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Objective To evaluate the effects of the recombinant human interleukin-1 receptor antagonist(rhIL-1ra)on a tol?uene-2,4-diisocyanate(TDI)-induced guinea pig allergic rhinitis (AR)model. Methods An AR model was established via sensiti?zation and challenge of two-step procedure using TDI in guinea pigs. Normal animals were treated only with the olive oil(TDI vehicle). Sixty adult guinea pigs were randomly divided into six groups(n=10):normal group,model group(rhIL-1ra vehicle),positive con?trol group(budesonide,25.6μg/kg),rhIL-1ra treated groups(rhIL-1ra 50,100 and 200μg/kg,respectively). From day 8 after sensi?tization,animals of all the groups were treated respectively with the agents or vehicle once a day for 14 days. During the observation pe?riod,the index of clinic score was recorded for every animal. At day 14 of the dosing,guinea pigs were sacrificed 30 min after the last TDI challenge and observation. Blood samples were taken from the abdominal aorta to prepare the serum for detection of histamine , and the nasal mucosase were dissected for histamine detection and histopathological observation. Results Compared with the guinea pigs in normal group,those in the model group exerted the typical symptoms of AR. It was shown that rhIL-1ra could improve nasal symptoms and cause a significant decrease in the instances of nasal sneezing as well. In addition,rhIL-1ra significantly reduced the concentrations of histamine in the nasal mucosa and IgE in the blood compared with those in the model group(P<0.05). Moreover, the pathological results showed that less edema,vasodilation and inflammatory cell infiltration were found in the nasal mucosa after rhIL-1ra application. Budesonide also showed the above effects with no significant difference compared with rhIL-1ra. Conclusion A guinea pig allergic rhinitis model is successfully induced by TDI. The results indicated that rhIL-1ra(50-200μg/kg)is effective in im?proving allergic rhinitis. Our findings indicated that rhIL-1ra might serve as a potential new drug for allergic rhinitis therapy.
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Objective To investigate the effects of omeprazole on the expression and the activity of CYP2C19 in HepG2 cells. Methods MTT assay was performed to screen the concentration range of omeprazole which did not inhibit HepG2 cell proliferation. Real-time quantitative polymerase chain reaction(Q-PCR)were carried out to determine the effects of omeprazole(0,0.1,1 and 10 mg/L)on the expression of CYP2C19 mRNA,Western blot was carried out to determine the effect of(0,0.1,1 and 10 mg/L)on the ex?pression of CYP2C19 protein. After incubation of different concentrations of omeprazole(0,0.1,1,10 and 100 mg/L)with microsomes prepared from insect cells expressing human P450 isozyme,the fluorescence of the samples was detected in a plate reader to analyze if omeprazole inhibited the CYP2C19 activity. Results Omeprazole could down-regulate CYP2C19 mRNA and protein expressions of HepG2 cells in a dose-dependent manner. When the concentration of omeprazole reached 10 or 100 mg/L,the fluorescence intensities obviously decreased,indicating inhibited CYP2C19 activity. Conclusion Omeprazole inhibits the expression and the activity of CYP2C19 in HepG2 cell. When omeprazole is used in combination with drugs activated via CYP2C19 metabolism,their interaction should be considered carefully.
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Objective To investigate the effect of activation of peroxisome proliferator-activated receptor β/δ ( PPARβ/δ)on protein synthesis and expression of angiotensin (Ang)Ⅱ-induced hypertrophic myocytes(MC) in vitro.Methods Hypertrophy in neonatal rat cardiac MC culture was established with AngⅡ, then the effect of GW0742 on hypertrophy was detected.The synthetic rate of protein in MC was detected by 3 H-leucine incorporation.mRNA and protein expression of atrial natriuretic IL-1βwas measured by reverse transcription-polymerase chain reaction ( RT-PCR) and Western-blotting. Results and Conclusion GW0742 could reduce the synthetic rate of protein in hypertrophic MC while down-regulating the mRNA and protein expression of IL-1β, but no changes were observed after treatment with DMSO.The result demonstrated that activation of PPAR beta/delta inhibited cardiac hypertrophy in vitro and this effect might be related to inflammatory factors.
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Objective To investigate the effect of simvastatin on the expression of interleukin-1β(IL-1β) and tumor necrosis factorα(TNF-α) in cardiomyocyte of aging rat. Methods Primary cultures of cardiomyocyte were got from aging rats. Myocyte were divided into control group, dimerthyl sulfoxide(DMSO) group, and simvastatin group, which were treated respectively by cell culture medium, DMSO and simvastatin (1 and 10 μmol/L). The expression of IL-1β、 TNF-α mRNA was evalulated by RT-PCR, and contents of IL-1βand TNF-αprotein were detected by Western blot. Results RT-PCR showed that, compared with the aging model group, IL-1β and TNF-α mRNA expression of solvent group was no significant change (P>0.05), the mRNA expression of IL-1βand TNF-αin 1 and 10μmol/L simvastatin group was significantly lower (P0.05). Conclusions Simvastatin down-regulates IL-1βand TNF-αgene expression.
