| ObjectiveBy observing and comparing blood pressure,behavior,serum,protein,gene and other indicators of rats in each group,we explored the mechanism of acupuncture lowering blood pressure and improving the vascular endothelial function through RhoA/ROCK signaling pathway.MethodsAll the rats were fed for 1 weeks before this experiment,the SPF level of 12 week old male spontaneously hypertensive rats(SHR)were randomly divided into model group,acupoint group and non-acupoint group,10 rats in each group.In addition,10 male Wistar rats(WKY)of the same age were used as blank group.The model group and the blank group did not given any interventions,only need to be fixed.The acupoint group and the non-acupoint group were fixed,the acupuncture intervention were carried out at the same time every day,and the acupuncture points were both bilateral.The acupoint group selected "Xin shu" "spleenshu" "Neiguan" and "Zusanli",and the non-acupoint group selected the non meridian points next to the above four points.Each time retaining needle 30 minutes,hand-manipulating of needle every 10 minutes,1 times operating 1min,the twist rate of about 180 degrees and frequency about 80 /min.1 times a day,continuously intervened 28 d.We measured the tail artery blood pressure and observed behavioral changes,watched morphological changes of the aorta by optical microscope,and observed the ultrastructural changes of the endothelium by electron microscopy,detected Serum levels of NO,NOS,ET-1,AngⅡ and TNF-α,Western Blot and RT-PCR were separately used to detected RhoA,Rock1,Rac1,Cdc42,eNOSprotein and gene expression level.Results1.Effect of acupuncture on blood pressure in spontaneously hypertensive ratsBefore the experiment,there was no significant difference in blood pressure between the model group,the acupoint group and the non acupoint group(P > 0.05).The blood pressure of the three groups was higher than that of the blank group,and the difference was significant(P < 0.05).During the whole experiment,the blood pressure of the rats in the blank group remained stable.After 1 week,the blood pressure of the rats in the model group was higher than that before,and the blood pressure of the rats in the non-acupoint group and the acupoint group was lower than that before.Compared with the model group,the blood pressure of the rats in the acupoint group decreased significantly,the difference was significant(P < 0.05).Compared with the non-acupoint group,the blood pressure of the rats in the acupoint group decreased slightly,and the difference was not statistically significant(P > 0.05).After 2 weeks,the blood pressure of the rats in the model group continued to rised,the blood pressure of the non-acupoint group increased slightly,and the blood pressure of the acupoint group continued to decline.Compared with the blood pressure in the model group and the non acupoint group,the blood pressure of the rats in the acupoint group was significantly decreased,and the difference was significant(P < 0.05).After 3 weeks,the blood pressure of the rats in the model group continued to rise.The blood pressure of the non-acupoint rats decreased slightly,and the blood pressure of the rats in the acupoint group was significantly lower than that before.Compared with the blood pressure in the model group and the non-acupoint group,the blood pressure decreased significantly in the acupoint group,and the difference was significant(P < 0.05).After the end of the experiment(4 weeks),blank group rats blood pressure group showed no significant difference(P > 0.05);compared with the control group,the blood pressure of rats in model group increased significantly,the difference was significant(P < 0.05);compared with the model group,non-acupoint group rats,rats of the acupoint group decreased significantly,the difference was significant(P < 0.05).2.Effect of acupuncture on behavior in spontaneously hypertensive rats(1)Effect of acupuncture on the irritability of spontaneously hypertensive ratsBefore the experiment,there was no significant difference in the irritability degree between the model group,the acupoint group and the non-acupoint group(P > 0.05).The irritability degree of the three groups of rats was higher than that of the blank group,with a significant difference(P < 0.05).During the whole experiment,there was no obvious change in the degree of irritability in the blank group.After 1 week of the experiment,the rats in the model group and non-acupoint group were more likely to be provoked,while those in the acupoint group were less likely to be provoked.There was no significant difference(P > 0.05)between the scores of irritability in model group,acupoint group and non-acupoint group.After 2 weeks of the experiment,the overall irritability of rats in the model group and non-acupoint group continued to increase,while that of rats in the acupoint group continued to decrease.There was no significant difference(P > 0.05)between the scores of irritability in model group,acupoint group and non-acupoint group.After 3 weeks of the experiment,the overall irritability of rats in the model group and non-acupoint group continued to increase,while that of rats in the acupoint group continued to decrease.Compared with rats in the model group and non-acupoint group,rats in the acupoint group were significantly decreased in irritability,and the difference was statistically significant(P < 0.05).After the end of the experiment(4 weeks),the total irritability of rats in the model group and non-acupoint group was increased significantly,and that of rats in the acupoint group was decreased significantly.Compared with rats in the model group and non-acupoint group,rats in the acupoint group were significantly decreased in irritability,and the difference was significant(P < 0.05).