OBJECTIVETo investigate the effects of fosinopril and valsartan on the expression of intercellular adhesion molecule-1 (ICAM-1) and nitric oxide (NO) induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells.

METHODSThe levels of NO, ICAM-1, and nitric oxide synthase (NOS) were determined using the nitrate reductase method, ELISA, immunohistochemical and image analyses.

RESULTSThe ox-LDL can significantly increase the expression of ICAM-1 and inhibit the expression of NO and NOS in a dose-dependent manner. Fosinopril and valsartan can significantly inhibit these roles of ox-LDL. The roles of fosinopril and valsartan were not significantly different.

CONCLUSIONFosinopril and valsartan inhibit oxidized LDL-induced expression of ICAM-1 and increase the expression of NO in human umbilical vein endothelial cells, which is one of the mechanisms of antiatherosclerosis.

Arteriosclerosis ; etiology ; prevention & control ; Cells, Cultured ; Endothelium, Vascular ; chemistry ; cytology ; drug effects ; Fosinopril ; pharmacology ; Humans ; Intercellular Adhesion Molecule-1 ; analysis ; Lipoproteins, LDL ; pharmacology ; Nitric Oxide ; analysis ; Nitric Oxide Synthase ; analysis ; Tetrazoles ; pharmacology ; Umbilical Veins ; cytology ; drug effects ; Valine ; analogs & derivatives ; pharmacology ; Valsartan

The effect of ginsenoside Rb1 on cardiomyocyte apoptosis after ischemia (30 min) and reperfusion (6 h) in rats was observed. The ischemia/reperfusion heart model was established by ligating left anterior descending branch of coronary artery in Wistar rats. The apoptotic cardiomyocytes were examined under transmission electron microscopy and counted by in situ nick end labeling (TUNEL) method and light microscopy. Results showed that (1) The apoptotic cardiomyocytes were found in ischemic regions in the ischemia/reperfusion group, but not in the sham-operating group under transmission electron microscopy; (2) The number of apoptotic cells were 134.45 +/- 45.61/field in the ischemia/reperfusion group, 0/field in the sham-operating group and 51.65 +/- 13.71/field in the ginsenoside Rb1-treated group. The differences were significant among the three groups (P < 0.01). It was concluded that myocardial ischemia-reperfusion could induce cardiomyocyte apoptosis, and ginsenoside Rb1 could significantly inhibit cardiomyocyte apoptosis induced by ischemia-reperfusion in rats, indicating that ginsenoside Rb1 could inhibit cardiomyocyte apoptosis induced by ischemia-reperfusion, thus alleviating ischemia-reperfusion injury.
Animals ; Apoptosis ; drug effects ; Female ; Ginsenosides ; pharmacology ; Male ; Myocardial Ischemia ; pathology ; Myocardial Reperfusion Injury ; pathology ; Myocytes, Cardiac ; pathology ; ultrastructure ; Panax ; chemistry ; Random Allocation ; Rats ; Rats, Wistar

OBJECTIVETo analyze the proteomic components of the sera from the patients with hepatocellular carcinomas (HCC), in search of the diagnostic markers of HCC.

METHODSImmobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2DE), silver staining, PDQuest 2DE analysis software, peptide mass fingerprint based on matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS) and SWISS-PROT database searching, were used to separate and identify the proteome of the sera from the patients with HCC.

RESULTSThe 2DE images were analysed by PDQuest 2DE software. The average matching rate was 70.2%. In IEF direction, the average deviation was (1.02 +/- 0.22) mm, in SDS-PAGE direction, the deviation was (0.97 +/- 0.14) mm. The twenty-three different protein spots were incised from silver staining gel and digested in-gel by TPCK trypsin. 15 peptide mass fingerprints (PMF) maps were obtained by MOLDI-TOF-MS. The typical peptide masses were searched in the SWISS-PROT database using PeptIdent software.

CONCLUSIONSGood reproducibility could be obtained by applying immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2DE) to separate and identify the proteome in serum. There is still the problem of efficiently removing a higher level of proteins and lipids from the serum. Identification by MOLDI-TOF-MS peptide mass fingerprint provides useful information for screening diagnostic markers of human HCC.

Blood Protein Electrophoresis ; Blood Proteins ; analysis ; Carcinoma, Hepatocellular ; blood ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Liver Neoplasms ; blood ; Proteome ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo investigate the mechanism of how vacuum sealing drainage (VSD) ameliorating ischemia reperfusion (I/R) injury in skeletal muscle I/R model.

