| Objective:Dracocephalum heterophyllum Benth Flavonoid（DHBF）is traditional Uyger medicine which is wildly used for the treatment of hypertension,coronary heart disease and myocardial hypertrophy and so on.In our study,DHBF was extracted and separated,and we could observe the effects of DHBF on myocardial hypertrophy and tension of thoracic aorta to rats and explore the possible mechanism by establishing the rat model of cardiac hypertrophy and isolated vascular ring.we also could provide the basis of theoretical for the development and effective use of Uygur local medicinal materials.Methods:（1）The method of ethanol was used to extract the total flavonoids in Dracocephalum Heterophyllum Benth,and The content of total flavonoids was determined by ultraviolet spectrophotometry;（2）Cardiomyocytes of the primary cultured neonatal rat were used as models,Myocardial hypertrophy was induced by AngⅡ(1μmol·L-1)or NE(2μmol﹒L-1).The cells were divided into normal cell control group,and AngⅡor NE model group,AngⅡ(1μmol·L-1)+Valsartan(50μmol·L-1)or NE+Prazosin(50μmol·L-1)group,AngⅡor NE+different concentrations of DHBF(10,25,50μmg·L-1)group.（1）CCK-8 method was used to observe the activity of myocardial cells,（2）RT-PCR technique was used to detect the expression of mRNA of c-fun,atrial natriuretic peptide（ANP）,brain natriuretic peptide（BNP）andβ-myosin heavy chain（β-MHC）,the internal factor were glyceraldehyde-3-phosphate dehydrogenase（GAPDH）;（3）Western blot was used to detect the protein expression of Ca N,Ca MK II,HDAC and NFAT-3 in myocardial cells.the internal factor of wereβ-actin.（4）Confocal laser scanning was to detect the surface area of myocardial cell and intracellular Ca2+concentrations([Ca2+]i).（5）enzymatic reaction of broken cells showed the activity of Ca2+-ATPase,;（6）colorimetric assays were used to assess nitric oxide（NO）levels and the activity of nitric oxide synthase（NOS）.These were all observe the influence of these factors on myocardial hypertrophy.（3）Male young Wistar rats（200-220g）were sacrificed by dislocation,thoracic aorta was removed quickly.With the model of isolated thoracic aorta ring of rat,vascular ring tension as index,we could observation the effect of DHBF(0.015,0.030,0.060,0.120,0.36 g·L-1)on vascular ring tension pre-contractioned by norepinephrine（NE）and potassium chloride（KCl）（3）Male young SD rats（220g250g）were sacrificed by dislocation,quickly remove the aorta.With isolated rat thoracic aorta model,vascular ring tension as index,the maximum amplitude of vasoconstriction is 100%by NE(1μmol·L-1)or KCl(60mmol·L-1).The change of vascular tension was reflected by the ratio between the amplitude of vascular tension and the maximal contraction amplitude induced by NE or KCl.We could observe the effect of DHBF((0.015,0.030,0.060,0.120,0.360 g L-1)to the vascular tension of intact endothelial and removal of vascular endothelium,resting state and vascular rings of intact endothelial which was pretreatment by L-NAME or MB that was pre-contraction by NE or KCl,and the ffects of DHBF to intracellular Ca2+release and extracellular Ca2+influx of the vascular tension of removal of vascular endothelium.Results:（1）In our study,the total flavonoids content was 27.44mg·g-1,and the extraction rate of total flavonoids was 11.11%.（2）Compared with the cell control group,cardiomyocytes were stimulated 72h by AngⅡ(1μmol·L-1),which could increase the expression of mRNA of c-Jun,ANP,BNP andβ-MHC all from（1.00±0.01）to（2.78±0.29）,（2.61±0.38）,（2.30±0.24）and（2.46±0.32）increased by 178%,161%,130%and 146%respectively（P<0.05）;decrease the survival rate of cardiomyocytes from（94.58±4.26）to（78.69±2.37）,reduced by 17.02%;The concentration of[Ca2+]i increased from（1.00±0.14）to（1.60±0.50）,increased by 60.00%and the activity of Ca2+-ATP decreased from（0.92±0.15）to（0.36±0.05）,decreased by 60.08%（P<0.05）;the protein expression of Ca MKII,HDAC,Ca N and NFAT-3 from（0.77±0.02）,（0.63±0.01）,（0.67±0.02）and（0.62±0.02）to（1.03±0.01）,（1.17±0.05）,（1.24±0.04）and（1.16±0.04）increased by 33.76%,85.71%,85.07%and 87.09%respectively（P<0.05）;the surface area increased from（1±0.01）to（1.42±0.19）,increased by 42.00%（P<0.05）;the concentration of NO decreased from（1.42±0.12）to（0.72±0.03）,by decreased 49.29%and the activity of NOS decreased from（0.82±0.05）to（0.46±0.11）,decreased by 43.90%（P<0.05）.DHBF which concentration was 10 to 50μmol﹒L-11 and Valsartan(50μmol.L-1)could inhibit the expression of mRNA of c-Jun,ANP,BNP andβ-MHC,the survival rate decreased and surface area increased that were induced by AngⅡ.It also could inhibit the concentration of[Ca2+]i increased,the activity of Ca2+-ATP decreased,the protein expression of Ca MKII,HDAC,Ca N and NFAT-3 increased the concentration of NO and the activity of NOS decrease(P<0.05,.and the effective of DHBF was better than Valsartan to inhibit to Cardiomyocyte hypertrophy（P<0.05）.