| Purpose:By observing the method of supplementing Qi,nourishing Yin and removing meridian obstruction with myocardial ischemia reperfusion rat myocardial cell apoptosis and the influence of JAK2/STAT3 signaling pathways regulating mechanism,to explore the ginseng and salvia granules for promoting blood flow(GSG)in the treatment of myocardial ischemia reperfusion injury in the mechanism of action,and to provide the scientific theoretical basis of science for the clinical application of supplementing Qi,nourishing Yin and removing meridian obstruction in the treatment of myocardial ischemia-reperfusion injury.Material and method:In this study,60 healthy male SD rats as the research object,body quality of 250 ~ 280 g,from Liaoning university of traditional Chinese medicine laboratory animal center,raised within clean level environment for 7 days.Intervention drug: the ginseng and salvia granules for promoting blood flow(By Ginseng 10 g,Astragalus membranaceus 20 g,Radix ophiopogonis,15 g,Angelica sinensis 15 g,Salvia miltiorrhiza 15 g,Ligusticum wallichii 20 g)with the traditional particle preparation methods,provided by the department of pharmaceutical preparation in liaoning university hospital of TCM.Diltiazem Hydrochloride as the controlled drug: manufactured by Tianjin production tanabe pharmaceutical co.,LTD.,30 mg/piece,approved by: H12020126.60 healthy SD rats were randomly divided into 6 groups: sham-operated group,model group,western medicine group(diltiazem hydrochloride),low dose group,medium dose group and high dose group.As GSG group,low dose equivalent to 9.37 g/(kg·d)content,the medium dose equivalent to 18.75 g/(kg · d)content,and the high dose equivalent to 37.5 g/(kg · d)content,all made for 6 ml solution,fill the stomach 2 times one day,every time 3ml.Positive drug control,giving diltiazem hydrochloride to fill the stomach,the dose of 4.23 mg/(kg · d),made from 6 ml solution,every time 3 ml.The sham group and control group were given normal saline lavage,every time 3 ml,2 times a day,a course of 4 weeks.At the end of the experiment,test the cardiac function;Preparation of serum specimens,cryopreserved standby;Myocardial tissue specimens,frozen for later use.The H9c2 rat myocardial cells were randomly divided into normal control group,hypoxia reoxygenation group,low dose group and high dose group of TCM,for 4 groups,and intervented with serum containing of shuangshen tongmai granules.Cell proliferation and apoptosis were detected after the experiment.Experiment 1: Using HE staining to observe the myocardial tissue pathology.TUNEL method to detect myocardial cell apoptosis.IHC method to detect P-JAK2,P-STAT3 protein expression.By the ALC-MPA more biological record blood flow signal analysis system to record the hemodynamic parameters: left ventricular systolic function(LVSF),left ventricular diastolic function(LVDF).Experiment 2: Using Western blot method-detect the myocardial tissue of rats P – JAK2,Bax,Bcl-2 and Caspase3 expression of protein.Experiment 3: Determined by MTT method was used to detect myocardial cell proliferation,the IF method for Bax,Bcl-2 protein expression.Statistical Analysis: SPSS 22.0 statistical software was used for statistical analysis.The data was expressed as the mean plus/minus standard deviation(?x±s).One-way analysis of variance was used for comparison between groups.All the data were tested for normality and homogeneity of variance and were in accordance with normal distribution and variance.P<0.05 was a significant difference in results,with statistical significance.Results: 1.The changes in groups of rats I lead electrocardiogram(ECG)before and after the molding.Each group of left anterior descending coronary artery ligation in rats after I lead ecg ST segment increased to different extent,ranged from 0.1 to 0.3 mv,prove that each group rat has copy with successful myocardial ischemia model.2.Hemodynamic detection in ratsHemodynamic parameters of rats after treatment are changed,which reflect the index of left ventricular systolic function(LVSF)significantly decreased(P<0.05).Whereas reflect the index of left ventricular diastolic function(LVDF)significantly increased(P < 0.05);Chinese medicine high dose group in the most obvious group in hemodynamic improement.Compared with the sham-operated group,the model group's left ventricular systolic function significantly reduced whereas left ventricular diastolic function increased significantly,the difference was statistically significant(P < 0.05).Compared with model group,each treatment group of the rats LVSF were significantly higher.LVDF in western medicine,each dose treatment group of Chinese medicine were significantly lower,differences was statistically significant(P < 0.05 or P < 