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Objective To evaluate the effect of kojic acid( KA) on the immune system of mice after exposure to gam-ma-irradiation.Methods Twenty male C57BL/6 mice were divided into normal group, irradiation group, low/high doses of KA pretreated groups.Mice in normal group did not receive any treatment,while mice in other groups were sc injected with a single dose of sterile distilled water or KA(75 and 300 mg/kg, respectively) 27 h prior to a sublethal dose(4 Gy, 138.54 and 140.30 cGy/min, respectively) of whole body gamma-irradiation.The injected volume was calculated by 0.2 ml/20 g.Forty mice were sacrificed at day 2 and day 8 post-irradiation, respectively.The splenic lymphocyte transformation and spleen and thymus indexes were determined.Histopathological sections were produced, and the morphological changes were also observed.Results The splenic lymphocyte transformation capacity and spleen and thymus indexes of mice pre-treated with 300 mg/kg elatve mass KA were increased significantly ( P<0.01) compared with the irradiation group.The morphological changes in the spleen and thymus of mice in 300 mg/kg KA pretreated group were better than in the irradia-tion group.The above parameters of mice in irradiation group were injured severely in comparison with the normal group. Conclusion Acute radiation can damage the immune system of mice obviously.KA can enhance the transformation capaci-ty of lymphocytes and has marked protective effect on the immune system of mice after irradiation.
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Objective To observe mRNA and protein expression of leukotriene B4(LTB4)receptor 2(BLT2)in mice during the course of development from colitis to colitis-associated cancer. Methods ICR mice were used to establish the animal model of colitis-associated colon cancer and randomly divided into control group and experimental group. We began to administer inducer on d0. Mice were sacrificed at 2,3,5,7,9,13,and 18w,respectively. Histopathological changes in colons were examined. The protein and mRNA expression of BLT2 in colons were measured by immunohistochemical assay and real-time quantitative PCR, respectively. Results Histological study showed that with the extension of time,the lesions of mice colons aggravated,first a mild inflammation,then atypical hyperplasia,and finally to colon cancer. Immunohistochemical staining showed that the expression of BLT2 protein was low in normal colon,but high in inflammatory lesions,especially in inflammatory cells. There was no BLT2 expression in atypical hyperplasia and cancer cells,while BLT2 was highly expressed in the stroma and lumans of cancer tissue. Compared with the control group,mRNA expression of BLT2 in the experimental group was significantly higher at 2,13 and 18w(P<0.05);and very significantly higher at 3,5,7 and 9w (P<0.01). Conclusion The mouse model of colitis-associated colon cancer was developed in this study. The expression of BLT2 changed in the course of colitis development,indicating that BLT2 may play an important role in the transition process from colitis to colon cancer.
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Objective To investigate the G protein-coupled receptor kinase 2 (GRK 2) level in peripheral blood lymphocytes with cardiac func-tion in elderly patients with acute myocardial infarction. Methods This study enrolled 40 patients with acute ST-segment elevation myo-cardial infarction (STEMI) and 40 patients with unstable angina. All patients were 65 years or older. Cardiac function was evaluated by echocardiography, and the GRK 2 level in peripheral blood lymphocytes was measured. Patients with STEMI were followed up for 2 years. Results The GRK 2 level in peripheral blood lymphocytes was significantly higher in patients with STEMI than in patients with unstable angina, and was negatively correlated with left ventricular ejection fraction, cardiac output, stroke volume, and left ventricular fractional shortening. The GRK 2 level was significantly elevated in some patients with acute STEMI and poor cardiac function. Conclusions In-creased GRK 2 level in patients with acute STEMI may contribute to poor myocardial systolic function and myocardial remodeling. Meas-urement of the GRK 2 level in peripheral blood lymphocytes may assist in the evaluation of cardiac function and myocardial remodeling in elderly patients with acute STEMI.
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Aim To establish a screening system of orphan G protein-coupled receptors (oGPCRs) for their ligands based on monitoring [Ca 2+]_i in engineered cells. Methods The whole ORF of a member of human oGPCR, designated human G-protein-coupled receptor c (hGPCRc), was amplified by RT-PCR from human colon tissue and its structure was analyzed with softwares. CHO-K_1 cells were transfected with the recombinant pcDNA 3.1(+)-hGPCRc to obtain engineered CHO-hGPCRc cells. As fluorescence probe, Fluo-3 was used in assaying the [Ca 2+]_i changes induced by different compounds in the CHO- hGPCRc cells.Results Bioinformatic analysis showed that hGPCRc was localized at 13q32.2, and its corresponding amino acids formed seven-transmembrane domains and was close to human P2Y_1 receptor. It was indicated that hGPCRc was a new member of human GPCR. CHO- hGPCRc cells expressing hGPCRc were obtained successfully but no one was able to activate hGPCRc among the tested compounds indicated by the [Ca 2+]_i changes. Conclusion Although hGPCRc was even though close to human P2Y_1 receptor, it can not be activated by the known compounds which activate the P2Y_1 receptor. hGPCRc might be a new member of purine receptor family but dose not belong to P2Y_1 subfamily.
