Experimental Study On The Effect Of Shuangshen Tongmai Granule On NF-κB Signal Pathway In Rats With Myocardial Ischemia Reperfusion

Purpose:To investigate the effects of Ginseng and Salvia Miltiorrhiza Granules（GSG）on cardiac function and classical signal transduction pathway of NF-kappa B in MIRI rats,and observed the effect of GSG-containing serum on NF-kappa B signal transduction pathway in Hypoxic-reoxygenation model of neonatal rat cardiomyocytes through in vitro cell experiment.We also discussed the protective effect and mechanism of GSG on MIRI,and provided important theoretical support for clinical medication.Material and method:Fifty SPF-grade 12-week-old SD rats,half male and half female,weighed（230±20）g.Fifty SD rats were randomly divided into five groups:normal group,model group,western medicine group,low dose group and high dose group.According to the surface area of human body and rat,the dosage of drugs in low and high doses was 7.31 g/kg,13.45 g/kg respectively.The normal group and model group were treated with the same amount of saline.The above groups were administered twice a day,15ml a time,continuously for four weeks.After ECG evaluation and screening,the left anterior descending coronary artery of rats was ligated for 30 min of ischemia and 120 min of reperfusion.The ECG was monitored throughout the operation to determine whether the model was successful or not and to monitor the cardiac function.Blood was taken from abdominal aorta of rats and serum was separated.The levels of glutathione peroxidase（GSH-PX）,catalase（CAT）,endothelin（ET）,myeloperoxidase（MPO）and inflammatory mediators IL-1beta,IL-6 and TNF-αwere measured by ELISA.Intercellular adhesion molecules（ICAM-1）and Vascular cell adhesion molecule（VCAM-1）were detected by immunohistochemistry.Apoptotic index of myocardial cells was assessed by TUNEL staining.Pathological changes of myocardial tissues were observed.The expression of nuclear transcription factor-kappa B（NF-κB）,IκBαprotein was detected by Western blot.Thirty SD rats were randomly divided into two groups:normal control group（10rats）,Ginseng and Salvia Miltiorrhiza Granules group（20 rats）,Chinese herbal medicine with 7.31 g/kg,13.45 g/kg granule solution,twice a day,twice a day for two weeks.The normal control group was given Ginseng and Salvia Miltiorrhiza Granules the same amount of saline.After 5 days of continuous gastric perfusion,blood was collected,abdominal aorta was found after anesthesia,blood was collected under negative pressure,supernatant was taken,then frozen at-80℃for reserve.Rat cardiomyocyte strains were inoculated into culture medium at 37℃for 24 hours in a 5%CO₂ incubator,and then subcultured to establish hypoxia-reoxygenation model of cardiomyocyte,which was divided into four groups:normal serum group,model group,low-dose serum group of GSG and high-dose serum group of GSG.Normal group added normal rat serum with the same concentration as drug-containing serum at 37℃,5%CO₂,90%N₂,5%O₂ into the normal group for 24 hours,model group placed myocardial cells in incubator（37℃,5%CO₂）for 6hours under hypoxia,then placed at 37℃,5%CO₂ medium for 12 hours after reoxygenation.Chinese medicine serum group added the best working concentration of Chinese medicine-containing serum（filtration,sterilization,4preservation）.Storage,dilutionto the appropriate concentration of about 1000 times.The activity of lactate dehydrogenase in cell culture medium of each group was detected by colorimetry according to the instructions of LDH detection kit.CCK-8 method was used to detect the activity of cardiac myocytes.The cultured cardiac myocytes were inoculated into 96-well plates with appropriate density.Six compound holes were set in each group.Cell culture medium was added to 100 microliters,CCK-8 and 10 microliters were added to each hole.The cells were placed in the incubator for4-5 hours.After completion,OD value was detected at 450 nm by enzyme labeling instrument,and apoptotic rate was detected by Annexin-V FITC/PI double staining.The apoptotic rate was detected by TUNEL and the changes of IL-1β,IL-6 and TNF-αin cell culture medium were detected by ELISA.The myocardial cells of each group were added with pharmaceuticals and reagents according to the above experimental methods.Immunofluorescence staining was used to observe the expression of NF-κB,IκBαand Iκκβ.The experimental data were processed and analyzed by SPSS17.0 software.