0.01).Compared with the model group,GSG-H group's LVSF was significantly higher,the LVDF was significantly lower,the difference was statistically significant(P < 0.01).3.The pathological changes of myocardial tissue were observed by HE stainingSham group: The myocardial cells were closely arranged without obvious edema,muscle fibers were not twisted,the nucleus was centered,the cell membrane was intact,and there was no obvious inflammatory infiltration.MIRI group: Myocardial cells edema,scattered nuclei,widened interstitial space,muscle fiber distortion,disorder,inflammatory cell infiltration,a large number of red blood cells in the intercellular space.DH group: Compared with the MIRI group,myocardial cell edema was significantly reduced,and myocardial fiber rupture was less.Inflammatory cell infiltration and red blood cell exudation were observed.GSG-L group: Compared with the MIRI group,myocardial edema was reduced,cell boundaries were clear,red blood cell exudation was more,and there was bleeding.GSG-M group: The myocardial cells were well arranged.Compared with the MIRI group,the myocardial cells had a smaller edema area,no obvious rupture of muscle fibers,and no obvious inflammatory infiltration.GSG-H group: Compared with the MIRI group,the damage was significantly reduced,with clear cell boundary,slight edema in tissue space,no rupture of muscle fibers,and less red blood cell exudation.4.Use TUNEL assay to detect myocardial cell apoptosisMicroscopically,the normal myocardial nuclei are blue and the apoptotic nuclei are tan.There were few apoptotic cells in the myocardial tissue of the sham group.In the model group,there were more and more apoptotic cells.Compared with the sham group,the apoptosis index of myocardial cells in the model group was significantly increased,and the difference was statistically significant(P<0.01).Compared with the model group,the apoptosis index of rat myocardial cells in each drug treatment group was decreased,and the difference was statistically significant(P<0.01).Compared with the DH group,the apoptosis index of myocardial cells in the low-dose Chinese medicine group was increased,and the difference was statistically significant(P<0.01).Compared with the DH group,The apoptosis index of myocardial cells increased in the medium dose group(P<0.05).5.Expression of P-JAK2 and P-STAT3 was detected by IHC assayCompared with the sham group,the mean optical density(IOD/area)of P-JAK2 protein and P-STAT3 protein expression in the model group was significantly decreased(P<0.01).Compared with the model group,P-JAK2 protein and P-STAT3 protein expression IOD/area were increased in each treatment group,and the difference was statistically significant(P<0.05 or P<0.01).Compared with the western medicine group,the expression of P-STAT3 protein IOD/area increased in the high-dose Chinese medicine group,and the difference was statistically significant(P<0.05).6.Expression of Bax and bcl-2 proteins in myocardial cells of each groupCompared with the sham group,Bax expression was significantly increased and bcl-2 protein expression was significantly decreased in the model group,the difference was statistically significant(P<0.01).Compared with the model group,Bax protein expression was decreased whereas bcl-2 expression was increased in all the treatment groups,the difference was statistically significant(P<0.01 or P<0.05).Compared with the model group,Bax expression was significantly decreased in the high-dose group of TCM,and bcl-2 protein expression was significantly increased in the western medicine group and the high-dose group of TCM,with statistically significant difference(P<0.01).Bax expression was significantly lower in the high-dose group than in the western group,with statistically significant difference(P<0.05).7.Expression of P-JAK2 in myocardial cells of each groupCompared with the sham group,P-JAK2 protein expression was significantly decreased in the model group,and the difference was statistically significant(P <0.01).Compared with the model group,P-JAK2 protein expression was increased in the western medicine group,the low dose group and the high dose group,with statistically significant difference(P<0.01).8.Expression of caspase-3 protein in myocardial cells of each groupCompared with the sham group,the expression of caspase-3 in the model group was significantly increased,and the difference was statistically significant(P<0.01).Compared with the model group,caspase-3 protein expression was decreased in each treatment group,and the difference was statistically significant(P<0.01 or P<0.05).Compared with the model group,the expression of caspase-3 in the high-dose Chinese medicine group was significantly decreased,and the difference was statistically