Objective: The study was designed to investigate the molecular mechanism of quercitrin on osteogenic differentiation and adipogenic differentiation of rBMSCs. Methods: rBMSCs were harvested from SD rats, and determination of alkaline phosphatase (ALP) activity, quantification of mineralization by Alizarin Red S staining, and the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) by RT-PCR after rBMSCs stimulated by osteogenic induction with (0.1–10) µg/mL of quercitrin, quantification of Lipid droplet by Oil Red O staining and the mRNA expression of adipogenic differentiation marker (PPARγ C/EBPα and aP2) by RT-PCR after rBMSCs stimulated by adipogenic induction with (0.1-10) µg/mL of quercitrin. Results: Quercitrin can up-regulate the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) and increase ALP activity and mineralization after osteogenic induction, on the other hand quercitrin can suppress the mRNA expression of adipogenic differentiation markers (PPARγ C/EBPα and aP2) and decrease lipid droplet after adipogenic induction. Conclusion: This study suggested that quercitrin not only stimulated osteogenic differentiation but also inhibited adipogenic differentiation of rBMSCs, which was associated with the up-regulation of Runx2, BMP-2, and OSX mRNA expression and the down-regulation of PPARγ C/EBPα and aP2 mRNA expression.

Objective: The study was designed to investigate the antitumor effect of Pulsatilla chinensis saponins components and the synergistic effect of its effect components on tumor glycolysis. Methods: NCI-H460 cell lines were cultured in vitro, MTT assay was used to detect the inhibitory rate of various components of Pulsatilla chinensis saponins. The compatibility and synergistic antitumor effect of different effect components were studied by Calcusyn 3.0 software. Biochemical assay and Elisa assay were used to detect the glycolysis related metabolites (pyruvate, lactic acid, and glucose) and enzymes (GLUT1, HK, PKM2, and LDHA). Energy metabolism related gene members from KEGG pathway database and key protein (ERK1/2, Ras, GLUT1, MCT4) were detected by qRT-PCR and Western blotting. Results: The antitumor results in vitro showed that PSD, R13 and PSA in Pulsatilla chinensis saponins had the strongest antitumor activity for human lung cancer NCI-H460 cells and the IC50 were 5.2, 4.6, and 7.9 μg/mL, respectively. Using Calcusyn 3.0 software to confirm the distribution ratio of three effective monomers in Pulsatilla chinensis saponins and determine the synergistic antitumor effect after compatibility. The results of biochemical and Elisa assay showed that the content of pyruvate, lacate, glucose, HK, PKM2 and LDHA were significantly decreased and the GLUT1 content was significantly increased compared with the blank group (P < 0.05),; Compared with each monomer group, the content of pyruvic acid, lactic acid, glucose, HK, PKM2 and LDHA in the combined group were significantly decreased and the content of GLUT1 was significantly increased (P < 0.05). Real-time PCR results show that the combinations group has the most nodes in the network diagram, and its target was more than each monomer group. Western blotting results showed that compared with the blank group, the combinations group significantly decreased the expression of ERK1/2, Ras, GLUT1, and MCT4 protein (P < 0.05). Conclusion: The combination of effect components of Pulsatilla chinensis saponins has synergistic antitumor effect on NCI-H460 cells and the antitumor mechanism may be associated with the regulation of glycolysis.

In the treatment of hypertensive crisis, the novel Rho kinase inhibitor DL0805-2 can rapidly lower systematic blood pressure, reduce pulmonary artery pressure, and has a significant protective effect on lung injury. This experiment intends to evaluate the efficacy of DL0805-2 against pulmonary arterial hypertension (PAH) and preliminarily reveals its underlying mechanism. Animal welfare and experimental procedures are in accordance with the provision of the Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Sciences. Sprague Dawley (SD) rats were randomly divided into DL0805-2 low, medium, and high dose groups (1, 3, and 10 mg·kg^-1), bosentan positive control group, model group, and blank control group. The drug was administered daily on the 7th day after model establishment by monocrotaline injection. On the 25th day of the experiment, relevant indicators were examined to observe the therapeutic effect of DL0805-2 on pulmonary hypertension. DL0805-2 significantly relieved the abnormal changes in the physiological parameters related to PAH induced by monocrotaline, including reducing right ventricular systolic pressure, alleviating cardiac damage caused by pressure overload, and reducing the levels of endothelin-1 and inflammatory factors in lung tissues. DL0805-2 also attenuated pulmonary arteries remodeling. It was preliminarily discovered that DL0805-2 exerts preventive and therapeutic effect on PAH through Rho-kinase pathway. Our results suggested that DL0805-2 had good therapeutic effects on monocrotaline-induced PAH rat model. It intervened early in the disease process, effectively prevented the development of the disease, and reduced the mortality of the diseased animals. The mechanism is related to Rho-kinase pathway.

AIMTo study the protective effect of sodium pyruvate on ischemia/reperfusion injury following hemorrhagic shock.

