The Mechanism Of Electroacupuncture Againsting Oxidative Stress To Improve Cognitive Impairment Via Regulating NOX In Spontaneously Hypertensive Rats With Chronic Cerebral Hypoperfusion

Objective To observe the relationship between the changes of cognitive function and the expression of NOX in cerebrovascular of spontaneously hypertensive rats(SHR)with chronic cerebral hypoperfusion(CCH)and to reveal the mechanism of electroacupuncture againsting oxidative stress to improve cognitive impairment via regulating NOX in spontaneously hypertensive rats with chronic cerebral hypoperfusion.Methods Fifty-five male spontaneously hypertensive rats(SHR)were randomly divided into control group,sham operation group,model group,ARB group and electroacupuncture group(EA group),and eleven rats in each group.Five groups of rats were duplicated CCH model by improved permanent bilateral occlusion of the common carotid arteries(2VO)(control group was only under anesthesia and sham operation group was only separated bilateral common carotid artery).The EA group was treated by electroacupuncture on the second day after operation and once a day for 4 weeks.The rats in EA group were fixed with a fixator and the acupoints of "Baihui" and "Dazhui" were stimulated by electroacupuncture.The size of acupuncture needle was 0.30 mm ×25mm.Electroacupuncture instrument of HANS-200 E was used with 15 Hz frequency and 1 m A current for 30 min.The ARB group was given candesartan(1mg/kg/day)by gavage.Control group,sham operation group and model group were given the normal saline(1mg/kg/day)by gavage for balance treatment.Observation indexes: The laser Doppler flowmetry was used to detect the region cerebral blood flow changes of rats before operation,after operation,1 week after operation,2 weeks after operation and 4 weeks after operation.Open field test,Morris water maze test and zero maze test were used to observe the behavioral changes such as anxiety and ability of learning and memory in rats.The levels of NOX1/2/4,3NT and SOD2 were measured by Western blotting in cerebrovascular.The levels of NOX1/2/4 and 3NT were detected by immunofluorescence in cerebral cortex,hippocampus CA1 and CA3 regions.Results1.Regional cerebral blood flow(rCBF)changes: There was no difference in region cerebral blood flow between control group and sham operation group at the same time point.The r CBF of model group,ARB group and EA group decreased significantly after operation,moreover,the r CBF of each group gradually increased with the prolongation of time.Compared with control group and sham operation group,the r CBF of model group,ARB group and EA group were significantly decreased after operation and at week 1,2,4 postoperatively(P<0.01).Compared with model group,the r CBF of ARB group and EA group increased significantly at 2 weeks and 4 weeks after operation.2.Open field test: The center area time and the center area entries between control group and sham operation group showed no obvious difference.Compared with control group and sham operation group,the center area time and the center area entries of model group were significantly decreased(P<0.01).Compared with the model group,the center area time and the center area entries of ARB group and EA group increased significantly(P<0.05,P<0.01).3.Zero maze test: Compared with control group and sham operation group,the residence time of the model group in the open arms was significantly decreased(P <0.01).The residence time in the open arms of ARB group and EA group were obviously longer than that of model group.4.Morris water maze test:(1)Escape latency: With the increase of training days,the escape latency of rats in each group showed a downward trend.Compared with control group and sham operation group,the escape latency of the model group was significantly increased at all time points(P<0.05,P<0.01).The escape latency of ARB group on the 2nd,3rd,4th and 5th day were significantly higher than those in control group and sham operation group(P<0.05,P<0.01).The latency of EA group increased significantly on the 4th and 5th day compared with control group and sham operation group(P<0.01).Compared with model group,the latency of ARB group decreased significantly on the 4th and 5th day(P<0.01),and the EA group significantly decreased on 3rd,4th and 5th day(P<0.05,P<0.01).(2)Results of spatial probe test:Compared with control group and sham operated group,the retention time in the quadrant of model group was significantly shortened(P<0.01).Compared with model group,the retention time in the quadrant of ARB group and EA group were significantly prlonged(P<0.05).5.Western blotting results: the relative expression level of SOD2 protein in model group was significantly lower than that in control group and sham operation group(P <0.01).Compared with model group,the relative expression level of SOD2 protein in ARB group and EA group were increased significantly(P<0.01).The relative expression level of 3-NT protein in model group was significantly higher than that in control group and sham operation group(P<0.01).Compared with model group,the protein expression level of 3-NT in ARB group and EA group were significantly decreased(P<0.05,P<0.01).Compared with control group and sham operation group,the relative expression level of NOX1,NOX2 and NOX4 protein in model group were significantly increased(P<0.01).Compared with model group,the relative expression level of NOX1,NOX2 and NOX4 in ARB group and EA group were decreased significantly(P<0.01).The relative expression level of NOX1,NOX2 and NOX4 protein had no significant difference between ARB group and EA group.6.Immunofluorescence results: 3-NT was co-expressed with vWF in cerebral cortex and the expression of 3-NT positive cells in model group was increased compared with control group and sham operation group.The expression of 3-NT positive cells in EA group and ARB group were slightly weaker than that in model group.NOX1/2/4 and CD31 were co-expressed in cerebral cortex,hippocampus CA1 and CA3,moreover,the expression of NOX1/2/4 positive cells were increased in model group compared with control group and sham operation group.The expression of NOX1/2/4 positive cells in EA group and ARB group were slightly weaker than that in model group.Conclusions1.The NOX-mediated oxidative stress may be the factor of inducing neurovascular injury and cognitive impairment in SHR with chronic cerebral hypoperfusion.2.Electroacupuncture can increase cerebral blood flood,improve anxious behavior and learning and memory ability of SHR with chronic cerebral hypoperfusion may be related to the down-regulation of the levels of NOX and vascular endothelial oxidative stress.