| Objective: Through the observation of qufengtongqiao decoction on positioning navigation,space exploration ability,hippocampus CA1 neurons,the expression of Ch AT,Tau,A?,VEGF protein,the concentration of IL-1,IL-6,TNF-? in vascular dementia(VD)rats,discussing the mechanism of Qufeng Tongqiao Decoction on vascular dementia.Methods :1.grouping:Rats were screened by water maze.Rats who were met the requirements were randomly divided into sham operation group,nimodipine group,model group,qufengtongqiao Decoction high,middle,low dose group.Each group had 10 rats.2.Drug delivery method:The high,middle and low dose groups were given qufengtongqiao Decoction 26.8g/kg/d,13.4 g/kg/d,6.7g/kg/d by gavage(According to the standard of equal volume 3ml,to calculate the concentration of drug in rats of high,middle and low dose groups).The model group and sham operation group were treated with normal saline,and the dose was 3ml.The nimodipine group was given nimodipine suspension6.25mg/kg/d.The rats were given the corresponding drugs for 30 days continuously,and once a day.3.Drawing method:To configure pentobarbital sodium concentration 1%,the rats were anesthetized intraperitoneally.Taking the supine position to fix the limbs of the rats in the disinfection experiment platform.Opening the chest to collect blood.Centrifuging and taking thesupernatant,the supernatant were stored in the refrigerator at-20 degrees centigrade.ELISA for testion of IL-1?,IL-6,TNF-? concentration..After taking the blood,the fresh brain tissues of some rats were removed rapidly and stored in the EP tube,using semi-quantitative PCR and Western Blot to test VEGF protein.Other rats had been done brain perfusion after the collection of blood.Using HE staining and immunohistochemistry to test Ch AT,Tau,A?protein.4.Observation index:Morris water maze was used to test the ability of navigation and space exploration.The morphology of neurons in hippocampal CA1 region was observed by HE staining and light microscope.The expressions of Ch AT,Tau and A? protein were tested by immunohistochemistry.The expression of IL-1?,IL-6 and TNF-? were tested by ELISA method.Semi-quantitative PCR were used to test the expression of VEGFm RNA.Western Blot was used to test the expression of VEGF protein.Results:1.Behavioral :Compared with the sham operation group,the EL of the high dose group,the middle dose group,the low dose group,the model group,the nimodipine group were prolonged,and the swimming time in quadrant I was shortened,the number of through the original platform was decreased,the difference was statistically significantcal difference(P<0.05).In the high dose group,the EL of rats decreased with the prolongation of training time,which was close to the sham operation group from the fourth day.Compared with the model group,the EL of the rats in the high dose group,the middle dose group,the low dose group and the nimodipine group was shortened with the prolongation of the training time,and the swimming time in quadrant I wasprolonged,the number of through the original platform was increased,the difference was statistically significantcal difference(P<0.05).Compared with the nimodipine group,the EL of the rats in the low dose group was shortened with the prolongation of the training time,the swimming time in quadrant I was prolonged,the number of through the original platform was increased,but the difference was not statistically significantstical difference(P >0.05),the EL of the rats in the high dose group,the middle dose group was shortened with the prolongation of the training time,the swimming time in quadrant I was prolonged,the number of through the original platform was increased,the difference was statistically significantcal difference(P<0.05).Among them,the behavior improvement of rats in high dose group was more obvious,compared with the middle dose group,the difference was statistically significant(P<0.05).2.HE staining :Observing the results under the same magnification microscope,the neurons of hippocampal CA1 area in the sham operation group,cells were arranged neatly,cytoplasmic red stain,the nucleus was large and round,nucleolus was clear and obvious.Hippocampal CA1 neurons in model group,the cell were arranged disorder,the number of cell decreased,the cell outline and the nucleus were fuzzy.Some groups couldn't even see cells.The cell of the high and middle dose groups were arranged rule,the cell outline,nuclear membrane and nucleolus were clear.The morphology of nerve cells in high dose group was better than that in middle dose group,the cells of he high dose groups were observed to be closer to normal morphology under microscope.The morphology of neurons in the low dose group and nimodipinegroup were between the model group and the middle dose group.3.Detection of Ch AT,Tau,A? protein:the positive cells of Ch AT,Tau and A? were all expressed as brown precipitate in cytoplasm.(1)Ch AT protein:Compared with the sham operation group,the MOD value of Ch AT protein in the model group and the each administration group were lower than those in the sham operation group(P<0.05).Compared with the model group,the MOD value of Ch AT protein in the each administration group were higher than those in the model group(P<0.05).Compared with the nimodipine group,the MOD value of Ch AT protein in the low dose group were higher than those in nimodipine group,but the difference was not statistically significantstical difference(P>0.05).The MOD value of Ch AT protein in the high dose group and the middle dose group were higher than those in the nimodipine group,the difference was statistically significant(P <0.05).Among them,the increase was most obvious in high dose group,compared with the middle dose,the difference was statistically significant(P<0.05).