The Improvement Effects Of Total Flavones Of Rhododendra On Ventricular Remodeling After Myocardial Infarction In Rats By Inhibition Of UTR-RohA-ROCK

Myocardial infarction?MI?after ventricular remodeling is considered to be the leading cause of heart failure and sudden cardiac death.At present,early drug intervention and treatment is still the primary methods for the treatment of ventricular remodeling,in addition to rebuilding blood supply and limiting the infarct range.As an active part extracted from the rhododendra,the total flavones of rhododendra?TFR?play an important protective role in acute myocardial ischemia and reperfusion injury,but its role in chronic myocardial ischemia is not clear,especially in ventricular remodeling after myocardial infarction.TFR contains flavonoids such as azalea,rutin,quercetin and hyperoside,and flavonoids have the anti-inflammatory,anti-oxidation and anti-fibrosis effects by regulating the cardiac urotensin ? receptor?UTR?pathway and blocking Rho associated coiled coil-forming kinase?ROCK?activity.Therefore,we hypothesized that TFR may have similar anti-fibrosis effects on chronic ventricular remodeling after infarction.The aim of this study was to investigate the effect of TFR in improving ventricular remodeling after myocardial infarction and focus on the relationship between TFR and UTR or ROCK.Purpose:1.To study the effect of TFR on ventricular remodeling and cardiac dysfunction after myocardial infarction in rats.2.To observe the effect of TFR on cardiomyocyte hypertrophy and fibroblast proliferation induced by hU? in primary neonatal rats.To study the mechanism of TFR on the pathway of UTR-RohA-ROCK and clarify the downstream MLCP pathway and whether it was related to calcium ion.3.To observe the effect of TFR on tension of different vascular in vitro and explore the possibility of the selective relaxation in rat coronary artery and improving myocardial perfusion of infarct area.Methods:1.An rat MI model was established by ligation of left anterior descending coronary artery.30¹20 mg/kg TFR was administrated by intragastric injection for 30 days.Left ventricular function and wall thickness were measured by echocardiography at 1 day and 4 weeks after operation.The rats were sacrificed at the end of the experiment,followed by weighing the body and heart weight,cutting off the heart below the ligament line,half of that was used for morphological testing:the cross-sectional area of myocardial cells was observed by hematoxylin-eosin?HE?staining,and the interstitial fibrosis was observed by sirius red staining.The other part of heart was used to detect the expression of UTR,RhoA,ROCK1 and ROCK2 proteins by Western blot.2.Primary neonatal rat cardiomyocytes and fibroblasts were cultured.After adding different concentration of hU? and TFR,microplate reader was used to detect cell viability.100 nM hU? was used to induce cell remodeling.After 10,30 and 90 mg/L TFR pretreatment,the morphological changes were observed by immunofluorescence;The expression of ANP mRNA and?-MHC mRNA were detected by RT-PCR;the fibroblast marker protein expression of?-SMA and SM22?in fibroblasts and the protein expression of UTR,RhoA,ROCK1,ROCK2,p-MYPT1 and p-MLC were detected by Western blot.Calcium Imager was used to detect the relative changes of intracellular calcium after TFR pretreatment.3.The protein expression of ROCK1 and ROCK2 in cardiomyocytes and fibroblasts were down-regulated respectively.The relationship between TFR pretreatment and down-regulation of ROCK1 and ROCK2 was observed.4.The appropriate vascular ring of rat coronary artery,mesenteric artery/vein,abdominal aorta and portal vein were isolated and prepared,respectively.Concentration gradient of 3.3²430 mg/L TFR was added successively,then the change in tension of blood vessel was measured.The effect of TFR after intravenous injection on the mean blood flow in the left ventricular surface region of normal or MI rats was observed by laser speckle flow imaging.Results:1.Compared with the Sham group,LVIDs,LVIDd,LVPWs and LVPWd were increased,LVFS and LVEF were decreased in MI model group.Cardiac function in rats after myocardial infarction may be improved after treatment of TFR 30,60 and 120 mg/kg/d or amlodipine 4 mg/kg/d for 4 weeks.2.The heart weight index?HWI?of the model group was significantly increased,and the index decreased gradually after treatment with TFR 30,60 and 120 mg/kg/d.Treatment of 30¹20 mg/kg TFR and 4 mg/kg amlodipine significantly reduced the average cross-sectional area of myocardial cells and myocardial fibrosis of left ventricular wall.3.TFR 30,60 and 120 mg/kg/d could significantly inhibit the up-regulation of fibrosis-related factors such as?-SMA,TGF-?1,MMP2 and collagen I in nonfarction areas after MI.The up-regulation effects of MI-induced expression level of UTR,GTP-RhoA,ROCK1 and ROCK2 were also significantly attenuated.4.10,30,90 mg/L TFR inhibited the increase in surface area of myocytes and fibrosis formation in fibroblasts induced by 100nM hU? in a dose-dependent manner.The intensity of intracellular green fluorescence was reduced,moreover,the expression of?