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Aim: To establish the standard hypoxia/reoxygenation (H/R) injury model of H9c2 myocardial cells and evaluate the intervention effect of notoginsenoside R, based on RTCA technology. Methods: H9c2 myocardial cells were inoculated into the E-Plate board, 3 wells by a group, then the growth curve and cell index of different hypoxia time, different reoxygenation time and notoginsenoside R, intervention were determined respectively, with the growth status of H9c2 myocardial cells reflected by cell index. Results: Cell survival rate was (48.82 ±5.32)% after hypoxia treatment for 4h and reoxygenation treatment for 16h with replacement of serum-free, glucose-free DMEM medium before hypoxia, and no significant difference was found between the results measured with MTT method. Notoginsenoside R, could protect the hypoxia/reoxygenation injury of H9c2 myocardial cells without time dependence. Conclusions: RTCA technology is an effective means to establish the standard H/R injury model of H9c2 myocardial cells, which also has some guiding significance in discovering new drug targets and mechanisms.
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Aim To assess the regulatory effects of Po-lygonatum sibiricum polysaccharides(PSP)on Toll-like receptor 4 (TLR4 )-myeloid differentiation factor 88 (MyD88)-nuclear factor κB(NF-κB)signaling path-way in anoxia /reoxygenation-H9c2 myocardial cells. Methods The H9c2 myocardial cells cultured in vitro were randomly divided into groups:control group (C group),hypoxia /reoxygenation group (H/R group), PSP group,TLR4 inhibitor group(TAK-242 group)and PSP +TLR4 inhibitor group(PSP +TAK-242 group). The cells were cultured in normal condition for 27 h in C group.The cells were subjected to 21 h hypoxia fol-lowed by 6 h reoxygenation in H/R.Definitely,the cells in TAK-242,PSP and PSP +TAK-242 groups were treated with PSP and TAK-242 with the final con-centration of 1 .5 g·L -1 and 1 μmol·L -1 for 1 2 h before 21 h hypoxia,then the cells were exposed to the normal culture condition for another 6 h.After the treatment,cell survival rate was tested by MTT method. The contents of tumor necrosis factor-α(TNF-α)and interleukin-1 β(IL-1 β)were determined by enzyme-linked immunosorbent assay(ELISA).The protein ex-pression levels of NF-κB and inhibitor κBα(IκBα) were detected by Western blot,and the expression lev-els of TLR4 and MyD88 mRNA were detected by fluo-rescence quantitative PCR method.Results Compared with H/R group,the cell survival rates were significant-ly increased,while the inflammatory cytokines contents and NF-κB protein expression were dramatically de-creased in groups PSP,TAK-242 and PSP +TAK-242, whereas the NF-κB expression was significantly down-regulated,and the IκBα protein expression was in-creased.The mRNA expression levels of TLR4 and MyD88 were markedly decreased.Conclusion PSP might protect H9c2 myocardial cells against H/R inju-ry,which may be associated with the inhibition of TLR4-MyD88-NF-κB pathway.
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Objective To establishment the model of angiotensin Ⅱ (AngⅡ) inducinged cardiomyocyte hypertrophy in H9c2 myocardial cells, and observinge the the changes of ubiquitin binding protein leveljunction protein ubiquitination level. Methods Myocardial cell isolated and subcultured H9c2 were cultured in vitro andmyocardial cell, they were divided into three groups in different AngⅡ concentration (10-8, 10-7, 10-6 mol/L), with in different hours (0, 6, 12, 24, 48, 72 h), and there is also a control group; measured the total protein contentlevel were measured useby BCA method , measured the number of cardiomyocytes in the same area were measured, and the relative surface area of myocardial cells were calculated. The expression of ubiquitin binding protein cardiomyocyte hypertrophy was time- and concentration-dependently induced by AngⅡ, wheremanifested, as total cellular protein content,relative cell surface area and the expression of ubiquitin binding proteinubiquitinated desmin significantly increasing compared with the control group (P < 0.05), which were most significant in 10-7 mol/L AngⅡtreated group for 48 h. Conclusions Cardiomyocyte hypertrophy model induced by 10-7 mol/L AngⅡ for 48 h , was successfully established , where H9c2 cardiomyocyte hypertrophy was induced by 10-7 mol/L AngⅡ in 48 h, and ubiquitin binding proteinubiquitinated desmin significantly increased in cardiomyocyte hypertrophy.
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Objective: To study the effects of Compound Granules of Yinxingye (Ginkgo leaf) on H2O2-induced cellular injury in H9c2 myocardial cells of rats. Methods: H9c2 cells were treated with H2O2 to establish a myocardial injury model, and then pretreated by Compound Granules of Yinxingye at the concentration of 25, 50, 100, and 200 μg/mL for 24 h. Cell viability was measured by methyl thiazolyl tetrazolium (MTT). Morphological changes were observed by phase contrast microscope. Lactic dehydrogenase (LDH), creatine kinase-MB (CK-MB), malondialdehyde (MDA), superoxide dismutase (SOD), and nitric oxide (NO) levels were detected by biochemical kits. Intracellular reactive oxygen species and calcium ion concentration and mitochondrial membrane potential were measured by laser confocal microscopy. Results: The exposure of H9c2 cells to 500 μmol/L H2O2 for 6 h could reduce cell viability significantly and induce oxidative stress injury. The Compound Granules of Yinxingye pretreatment could improve the cell viability, improve the damage cell morphology change, reduce LDH, CK, MDA, and NO levels in the cell supernatant, increase SOD activity. ROS generation and concentration of intracellular calcium could be reduced and the in mitochondrial membrane potential declined. Conclusion: The Compound Granules of Yinxingye has the protective effect on H2O2-induced cellular injury in H9c2 myocardial cells.
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Aim Tostudytheeffectsofferulicacid (FA) on doxorubicin (DOX) induced cellular injury inH9c2ratmyocardialcells.Methods H9c2cells were treated with 1μmol·L-1 DOX treated for 24 h to establish a myocardial injury model. 10, 20, 40μmol ·L-1 FA was added 2 h before DOX treatment. Cell viability was measured by cell counter kit ( CCK-8 ) . Morphological changes were observed by phase contrast microscope. LDH, CK, MDA, SOD levels were detec-ted by biochemical kits. Intracellular level of reactive oxygen species ( ROS) was examined by DCF-DA stai-ning with flow cytometry. Cellular apoptosis was detec-ted by AO-EB staining and DNA agarose gel electro-phoresis. The expression of caspase-3, Bax, Bcl-2 was evaluatedbyWesternblot.Results Exposureof H9c2 cells to DOX led to decrease in cell viability, in-crease in stress and apoptosis. FA pre-treatment im-proved cell viability in a dose-dependent manner, at-tenuated leakage of LDH and CK, and reversed mor-phological changes induced by DOX. FA suppressed DOX-induced oxidative stress as evidenced by reducing ROS and MDA generation and increasing SOD enzyme activity. FA depressed myocardial apoptosis by down-regulating pro-apoptotic protein caspase-3 and Bax, whereas up-regulating apoptosis inhibitory protein Bcl-2.Conclusions FAhasaprotectiveeffectonDOX-induced injury in H9c2 cells. This protection may re-sult from inhibition of myocardial oxidative stress and apoptosis.