The Research Of Mechanism Of Total Flavonoids Of Rhododendron Blocking Rho Kinase-induced Vasodilation In Rats

Rho kinases?ROCKs?are the major effector molecules of the small-molecule guanylate-binding protein RhoA.They are expressed in vascular endothelial cells and smooth muscle cells.There are two subtypes of ROCK,it's ROCK1 and ROCK2.There is a certain homology in the domain of amino acid sequence and kinase.ROCK has a certain physiological role in cardiovascular diseases such as atherosclerosis,heart failure,pulmonary hypertension,and vascular remodeling.At rest,RhoA is located in the cytoplasm,when it's activated,it can be transferred to the membrane and activate downstream ROCK to cause phosphorylated of myosin binding subunit?MBS?of myosin light chain dephosphorylation?MLCP?.It lead to inactivate of MLCPh,phosphorylated myosin light chain?MLC?can not dephosphorylate,and ROCK can also directly phosphorylate MLC,hereby increasing smooth muscle contractility,eventually causing sputum response in the coronary arteries,triggering a series of Cardiovascular disease.Rho kinase inhibitor Y27632 can improve vasospasm by relaxing vascular.The Rhododendron's effective partextracted is Total flavones of rhododendra?TFR?,and rutin,quercetin and hyperoside are the main components.We have previously reported that TFR had vasodilation,and the mechanism involved decrease of RhoA activity in vasculars.However,whether TFR improves the vasospasm by blocking Rho kinase and exerting vasodilated remains to be further explore.In this study,the expression of ROCK1 and ROCK2 protein in rats was down-regulated by siRNA in vivo interference technique,and the changes of vasodilation of TFR were observed.And the relationship between vasodilation and ROCK blockade and ROCK subtype was discussed.Purpose:1.To observe the Rho kinase blocking mechanism of vasodilation by TFR on rat.2.To explore the relationship between the vasodilation by TFR on rat and the subtypes of ROCK.Methods:1.Transfection model was established using interference technology in vivo.1.1 Coronary artery,mesenteric artery,mesenteric vein and abdominal aorta were taken separately.Each blood vessel was divided into 5 groups:it's control group,negative siRNA interference group,transfection reagent,ROCK₁-siRNA interference group and ROCK₂-siRNA interference group.1.2 Western blot was used to determine the expression of ROCK1 and ROCK2proteins in vascular tissues.2.Establish muscle function tension test,using TFR and Y27632 to detect the relaxation of normal and the vascular after interfered.2.1 Experimental groupingCoronary artery,mesenteric artery,mesenteric vein and abdominal aorta were taken respectively.Each blood vessel was divided into 9 groups:It‘s vehicle group,TFR group,ROCK₁-siRNA+vehicle group,ROCK₁-siRNA+vehiclegroup,ROCK₁-siRNA+TFR group,ROCK₂-siRNA+vehicle group,ROCK₂-siRNA+TFR group,Y27632group,ROCK₁-siRNA+Y27632 group and ROCK₂-siRNA+Y27632 group.Results:1.Compared with the control group,ROCK1 protein expression in ROCK₁-siRNA transfection group and ROCK2 protein expression in ROCK₂-siRNA transfection group were significantly lower,the difference was statistically significant?P<0.01?.While compared with the control group,the expression of ROCK1and ROCK2 protein of the transfection reagent group and the negative siRNA group in each vascular tissue was not significantly decreased,and the difference was not statistically significant.2.Compared with the ROCK₁-siRNA+vehicle group,Y27632 can induce the relaxation effect of vascular after ROCK1 interfered.Compared with normal rat vascular,Y27632can attenuate the relaxation effect of vascular after ROCK1 interfered.3.Compared with the ROCK₂-siRNA+vehicle,Y27632 can induce the relaxation effect of rat coronary artery,mesenteric artery and abdominal aorta after ROCK2 interfered,while the relaxation effect of mesenteric vein was not obvious.Compared with the normal rat vascular,Y27632 reduced the relaxation of vascular after ROCK2 interfered.4.Compared with the ROCK₁-siRNA+vehicle group,TFR can induce the relaxation effect of vascular after ROCK1 interfered.Compared with normal rat blood vessels,TFR can attenuate the relaxation effect of vascular after ROCK1 interfered.5.Compared with the ROCK₂-siRNA+vehicle group,TFR can induce the relaxation in the coronary,mesenteric and abdominal aorta of rats with ROCK2 interfered,but there's no significant relaxation effect on the mesenteric vein which ROCK2 interfered.6.The vasodilation of Y27632 is related to the blockade of ROCK,and the relaxation effect on mesenteric vein selectively acts on ROCK2,and the relaxation effect on mesenteric artery is more closely related to ROCK1.7.The vasodilation of TFR on vascular is related to the blockade of ROCK,and the relaxation effect on mesenteric artery,mesenteric vein and abdominal aorta selectively acts on ROCK2,and the relaxation effect on coronary artery is more closely related to ROCK1.Conclusions:1 ROCK1,ROCK2 down-regulation can weaken the vasodilation of Y27632 and TFR.2 The vasodilation of TFR on rat may be related to the blockade of ROCK.The relax-ation of coronary artery is related to the blockade of ROCK1.The relaxation of mese-nteric artery,mesenteric vein and abdominal aorta may be more related to ROCK2.