| Research purposes:1 From oxidative stress, using the in vitro cultured rat myocardial cell lines, primary cell and reveals Yixinjiedu formula improve cardiac function, and postpone the main effect of ventricular remodeling.2 From the key enzyme of oxidative stress mediated heart failure--the main subtypes NOX2 NADPH oxidase, NOX4 subunits of the transcription, translation and translation level, after explaining Yixinjiedu formula main effect of inhibition of NADPH oxidase activity and mechanism.3 Oxidative stress pathways through testing the change of cell signal transduction related molecules, explore NOX2, NOX4 genes in heart failure rat myocardial cells of the protective mechanism of molecular signals.4 To develop new, can be used for clinical prevention and treatment of heart failure of coronary artery disease, low toxicity and high efficiency of ROS inhibitors provide available experimental basis.The research method and content:1 Yixinjiedu formula of primary cultured rat cardiomyocytes contractile force:myocardial cell isolation and culture.Yixinjiedu formula drug-containing serum and blank serum preparation.Use AngⅡ stimulate the NADPH oxidase activity, normal rat serum and medicated serum intervention. Observe target the original beats generation of myocardial cells.2 Yixinjiedu formula the expression of NADPH oxidase H9c2 cells induced by AngⅡ mechanism research:To cultivate rat H9c2 cells, cell density in 1 × 105/mL vaccination in well culture plate, cells grow to 85% to 90% at the bottom of the bore, choose good growth state of the cell, into serum free medium synchronization 24 h, then on with the experiment. DCFH-DA determination of ROS levels.Western blot and PCR method to detect NOX2 and NOX4 expression levels.3 Yixinjiedu formula control NOX2 (or NOX4) overexpression of H9c2 cells of NADPH oxidase activity:H9c2 cell transfection empty plasmid vector (pEGFP C1) or contained can express NOX2 (or NOX4) on the open reading frame of the recombinant plasmid vector control group, By FCM and western blot method detection NOX2 (or NOX4) subunits expression level to verify that the transfection efficiency.Each group was given Yixinjiedu formula drug-containing serum, Through the NADPH oxidase activity detection kit NADPH oxidase activity of myocardial cells.4 Yixinjiedu formula of NOX2 (or NOX4 subunits) the influence of NADPH oxidase activity of RNAi H9c2 cells:Design specific N0X2 (or NOX4 subunits) small interference RNA, establish NOX2 RNAi (or NOX4 RNAi) H9c2 cell model. Gene silencing effect by qPCR and western blot method detection NOX2 (or NOX4 subunits) verified expression level. After transfection the cells were divided into group given different drug intervention, through the NADPH oxidase activity detection kit NADPH oxidase activity of myocardial cells.5 Yixinjiedu formula of NOX2 H9c2 cells (or NOX4 subunits) report the effects of gene expression:Build pGL3-NOX2 (or pGL3-NOX4) recombinant plasmid transfection H9c2 cells. After transfection the cells were divided into group given different drug intervention, Experiment end point detection luciferase report gene activity, to analyze benefit heart detoxification medicated serum of NOX2 influence of transcription.6 Oxidative stress on the Yixinjiedu formula pathway of cellular signal transduction molecules:Through western blot method to detect each group H9c2 cardiac muscle cell signal transduction pathways related molecular changes.Include AT1, p22, p47, MMP3, STAT3, MMP9.The results of the study:1 Giving AngⅡ stimulation after 24 h, model group abnormal myocardial cell rate down, compared with normal control group compared with model group, Yixinjiedu formula medicated serum levels of high, medium and low dose group can significantly improve the myocardial cells caused by AngⅡ pulsation frequency decreased, compared with AngⅡ group, high dose drug-containing serum group can significantly increase myocardial cell pulse frequency (P<0.05), and Yixinjiedu formula high,low dose drug-containing serum group can improve the myocardial cell pulse amplitude(P>0.05).2 Compared with AngⅡ model group, the Yixinjiedu formula drug-containing serum of each dose group can obviously inhibit AngⅡ cause the rise of intracellular ROS level, and small dose group of the most significant, the difference was statistically significant (P< 0.05);Medicated serum every dose group of NOX2 and NOX4 subunits mRNA expression significantly lower than the model group, compared with model group, NOX2 subunits mRNA expression of middle dose group, low dose group and the DPI control group difference was statistically significant (P<0.05;P<0.01); Yixinjiedu formula each dose group of NOX2 H9c2 cardiac muscle cells caused by AngⅡ and NOX4 protein expression compared with model group, the results are statistics show:low dose group of NOX2 and NOX4 subunits expression of the ratio of optical density in the band lower than the model group, the difference was statistically significant (P<0.01;P<0.05).mRNA and protein level of change has good consistency, the expression levels of regulation on the Yixinjiedu formula party may give priority to with the transcription regulation.3 Successfully constructed NOX2 and NOX4 subunits expression plasmid and constructed NOX2, NOX4 H9c2 cell model. Observed the Yixinjiedu formula drug-containing serum of NOX2 or NOX4 express H9c2 cells of NADPH oxidase activity influence, the results show that after NOX2 expression system, the heart of detoxification, low dose group of NADPH oxidase activity decreased significantly (P<0.01);After NOX4 expression system, Yixinjiedu formula dose group of NADPH oxidase activity decreased significantly (P<0.05).Further evidence that benefits the Yixinjiedu formula drug-containing serum on regulating effects of NADPH oxidase activation link, and the regulation effects of NOX2 type NADPH oxidase activation is most significant. It’s a matter with NOX2 and characteristic.4 Successfully constructed NOX2, NOX4 subunits small interference RNA expression plasmid and build NOX2 or NOX4 