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Objective To construct luciferase reporter vector containing full-length high-mobility group box 1 ( HMGB1, GenBank NM-010439) promoter for the screening of medicine. Methods The full-length HMGB1 promoter was amplified by polymerase chain reaction ( PCR) , and then was inserted into GV238 vector to construct plasmid GV238-HMGB1-P-Luc. GV238-HMGB1-P-Luc combined with internal reference plasmid pRL was co-transfected into Hela cells ( GV238-HMGB1-P-Luc group, which served as positive control group) . Plasmid pGL3-basic combined with pRL was co-transfected into Hela cells (pGL3-basic group, which served as negative control group) . Additionally, lipopolysaccharides ( LPS, 0.2 μg/mL) was used as the activator for the positive control group (LPS group), and then sodium butyrate (SB, 10 mmol/L) was used as the inhibitor for LPS group ( SB group) . At the end of experiment hour 24, luciferase activity was detected. Results The results of digestion, amplification, sequencing and identification showed that the full length of HMGB1 promoter was 2 140 bp, and the DNA sequence was correct, without mutation. Luciferase activity in GV238-HMGB1-P-Luc group was increased as compared with that of the pGL3-basic group ( P<0.05) . Luciferase activity in the LPS group was increased ( P<0.01, compared with that of GV238-HMGB1-P-Luc group) , and then was decreased after the administration of SB ( P<0.01, compared with that of the LPS group) . Conclusion A model of luciferase reporter vector containing HMGB1 promoter has been successfully constructed. Its activity can be increased by LPS, and then is in hibited by SB. The model can be used for further screening of medicine with the activities of regulating HMGB1 promoter.
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Objective To observe the effects of Niupo Zhibao pellet, a compound traditional Chinese herbal medicine,on helper T cell(Th cell)expression in rats with endotoxin-induced rapid pulmonary fibrosis (RPF). Methods Two hundred and twenty-four Wistar rats were randomly divided into four groups:normal control,model,dexamethasone(DXM)and Niupo Zhibao pellet(NW)groups(each n=56). By using endotoxin three-hit regimen,the RPF model was established. Three days before and 7 days after the establishment of models in NW group,they were administered with the pellet by intragastric feeding,50 mg/100 g of distilled water twice a day,a total of 10 days medication. Rats in DXM group received DXM intraperitoneal injection,3.0 mg/kg once a day for consecutive 7 days. Rats in normal control group were administered with the same volume of distilled water by intragastric administration. On 1,3,7,9,14,21,28 days after the administration,blood and bronchoalveolar lavage fluid(BALF)specimens were collected. The contents of γ-interferon(IFN-γ)and interleukin-4(IL-4) in the serum and BALF were investigated by enzyme-linked immunosorbent assays(ELISA). The lung tissue was stained by hematoxylin-eosin(HE)and Van Gieson respectively. Morphological changes in lung tissue were observed under light microscope. Results HE staining showed that the pulmonary interstitial tissues of rats in model group were thickened,there was a large amount of inflammatory exudates,and micro thrombi were seem in alveolar space. The pulmonary interstitial tissues in rats of DXM group were thickened too,but the inflammatory exudate in alveolar space was much less. Compared to rats in DXM group,the rats in NW group had slightly more inflammatory exudate and their pulmonary interstitial tissues were basically normal. Van Gieson staining showed that the expression of collagen fiber in model group was obvious,that of DXM group less than the former one but not significant,while that in the NW group was reduced markedly. ELISA assays demonstrated that the levels of IFN-γin BALF and serum in model group were increased significantly,those in DXM group were lowered to the minimum on the 7th day,raised to the levels before treatment on the 14th day,and gradually declined on the 21st day. In the NW group,the IFN-γwas consistently at a high level,and then gradually declined at a slow rate. After 7 days of drug administration,the IFN-γ levels in BALF and serum at various time points in NW group were obviously higher than those in model and DXM groups〔BALF(ng/L):140.47±4.22 vs. 149.23±8.35,90.67±6.65;serum(ng/L):140.47±4.15 vs. 100.43±11.05,99.35±7.85,P<0.05 or P<0.01〕. The levels of IL-4 in BALF and serum in each group increased significantly,reached to their maximum on the 7th day,and then gradually decreased. After the drug administration for 28 days,those levels in NW group were obviously lower than those in model and DXM group〔BALF(ng/L):6.60±1.05 vs. 7.20±1.25,8.55±1.05,serum(ng/L):6.75±1.05 vs. 7.21±1.25,8.25±1.15,P<0.05 or P<0.01〕. Conclusion Niupo Zhibao pellet can suppress inflammation,ameliorate injury of lungs and inhibit lung fibrosis by promoting IFN-γsecretion,restraining IL-4 secretion and adjusting the imbalance of Th cells.