（Mean+SD）,one-way ANOVA,q-test,Chi-square test and P<0.05 were used to compare the two groups.Results:Effect of Ginseng and Salvia Miltiorrhiza Granules（GSG）on Cardiac Function in MIRI Rats.1.1 Effect of GSG on Hemodynamics of Rats in Different Groups.Effects of GSG on cardiac hemodynamics in MIRI rats.Compared with the model group,the LVSP of GSG group increased significantly（p<0.05）,the maximum left ventricular ascending rate（+dp/dtmax）increased（p<0.05）,the LVDP of GSG group decreased significantly（p<0.05）,the maximum left ventricular descending rate（-dp/dtmax）decreased（p<0.05）,and the heart rate change（D-value）of GSG group and western medicine group was smaller（p<0.05）.1.2 Effect of GSG on serum GSH-PX,CAT,ET and MPO in MIRI rats.After GSG intervention,ET and MPO decreased,GSH-PX and CAT increased,and there was significant difference compared with model group（p<0.05）.Compared with the sham-operated group,the apoptotic index of myocardial cells in the model group increased significantly（p<0.05）,while the apoptotic index of myocardial cells in the western medicine group and GSG groups decreased significantly（p<0.05）.1.3 Effect of GSG on myocardial pathological changes in MIRI rats.In the sham-operated group,myocardial fibers were arranged tightly and regularly,interstitial edema was slight,only slight congestion,micro-exudation of surrounding tissues and slight dilation of blood vessels were observed.In the model group,the myocardial fibers were distorted and broken,the cells were swollen,the interstitial space was edema,the nuclei were disordered,the myocardial cells were hypertrophic and the staining was loose.In western medicine group,myocardial fibers were less broken,arranged disorderly,myocardial interstitial loosening,myocardial interstitial vasodilation,surrounding edema and exudation were observed.The arrangement of myocardial fibers in the low dose group was more regular than that in the MIRI group,with edema and exudation in the interstitial space and infiltration of inflammatory cells.The myocardial fibers in the high dose group of GSG were arranged more regularly,the breakage of myofibers was less,and the edema of interstitial tissue was lighter.2 Effect of GSG on NF-κB Signaling Pathway in MIRI Rats2.1 Effects of GSG on serum levels of inflammatory mediators IL-1β,IL-6 and TNF-α.The levels of IL-1β、IL-6 and TNF-αin the sham-operated group were not significantly increased,while the levels of IL-1β,IL-6 and TNF-αin the model group were increased.Compared with the model group,the contents of IL-1β,IL-6 and TNF-αin the GSG group were significantly decreased.2.2 Effect of GSG on VCAM-1 and ICAM-1 contents in myocardium.VCAM-1 content changes:sham operation group myocardial tissue can be seen a small number of stained cells,model group myocardial tissue stained cells more,more dense,western medicine group stained cells less than model group,The low-dose group of GSG stained cells less than model group,the high-dose group of GSG stained cells least.Changes of ICAM-1 content:In sham-operated group,tiny stained tissues were observed in myocardial tissue,in model group,the stained parts of myocardial tissue were larger and more dense,in western medicine group,the stained tissues were less than those in model group,in low-dose group,the stained tissues were less than those in model group,and in high-dose group,the stained tissues were the least.2.3 Effect of GSG on apoptotic rate of rat cardiomyocytes.Myocardial apoptotic index,no obvious apoptotic cells were found in myocardial tissue of sham-operated