| Objective: Shenfu Decoction is a commonly used classic prescription for the treatment of ischemic heart disease.It mainly treats ischemic heart disease with heart and kidney yang deficiency and deficiency syndrome of yang qi.Its curative effect has been widely confirmed clinically.However,due to the complex and diverse active ingredients of Shenfu Decoction,its protective mechanism against ischemic myocardium is not comprehensive.In the previous experiment,the effective regulation of active ingredient in Shenfu Decoction ginsenoside Rg1,Re,aconitine Aconine monomer and compatibility on the calcium ion regulation of cardiomyocytes was studied.In this study,the protective effects and molecular mechanisms of the three monomer in Shenfu Decoction of antioxidant stress on H9C2 cardiomyocytes injured by hypoxia and reoxygenation was investigated.The main indicators include antioxidase proteases Prx-3,SOD2,reactive oxygen species(ROS),pro-apoptotic factor Bax and antioxidant-related upstream signaling pathway factor Nrf-2.By investigating the changes of various oxidative stress-related protein activities,the regulation effect and possible mechanism of the three active components of Shenfu Decoction on anti-oxidation-related protein markers were explored.The objective of this study is to provides a scientific basis from the microscopic point of view for the theory of tonifying qi and warming yang of of Shenfu Decoction in the treatment of ischemic cardiomyopathy.The experimental results show that the Rg1/Re compatibility group can up-regulate the expression of Prx-3/SOD2 and decrease the level of ROS production;the Rg1/Aconine combination can activate the Nrf-2 signaling pathway and down-regulate the ROS level;the Aconine group can up-regulate the expression of Prx-3 and decrease ROS levels.It can be inferred that the three effective forms of Shenfu Decoction can exert a certain degree of anti-oxidative stress to protect the ischemic myocardium.Method: 1.Preparation of myocardial cell model of hypoxia-reoxygenation injury: H9C2 cardiomyocyte cell line was selected,hypoxia culture stage: using hypoglycemic serumfree medium,95% N2+5% CO2 mixed gas to simulate hypoxic environment to hypoxic chamber,After ten minutes of ventilation,the cells were cultured under the simulated hypoxic conditions,and then the cells treated by hypoxia were transferred to fresh complete medium and cultured in a normal incubator of 5% CO 2 at 37 ° C for reoxygenation.Finally,the CCK-8 cell survival rate was tested(survival rate was maintained at 50-60%).2.Grouping:(1)normal group(no drug,no DMSO,no hypoxia reoxygenation);(2)DMSO group(no drug,DMSO 2μl/ml,no hypoxia reoxygenation);(3)DMSO+HR group(no drug,DMSO 2μl/ml,hypoxia reoxygenation);(4)Rg1+HR group(Rg1 200μM,hypoxia reoxygenation);(5)Re+HR group(Re 200μΜ,hypoxia reoxygenation);(6)Aconine+HR group(Aconine 100μΜ,hypoxia reoxygenation);(7)Rg1+Re+HR group(Rg1 200μM + Re 200μΜ,hypoxia reoxygenation);(8)Rg1+ Aconine+HR group(Rg1 200μM + Aconine 100μΜ,hypoxia reoxygenation);(9)Re+ Aconine+HR group(Re 200μM + Aconine 100μΜ,hypoxia reoxygenation);(10)Rg1+Re+ Aconine+HR group(Rg1 200μM+ Re 25μΜ+ Aconine 100μΜ,hypoxia reoxygenation)3.Western blotting: The cells were lysed,the supernatant was taken and a 1/4 load buffer was added,and denatured at 95 ° C for 10 minutes in a water bath,then quenched on ice,and the sample preparation was completed.The desired concentration of the separation gel is prepared according to the size of the target protein molecule,and the concentrated gel is prepared,sampled,electrophoresed,and transferred.The 5% skim milk was sealed at room temperature for 1 hour,then the β-actin primary antibody was placed in the inner membrane,and the target protein membrane was placed in the corresponding primary antibody,and incubated at 4 ° C in the refrigerator overnight.The primary antibody was discarded,and TBST was washed 3 times for 10 minutes,then placed in a secondary antibody and incubated for 1 hour at room temperature on a shaker.Discard the secondary antibody and wash the TBST 3 times for 10 minutes.The developer solution A liquid B is prepared in a ratio of 1:1,and is applied to the film in the dark,and then photographed in a chemiluminescent gel imager.4.Cellular immunofluorescence: After the cell