(2)Effect of acupuncture on the rotation tolerance time of spontaneously hypertensive ratsBefore the experiment,there was no significant difference in the rotation tolerance time between the model group,the acupoint group and the non-acupoint group(P > 0.05).The rotation tolerance time of the three groups was lower than that of the blank group,and the difference was significant(P < 0.05).During the whole process,the rotation tolerance time of the rats in the blank group was not significantly changed.After 1 week,the rotation tolerance time of the rats in the model group decreased and the rotation tolerance time of the rats in the non-acupoint group and the acupoint group increased.Compared with the rotation tolerance time of the model group and the non-acupoint group,the rats in the acupoint group increased obviously,but the difference was not significant(P > 0.05).After 2 weeks,the rotation tolerance time of the rats in the model group decreased and the rotation tolerance time of the rats in the acupoint group and the non-acupoint group increased.Compared with the rotation tolerance time of the rats in the model group,the acupoint group increased significantly,the difference was statistically significant(P < 0.05),compared with the rotation tolerance time of the non-point group rats,the acupoint group increased significantly,but the difference was not statistically significant(P > 0.05).After 3 weeks of intervention,the rotation tolerance time of the rats in the model group was not significantly changed,and the time of rotation tolerance in the acupoint group and the non-acupoint group was increased.Compared with the rotation tolerance time of the model group and the non-acupoint group,the rats in the acupoint group were significantly increased,and the difference was significant(P < 0.05).After the end of the experiment(4 weeks),the rotation tolerance time of the rats in the model group decreased slightly,and the rotation tolerance time of the acupoint group and the non acupoint group increased.Compared with the rotation tolerance time of the model group and the non acupoint group,the rats in the acupoint group were significantly increased,and the difference was significant(P < 0.05).3.Effect of acupuncture on the morphology of aorta in spontaneously hypertensive ratsObserved by optical microscope,the blank group aortic morphology and normal endometrial endothelial cells were smooth and complete,continuous,regular shape,endothelial tight junction,membrane elastic fibers were normal without thickening,vascular smooth muscle cells arranged orderly;the boundary of three layers in model group is not clear,serious endometrial thickening,edema,irregular endothelial cell injury tear film,elastic fiber fracture,vascular smooth muscle cells arranged in disorder;acupuncture group compared with the normal aortic morphology,the boundary is clear,the lining is still intact,no obvious thickening,membrane elastic fibers and smooth muscle cells arranged in disorder without obvious pathological injury,have different degrees of improvement;non-acupoint group aortic wall boundary not clear,endometrial hyperplasia and hypertrophy,shedding of endothelial cell protrusions,serious,elastic membrane fiber thickening,smooth muscle cells arranged irregularly.4.Effect of acupuncture on the ultrastructure of vascular endothelium in spontaneously hypertensive ratsObserved by electron microscope,the aortic endothelial cells in the blank group were arranged tightly,the morphology was normal,the intima was flat,the smooth muscle nuclei chromatin was normal,and mitochondria,endoplasmic reticulum,and Golgi complex were seen under microscope.Endothelial cell injury model rats into smooth muscle cells increased significantly;the loose structure of the subendothelial,elastic membrane rupture,wall collagen fibers;heterochromatin increased wall,vacuolar rod mitochondria and endoplasmic reticulum swelling,abnormal cell organelles.The endothelial structure of the acupoint group was normal,the insertion of smooth muscle cells was obviously reduced,and the abnormal organelle decreased obviously.The endothelial cells in non-acupoint group were severely damaged,the endothelium structure loosened,the elastic membrane ruptured,the collagen fibers increased,the gap between the flat cells and the arterial wall widened,the endoplasmic reticulum was swollen,and the abnormal organelles increased.5.Effects of acupuncture on serum NO,NOS,ET-1,AngⅡand TNF-α in spontaneously hypertensive ratsCompared with the content of serum NO and NOS in the blank group,the model group was significantly lower than that of the model group,and the difference was significant(P < 0.05).Compared with the content of NO and NOS in the model group and the non-acupoint group,the acupoint group was significantly increased,and the difference was significant(P < 0.05).Compared with the control group of rats serum ET-1,AngⅡ and TNF-αin model group was significantly increased,the difference was significant(P < 0.05);compared with the content of ET-1,AngⅡ,TNF-αin the model group and the non-acupoint group rats,acupoint group significantly decreased,the difference was statistically significant(P < 0.05).6.Effects of acupuncture on protein and gene expression levels of RhoA,Rock1,Rac1,Cdc42,eNOSCompared with protein and gene expression levels of RhoA,Rock1,Rac1,Cdc42,eNOS in the blank group rats,the expression level of model rats increased significantly,the difference was significant(P < 0.05);compared with eNOS protein and gene expression levels of blank group rats,the expression level of model rats decreased significantly,the difference was significant(P < 0.05);compared with the model group,RhoA,Rock1,Rac1,Cdc42 protein and gene expression level of non-acupoint group rats,the expression of acupuncture group rats significantly decreased,the difference was significant(P < 0.05);compared with the model group,eNOS protein and its gene expression level of non-acupoint group rats,expression of acupuncture group rats increased significantly,the difference was significant(P < 0.05);There was no significant difference between model group and non-acupoint group in the protein and gene expression level of RhoA,Rock1,Rac1,Cdc42,eNOS(P > 0.05).ConclusionAcupuncture may increase the content of NO and activity of NOS,reduce expression level of ET-1,AngⅡ,TNF-α,decrease the protein content and gene expression level of RhoA,Rock1,Rac1,Cdc42,increase the protein content and gene expression of eNOS,reduce the blood pressure of rats,inhibit RhoA/Rock signaling pathway,improve vascular endothelial function. |
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