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To observe the effects of Niupo Zhibao Pellet, a compound traditional Chinese herbal medicine, on high-mobility group box-1 protein (HMGB1) expression in lung tissues of rats with endotoxin shock.
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BACKGROUND: Stem cell differentiation potential is strongly correlated with culture condition. The alteration in scaffold material surface function, three dimensional (3D) structure, and addition of growth factors can control stem cell proliferation and differentiation.OBJECTIVE: To develop 3D macroporous scaffolds with optimal porosity and porous structure to provide a microenvironment that promotes the growth of multi-potent stem cells.DESIGN: Repetitive measurement.SETTING: Department of Anatomy, College of Basic Medicine, Guangzhou University of Chinese Medicine.MATERIALS: Healthy adult SD rats were provided by the Experimental Animal Center in Guangzhou University of Chinese Medicine. Chitosan and basic fibroblast growth factor (bFGF) were purchased from Sigma Corporation (St. Louis,MO).METHODS: The experiment was performed at the Department of Anatomy, College of Basic Medicine, Guangzhou University of Chinese Medicine from March 2003 to December 2006. Using a freeze-drying method, 3D macroporous scaffolds made of different ratios of chitosan-gelatin with bFGF were fabricated that could release bFGF with controlled porosity and porous structure. Bone marrow was obtained from the femur and tibia of SD rats, and mesenchymal stem cells (MSCs) were isolated, cultured and seeded on the scaffolds with bFGF. MSCs seeded on scaffolds with no bFGF served as control. The procedure during experiment was accorded with animal ethical requirements.MAIN OUTCOME MEASURES: 3D structure and release performance of the scaffolds were observed by ELISA and scanning electron microscope; the effect of 3D macroporous scaffolds that released bFGF on MSC growth and viability were observed by HE staining, MTT, cell counting and SEM.RESULTS: There was no significant difference in pore size between scaffolds with and without bFGF (P ＞ 0.05). Scaffolds with bFGF significantly improved MSC survival rate, promoted cell adhesion, proliferation, and viability compared with scaffolds without bFGF (P ＜ 0.05).CONCLUSION: The results suggest that 3D macroporous scaffolds with bFGF release improve MSC survival on scaffolds,and lay a foundation for its application in tissue engineering.
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OBJECTIVE: To investigate the effect of Niupo Zhibao Pellet (NPZBP) on the expression of neuronal nitric oxide synthase (nNOS) in the brain of endotoxin-induced shock rats. METHODS: SD rats were randomly divided into normal control group, endotoxin-induced shock model group and NPZBP-treated group. Lipopolysaccharide (LPS) (1.5 mg/kg i.v.) and tsD-galactosamine (D-GalN) (100 mg/kg i.p.) were administered to the rats in endotoxin-induced shock model group, as well as to the rats in NPZBP-treated group after seven-day treatment, to induce the shock. The expression of nNOS in the brain of the rats in each of the 3 groups was measured by immunohistochemical methods. RESULTS: In the 3 groups, nNOS immuno-positive cells distributed widely in layer II, III, IV of the cerebral cortex, the molecular layer of hippocampus, the polymorphic layer of the dentate gyrus, the reticular formation of brain stem, and the molecular, granular and Purkinje cell layer of the cerebellar cortex. The number of immuno-positive cells in the NPZBP-treated group was slightly higher than that of the normal control group, and significantly lower than that of the model group (P<0.05) in many regions of the brain, including cerebral cortex, hippocampus, brain stem and cerebellar cortex. CONCLUSION: NPZBP can inhibit the over-expression of nNOS in wide area of the brain in endotoxin-induced shock rats.