culture is completed,the culture solution is discarded,washed with PBS,and fixed with methanol for 10 minutes.The methanol was discarded,washed with PBS for 3 minutes for 3 minutes,incubated with Triton 100 for 15 minutes,Triton 100 was discarded,and 5% BSA was added for 1 hour.Add primary antibody and store in a refrigerator at 4 °C overnight.The cells were washed 3 times with PBS for 10 minutes,and protected from light,and incubated with fluorescent secondary antibody for 1 hour.Wash in PBS 3 times for 15 minutes.DAPI or glycerol was added dropwise to the slide,and the cell climber was applied face down.Temporary storage in the wet box,taking a photo with a fluorescence microscope.Result: 1.Results obtained by CCK-8 cell viability assay(survival rate is maintained at 50-60%),selected modeling conditions: sugar-free serum-free medium,95% N2+5% CO2 mixed gas simulation Oxygen environment was applied to ventilate the hypoxic chamber for ten minutes.After culturing the cells for 24 hours under hypoxic conditions,the cells with hypoxia were transferred to fresh complete medium and cultured in a normal incubator with 5% CO2 and 37 °C for3 hours.2.Results of Western blotting detection of Prx-3: the level of Prx-3 in the DMSO+HR model group was significantly decreased(P<0.05,N=6),and the Prx-3 level is significantly increased in Aconine+HR,Rg1+Re+HR,Rg1+Aconine+HR groups which cardiomyocytes was injured by hypoxia-reoxygenation(P<0.01,N=6).3.Results of Cellular immunofluorescence method to detect each indicator: 1)Prx-3: DMSO+HR group(model group)Prx-3 content was significantly lower than DMSO(blank group)group(P<0.05,N=4);the Prx-3 level were all effectively increased Re+HR group,Rg1+Re+HR group,Aconine +HR group and Rg1+Re+Aconine+HR group(P<0.05),and the difference was statistically significant.In particular,the Prx-3 protein content was significantly increased in the Rg1+Re+HR group(P<0.001).2)SOD2: There was a significant difference between DMSO+HR group and DMSO blank group(P<0.05,N=4),and SOD2 content decreased.Only Rg1+Re+HR group in the drug treatment group could increase the SOD2 content of cardiomyocytes injured by hypoxiareoxygenation(P<0.01,N=4).3)ROS: The ROS level was significantly increased in DMSO+HR group compared with DMSO blank group(P<0.05,N=4).The ROS levels of myocardial cells in hypoxia-reoxygenation injury were decreased in all testing groups,with P values less than 0.01,which was statistically significant compared with the DMSO+HR group,except for Re+Aconine+HR group(P>0.05).4)Bax level was significantly increased in DMSO+HR group compared with DMSO blank group(P<0.05,N=4),therefore,Bax level was observed unchanged after application of the tested drug in damaged H9C2 cardiomyocytes.5)Nrf-2: the Nrf-2 signal was significantly decreased in DMSO+HR group compared with the DMSO blank group(P<0.05,N=4).In the drug treatment group,only the Rg1+Aconine+HR group had enhanced Nrf-2 activity and the difference was statistically significant(P<0.05,N=4).Conclusion: 1.Cell modeling: cell modeling protocol was improved and upgraded as follows: 95% N2+5% CO2 mixed gas was exchanged for ten minutes to simulate the hypoxic environment,then the cells were cultured for 24 hours under hypoxia,and reoxygenated after the end of hypoxia.Incubate for 3 hours.2.The Rg1·200μM+Re·200μM+HR group was initially determined to have relatively stable anti-oxidative stress.The mechanism of action may be the results of the reduction the ROS production level of myocardial cells induced by hypoxia-reoxygenation and improvement of Prx-2.,SOD2 level.3.Aconine·100μM+HR group showed up-regulation of Prx-3 in myocardial cells damaged by hypoxia-reoxygenation,and down-regulation the level of ROS in cells,which can be considered to have anti-oxidative stress effect.4.With comprehensive analysis from the experiment results,the effect of Rg1·200μM+Aconine·100μM+HR group on reducing the level of ROS production in myocardial cells induced by hypoxia-reoxygenation may be related to the activation of Nrf-2 signaling pathway.5.In this research,the different doses of Rg1,Re,Aconine and compatible active ingredients of each group of Shenfu Decoction were not effective in regulating the proapoptotic factor Bax. |
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