group,apoptotic cells in myocardial tissue of model group were more and more dense,apoptotic cells in western medicine group were less than model group,apoptotic cells in low-dose group of GSG were less than model group,apoptotic cells in high-dose group were less than model group.2.4 Effect of GSG on the expression of NF-κB and IκBαin MIRI rats.Compared with sham-operated group,the expression of NF-κB protein in model group was significantly increased（p<0.05）,the expression of IκBαprotein was significantly decreased（p<0.05）.After GSG and Hexinshuang were given,the expression of NF-κB protein in GSG groups was significantly decreased（p<0.05）,and the expression of IκBαprotein was significantly increased（p<0.05）.3 Effect of GSG Serum on NF-κB Signaling Pathway in Rat MIRI Cardiomyocytes.3.1 Changes of LDH activity in cell culture medium of each group.The LDH activity of cardiomyocyte culture medium was 93.47U/L in the normal group,241.54U/L in the model group and 175.02U/L in the low dose group of GSG serum and 175.02U/L in the high dose group of GSG serum,respectively.LDH activity in liquid was 142.77 U/L,which was significantly lower than that in model group（p<0.05）.3.2 Changes of myocardial cell viability in each group.The survival rate of myocardial cells in the normal group was 80.65%,that in the model group was 42.14%,that in the model group was significantly lower than that in the normal group（p<0.05）,that in the low-dose group was 58.32%,that in the model group was significantly higher（p<0.05）,and that in the high-dose group was 60.41%,which was significantly different from that in the model group（p<0.05）.3.3 Changes of apoptotic rate of myocardial cells in each group.The apoptotic rate of cardiomyocyte in normal group was 2.84%,and that in model group was 26.62%,which was significantly different from that in normal group（p<0.05）;the apoptotic rate of cardiomyocyte in low dose group was 14.38%,and that in high dose group was 6.16%,which was significantly different from that in model group（p<0.05）.3.4 Changes of IL-1β,IL-6 and TNF-αcontents in supernatants of cardiomyocyte culture media in each group.Compared with the normal group,the contents of IL-1β,IL-6 and TNF-αin the model group increased significantly（p<0.05）.Compared with the model group,the contents of IL-1β,IL-6 and TNF-αin the serum group of GSG decreased significantly（p<0.05）.3.5 Changes of expression of protein kinase NF-κB,IκBαand Iκκβin myocardial cells of each group.Compared with the normal group,the green fluorescence intensity of cardiac myocytes in the model group increased significantly,indicating that the expression of NF-κB and Iκκβprotein kinase increased（p<0.05）,the IκBαdecreased,suggesting that the content of NF-κB and Iκκβincreased,compared with the model group,the green fluorescence intensity in the serum treatment group of GSG decreased,indicating that the Chinese medicine GSG containing pharmaceutical Qing inhibited the expression of NF-κB and Iκκβprotein,Enhance the IκBα,the serum group of GSG could reduce the expression level of NF-κB and Iκκβprotein kinase（p<0.05）.Enhance the IκBα（p<0.05）.Conclusion:1 Ginseng and Salvia Miltiorrhiza Granules can protect the cardiac function of ischemia-reperfusion rats,improve various cardiac function indicators,reduce the incidence of arrhythmia,and reduce oxidative damage of myocardial cells.2 Ginseng and Salvia Miltiorrhiza Granules can reduce the levels of IL-1β,IL-6 and TNF-α,which are key inflammatory mediators in the signal transduction pathway of NF-κB,decrease the expression of protein kinase NF-κB,increase the expression of anti-apoptotic protein IκBα,and decrease the apoptotic rate of myocardial cells.3 The serum containing Ginseng and Salvia Miltiorrhiza Granules can reduce the content of inflammatory factors IL-1β,IL-6 and TNF-α,reduce the content of protein kinase NF-κB and Iκκβ,and increase the content of IκBα,so as to improve the survival rate of hypoxic-reoxygenated cardiomyocytes and inhibit the apoptosis rate.