METHODSThirty New Zealand white rabbits were divided into three groups: control (sham operation) group, I/R group, VSD+ I/R group.The ischemia of the left hind limb of the animal was induced by clamping the common femoral artery and vein. After 4 hours of ischemia, the clamp was removed and the hind limp underwent 6 hours reperfusion. VSD treated animals received the treatment at the beginning of reperfusion. The concentrations of myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) in muscular tissues were assayed. HE stained pathological section was used to evaluate the degree of edema of muscular tissues, and the immunohistochemistry was used to detect the percentage of positive cells expressing high mobility group protein B1 (HMGB1). Q-RT-PCR and Western Blot were used to detect the mRNA levels and protein expression of HMGB1 in myocyte respectively. The experimental data was tested using variance analysis.

RESULTSThe levels of inflammatory factors and antioxidant factors in muscular tissues were significantly different in the I/R group compared to the VSD group and control group (the levels of MPO in I/R group, I/R+ VSD group and control group were 0.91±0.22, 0.53±0.08, 0.31±0.10, respectively, F=26.48, P=0.000; MDA were 2.04±0.92, 1.65±1.02, 1.01±0.12, F=4.250, P=0.040; SOD were 35.97±9.23, 55.99±18.97, 61.83±14.91, F=5.240, P=0.020; CAT were 31.42±16.27, 48.50±17.86, 75.95±13.09, F=9.720, P=0.002; GSH were 1.48±0.90, 3.54±1.88, 3.84±2.08, F=5.240, P=0.020). HE staining showed an increased intercellular space ratio in the I/R group (F=16.47, P<0.05). Immunohistochemistry staining showed that percentage of HMGB1 positive myocytes in control, I/R and I/R+ VSD group are 1.94%, 18.63% and 61.36%, respectively. There was significant difference among groups (F=853.886, P<0.01). A significantly inhibited HMGB1 expression by VSD therapy was also validated by the results of Q-RT-PCR (F=50.653, P<0.01) and Western blot (F=963.489, P<0.01).

CONCLUSIONThe results from the present research suggest that VSD may attenuate skeletal muscles I/R injury by increasing the cellular antioxidative stress reaction and inhibiting the reactive oxygen species as well as the inflammatory mediators.

Animals ; Antioxidants ; metabolism ; Catalase ; metabolism ; Drainage ; methods ; HMGB1 Protein ; metabolism ; Malondialdehyde ; metabolism ; Muscle, Skeletal ; physiopathology ; Oxidative Stress ; Peroxidase ; metabolism ; Rabbits ; Reperfusion Injury ; therapy ; Superoxide Dismutase ; metabolism ; Vacuum

The characteristics of purinoceptors in the membrane of rat trigeminal ganglion (TG) neurons were studied by using whole- cell patch clamp technique. The results showed that most of neurons examined (78.9%, 142/180) were responsive to ATP in a concentration-dependent manner; the others (21.1%, 38/180) were ATP insensitive. Of the ATP-sensitive cells, the majority (95.1%, 135/142) responded to ATP with an inward current, a few (2.1%, 3/142) with an outward current, and the rest (2.8%, 4/142) with biphasic current. Small sized cells (<30 mum) responded to ATP with a rapid desensitizing inward current and were highly sensitive to vanilloid; the medium sized cells (30~40 mum) responded to ATP with slow desensitizing inward current and were not sensitive to vanilloid; while the majority of large sized cells (>40 mum) did not respond to ATP and vanilloid. The waveform of ATP-activated inward currents was related to the cell diameter. The I-V curves for both small and medium sized cells manifested obvious inward rectification. Furthermore, we studied the kinetic features of ATP-activated currents and the effects of P2 purinoceptor agonists and antagonists on I(ATP). The findings suggest that ATP receptor-ion channels are expressed differently among different types of rat TG neurons.
Adenosine Triphosphate ; metabolism ; Animals ; Animals, Newborn ; Neurons ; metabolism ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2X ; physiology ; Trigeminal Ganglion ; cytology ; metabolism ; physiology

To explore the anti-apoptotic role of electroacupuncture (EA) and its molecular mechanisms after cerebral ischemia/reperfusion (IR) of rats, by using animal model of middle cerebral artery occlusion (MCAO), the changes of the cleavage of PARP were observed by Western blot and the mRNA of heat shock protein (Hsp) 70 and Hsp90 beta detected by competitive RT-PCR after cerebral IR and EA treatment. The results were as follows: (1) The cleavage of PARP was increased in ischemic hippocampus, and EA treatment could attenuate the level of the cleavage remarkably; (2) The mRNA expression of Hsp70 was increased in the ischemic cortex and hippocampus and was further increased after EA treatment; (3) The mRNA expression of Hsp90 beta was decreased in ischemic cortex and hippocampus and the decrease was relatively slight after EA treatment. The above results demonstrated EA treatment could protect neurons from apoptosis after cerebral IR. One of the molecular mechanisms was the promotion of the inducible expression of Hsp70 and the improvement of the inhibition of the expression of Hsp90.
Animals ; Apoptosis ; Brain ; metabolism ; Brain Ischemia ; genetics ; metabolism ; Electroacupuncture ; Female ; HSP70 Heat-Shock Proteins ; biosynthesis ; genetics ; HSP90 Heat-Shock Proteins ; biosynthesis ; genetics ; Male ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; genetics ; metabolism