METHODSRat models of hemorrhagic shock were built up. When the shed blood was infused, the rats were also randomly provided by one of normal saline, glutathione and sodium pyruvate. Rats were killed 3 hours after the reperfusion, the activity of plasma lactate dehydrogenase (LDH) and glutamic-oxaloacetic transaminase (GOT), the level of tissue malondialdehyde (MDA) and the activity of tissue myeloperoxidase (MPO) were detected. Biopsy specimens were obtained to investigate morphological changes of the myocardial, hepatic, lung and renal tissue.

RESULTSThe activity of plasma LDH and GOT, the level of MDA of hepatic, lung and renal tissue and the activity of MPO of myocardial, lung and renal tissue decreased remarkably in group given sodium pyruvate compared with group given normal saline, and the effect of group given sodium pyruvate was more remarkable than group given glutathione.

CONCLUSIONThese data support the view that sodium pyruvate shows protective effect on ischemia/reperfusion injury following hemorrhagic shock. It is possibly relevant to scavenging of oxygen free radicals, reduction of neutrophil, and anti-inflammatory response.

Animals ; Aspartate Aminotransferases ; blood ; Disease Models, Animal ; Kidney ; metabolism ; pathology ; L-Lactate Dehydrogenase ; blood ; Liver ; metabolism ; pathology ; Lung ; metabolism ; pathology ; Male ; Malondialdehyde ; analysis ; Peroxidase ; analysis ; Protective Agents ; pharmacology ; Pyruvic Acid ; pharmacology ; Rats ; Rats, Wistar ; Reperfusion Injury ; blood ; pathology ; prevention & control ; Shock, Hemorrhagic ; blood ; pathology

OBJECTIVESTo evaluate the efficacy and toxicity of the B-NHL-BFM-90 protocol in the treatment of Chinese childhood and adolescent B-cell non-Hodgkin's lymphomas (B-NHL).

METHODSForty-two untreated childhood and adolescent B-NHL were enrolled in the present study. Of them 18 cases were Burkitt's lymphoma, 16 diffuse large B cell lymphoma and 8 anaplastic lymphoma. There were 10 cases in stage II and 32 in stage III/IV. The patients were grouped by risk factors into low, medium and high risk groups. All patients were treated with the B-NHL-BFM 90 (Berlin-Frankfurt- Münster) protocol. The low risk group received A, B courses for 4 cycles, the medium risk group AA, BB courses for 6 cycles, and the high risk group AA, BB, CC courses for 6 cycles.

RESULTSComplete remission (CR) was obtained in 37 patients (88%), and partial remission (PR) in 5 (12%). Of the 5 PR patients, I received autologous hematopoietic stem cell transplantation, 3 received radiotherapy for residual disease and 1 just under watching. Major toxicity was myelosuppression and mucositis, especially in AA, BB and CC cycles, but was tolerant and manageable. Median follow-up was 20 (4 - 89) months. Kaplan-Meier method was used to analyse survival data. Two year event free survival (EFS) for all patients was 86. 24%, being 100% for stage II and 80.95% for stage III/IV.

CONCLUSIONShort term and intensive chemotherapy can improves the efficacy and survival rate of childhood and adolescent B-NHL, especially for advanced stage patients.

Adolescent ; Adult ; Antineoplastic Combined Chemotherapy Protocols ; administration & dosage ; adverse effects ; Child ; Child, Preschool ; Feasibility Studies ; Female ; Follow-Up Studies ; Humans ; Infant ; Lymphoma, B-Cell ; drug therapy ; Male ; Retrospective Studies ; Treatment Outcome

OBJECTIVETo develop a new DNA chip with coloration, which can be used for rapid and economical detection of the genotyping of hepatitis C virus (HCV).

METHODSProbes and primers were designed according to the sequence of HCV 5' non-coding region (5' NCR) to fabricate DNA chip. Experimental group consisted of 60 positive serum samples and control group consisted of 20 negative serum samples. To obtain the aimed gene, then they were hybridized with DNA chip. Finally, the results showed in a nylon film. The results of DNA sequencing of samples were used as the control in double blind experimental.

RESULTSUsing DNA chip, HCV was detected in positive of all serum specimens of experimental group and negative in control group. The determination of HCV genotype by DNA chip showed corresponding rate of 96.7% with those by sequence assay.

CONCLUSIONIt showed higher specialty and sensitivity using DNA chip to detect the genotype of HCV. It would be valuable for the clinical genotyping of HCV

5' Untranslated Regions ; genetics ; Base Sequence ; Genotype ; Hepacivirus ; classification ; genetics ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; Sequence Analysis, DNA

OBJECTIVETo construct an expression vector of anti-MM scFv-tP fusion protein and test its expression efficiency and function.

METHODSThe truncated protamine (tP) gene sequence was added to the gene of single chain antibody against the specific antigen on the surface of malignant melanoma tumor cells using PCR. A GST-fusion expression vector was constructed and the soluable protein was expressed in the E.coli system. After cleavage and purification, the purified fusion protein was obtained. The binding activity of Anti-MM scFv-tP and siRNA was detected by EMSA. Flow cytometry and confocal microscopy were used to detect the cell surface antigen binding activity of the fusion protein.

