Effect Of Zhenwu Decoction-containing Serum On The Apoptosis Of Rat Cardiomyocytes Induced By Isoproterenol

Purpose:By using serum pharmacology,observe the effect of ZhenWu Decoction-containing serum on isoproterenol-induced oxidative damage of rat cardiomyocytes and its effect on the expression of apoptosis-related proteins from different angles,and investigate the effect of Zhenwu Decoction on oxidative damage of myocardial cells.And the protective mechanism of apoptosis.For the clinical application of Zhenwu Decoction in the treatment of heart failure provides a scientific theoretical basis.Materials and Methods:For experimental animals,20 healthy Wister rats(individual or male)with fresh 1-3 days of age were used for the isolation and culture of cardiomyocytes.In addition,10 healthy male Wistar rats aged 3 months(250-270 g)were used to make Zhenwu Decoction containing serum.The experimental medicines included Jingzhenwu Decoction.Its five-flavored Chinese herbal medicines consisted of Radix Astragali,Atractylodes Macrocephala,Radix Paeoniae Alba,Aconite and Ginger.Provided by the Chinese Herbal Medicine Bureau of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,prepared by the preparation room and decocted,according to the equivalent dose conversion method of humans and animals,the rat dose was converted,and its dose group was equivalent to 18.9 g/kg of crude drug.·d).Captopril has a dosage of 12.5 milligrams per tablet.The drug production lot number is the national drug license number H3 1022986,and the manufacturer is Sino-US Shanghai Squibb Pharmaceuticals.In addition,the main experimental reagents required are isoproterenol and the like.First,Zhenwu Decoction containing serum and Captopril-containing serum were prepared.Prepare Zhenwu decoction containing drug serum preparation method:take normal Wistar rats to irrigate Zhenwu Decoction and captopril,and measure,method and reference equivalent to the in-vivo test part(in this experiment,the calculation method of rat dose is:Calculated from the body surface area of humans and rats,according to the equivalent dose coefficient conversion method,calculate the dose of rats,the formula is:Rat’s drug dose(mg/kg)＝Adult’s medication Dosage(mg/d)/60 kgx 7),rats were gavaged for 7 consecutive days.One hour after the last gavage,blood was taken from the abdominal vena cava,and the serum wasseparated under aseptic conditions and inactivated by heating(according to the method recommended by the Task Force of the National Natural Science Foundation of China"Methodology Study of Chinese Herbal Compound In Vitro Experiment").Cardiomyocytes were then isolated and cultured:they were sterilized with 75%alcohol,and the thoracic cavity was opened under aseptic conditions in a clean bench,and the excised portion of the ventricle was placed in a petri dish containing pre-cooled PB S.The residual blood was removed by pipetting with a pipette and transferred to another petri dish.A suitable amount of 0.06%trypsin(0.125%trypsin and 0.1%collagenase Ⅱ)was added.The ventricular muscles were cut into pieces of 1 mm 3 in size,transferred to a 50 ml Erlenmeyer flask,digested at 37℃,and stirred every 1 min with a magnetic stirrer.Digest for 10 minutes,discard the first digestion supernatant,add appropriate amount of 0.06%pancreatin,continue digestion for 8 min,repeat 5 times,collect the supernatant for each digestion,and add equal volume of 10%FB S medium to stop the digestion.After centrifugation at 1500r for 8 min,the supernatant was discarded,an appropriate amount of the culture solution was added,and the cell suspension was prepared by pipetting.The cell suspension was placed in a 5%CO 2 incubator at 37°C.in a static culture.We then used a differential number for 1.5 hours depending on the time of attachment of fibroblasts and cardiomyocytes.After 48 hours,the solution was changed and 200 v 5-brdu 0.1 mmol/L was added to inhibit the proliferation of fibroblasts,and the cells were statically cultured in a 37℃,5%CO 2 incubator.Take a group of cells with good logarithmic growth phase and good growth status.The experimental groupings were divided into four groups:blank control group,model control group,captopril group and Zhenwu Decoction group.In the blank control group,the medium was changed to DMEM containing 10%blank rat serum,and the culture was continued in the next step and continued for 48 hours.Model control group:The medium was changed to DMEM containing 10%blank rat serum and isoproterenol at a concentration of 1μmol/L and cultured for 48 h.Captopril group:DMEM medium containing 10%blank rat serum,10 μmol/L isoproterenol,and DMEM medium at a concentration of μmol/L captopril were used to continue the culture for 48 hours.Zhenwu Decoction group:The medium was changed to DMEM medium containing 10 μmol/L isoproterenol plus 10%Zhenwu Decoction serum for 48 h.This experimental study was based on in vivo studies of pre-protocol animal experiments and was transferred to in vitro experimental studies.Firstly,through cell extraction and culture,isoproterenol on cell growth inhibition assay,immunofluorescence on mitochondrial membrane potential,and MTT assay on absorbance,the effect of Zhenwu Decoction-containing serum on cardiomyocyte survival and mitochondrial membrane potential was investigated.The serum containing Tang-Medicine has a protective effect on isoproterenol-induced oxidative damage of rat myocardial cells.This study lays a foundation for further research on the anti-apoptosis mechanism of Zhenwu Decoction serum and its anti-apoptosis mechanism.Then,the anti-apoptosis effects of Zhenwu Decoction serum were elucidated from different aspects by different methods from the aspects of morphological detection and quantitative detection.They were:immunofluorescence detection of mitochondrial membrane potential(△Ψ)and rhodamine 123(rhodamine 123).The mitochondria were stained to detect the mitochondrial membrane potential;AO/EB double fluorescent staining was used to detect apoptosis;TUNEL was used to determine the MDA content of the cultured cells and the number of apoptotic cells was counted;flow cytometry was used to detect the cell cycle distribution.Apoptosis rate.The expression of Bcl-2,Bax,Caspase-3,Sirt-1 and other related proteins in cultured cardiomyocytes