To observe the effect of multiple electroacupuncture (EA) on the pain threshold and the regulation of N-methyl-D-aspartate (NMDA) receptor in dorsal root ganglia (DRG) of neuropathic pain rats. Rats were prepared with a unilateral chronic constriction injury (CCI) to the sciatic nerve. EA was done in acupoints "Huan Tiao" and "Yang Ling Quan" for 30 min every day and the thermal thresholds were detected after EA at 3, 5, 7, 10, 14 days after operation. On day 14 after nerve injury, the in situ hybridization method was used to investigate the change of NMDA R1 mRNA in L4-L5 DRG. The thermal threshold reduced significantly from day 3 after operation in CCI rats. After multiple EA treatment, the ipsilateral thermal hyperalgesia relieved gradually and the thermal threshold had no difference with control side after day 5 (P > 0.05). From Day 7 after operation, the thermal threshold at each time point were significantly different compared with CCI group respectively (P > 0.05). Moreover the EA had accumulative effect. On Day 14 after operation, the NMDAR1 mRNA positive neurons and the mean optic density in ipsilateral L4-5 DRG were less than that of control side (P < 0.05), mainly in medium and small neurons. After EA treatment, the NMDAR1 mRNA positive neurons in ipsilateral DRG had no considerable difference comparing with those of control side, significantly increased comparing with CCI group (P < 0.05). It's concluded that the NMDA receptors in DRG relate closely with the generation and development of neuropathic pain. The multiple EA treatment can attenuate the thermal hyperlagesia of neuropathic pain rats and regulate the NMDA receptor.
Animals ; Electroacupuncture ; Ganglia, Spinal ; metabolism ; Hyperalgesia ; physiopathology ; Male ; Neuralgia ; metabolism ; physiopathology ; Pain Measurement ; Pain Threshold ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; biosynthesis ; genetics ; Sciatic Nerve

OBJECTIVETo investigate the expression profiles of myocardin gene during the differentiation of bone marrow-derived mesenchymal stem cell to smooth muscle cells in the conditional medium combined with a high concentration of fetal bovine serum (FBS).

METHODSMarrow-derived mesenchymal stem cells were isolated and purified from mouse femoral bone and shinbones using differential adherent methods. Cells at the third passage were induced by 20% FBS in conditioned medium, conditioned medium alone, 20% FBS or 10% FBS alone respectively. Mouse aortic smooth muscle cells were cultured as the positive control. Levels of mRNA and protein expression of myocardin and several smooth muscle cells marker genes were determined by immunofluorescence, RT-PCR and Western blot before and 3, 7, 10, 14 d after the induction. The presence of smooth muscle myofilaments was detected by using transmission electron microscope.

RESULTSNaive bone marrow-derived mesenchymal stem cells displayed multiple morphological forms including fusiform, polygon, oval, and micro-spherical, as compared to the single macro-spindle form after the induction. Typical appearance of peak valley was displayed on the 21st day after induction. At the same time, the expression of smooth muscle marker genes was reinforced along with an up-regulation of myocardin expression. Immunofluorescence study showed that the cells expressing myocardin and smooth muscle marker genes such as alpha-SMA and SM-MHC increased. Fluorescence domain of myocardin translocated from cytoplasm to nucleus and the amounts of double positive cells for myocardin with alpha-SMA or SM-MHC also increased. RT-PCR confirmed that the mRNA expression of myocardin increased gradually and remained stabilized after achieving its peak on the 7th day after induction. The expression of smooth muscle marker genes, alpha-SMA and SM22alpha, remained stable on the 10th day of induction. It was also confirmed by Western blot that the protein expression of both myocardin and alpha-SMA were markedly increased during the induction. Finally, transmission electron microscopy revealed the presence of myofilament on the 21st day after induction.

CONCLUSIONSBone marrow-derived mesenchymal stem cells can be effectively induced into smooth muscle-like cells by conditioned medium combined with 20% FBS. Myocardin plays an important role in the differentiation process of bone marrow-derived mesenchymal stem cells to the peripheral smooth muscle cells.

Animals ; Bone Marrow Cells ; cytology ; physiology ; Cattle ; Cell Differentiation ; physiology ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mice ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Nuclear Proteins ; genetics ; metabolism ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Trans-Activators ; genetics ; metabolism ; physiology ; Up-Regulation

OBJECTIVETo investigate the effects of cAMP-response element binding protein-1 (CREB-1) on transforming growth factor-b3 (TGF b3) mRNA expression and promoter activity in hepatic stellate cells (HSCs).