(2)Tau protein:Compared with the sham operation group,the MOD value of Tau protein in the model group and the each administration group were higher than those in the sham operation group(P<0.05).Compared with the model group,the MOD value of the Tau protein in the each administration group were lower than those in the model group(P<0.05).Compared with the nimodipine group,the MOD value of the Tau protein in the low dose group were lower than those in the nimodipine group,but the difference was not statistically significantstical difference(P>0.05).The MOD value of Tau protein in the high dose group and the middle dose group werelower than those in the nimodipine group,the difference was statistically significant(P<0.05).Among them,the decrease was most obvious in high dose group,compared with the middle dose,the difference was statistically significant(P<0.05).(3)A? protein :Compared with the sham operation group,the MOD value of A? protein in the model group and the each administration group were higher than those in the sham operation group(P<0.05).Compared with the model group,the MOD value of A? protein the each administration group were lower than those in the model group(P<0.05).Compared with the nimodipine group,the MOD value of the A? protein in the low dose group was lower than those in the nimodipine group,but the difference was not statistically significantstical difference(P>0.05).The MOD value of A? protein in the high dose group and the middle dose group were lower than those in nimodipine group,the difference was statistically significant(P<0.05).Among them,the decrease was most obvious in high dose group,compared with the middle dose,the difference was statistically significant(P<0.05).4.Detection of IL-6,IL-1?,TNF-?:Compared with the sham operation group,the concentrations of IL-6,IL-1?,TNF-? in the model group and the each administration group were significantly higher than those in the sham operation group(P<0.05).Compared with the model group,the concentrations of IL-6,IL-1?,TNF-? in the each administration group were significantly lower than those in the model group(P<0.05).Compared with the nimodipine group,the concentrations of IL-6,IL-1?,TNF-? in the high dose group,the middle dose group,the low dose group were lower than those in thenimodipine group(P<0.05).Among them,the decrease was most obvious in high dose group,compared with the middle dose,the difference was statistically significant(P<0.05).5Detection of VEGFm RNA expression:Compared with the sham operation group,the expression of VEGFm RNA in the model group and the each administration group were higher than those in the sham operation group(P<0.05).Compared with the model group,the expression of VEGFm RNA in the each administration group were higher than those in the model group(P <0.05).Compared with the nimodipine group,the expression of VEGFm RNA in the low dose group were higher than those in the nimodipine group,but the difference was not statistically significantstical difference(P>0.05).The expression of VEGFm RNA in the high dose group and the middle dose group were higher than those in nimodipine group,the difference was statistically significant(P<0.05).Among them,the increase was most obvious in high dose group,compared with the middle dose,the difference was statistically significant(P<0.05).6.Detection of VEGF protein expression:Compared with the sham operation group,the expression of VEGF protein in the model group and the each administration group were higher than those in the sham operation group(P<0.05).Compared with the model group,the expression of VEGF protein in the each administration group were higher than those in the model group(P<0.05).Compared with the nimodipine group,the expression of VEGF protein in the low dose group were higher than those in the nimodipine group,but the difference was not statistically significantstical difference(P>0.05).The expression of VEGF protein in the high dose group and themiddle dose group were higher than those in nimodipine group,the difference was statistically significant(P<0.05).Among them,the increase was most obvious in high dose group,compared with the middle dose,the difference was statistically significant(P<0.05).Conclusion:1.The learning and memory ability of vascular dementia rats was decreased.The morphology of neurons in the hippocampal CA1 region of vascular dementia rats was disordered.The expression of Ch AT protein was decreased,the expression of Tau and A? were increased,the concentrations of IL-6,IL-1? and TNF-? were elevated,the expression of VEGF was increased.2.Qufengtongqiao decoction can effectively improve the learning behavior of VD rats,and protect morphology of neurons in hippocampal CA1 region.3.The mechanism of Qufengtongqiao decoction on rat model of vascular dementia may be related with the following:(1)Qufengtongqiao decoction could up regulate the expression of Ch AT in VD rat,and improve the function of cholinergic system.(2)Qufengtongqiao decoction can inhibit hyperphosphorylation of Tau protein in VD rat brain,decrease the expression of A? protein,reduce the damage of neurons.(3)Qufengtongqiao decoction can reduce the concentration of IL-6,IL-1?,TNF-?,inhibit the inflammatory response in ischemic region of VD rats.(4)Qufengtongqiao decoction can stimulate the expression of VEGF,promote angiogenesis in ischemia,repair the ischemic nerve.4.There is a dose effect relationship in Qufengtongqiao decoction improving the learning and memory ability of vascular dementia rats. |
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