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<p><b>OBJECTIVE</b>To investigate the effect of peroxisome proliferator-activated receptors (PPARs) activators on plasminogen activator inhibitor 1 (PAI-1) expression in human umbilical vein endothelial cells and elucidate a possible mechanism.</p><p><b>METHODS</b>Human umbilical vein endothelial cells (HUVECs) were obtained from normal fetus, and cultured conventionally. Then the HUVEC were exposed to fatty acids and prostaglandin J(2) in varying concentrations with fresh media. RT-PCR and ELISA were used to determine the expression of PPAR and PAI-1 in HUVECs. Transient co-transfection of PAI-1 promoter and PPARalpha gene or PPARgamma gene to ECV304 was performed.</p><p><b>RESULTS</b>PPARalpha, PPARdelta and PPARgamma mRNA in HUVECs were detected by RT-PCR. Treatment of HUVECs with PPARalpha and PPARgamma activators-linolenic acid, linoleic acid, oleic acid and prostaglandin J(2), but not with stearic acid could augment PAI-I mRNA expression and protein secretion in a concentration-dependent manner. Proportional induction of PAI-1 promoter activity was observed through increasing amounts of PPARalpha DNA in HUVECs through a transient gene transfection assay, although the mRNA expression of the 3 subtypes of PPAR with their activators were not changed compared with controls.</p><p><b>CONCLUSIONS</b>HUVECs express PPARs. PPARs activators may increase PAI-1 expression in endothelial cells (EC). Although PPARs expression was not enhanced after being stimulated by their activators in EC, the functionally active PPARalpha is probably involved in regulating PAI-1 expression in EC.</p>
Subject(s)
Humans , Cells, Cultured , Fatty Acids , Pharmacology , Gene Expression Regulation , Plasminogen Activator Inhibitor 1 , Genetics , Prostaglandin D2 , Pharmacology , RNA, Messenger , Receptors, Cytoplasmic and Nuclear , Genetics , Physiology , Transcription Factors , Genetics , Physiology , Transcription, GeneticABSTRACT
<p><b>OBJECTIVE</b>To investigate the reasons for the rarity of metastases in skeletal muscle.</p><p><b>METHODS</b>By injecting tumor cells (Walker256 rat carcinosarcoma) through the iliac artery (experimental group) and the tail vein (control group), animal models of blood-borne metastases were established. The quadriceps femoris muscle and lungs were observed grossly and microscopically. Immunohistochemistry was applied to investigate the expression of vascular cell adhesion molecule-1 (VCAM-1) in the microvascular endothelium of these organs. Primary culture of rat skeletal muscle cells was established and conditioned medium (MCM) was collected. Effects of MCM on several tumor cell lines and the biochemical characteristics of skeletal muscle delivered tumor factor(s) were tested by MTT assay. Apoptosis and morphological examination were carried out to investigate the antitumor mechanisms of MCM.</p><p><b>RESULTS</b>In the experimental group, there were no definite metastases observed in muscle cells. In the control group, lung metastases were present in the lungs of all rats that were sacrificed at the 14th day or died spontaneously (17 rats in all). There was no significant difference between the increase in VCAM-1 in quadriceps femoris muscle 7 days after iliac artery injection and that in lungs 7 days after tail vein injection (P > 0.05). In vitro studies showed that the proliferation of tumor cell lines of mouse SP2/0 myeloma, rat Walker256 carcinosarcoma or human chronic granulocytic leukemia K562, human acute lymphatic leukemia HL-60, LS-174-T colon adenocarcinoma, PC3-M prostatic carcinoma and lung giant cell carcinoma with different metastatic potency (PLA801-C with low metastatic potency, PLA801-D with high metastatic potency) was significantly inhibited when cultured with MCM (P < 0.01 - 0.05). Proliferation of malignant cells showed a dose-dependent decrease, to a certain degree. Proliferation of normal rabbit joint epiphysial disk cells (RGP-2) were not affected by MCM. Proliferation of lung giant cell carcinoma cells with high metastatic potency showed a significant decrease even when cultured in highly diluted MCM (6.25% of primary MCM), when compared with the strain of low metastatic potency. Following ultrafiltration, boiling at 100 degrees C, and treatment with trypsin, skeletal muscle delivered tumor factor(s) were found to be a low molecular weight (MW <or= 10.0 KDa) component which was trypsin resistant but not heat resistant. The factor(s) did not induce apoptosis in K562 cells but caused direct destruction of the cytoplasmic membrane.</p><p><b>CONCLUSIONS</b>The rarity of metastases in skeletal muscles, generally accepted in the clinical setting, can be reproduced in an animal model. It does not seem to be related to VCAM-1 expression in the microvessels of these organs. Skeletal muscle delivered factor(s) play a key role in the mechanism of the rarity of metastases in skeletal muscle.</p>