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OBJECTIVE:To establish the method to identify the polysaccharide element in Holothuria atra Jeager by spectroscopy.METHODS:The purity of polysaccharide in Holothuria atra Jeager was determined by electrophoretic method;the infrared absorption spectrum,ultraviolet absorption spectrum,carbon spectra and hydrogen spectra of NMR were identified in structure by infrared spectrophotometry,ultraviolet spectrophotometry and NMR spectrometry respectively.RESULTS:The gel band after electrophoresis took blue color and the results of spectrum determination accountable for each other.CONCLUSION:Uniformly constituent of polysaccharose was found in the Holothuria atra Jeager,which was an acidic polysaccharose with vitriolic esterfunction,without glucopeptide;?-configuration,?-glucopyranose was found and the intramolecular hydrogen bond was formed by hydroxide radical.
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<p><b>OBJECTIVE</b>To explore the effect of jianxin pinglu pill (JPP) on arrhythmia and aquaporin 4 (AQP4) in rats with myocardial ischemia/reperfusion (I/R) injury.</p><p><b>METHODS</b>The effects of JPP on arrhythmia, mortality and AQP4 on I/R injured rats model induced by blocking left coronary artery were observed using II lead of ECG, HE stain and AQP4 immunohistochemical stain.</p><p><b>RESULTS</b>JPP showed significant effect in lowering the arrhythmia occurrence and mortality, reducing myocardial ischemic edema and injury, strengthening AQP4 expression in myocardial tissue.</p><p><b>CONCLUSION</b>JPP has the effect of preventing I/R induced arrhythmia, it might be related with its action in up-regulating AQP4 expression level in myocardium and reducing the intracellular edema.</p>
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Animais , Feminino , Masculino , Ratos , Antiarrítmicos , Farmacologia , Aquaporina 4 , Aquaporinas , Arritmias Cardíacas , Metabolismo , Medicamentos de Ervas Chinesas , Farmacologia , Traumatismo por Reperfusão Miocárdica , Metabolismo , Ratos Sprague-Dawley , Equilíbrio HidroeletrolíticoRESUMO
To investigate the osteogenesis characteristics of cultured rat mesenchymal stem cells (MSCs) under bone induction condition.
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OBJECTIVE: To observe the effect of electro-acupuncture of Neiguan (PC 6) on mean systemic arterial blood pressure and plasmic concentrations of NO and TNFalpha in endotoxin shock rats. METHODS: The model of endotoxin shock was induced by lipopolysaccharide (1.5 mg/kg i.v.) and D-galactosamine (100 mg/kg i.p.). Aminoguanidine (100 mg/kg i.p.) and electro-acupuncture of bilateral Neiguan (PC 6) were administered. A catheter was inserted into the right subclavian artery to record the change of blood pressure and the blood was abstracted out and centrifuged to determine the NO and TNFalpha concentrations. RESULTS: Electro-acupuncture of Neiguan (PC 6) retrieved the blood pressure and reduced the plasmic NO and TNFalpha concentrations. CONCLUSION: Electro-acupuncture of Neiguan (PC 6) expresses an anti-endotoxin shock effect by repressing the plasmic NO and TNFalpha concentrations smoothly and retrieving the blood pressure stably.
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[Objective] To observe the effect of serum containing Carapax et Plastrum Testudinis (CPT) in inducing the differentiation of mesenchymal stem cells (MSC) of adult rats into neurons. [Methods] The fifth generation of MSC were induced by the serum containing CPT (sero-CPT) and the expressions of neurofilament (NF) protein and glial fibrillary acidic protein (GFAP), the marks of neuron-like cells were detected by immunohistochemical method. [Results] The expression of NF protein was positive after the stimulation of sero-CPT and the expression reached the peak 12 hours after induction. [Conclusion] Sero-CPT can induce the differentiation of MSC into neurons in vitro.