METHODSFreshly isolated HSCs from rats were divided into six groups: CREB-1 expression plasmid transfected group (C), siRNA-CREB-1 plasmid transfected group (S), negative control group (N), forskolin treated group (F), H-89 treated group (H), and blank group (B). Rats in each group were further sub-divided according to whether (+) or not (-) they were exposed to exogenous TGF b3. TGF b3 mRNA expression was measured by real time quantitative PCR. HSCs of the C, S, N, F, H and B groups were transfected with the TGF b3 promoter luciferase reporter plasmid (PGL3-TGF b3-P; W group), the TGF b3 promoter luciferase reporter plasmid with CRE mutation (PGL3-basic-TGF b3P-mCRE; M group) and the renilla luciferase control plasmid (pRL-SV40; control group). TGF b3 promoter activity was assessed by luciferase reporter assays.

RESULTSCompared to N(-), the TGF b3 mRNA expression was reduced to 0.69+/-0.15 in S(-) (P less than 0.05) and increased to 4.68+/-2.76 in C(-) (P more than 0.05). Compared to B(-), the TGF b3 mRNA expression was reduced to 0.57+/-0.08 in H(-) (P less than 0.05). The differences between N(+) and N(-), S(+) and S(-), B(+) and B(-), and H(+) and H(-) were all significant (P less than 0.05). The values of TGF b3 promoter activity in S(W), N(W), and C(W) were 0.062+/-0.013, 0.122+/-0.011, and 0.165+/-0.016 (P less than 0.05), but the changes of TGF b3 promoter activity in S(M), N(M), and C(M) were not significant (P more than 0.05). The values of TGF b3 promoter activity in H(W), B(W), and F(W) were 0.154+/-0.010, 0.188+/-0.016, and 0.276+/-0.031 (P less than 0.05), but the changes of TGF b3 promoter activity in H(M), B(M), and F(M) were not significant (P more than 0.05).

CONCLUSIONIncreased levels of CREB-1 mRNA or p-CREB-1 up-regulate the TGF b3 mRNA expression and promoter activity in rat HSCs. The CRE site in the TGF b3 promoter is critical for this effect, and the gene's activity becomes significantly decreased when the site is missing. Exogenous TGF b3 enhances expression of endogenous TGF b3 in rat HSCs.

Animals ; Cells, Cultured ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Hepatic Stellate Cells ; metabolism ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; Rats ; Transforming Growth Factor beta3 ; genetics

To comparatively investigate ultrastructural characteristics and expressions of AFP (alpha-fetoprotein) and Tn (Thomsen-Friedenreich-related antigen) protein in AFP negative (AFP-) and AFP positive (AFP+) primary hepatocellular carcinoma. Fourty-three cases of AFP- and AFP+ hepatocellular carcinoma (HCC) tissues and five cases of normal liver tissues were divided into three groups: control group (normal liver tissue, n=5); AFP+ HCC group (the serum AFP level was higher than 10 ng/ml, n = 22); AFP- HCC group (the serum AFP level was lower than 10 ng/ml, n=21). The ultrastructural morphology was studied by transmission electron microscopy, the expressions of AFP and Tn protein were detected by immunohistochemistry and cell image analysis. 1. The immunohistochemical study showed that (1) the expression intensity and positive rate of Tn protein in AFP- HCC group were markedly higher than that in AFP+ HCC group (P<0.01); (2) The expression intensity of AFP in AFP- HCC group was lower than that in AFP+ HCC group (P<0.01). 2. The transmission electron microscopy demonstrated that some AFP- HCC cells linked closely with each other, others dispersed loosely just as cultured cells, the remarkable morphologic features in AFP- HCC cells were simple organelles, but they were abundant in the free polyribosomes. In AFP+ HCC group, all the HCC cells linked closely together and were rich organelles in their cytoplasm, especially the rough endoplasmic reticula. In addition, mitochondria and Golgi complex were obviously observed. (1) The AFP and Tn protein had discrepancy distribution in AFP- and AFP+ HCC tissues, Tn protein may be one of the early diagnostic indicators in AFP- HCC; (2) The synthetic locations of the AFP and Tn protein were different in hepatocarcinoma cells by ultrastructural observation.
Antigens, Tumor-Associated, Carbohydrate ; biosynthesis ; genetics ; Biomarkers, Tumor ; Carcinoma, Hepatocellular ; metabolism ; ultrastructure ; Female ; Humans ; Immunohistochemistry ; Liver Neoplasms ; metabolism ; ultrastructure ; Male ; Tumor Cells, Cultured ; alpha-Fetoproteins ; biosynthesis ; genetics