Subject(s)
Animals , Humans , Rats , Cell Division , Immunohistochemistry , Muscle Neoplasms , Pathology , Muscle, Skeletal , Physiology , Rats, Wistar , Tumor Cells, Cultured , Vascular Cell Adhesion Molecule-1ABSTRACT
AIM: To investigate the expression of peroxisome proliferator-activated receptors (PPARs) in human endothelial cells, and their effects on plasminogen activator inhibitor-1 transcription. METHODS: The expression of three types of PPARs in mRNA level were detected in human umbilical vein endothelial cells(HUVECs) by using RT-PCR. Cultured endothelial cells line-ECV304 were transfected with PAI-1 promoter controlling CAT reporter gene and co-transfected with varying doses (250, 500, 1 000 ng) of expression vectors PPAR? or PPAR?.The transcripton activity of PAI-1 promoter were detected with ELISA. RESULTS: There were all three types of PPARs mRNA expression in HUVECs, while the expression of PPAR? was less than that of PPAR?( P
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The safety of hEPO gene therapy was assessed. No env gene of retrovirus was detected in the EPO-transgenic myoblasts and fibroblasts by PCR detection. Malignant transformation of transgenic cells was ruled out by various examinations including cell morphology, chromosome karyotype, soft-agar test, nude mice test and pathology. All the assessments primarily demonstrated that EPO gene therapy mediated by retrovirus was safe.
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Aim To investigate the effect of atorvastatin on AngⅡ-induced hypertrophic myocytes in vitro.Methods Hypertrophy in neonatal rat cardiac myocytes(MC) was established via culture with angiotensin Ⅱ(AngⅡ),then the effect of atorvastatin on the hypertrophy was detected.The surface area of MC was analyzed with the aid of NIH Image J software,and the synthetic rate of protein in MC was detected with()~3H-leucine incorporation.mRNA expression of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP) and Corin was measured using reverse transcription-polymerase chain reaction(RT-PCR).Results The surface area,()~3H-leucine incorporation and mRNA expression of ANP,BNP and Corin in hypertrophic myocytes were decreased after treatment of atorvastatin in a dose-dependent manner,but no change was found in the myocytes treated with DMSO.Conclusion Atorvastatin inhibits cardiac hypertrophy in vitro and the role might be independent of the cholesterol lowering effect of atorvastatin.
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AIM To investigate the effects of pioglitazone on cardiac hypertrophy in vitro. METHODS Hypertrophy in neonatal rat cardiac myocytes (MC) and cardiac nonmyocytes (NMC) was established with angiotensinⅡ (AngⅡ). mRNA expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was measured by reverse transcription polymerase chain reaction (RT PCR). MTT assay and 3H TdR uptake was used to estimate proliferation of NMC. The surface area of MC was analyzed by the aid of NIH Image J software, and the synthetic rate of protein in MC was detected by 3H leucine incorporation. RESULTS In the condition of hypertrophy, increases of surface area,mRNA expression of ANP and BNP and 3H leucine incorporation in MC and an increase of proliferation in NMC were detected, but no changes in mRNA expression of ANP and BNP in NMC. Pioglitazone inhibited the changes above and reduced mRNA expression of ANP and BNP in NMC in a dose dependent manner. CONCLUSION The results demonstrate that pioglitazone inhibits cardiac hypertrophy in vitro and it suggests that pioglitazone has a potential role in the prevention and treatment of cardiac diseases such as cardiac hypertrophy.
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The initial definition of stress and its development were briefly retrospected with elucidating the significance of stress in life sciences study. Stress is involved in a variety of physiological and pathophysiological processes. Description of the advances focusing on the relationships between stress and several body systems including nervous system, immune system and cardiovascular system etc. and on the stress molecules and signal transduction was carried out. The ultimate aim of the review is to emphasize the importance and the distinct position of stress during the development of modern bio-medicine, and to further attract more attention to the research field of stress from more scientists.
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G protein-coupled receptors(GPCRs) are the largest and most diverse group of trans-membrane proteins involved in signal transduction. They have been playing key roles in drug discover-y. Increasing orphan GPCRs (oGPCRs) whose endogenous ligands and functions are still to be identified have been discovered in recent years. It is obvious that oGPCRs might be the most important targets for innovating drugs.