significant(P<0.01).9.Comparison of proliferation activity of rat myocardial cells in each groupCompared with the normal control group,the myocardial cell activity in the hypoxia/reoxygenation group was significantly decreased,and the difference was statistically significant(P<0.01).Compared with the hypoxia/reoxygenation group,the myocardial cell activity was significantly increased in the low-dose group and the high-dose group,and the difference was statistically significant(P<0.01).Compared with the low-dose group,the myocardial cell activity was significantly increased in the high-dose group,with statistically significant difference(P<0.01).10.Comparison of the apoptosis rate of myocardial cells in each groupCompared with the normal control group,the apoptosis rate of myocardial cells in the hypoxia/reoxygenation group was significantly increased,and the difference was statistically significant(P<0.01).Compared with the hypoxia/reoxygenation group,the apoptosis rate of myocardial cells in the low-dose group and the high-dose group was significantly reduced,and the difference was statistically significant(P<0.01).Compared with the low-dose group,the apoptosis rate of myocardial cells in the high-dose group was significantly reduced,with statistically significant difference(P<0.01).11.Morphological observation of myocardial cellsThe morphology of cultured myocardial cells was observed by inverted microscope.The myocardial cells in the normal control group were triangular,fusiform or irregular with clear nucleoli.Myocardial cells in hypoxia/reoxygenation group were round or oval with unclear nucleoli.The myocardial cells in the low-dose group showed oval or irregular shape with unclear nucleoli,and the activity of myocardial cells was slightly higher than that in the hypoxia/reoxygenation group.Myocardial cells in the high-dose group were oval,fusiform or irregular,with slightly clear nucleoli,and the activity of myocardial cells was higher than that in the hypoxia/reoxygenation group.12.Bcl-2 protein expression was detected by immunofluorescenceCompared with the normal control group,bcl-2 vimentin expression was significantly reduced in each group,the nuclear shape was irregular,and the fluorescence intensity was significantly reduced.In the hypoxia/reoxygenation group,a few myocardial nuclei were sunken inward in the middle and constricted into two nuclei.Compared with the hypoxia/reoxygenation group,the expression of bcl-2 vimentin in the low-dose group showed an increased trend,with regular nuclear shape and increased fluorescence signal.Compared with the hypoxia/reoxygenation group,bcl-2 vimentin expression was significantly increased in the high-dose Chinese medicine group,with regular nuclear shape and strong fluorescence signal.13.Bax protein expression was detected by immunofluorescenceCompared with the normal control group,Bax vimentin expression,nuclear shape and fluorescence intensity were significantly increased in each group.In the control group,a few myocardial nuclei were constricted into multiple nuclei.Compared with the hypoxia/reoxygenation group,Bax vimentin expression in the low-dose group of traditional Chinese medicine was decreased,and the nuclear shape was irregular,and the fluorescence signal was weakened.Compared with the hypoxia/reoxygenation group,Bax vimentin expression was significantly decreased in the high-dose group,with irregular nuclear shape and weak fluorescence signal.Conclusion: 1.The method of supplementing Qi,nourishing Yin and removing meridian obstruction can obviously improve left ventricular function in rats with myocardial ischemia-reperfusion injury.2.The method of supplementing Qi,nourishing Yin and removing meridian obstruction can effectively improve the apoptosis of myocardial cells caused by myocardial ischemia-reperfusion injury,and the mechanism may be related to the up-regulation of P-JAK2 and P-STAT3 protein expressions.3.The method of supplementing Qi,nourishing Yin and removing meridian obstruction may play a protective role in apoptosis which induced by myocardial ischemia-reperfusion injury by activating JAK2/STAT3 signaling pathway.4.The method of supplementing Qi,nourishing Yin and removing meridian obstruction can up-regulate the protein expressions of P-JAK2 and Bcl-2,and down-regulate the protein expressions of Bax and Caspase-3 in rats with myocardial ischemia-reperfusion injury.5.The method of supplementing Qi,nourishing Yin and removing meridian obstruction can improve the proliferation activity of cells and reduce the apoptosis rate of cardiomyocytes.The myocardial cells were protected by down-regulation of Bax protein and up-regulation of bcl-2 protein. |
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