cholesterol. According to the analytical results of PCA of the rats' urine samples, the CR group was the most close to the normal group, with no significant difference in CR and RR group of different combination ratios. Twelve differentiated metabolites were identified to be related to type II diabetes. Compared with the normal group, the CR-treated group showed significant increase in seven differentiated metabolites. Among CR and RR drugs with different combination ratios, CR played a major role and thus acted as the monarch drug. Whereas RR served as the ministerial drug and assisted CR to show the efficacy. This study laid a foundation for the explanation of the combination mechanism of traditional Chinese medicines.
Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Type 2 ; blood ; drug therapy ; urine ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Male ; Rats ; Rats, Wistar ; Rehmannia ; chemistry

Objective To study the relationship between coronary arteriographic and fluorescence fundus angiographic characteristics in type 2 diabetic patients with coronary heart disease.Methods The study was carried out by the analysis of the data from coronary arteriography and fluorescence fundus angiography in 203 type 2 diabetic patients with coronary heart disease in different groups divided according to age or total cholesterol level. Logisitic regression analysis was applied to explore various risk factors to angiographic characteristics.Results With advancing age,there were more involvement of 3 coronary vessels or the left main branch along with stageⅢretinopathy,but less single vessel diseases in the coronary arteries and less stageⅠretinopathy.The difference in coronary angiographic and fluorescence fundus angiographic characteristics between groups with different total cholesterol levels was not significant.Logistic regression analysis suggested that coronary artery diaease was related to age,sex and blood glucose and triglyceride levels while diabetic retinopathy was related to blood glucose level and age.Conclusion There is great difference in coronary arteriography and fluorescence fundus angiography among different age groups.Aging may aggravate the lesions both in the coronary arteries and fundal vessels in type 2 diabetic patients with coronary heart diseease.

The DNA fragments of ag85b、esat-6、hsp65、mpb64 and ag85b-esat-6、hsp65-esat-6、mpb64-esat-6 were amplified by PCR and SOE technique.These seven fragments were inserted into pCDNA3.1(+)vector to construct recombinant plasmids pCA、pCE6、pCH、pCM、pCAE、pCHE and pCME.The seven plasmids were transfected into SP2/0 cell in vitro to detect the expression of target genes.BALB/c mice were intramuscularly vaccinated with the seven plasmids and the control vector pCDNA3.1(+)and PBS respectively.The serum antibodies and the spleen lymphocyte proliferation(SLP)and secreted IFN～? of spleen were tested.The results of indirect ELISA showed the levels of antibodies in all recombinant plasmids groups were significantly higher than the two control groups(P

AIMTo study the effects of PKC activation on apoptosis during ischemia/reperfusion in L-6TG rat skeletal myoblasts.

METHODSCultured L-6TG cells were divided into 3 groups: control group (C), ischemia/reperfusion group (I/R), PMA + ischemia/ reperfusion group (PMA), SOD, XOD and free calcium and mitochondrial respiration in L-6TG cell were evaluated in each group. Apoptosis was detected by flow cytometer with PI staining method and agarose gel electrophoresis, the immunohistochemical method was used to determine the expression of caspase-3.

RESULTSCompared with I/R group, in PMA group, XOD , free calcium in L-6TG cell and apoptotic percentage all decreased significantly, while SOD and mitochondrial respiration in L-6TG cell increased. DNA fragmentation analysis of L-6TG cell showed no laddering pattern. The expression of caspase-3 was down regulated significantly.

CONCLUSIONActivation of PKC can lessen ischemia/reperfusion injury and apoptosis through lessening oxidative injury and mitochondrial injury, adjusting calcium dyshomeostasis and down expression of caspase-3.

Animals ; Apoptosis ; Calcium ; metabolism ; Caspase 3 ; metabolism ; Cells, Cultured ; Mitochondria ; metabolism ; Myoblasts, Skeletal ; cytology ; metabolism ; Oxidative Stress ; Protein Kinase C ; metabolism ; Rats ; Reperfusion Injury ; metabolism ; pathology

BACKGROUNDInfections caused by gram-negative bacteria (GNB) often lead to high mortality in common clinical settings. The effect of traditional antibiotic therapy is hindered by drug-resistant bacteria and unneutralizable endotoxin. Few effective methods can protect high risk patients from bacterial infection. This study explored the protection of adeno-associated virus 2 (AAV2)-bacteriacidal permeability increasing protein 700 (BPI(700))-fragment crystallizable gamma one 700 (Fc gamma1(700)) chimeric gene transferred mice against the minimal lethal dose (MLD) of E. coli and application of gene therapy for bacterial infection.