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[ Objective ] To investigate the mechanism of Jianxin Pinglu Pill (JPP) in counteracting arrhythmia. [Methods] Ninety rats were randomized into 3 groups: model (normal saline), high-dose JPP (1.6 g/kg) and low-dose JPP (0.8g/kg) . The treatment lasted 7 days. Rat models with myocardial ischemia /repermsion were established by blocking left coronary artery. Ⅱ lead of electrocardiogram was recorded, connexin 43 (Cx 43) was detected by immunohistochemical assay. The effect of JPP on incidence of arrhythmia, death rate and the expression of connexin 43 in the myocardium was observed. [Results] JPP could decrease the incidence of arrhythmia and death rate obviously and enhance the expression of connexin 43 in myocardium. [ Conclusion ] JPP can counteract the damage of myocardial ischemia /reperfusion to enhance Cx 43 expression, and this is one of its possible mechanism.
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Objective: To explore the effect of Plastrum Testudinis on the three subtypes of NOS (nNOS,iNOS,eNOS) expression in rats with focal cerebral ischemia. Methods:Rat model of focal cerebral ischemia was set up by inserting nylon thread into the internal carotid artery . The expressions of nNOS, iNOS and eNOS was detected by immunohistochemical method to observe the effects of Plastrum Testudinis. Results: Expressions of nNOS and iNOS in cerebral cortex and caudate-putamen of Plastrum Testudinis treatment group were significantly lower than those of model control group (P
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Objective To investigate the effect of Plastrum Testudinis on neural stem cell after focal cerebral ischemia.Methods Rat models of focal cerebral ischemia were established by inserting nylon thread into the anterior cerebral artery.Immunohistochemical method was used to detect the expression of the neuroepithelial stem cell protein(Nestin),neurologic deficit and histological features were also observed.Results Plastrum Testudinis can decrease the neurologic dificit score and promote the expression of Nestin.Conclusion Plastrum Testudinis can alleviate the neurological symptoms and promote the proliferation of neural stem cell after focal cerebral ischemia.
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[Objective] To observe the effect of extract of Carapax et Plastrum Testudinis (ECPT) on nuclear receptor in the proliferation of rat mesenchymal stem cell (MSC) in vitro. [Methods] The rat MSC dissociated from bone marrow by density gradient method were cultured and identified by marking of bromodeoxyuridine (Brdu) and staining of CD44. Then different doses of ECPT (3333, 333.3 and 33.33 ?g/mL) were respectively added into in-vitro cultured MSC for 12, 24, 72 and 120 hours. The expression of retinoic acid receptor-?(RAR?), vitamin D receptor (VDR), estrogen receptor (ER), glucocorticoid receptor (GR), thyroid hormone receptor-?(TR?) and peroxisome proliferator-activated receptor ?(PPAR?) in MSC was detected by immunohistochemistry and immunofluorescence methods. [Results] The number of RAR?-and VDR-positive cells in ECPT groups was higher than that in the control group (P
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Objective To investigate the effects of kidney-tonifying Plastrum Testudinis on o steogenesis of cultured rat mesenchymal stem cells(MSCs ).Methods MSCs were isolated from adult rats wi th density gradient separation meth od.Osteogenesis of MSCs in the culture a dded with different concentrations of serum containing Plastrum Testudinis was evalu-ated by detecting bone glaprotein(BGP)level and observing the morphologic al features of MSCs and by alkaline ph os-phatase(ALP)and Von Kossa immunohistochemical m ethods.Results Morphological examination showed t hat the MSCs attachment formed soon after seedin g ,and grew into colonies with the appearance of fibroblastic cells.Seru m containing Plastrum Testudinis promoted the expression of alkaline phosphatase(ALP)in MSCs ,and increased BGP level and the number of calcified deposition in dose -dependant manner,the difference being significant in comparison wi th the control groups(P