ABSTRACT
This study is to investigate if madecassoside can protect against myocardial reperfusion injury in rabbit heart in vivo. The ischemia reperfusion model was established. Left ventricular function and ECG were monitored at the ischemia and reperfusion period. The infarct areas were expressed as percentage. The levels of LDH, CK, MDA and SOD were measured and C-reactive protein (CRP) in serum was measured by ELISA kit. Cardiomyocyte apoptosis were measured by TUNEL staining. A monoclonal rabbit anti-goat Bcl-2 proteins as primary antibody was used for Bcl-2 immunohistochemical staining. Treatment with madecassoside (3.2, 1.6 and 0.8 mg x kg(-1)) i.v. during ischemia reperfusion injury attenuated myocardial damage, that is, characteristic of decreasing infarct size, decreasing LDH and CK release. Activities of SOD were diminished and MDA level increased obviously in control group whereas pretreatment with madecassoside significantly blunted the decrease of SOD activity, markedly reduced the levels of MDA, CRP and cardiomyocyte apoptosis, and upregulated the expression of Bcl-2. Madecassoside has the protective effect against myocardial ischemia reperfusion injury, and effects of anti-lipid peroxidation, enhancement of SOD activity, anti-inflammation and anti-apoptosis.
Subject(s)
Animals , Male , Rabbits , Apoptosis , C-Reactive Protein , Metabolism , Cardiotonic Agents , Pharmacology , Centella , Chemistry , Creatine Kinase , Blood , Electrocardiography , L-Lactate Dehydrogenase , Blood , Lipid Peroxidation , Malondialdehyde , Blood , Myocardial Reperfusion Injury , Metabolism , Pathology , Myocardium , Metabolism , Pathology , Myocytes, Cardiac , Pathology , Plants, Medicinal , Chemistry , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Random Allocation , Superoxide Dismutase , Blood , Triterpenes , PharmacologyABSTRACT
<p><b>AIM</b>To establish a sensitive and efficient reporter gene-based screening model for finding agonists of estrogen receptor beta subtype.</p><p><b>METHODS</b>A recombinant vector pTAL-ERE-SEAP was constructed by inserting a synthetic sequence composed of five estrogen responsive elements in front of promoter of pTAL-SEAP vector. pTAL-ERE-SEAP was then transfected into human embryonic kidney (HEK293) cells. G418 (200 microg x mL(-1)) was added to select positive clones that can be induced by E2 to express reporter gene SEAP. The speciality was tested by several ligands of relative nuclear receptors of the same family. The stability of the model, the time-effect relationship, the dose-response relationship, and the immunocytochemistry staining of ERbeta expression after transfection were observed. 2 622 compounds were screened by using this model.</p><p><b>RESULTS</b>Stably transfected clones were obtained. The expression levels of reporter gene SEAP of positive clones was induced by E2 in a dose-response and time-effect relationship manners. The Z' factor value was 0.7. The expression levels of dexamethasone and other ligands were low. The result of immunocytochemistry staining showed the expression of ERbeta. E2 had no proliferating effects on stably transfected clones.</p><p><b>CONCLUSION</b>Stably transfected positive clones transfected with recombinant vector pTAL-ERE-SEAP were obtained. The positive clones may be used to screen for agonists of estrogen receptor beta subtype by measurement of luminescent value of expressed SEAP in wells of microlitre plate.</p>
Subject(s)
Humans , Alkaline Phosphatase , Genetics , Metabolism , Cell Line , Estradiol , Pharmacology , Estrogen Receptor beta , Genetics , Metabolism , Gene Expression Regulation , Genetic Vectors , Immunohistochemistry , Promoter Regions, Genetic , Genetics , Recombinant Fusion Proteins , Genetics , Metabolism , Response Elements , Genetics , TransfectionABSTRACT
<p><b>OBJECTIVE</b>AIM To establish a drug screening model based on transcriptional regulation of estrogen responsive element (ERE) and use it to screen compounds for discovering new ligands of estrogen receptor (ER) subtypes.</p><p><b>METHOD</b>A recombinant reporter vector pERE-TAL-SEAP was constructed by inserting a synthetic sequence composed of five tandem copies of EREs upstream of promoter of the reporter vector pTAL-SEAP. The pERE-TAL-SEAP and the internal control plasmid pCMV were transiently co-transfected into Hela cells expressing ER subtype or ER subtype, and the effects of pure ER agonists 17estradiol, phytoestrogen genistein and pure ER antagonist ICI182, 780 on reporter gene SEAP expression were observed.</p><p><b>RESULT</b>In the Hela cells expressing ER alpha or ER beta subtype, the expression of SEAP gene were induced in a dose dependent manner by 17-estrodiol with a maximal effect at approximately 10 nmol.L-1 and with EC50 of (80.58 +/- 8.51) pmol.L-1 and (103.90 +/- 5.29) pmol.L-1, respectively, so done by phytoestrogen genistein with a maximal effect at 1 mumol.L-1 and with EC50 of (10.86 +/- 0.75) nmol.L-1 and (39.38 +/- 2.26) nmol.L-1, respectively. The maximal level induced by estrodiol and genistein were about 7-14 fold higher than that of vehicle. The pure antiestrogen ICI182, 780 at concentration of 1 mumol.L-1 completely blocked the inductions of 17-estrodiol and genistein.</p><p><b>CONCLUSION</b>The cellular drug screening model can be established by transfecting reporter vector pERE-TAL-SEAP in Hela cell lines expressing ER alpha or ER beta. The cell lines can be used to screen compounds with estrogenicity by testing SEAP activity in the culture media of cells growing in microtitier wells. The system should provide an efficient model for screening and analyzing the activity of large numbers of ligands of ER.</p>