RNAi model. Observed the Yixinjiedu formula drug-containing serum of NOX2 or NOX4 subunits H9c2 cell model of NADPH oxidase activity of RNA interference effects, the results show that the NOX2 or NOX4 subunits of RNA silencing, NADPH oxidase activity of myocardial cells were significantly lower, prompt NOX2 and NOX4 type are the main form of myocardial cell NADPH oxidase, prompt NOX2 and NOX4 NADPH oxidase is the function of Yixinjiedu formula.5 Cloned NOX2 (or NOX4) promoter fragment, build NOX2 (or NOX4), the report gene expression vector constructed NOX2 H9c2 cardiac muscle cells, NOX4 report gene expression model. Observed the Yixinjiedu formula drug-containing serum of NOX2/NOX4 report gene expression of NADPH oxidase activity influence H9c2 cells, the results show that this study by transient transfection technique, eukaryotic cells found in H9c2 cells, the NOX2 (or NOX4) are active promoter fragment, after AngⅡ stimulate NOX2 (or NOX4) report gene expression quantity increased obviously, namely AngⅡ can make this increased transcription of two genes expression carrier activities.In Yixinjiedu formula, the genome of NOX4 report luciferase expression quantity compared with model group, the difference was statistically significant (P<0.05, P<0.01), report NOX2 genome luciferase expression level relatively compared with model group, there was no statistically significant difference, that the party of NOX4 gene transcription regulation is NOX2 is more apparent.So Yixinjiedu formula is by regulating gene transcription level intervention NOX2 and NOX4 NADPH oxidase activity.6 By detecting oxidative stress pathways downstream key protein:AT1 receptor, STAT3, MMP9, MMP3, p22phox, p47phox protein expression is found that Yixinjiedu formula medicated serum levels of high, medium and low dose group of H9c2 cardiac muscle cells caused by AngⅡ 6 protein expression level lower in the model group, the results are statistics show that the low dose group, DPI, six protein expression level were lower than that of model group, the difference was statistically significant (P<0.01, P<0.05);Medium and high dose group of STAT3, MMP3, p47phox, p22phox protein expression levels are lower than the model group, the difference was statistically significant (P<0.01, P<0.05).p22phox, p47phox protein expression changes suggest Yixinjiedu formula for complex formation and NADPH oxidase activation link has a regulatory role; Reduce the expression levels of AT1 receptor play its role in myocardial protection in the process of myocardial hypertrophy; Inhibit the expression of STAT3 protein, delay the process of myocardial hypertrophy; At the same time reducing the activity of MMPs, inhibit myocardial remodeling and protect myocardial cells. The party may through regulating NADPH oxidase activity, give play to inhibit myocardial cell hypertrophy and myocardial fibrosis, protect myocardial inhibition.ConclusionYixinjiedu formula party mainly applies to NOX2 and NOX4 NADPH oxidase, possible links including:1 Through the generation of the myocardial cells of the experimental results confirmed that the compound can indeed enhance myocardial cell contraction force, improve the myocardial contraction amplitude and frequency, combined with previous animal studies, the party in improving the blood circulation, reduce myocardial oxygen consumption, regulating cardiac energy metabolism in effect is remarkable.2 In H9c2 cells, AngⅡ can activate the NADPH oxidase, generate ROS, causing cell oxidative stress reaction, and through regulating NOX2 and NOX4 subtypes of NADPH oxidase activity, reduce myocardial ROS levels may be a Yixinjiedu formula play the important mechanism of the effect.3 Observe the Yixinjiedu formula drug-containing serum of NOX2 or NOX4 expression of NADPH oxidase activity influence H9C2 cells. Yixinjiedu formula in NOX2 expression system is probably by inhibiting the expression of NOX4 work; In NOX4 expression system is probably by inhibiting the expression of NOX2 work. Yixinjiedu formula party can reduce NOX2 or NOX4 subunits expression model of the NADPH oxidase activity, reveal the role is intervention NOX2 or NOX4 subtypes of NADPH oxidase gene translation, inhibition of NADPH oxidase gene expression is another way to reduce NADPH oxidase activity4 Observed the Yixinjiedu formula drug-containing serum of NOX2 or NOX4 subunits H9C2 cell model of NADPH oxidase activity of RNA interference, the influence of the results showed that the NOX2 or NOX4 subunits of RNA silencing, NADPH oxidase activity of myocardial cells were significantly lower, prompt NOX2 and NOX4 inhibition translation level expression of NADPH oxidase.5 Through technique of transient transfection, found in H9c2 cells, the NOX2 (or NOX4) are active promoter fragment, after AngⅡ stimulate NoX2 (or NoX4) report gene expression quantity increased obviously, namely Angll can make this increased transcription of two genes expression carrier activities.In Yixinjiedu formula sides under the action of reduced, so Yixinjiedu formula is by regulating gene transcription level intervention NoX2 and NoX4 NADPH oxidase activity.6 May through multiple detoxification effect components, its role in the NADPH oxidase activity regulation of multiple links consolidation effect, shown as the upstream signal transduction pathways, transcription, translation modification after the readjustment of multiple links such as integrated effect. The role of these links must involve more than one effect ingredient, may also involve the NADPH oxidase regulation network of multiple targets. By reducing the transmembrane subunits p22phox and activation subunits p47phox expression, assembly and adjust the NADPH oxidase activation;Reduce the AT1 receptor content, inhibit AngⅡ induced NOX2 or NOX4 expression level increased; By regulating the NADPH oxidase activity, reduce the downstream MMP3 and the expression of MMP9 and STAT3 protein level, thereby inhibiting ventricular remodeling, myocardial protection. |
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