（3）Compared with the cell control group,cardiomyocytes were stimulated 48h by NE(2μmol·L-1),which could increase the expression of mRNA of c-Jun,ANP,BNP andβ-MHC all from（1.00±0.01）to（3.02±0.36）,（2.87±0.31）,（2.34±0.26）and（2.27±0.22）increased by 202%,178%,134%and127%respectively（P<0.05）;decrease the survival rate of cardiomyocytes from（94.48±4.19）to（75.62±2.24）,reduced by19.96%;The concentration of[Ca2+]i increased from（1.00±0.01）to（1.72±0.49）,increased by 72.00%and the activity of Ca2+-ATP decreased from（1.01±0.14）to（0.41±0.06）,decreased by（P<0.05）;the protein expression of Ca MKII,HDAC,Ca N and NFAT-3 from（0.79±0.01）,（0.62±0.01）,（0.69±0.02）and（0.64±0.02）to（1.21±0.05）,（1.19±0.04）,（1.27±0.06）and（1.18±0.06）,increased by53.16%,57.00%,71.01%and 44.00%respectively（P<0.05）;the surface area increased from（1±0.01）to（1.49±0.21）,increased by 49.00%（P<0.05）;the concentration of NO decreased from（1.36±0.11）to（0.65±0.05）,by decreased 39.53%;and the activity of NOS decreased from（0.86±0.06）to（0.52±0.10）,decreased by 43.90%（P<0.05）.DHBF which concentration was 10 to 50μmol·L-11 and Valsartan(50μmol·L-1)could inhibit the expression of mRNA of c-Jun,ANP,BNP andβ-MHC,the survival rate decreased and surface area increased that were induced by NE.At the same time,it also could inhibit the concentration of[Ca2+]i increased,the activity of Ca2+-ATP decreased,the protein expression of Ca MKII,HDAC,Ca N and NFAT-3 increased the concentration of NO and the activity of NOS decrease（P<0.05）.and the effective of DHBF was better than Valsartan to inhibit to Cardiomyocyte hypertrophy（P<0.05）.（4）Compared with the control group,the endothelium intact group had a significant relaxation effect on the thoracic aortic rings of rats precontracted by NE or KCl.（P<0.05）in a concentration-dependent manner,and there was no significant difference between the two groups（P>0.05）.Compared with the aortic rings of intact endothelial vessels which was preincubated by L-NAME or MB for 15min,vasoconstriction induced by NE,and they were interved by DHBF(0.015,0.030,0.060,0.120,0.36 g·L-1)with isoflavone.the effect of DHBF on vasodilation of precontracted vascular rings was significantly inhibited stimulated by NE（P<0.05）.（5）Compared with the control group,the blood vessels which were preincubated by DHBF for 15mins.Different concentrations of DHBF(0.015,0.030,0.060,0.120 and 0.36g·L-1)could significantly inhibit the intracellular calcium release during the contraction of vascular smooth muscle cells induced by NE（P<0.05）.（6）Compared with the control group,in the calcium free K-Hsolution,different concentrations of DHFF(0.030,0.060,0.120 g·L-1)were incubated for 30mins and the cumulative concentration of CaCl2,DHFF was added in a concentration-dependent manner to make the calcium chloride dose-effect curve significantly lower than that of the control group（P<0.05）.Conclusions:（1）DHBF has obvious inhibitory and protective effects on Hypertrophic cardiomyocytes induced by Ang II and NE,the mechanism of action may be related to promoting the release of NO,regulating the concentration of[Ca2+]i and the activity of Ca2+-ATP.signal transduction pathways of Ca N-NFAT-3 and Ca MK II-HDAC is blocked that are mediated by Ca2+.（2）DHBF have vasodilator effect on the intact or endothelium-free endothelium precontracted by NE and KCl,which is concentration-dependent and partly dependent on endothelial cells.It may be related to the endothelium-dependent NO-c GMP signaling pathway.（3）DHBF can significantly decrease the calcium chloride dose-effect curve in a concentration-dependent manner,and inhibit the release of calcium ions in vascular smooth muscle cells induced by NE,which may be related to the inhibition of ROCC,VOCC activity on vascular smooth muscle.Blockage of L-type calcium channels,opening of calcium channels and inhibition of intracellular calcium release are related to vasodilation. |
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