METHODSAfter AAV2-BPI(700)-Fc gamma1(700) virus transfection, dot blotting and Western blotting were used to detect the target gene products in Chinese hamster ovary-K1 cells (CHO-K1cells). Reverse transcription-polymerase chain reaction and immunohistochemical assay were carried out to show the target gene expression in mice. Modified BPI-enzyme linked immunosorbent assay was used to identify the target gene products in murine serum. The protection of BPI(700)-Fc gamma1(700) gene transferred mice was examined by survival rate after MLD E. coli challenge. Colony forming unit (CFU) count, limulus amebocyte lysate kit and cytokine kit were used to quantify the bacteria, the level of endotoxin, and proinflammatory cytokine.

RESULTSBPI(1-199)-Fc gamma1 protein was identified in the CHO-K1 cell culture supernatant, injected muscles and serum of the gene transferred mice. After MLD E. coli challenge, the survival rate of AAV2-BPI(700)-Fc gamma1(700) gene transferred mice (36.7%) was significantly higher than that of AAV2-enhanced green fluorescent protein (AAV2-EGFP) gene transferred mice (3.3%) and PBS control mice (5.6%). The survival rate of AAV2-BPI(700)-Fc gamma1(700) gene transferred mice treated with cefuroxime sodium was 65.0%. The bacterium number in main viscera, the levels of endotoxin and proinflammatory cytokine (tumor necrosis factor-alpha and interleukin-1beta) in serum of the AAV2-BPI(700)-Fc gamma1(700) gene transferred mice were markedly lower than that of PBS control mice (P < 0.01).

CONCLUSIONSAAV2-BPI(700)-Fc gamma1(700) gene transferred mice can resist MLD E. coli infection through expressing BPI(1-199)-Fc gamma1 protein. Our findings suggested that AAV2 mediated BPI(700)-Fc gamma1(700) gene delivery could be used for protection and treatment of clinical GNB infection in high-risk individuals.

Animals ; Anti-Bacterial Agents ; therapeutic use ; Antimicrobial Cationic Peptides ; Blood Proteins ; CHO Cells ; Cricetinae ; Dependovirus ; genetics ; Disease Models, Animal ; Escherichia coli Infections ; therapy ; Gene Transfer, Horizontal ; Genetic Therapy ; Mice ; Mice, Inbred BALB C ; Proteins ; genetics ; Receptors, IgG ; genetics ; Recombinant Fusion Proteins ; genetics

AIMTo investigate the effect of pretreatment with taurine on liver injury changes and the change of tumor necrosis factor alpha and NFkappaB expression following rats limbs ischemia/reperfusion.

METHODSThe model of limbs ischemia/reperfusion injury on rats was adopted in the experiment. Wistar rats were randomized into 4 groups (n = 10): Control group, T group, I/R group and TR group. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and MDA in the plasma, MDA, MPO, calcium in liver tissues were measured by colorimetric method. The content of TNF-alpha in plasma and liver tissues was determined by radioimmunoassay. The morphologic changes were observed with HE staining. The expressions of NF-kappaBp65 in liver tissues were tested by immuno-histochemistry method.

RESULTSIt was found that against the control group, the test values of ALT, AST, et al. and expressions of TNF-alpha, NF-kappaB increased in I/R group and TR group, but values of those in TR group were lower than in I/R group.

CONCLUSIONTaurine can decrease the levels of TNF-alpha and NF-kappaB. It can mitigate the liver injury after limb ischemia/reperfusion injury in rats.