-MHC mRNA and ANP mRNA in cardiomyocytes and the expression of?-SMA and SM22?in fibroblasts were inhibited in varying degrees.5.The 100 nM h U?-induced increased levels of UTR,GTP-RhoA,ROCK2 and p-MYPT1 and p-MLC in cardiomyocytes were attenuated by 10⁹0 mg/L TFR pretreatment in different degrees,however,there were statistical differences between TFR-treatment group and vehicle control group?P<0.01?.The effect of SB 706375?as a positive control drug?on protein expression was similar to that of high dose of TFR.The effect of TFR on UTR-RhoA-ROCK pathway after 100 nM hU?-induced fibroblast proliferation was similar to that of cardiomyocytes.The differences of an inhibiting effect of the up-regulation of ROCK1 protein and an insignificant effect on ROCK2protein level were observed.6.ROCK2-siRNA pretreatment could significantly weaken the increased surface area of cardiomyocytes induced by 100 nM hU? and decrease the enhanced intracellular green fluorescence,inhibit the hypertrophy gene expression of?-MHC mRNA and ANP mRNA.However,ROCK1-siRNA pretreatment did not produce similar results and 90mg/L TFR pretreatment had similar effect with that of ROCK2-siRNA.Combined with ROCK2-siRNA,the cell surface area and the level of hypertrophy gene expression in group hU? pretreated with TFR were further reduced,and combined with ROCK1-siRNA,similar inhibition effect still exists in group hU? pretreated with TFR.7.ROCK1-siRNA pretreatment could significantly inhibit fibroblast formation induced by 100 nM hU? and remarkable intracellular green fluorescence,down-regulate the expression of fibrosis marker protein like?-SMA and SM22?.However,ROCK2-siRNA pretreatment did not produce a consistent results and the effect of 90mg/L TFR pretreatment was similar to that of ROCK1-siRNA.Combined with ROCK1-siRNA,the inhibitory effect in group hU? pretreated with TFR was more obvious,the combination of ROCK2-siRNA after TFR incubation had similar inhibitory effect.8.After neonatal cardiomyocyte hypertrophy and fibroblast proliferation induced by100 nM hU?,Fluo-8 AM-labeled calcium fluorescence intensity was significantly enhanced with a relative increase in calcium concentration.High fluorescence intensity induced by hU? was reduced to varying degrees after 10,30 and 90 mg/L TFR pretreatment,the concentration of relative calcium ion was also reduced.9.After acute MI,we observed an increase in the yellow area and a decrease in the red area from the infarct area of the blood perfusion imaging.The corresponding average blood flow curve in a real-time and dynamic form was decreased significantly and the average blood flow rate was 64.6±18.7%.After intravenous administration of TFR 30mg/kg,the yellow area was lessened and the corresponding average blood flow curve was elevated,also the mean blood flow rate was increased to 45.6±13.5%.Interestingly,there was no significant difference in perfusion and mean blood flow curve before and after TFR treatment in Sham group.10.The relaxation effect of 3.3²430 mg/L TFR on coronary arteries was significantly attenuated,and the EC₅₀ was increased from 331.8±7.2 mg/L to 480.1±1.8 mg/L,E_max was decreased from 97.4±3.7%to 90.9±1.9%.11.After adding the increasing concentration,3.3²430 mg/L TFR induced relaxation in different degrees in coronary arteries precontracted with U46619,PE and high potassium.The effect of TFR-induced relaxation in coronary arteries with and without endothelium was different,low concentration of TFR induced an endothelium dependent relaxation.After being precontracted with U46619,EC₅₀ of TFR-induced relaxation was 331.8±7.2 mg/L and 469.7±2.5 mg/L respectively,while E_max?the maximal diastolic rate?was 97.4±3.7%and 95.0±2.8%respectively.EC₅₀ of TFR-induced relaxation in coronary arteries precontracted with PE was increased significantly,while E_max had a significant decrease?P<0.01?.Not E_max but EC₅₀ was further increased after being precontracted with high potassium?P>0.05?.12.The EC₅₀ of TFR-induced relaxation in superior mesenteric artery was higher than that of the coronary artery,while the E_max was decreased significantly?P<0.01?.Meanwhile,the EC₅₀ of TFR-induced relaxation in the aorta was further increased?P<0.01?,and E_max was decreased further?P<0.01?.Conclusion:1.TFR could suppress ventricular remodeling effectively and ameliorate left cardiac function in rats after myocardial infarction.2.TFR attenuated hU?-induced cardiomyocyte hypertrophy and fibroblast proliferation,which may be associated with calcium-dependent UTR-RhoA-ROCK-MLCK blocking mechanisms.Among them,RCOK2 was an important target for reversing cardiomyocyte hypertrophy.However,RCOK1 may play an important role in fibroblast proliferation.3.TFR could relax the coronary artery in vitro selectively,improve myocardial perfusion in the infarct area after myocardial infarction.