RESULTSThe expression vector of Anti-MM scFv-tP fusion protein was successfully constructed. The soluable protein could be expressed in the E.coli system, and the purified fusion protein was obtained. The anti-MM scFv-tP fusion protein retained siRNA binding ability and could directly target malignant melanoma (MM) LiBr cells.

CONCLUSIONThe recombinant GST- Anti-MM-scFv-tp expression vector was successfully constructed. The fusion protein retains siRNA binding ability and can directly target LiBr cells to provide a reliable tool for further study.

OBJECTIVE: To establish a new animal model of grading skeletal muscle contusions that could be controllable and repetitive. METHODS: The rats' gastrocnemius was injured by a new weight-dropping device designed. The force acting on gastrocnemius with a comparatively constant duration and inducing elastic deformation of the gastrocnemius was expressed with velocity (v) and deformation (DF). Instant velocity was changed to create gastrocnemius contusions. Pathological changes of gastrocnemius were graded by the gross and histological examinations of 39 rats. RESULTS: At low level of impact (v: 2 m/s, DF: 5.5 mm), mild injuries were detected in epimysium and superficial layer of gastrocnemius. At moderate level of impact (v: 2.5 m/s, DF: 6.5 mm), the injuries were observed in epimysium and whole gastrocnemius. At high level of impact (v: 3 m/s, DF: 7.5 mm), severe injuries were seen deeper to soleus with more extensive skeletal muscle damage. CONCLUSION Grading of skeletal muscle blunt force contusion is created by parameter of velocity and muscle deformation. The model could be used for further research on skeletal muscle contusions.
Animals ; Contusions/classification* ; Disease Models, Animal ; Forensic Pathology ; Male ; Muscle, Skeletal/injuries* ; Rats ; Rats, Sprague-Dawley ; Wounds, Nonpenetrating

OBJECTIVE: To investigate the changes of phospho-JNK (p-JNK) during the incised wound healing of the skin in mice and to explore the rule of the time-dependent change of p-JNK in wound age determination. METHODS: The changes of p-JNK expression in incised skin wound were detected by immunohistochemistry and Western blot. RESULTS: There was a minimal baseline staining of p-JNK in control mouse skin. Changes of p-JNK expression were mainly detectable in neutrophils in the wound specimens from 3 hours to 12 hours after injury. Afterwards, the p-JNK positive cells were mostly mononuclear cells and fibroblasts between post-injury day 1 and day 5, whereas the p-JNK positive cells were mostly fibroblasts between post-injury day 7 and day 14. Morphometrically, the ratio of the p-JNK positive cells to the total increased gradually in the wound specimens from 3 hours to day 1, and maximized at day 1 with a slight decrease from post-injury day 3 to day 5. The ratio showed a second peak in the specimens of day 7, and then decreased gradually from post-injury day 10 to day 14. The changes of p-JNK expression were observed throughout the wound healing stages by Western blot as well, with a peak expression occurring between 12 hour and day 3 after injury. CONCLUSION p-JNK may play a pivotal role in inducing apoptosis of neutrophils, mononuclear cells, and fibroblasts during skin wound healing and meanwhile, p-JNK may be a potentially useful marker for wound age determination.
Animals ; Biomarkers ; Female ; Forensic Medicine ; JNK Mitogen-Activated Protein Kinases/metabolism* ; Male ; Mice ; Phosphorylation ; Random Allocation ; Skin/injuries* ; Time Factors ; Wound Healing ; Wounds, Penetrating/enzymology*

OBJECTIVE: To investigate the expression of cannabinoid receptor I (CB1R) during mice skin incised wound healing course and time-dependent changes of CB1R in wound age determination. METHODS: The changes of CBIR expression in skin incised wound were detected by immunohistochemistry and Western blotting. RESULTS: The control group showed a low expression of CB1R detected mainly in epidermis, hair follicles, sebaceous gland and dermomuscular layer. CB1R expression was undetectable in neutrophils in the wound specimens from 6h to 12h post-injury. CB1R positive cells were mostly mononuclear cells (MNCs) and fibroblastic cells (FBCs) from 1 d to 5 d post-injury. CB1R positive cells were mostly FBCs from 7 d to 14d post-injury. The ratio of the CB1R positive cells increased gradually in the wound specimens from 6 h to 3 d post-injury, reached peak level at 5 d, and then decreased gradually from 7d to 14 d post-injury. The positive bands of CB1R were observed in all time points of the wound healing course by Western blotting. The expression peak showed at 5 d post-injury. CONCLUSION CB1R is activated during the wound healing course. The expression of CB1R is found in mononuclear cells, which could be involved in inflammation reaction. CBIR is observed in fibroblastic cells, which could participate in the wound healing. CB1R may be a potentially useful marker for determination of wound healing age.
Animals ; Blotting, Western ; Disease Models, Animal ; Fibroblasts/metabolism* ; Forensic Pathology ; Immunohistochemistry ; Male ; Mice ; Monocytes/metabolism* ; Random Allocation ; Receptor, Cannabinoid, CB1/metabolism* ; Skin/metabolism* ; Staining and Labeling ; Time Factors ; Wound Healing ; Wounds and Injuries/pathology*