Animals ; Extremities ; blood supply ; Ischemia ; physiopathology ; Liver ; blood supply ; Male ; NF-kappa B ; genetics ; metabolism ; Protective Agents ; pharmacology ; Random Allocation ; Rats ; Rats, Wistar ; Reperfusion Injury ; metabolism ; prevention & control ; Taurine ; pharmacology ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

This study was purposed to explore the inhibitory effect of Curcumin on growth of retinoic acid-resistant acute promyelocytic leukemia (APL) cells and its mechanism. The NB4-R1, an APL cell line resistant to retinoic acid, was used as a model. The growth level of NB4-R1 was detected by MTT assay, the morphologic features of cells were observed by light microscopy, the mitochondrial transmembrane potential was determined by flow cytometry, the expressions of apoptosis-related proteins procaspase 3, caspase 3, PARP and BCL-XL were measured by Western blot. The results indicated that the sensitivity of NB4-R1 to Curcumin was consistent with NB4 though NB4-R1 was resistant to retinoic acid, Curcumin displayed inhibitory effect on growth of NB4-R1 in time-and concentration-dependent manners. The morphologic observation showed existence of apoptotic bodies in NB4-R1 cells treated with 20 micromol/L of Curcumin. The flow cytometry indicated that the mitochondrial transmembrane potential in NB4-R1 cells treated with 20 micromol/L of Curcumin obviously decreased. The Western blot detection revealed that expressions of pro-caspase 3 and BCL-XL were down-regulated, expressions of caspase 3 and sheared PAPP were up-regulated in NB4-R1 cells treated with 20 micromol/L of Curcumin. It is concluded that the Curcumin can inhibit the growth and induce the apoptosis of NB4-R1.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Curcumin ; pharmacology ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Tretinoin ; pharmacology ; bcl-X Protein ; metabolism

Animals ; Extremities ; blood supply ; Heparin ; pharmacology ; Male ; Mesentery ; blood supply ; Microcirculation ; drug effects ; Rats ; Rats, Wistar ; Reperfusion Injury ; Splanchnic Circulation ; drug effects

In order to investigate the effect of curcumin combined with all-trans retinoid acid (ATRA) on differentiation of ATRA-resistant acute promyelocytic leukemia (APL) cells and its molecular mechanism, the NB4-R1, an ATRA-resistant APL cells, was used as a model, counting of NB4-R1 and cell morphologic observation were performed, the effect of curcumin alone or combined with ATRA on proliferation, differentiation of NB4-R1 cells was detected by flow cytometry (FCM), the change of AKT phosphorylation in cell differentiation was detected by Western blot. The results showed that ATRA had no influence on NB4-R1 cell proliferation, but enhanced the inhibitory effect of curcumin on NB4-R1 cell growth; the curcumin or ATRA alone did not affect NB4-R1 differentiation; curcumin combined with ATRA could obviously induce CD11b expression; the cell morphology showed obvious differentiation characteristics. ATRA could promote phosphorylation of AKT in NB4 cells at short time, but not had effect on phosphorylation of AKT in NB4-R1 cells; the curcumin could enhance the phosphorylation of AKT in NB4-1R cells, the curcumin combined with ATRA could further enhance the phosphorylation of AKT. It is concluded that PI3K/AKT pathway inactivation may be one of the factors of drug resistance in APL and curcumin promotes differentiation of NB4-R1 through activating PI3K/AKT pathway.
Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Curcumin ; pharmacology ; Drug Resistance, Neoplasm ; drug effects ; Humans ; Leukemia, Promyelocytic, Acute ; pathology ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; Tretinoin ; pharmacology

This study was aimed to investigate the suppressive effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cell line and to explore its mechanism. C57BL/6 Mouse Treg cells were isolated by MACS (magnetic cell sorting). The purity and the expression of Foxp3 were detected by flow cytometry. The suppressive effect of sorted Treg cells on EL4 cells was detected by MTT assay. The secretion of TGF-beta1 and IL-10 was examined by enzyme-linked immunosorbent assay (ELISA). The results showed that CD4(+)CD25(+) T cells could be successfully isolated by MACS with the purity reaching 91.6% and the expression level of Foxp3 was 78.9%. The ratio of viable cells was more than 95%. Regulatory T cells could suppress the proliferation of EL4 cells effectively in the presence of antigen presenting cells (APCs). And the suppressive effect was most significant at 1:1 ratio. In addition, the suppression still existed without APCs. TGF-beta1 and IL-10 could not be detected by ELISA. It is concluded that the Treg cells can suppress T lymphoma cell in vitro. The suppressive effect of Treg cells works in dose-dependent manner, but not in cytokine-dependent manner. The mechanism of this suppression may take effect through cell-cell contact.
Animals ; Cell Line, Tumor ; Cell Proliferation ; Cell Separation ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Interleukin-10 ; metabolism ; Lymphoma ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; immunology ; Transforming Growth Factor beta1 ; metabolism