| Heart failure is the endpoint of many kinds of cardiovascular diseases.Because of high incidence and high mortality,poor prognosis,it has become the world’s medical problems.Oxidative stress is one of the most important pathological steps.Pre-study showed that,Qishenkeli can improve the cardio function by its anti-oxidative capacity,Qishenkeli also can regulate the NADPH oxidase activity.Kaempferol and formononetin may be the potential active components that paly a role in regulating NADPH oxidase activity.ObjectiveTo start with oxidative stress,the cardiac cell line H9c2 was cultured in vitro to reveal the main role of kaempferol and formononetin in improving cardiac function and reducing the level of reactive oxygen species(ROS).To provide available experimental basis for developing new low toxicity and efficient NADPH inhibitors which can be used in prevention and treatment of heart failure.Methods1 Cell viability was determined by MTT method:H9c2 cells were cultured in vitro and the cell density of 1×105/ml into cell culture plates,growing up to 75%confluence,then different concentrations of kaempferol and formononetin 10-4-10-8M were added in the cell culture medium to discovery the effect on cell growth.The difference is also comparised between dose and predose with the same situation.Cell hypoxia model was constructed by keeping model groups cells cultured in Earles balanced salt solution in a hypoxia incubator.The tester was used to calculate the time and determine the best modeling time.The effects were compared between different concentration of kaempferol and formononetin treatment.2 DCFH-DA staining was used to measure ROS level.The H9c2 cells were induced by hypoxia were stained with DCFH-DA.The inverted fluorescence microscope was used to select 5 non repeated regions for recording.The differences between the different groups were compared.3 Intracellular ROS level was measured by flow cytometry:The H9c2 cells were induced by hypoxia were digested by 0.05%trypsin and then stained with 10μM DCFH-DA.After washing,the cells were resuspended with 200μl PBS buffer,then filtered with 200-400 nylon mesh filter,and then detected by the machine.4 NADPH oxidase related subunits Nox4,P67phox,P47phox,P22phox mRNA expression level were measured by Real-Time PCR.The process include:collected H9c2 cell,cleaved with Trizol,extracted RNA,reversing transcription,obtained cDNA,and finally determined by the Real-Time PCR machine.5 Western blot was used to detect the expression of NADPH oxidase related subunits Nox4,P67phox,P47phox,P22phox:H9c2 cells were colected by cell scraper,lysis and sonication extraction,electrophoresis,electroporation,coloration,and photographed for protein analysis.Results1 MTT results showed that:compared with the normal group,when treated with kaempferol and formononetin of the concentration 10-5M and above,the cell growth was inhibited(P<0.01);When pretreated for 24h,cell growth state is superior to drug-treatment group,cell growth activity statistical differences were found between the two groups(P<0.05);the cell growth of hypoxia model was inhibited more obviously with modeling time increased,there were statistically significant differences(P<0.01)between the normal group and the hypoxia model group of 8h,12h,24h;With the same treating time of hypoxia,different concentrations of kaempferol and formononetin showed different effects on the cells,it also showed that 10-6M would be the most effective concentration.2 Fluorescence microscopy results showed that:when induced by hypoxia for 8h,high intensity green fluorescence was observed under microscope in the cells,black particles were found increased in the cytoplasm of internal,the cell body was found curled up and bright,cells were changed obviously compared with normal group,the number of cells was significantly reduced;when pretreated with the kaempferol and formononetin,cells activity were improved significantly,thus kaempferol and formononetin could improve the growth state of H9c2 myocardial cells,increase cell viability and attachment.3 Flow cytometry results showed that:compared with the normal group,the fluorescence intensity of the model group was significantly enhanced(P<0.01).Compared with the model group,fluorescence intensity of kaempferol group,formononetin group and positive drug oleander group are significantly reduced(P<0:01),it showed that kaempferol and formononetin could reduce the level of ROS in cells.4 Real time PCR results showed that:compared with the normal group,the NOX4,P67phox,P47phox and mRNA increased significantly in the hypoxia model of 4h(P<0.01),by kaempferol intervention,mRNA of NOX4 and P67phox were decreased by 33%(P<0.05)and 70%(P<0.01)compared with the model group,by formononetin intervention,mRNA of NOX4 and P67phox were decreased by 13%(P>0.05)and 69%(P<0.01)compared with the model group,it showed that kaempferol and formononetin can make the expression level of mRNA for NOX4 and P67phox.Another time,after hypoxia model for 8h,compared with the normal group,mRNA of NOX4,P67phox and P47phox increased significantly in model group(P<0.01),by kaempferol intervention,mRNA of NOX4,P67phox and P47phox were decreased by 80%(P<0.01),53%(P<0.01)and 45%(P<0.05)compared with the model group,by formononetin intervention,the mRNA of NOX4,P67phox and P47phox decreased by 80%(P<0.01),18%(P>0.05),55%(P<0.05),indicating that kaempferol and formononetin were able to reduce the expression of mRNA of NOX4,P67phox,and P47phox.5 Western blot results showed that:compared with the normal group,the P67phox and P47phox protein levels of the hypoxia model of 4h were significantly increased(P<0.01),by kaempferol intervention,the protein content of P67phox and P47phox were decreased by 21%(P>0.05),19%(p<0.05)and 26%(P<0.05)compared with the model group,by formononetin intervention,the protein content of NOX4,P67phox and P47phox were decreased 9%(P>0.05)and 5%(P<0.01)compared with the model group,it shows that kaempferol and formononetin can reduced Protein expression of NOX4 and P67phox.In the meantime,after hypoxia model of 8h,compared with the normal group,protein expression of NOX4,P67phox and P47phox were significantly increased in the model group(P<0.01),by kaempferol intervention,the protein content of P67phox and P47phox were decreased by 32%(P<0.05),22%(P<0.05)and 30%(P<0.05)compared with the model group.By formononetin intervention,the protein content of NOX4,P67phox and P47phox were decreased by 35%(P<0.011)65%(P<0.01)and 24%(P>0.05)compared with the model group.It shows that kaempferol and formononetin can reduced protein expression of NOX4,P67phox and P47phox.ConclusionInduced by hypoxia,H9c2 cell proliferation was decreased;the level of reactive oxygen species increased when H9c2 cells induced by hypoxia,especially obvious in hypoxia 8h.Cell growth and proliferation can be significantly improved by pretreating with kaempferol and formononetin.Pretreated with 24h has the best effects and both Kaempferol and formononetin could protect cells effectively at the concentration of 10-6M and in the same time the expression of the active oxygen species were markedly decreased;Kaempferol can decrease mRNA and protein level of Nox4,P67phox,P47phox and the formononetin can decrease mRNA level of P67phox,P47phox and significantly decrease the level of Nox4,P67phox protein.The results suggest that both kaempferol and formononetin can protect the cardiac cells by decreasing the level of the reactive oxygen species in the cells,the effect of decrease the expression of Nox4、P67phox、P47phox also related to this.This research also provide experimental basis for finding new anti-oxidative stress ideas for clinical treatment of heart failure. |
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