was determined by Western-blot method.The protective mechanism of Zhenwu Decoction-containing serum on cardiomyocyte oxidative damage was studied in terms of protein expression.Anti-heart failure treatment provides experimental and theoretical basis.Statistical analysis:Statistical analysis SPSS 13.0 statistical software was used for the statistical data,and x2 test was used for count data.The analysis of measurement data was expressed as mean±standard deviation(±s).The analysis among multiple groups was performed using one-way ANOVA.Methods:The comparison between the two groups was statistically significant with the q test,α=0.05,P<0.05.Results:1.Isoproterenol has an inhibitory effect on the growth of cardiomyocytes.After isoproterenol 10μmol/L is applied to cardiomyocytes for 6 hours,it can significantly reduce the cell survival rate.After 48 hours of cardiomyocytes in each group and the corresponding interventions during the screening of serum concentration of Zhenwu Decoction,isoproterenol group,isoproterenol+40%drug-containing serum group,isoproterenol+20%drug-containing serum The activity of cells in the group of isoproterenol+10%drug-containing serum and isoproterenol+5%drug-containing serum was lower than that in the blank group(P<0.05);isoproterenol+card Topiraline,isoproterenol+40%drug-containing serum,isoproterenol+20%drug-containing serum,isoproterenol+10%drug-containing serum and isoproterenol+5%The activity of serogroup cells was higher than that of isoproterenol group(P<0.05);isoproterenol+20%drug-containing serum group,isoproterenol+10%drug-containing serum group and isoproterenol+captop The survival rate of the Lee group was similar to that of the cell and the difference was not statistically significant.Different concentrations of Zhenwu Decoction containing serum act on the cells,and as the concentration of drug-containing serum increases,the cell survival rate increases,but at the same time we consider that the higher the concentration of the drug-containing serum,the greater the cytotoxicity may be,and 10 The drug-containing serum group and captopril have similar effects on cell viability,which provides experimental basis for selecting 10%drug-containing serum as the optimal concentration.2.Immunofluorescence detection of mitochondrial membrane potential(△Ψ):Compared with the normal group,the mitochondrial membrane potential of the myocardium significantly changed,and the mitochondrial fluorescence was significantly increased in the model group compared with the normal group,while the Zhenwu Tang,Western medicine and model group were significantly improved,and the mitochondrial fluorescence was expressed.Weakened.3.Apoptosis was detected by double fluorescent staining with AO/EB:In the blank control group,the nuclear chromatin structure of most cells was normal,the cell membrane was intact,and the coloration was uniform;in the control group,a large number of necrotic cells and late apoptotic cells were seen,and membrane damage was observed.The nuclear membrane is intact,and the red and orange colored chromatin are dense patches or fragments;the Zhenwu Tang group has normal nuclear chromatin structure,complete cell membrane,and more nuclear staining,bright green,and dense patches or debris early.Apoptotic cells and scattered late apoptotic cells and necrotic cells;captopril group showed a large number of nuclear staining,bright green,dense patches,or debris-like early apoptotic cells and nuclear staining of orange-red compact patches or fragments.Late apoptotic cells.After TUNEL analysis and flow cytometry,the apoptotic rate of isoproterenol group increased significantly,while the apoptotic rate of cardiomyocytes in isoproterenol+captopril group and isoproterenol+drug-containing serum group was significantly lower.Isoprenaline group.4.TUNEL analysis:The amount of MDA can often reflect the degree of lipid peroxidation of cells,indirectly reflecting the degree of cell damage.Our experimental results showed that the content of MDA in the model group was significantly higher than that in the blank group(P<0.05).The MDA content in the cardiomyocytes after the addition of Zhenwu Decoction was significantly lower than that in the model group(P<0.05).Flow cytometry results showed that the apoptosis rate of the model control group was significantly higher than that of the blank control group(P<0.01),and the apoptotic rate of the captopril group and the Zhenwu Tang group compared with the model control group.Significantly decreased,there is a significant difference(P<0.05),Zhenwu Decoction group and captopril group had no significant difference(P>0.05).5.The expression of Bax,Bcl-2,Caspase-3 and Sirt-1 protein in cardiomyocytes of each group was detected by Western blotting.The expression of Bcl-2 protein and the expression of Bax protein were increased in the model control group.Bcl-2/Bax The ratio was significantly lower than that of the blank control group(P<0.01).Compared with the model control group,the expression of Bcl-2 protein was increased and the expression of Bax protein was decreased in Zhenwu Decoction group and captopril group(P<0.05).Caspase-3 in cardiomyocytes of the model control group was significantly decreased compared with the control group(P<0.01);the expression of Caspase-3 protein was decreased in the Zhenwu Decoction group and the captopril group compared with the model control group.(P<0.05);Sirt-1 protein expression was increased in Zhenwu Decoction group and captopril group compared with model control group(P<0.05).Conclusion:1.Zhenwu Decoction containing serum can significantly improve the mitochondrial membrane potential of myocardial cells in oxidative inj ury.2.The serum containing Zhenwu Decoction can significantly reduce the MDA content of myocardial cells in oxidative inj ury rats.3.Zhenwu Decoction containing serum can significantly inhibit the apoptosis of myocardial cells induced by oxidative inj ury.4.The serum containing Zhenwu Decoction can significantly reduce the expression of Bax protein and increase the expression of Bcl-2 protein in oxidatively injured rat cardiomyocytes.5.The serum containing Zhenwu Decoction can significantly reduce the expression of Caspase-3 protein and increase the Sirt-1 protein expression in oxidatively injured myocardium.