Study On The Effect And Mechanism Of Hippocampal Circular RNA 0001724 In Improving Learning And Memory In Cerebral Palsy Rats By Massage

Objective:To observe the effect of massage on the expression of hippocampal and peripheral blood BDNF、Trk B and mmu＿circ＿0001724 in the hippocampus of cerebral palsy rats,and explore the molecular mechanism of massage in promoting learning and memory in cerebral palsy rats,and provide experimental evidence for clinical application of massage to improve learning and memory for cerebral palsy children.Method:the 52 newborn SD rats were randomly divided into the Blank Control Group(n=12),the Sham Group(n=12)and the Model Group(n=28).The Model Group were prepared by left carotid artery ligation combined with hypoxia.The sham Control Group only separated the left common carotid artery.24 rats with successful modeling were randomly divided into the Massage Group(n=12)and the Model Control Group(n=12).The feeding environment and methods of the four groups were the same.At the second day after modeling,the Massage Group were intervented by massage,15 min /time,1 time/day,6times/week,for 4weeks.The other three groups without any intervention,regular feeding.The growth and development situations of four groups(weight,body and tail length,eye opening time)were recorded,and conducted righting reflection experiments and Morris Water Maze(MWM)behavioral experiments at the same time.All rats were killed to take peripheral blood and brain hippocampus tissue,Using ELISA and WB method to detect the expression of BDNF and Trk B proteins in the serum and hippocampus,Choosing q PCR to detect the expression of circular RNA in the left hippocampus,and HE staining to observe the morphological changes of the brain tissue in the CA1 region of the left hippocampus.Result:1.After modeling,the model was verified by the righting reflex experiment.The average correction time of the Model Group was significantly longer than that of the Blank Control Group and the Sham Group(P<0.001),indicating that the model of cerebral palsy was successfully prepared.2.After 4 weeks of intervention,the average weight of the Massage Group was higher than the Model Control Group(P<0.05),the average length of body and tail of the Massage Group was longer than the Model Control group(P<0.05);Compared with the Blank Control Group had no statistical differences(P>0.05).The time of eye opening of the Massage Group was earlier than the Model Control Group(P<0.01),and later than the Blank Control Group(P<0.05),indicating that massage can improve the abnormal growth and development of cerebral palsy rats.3.The MWM showed that in the positioning navigation experiment: the swimming distance and the latency period of the Model Group were longer than the Blank Control Group(P<0.05).The Model Group needed more distance and time to find escape platform,indicating that the cerebral palsy rats had defects in spatial positioning learning ability.The Massage Group had fewer swimming distances and shorter latency period than the Model Group(P<0.05).The results of space exploration in each group were compared.Compared with the Blank Control Group,the Model Control Group had longer test latency period(P<0.001),the times to cross the platform was reduced(P<0.001)and the percentage of distance to the platform quadrant was reduced(P<0.001).The latency period(P<0.001),the times of crossing the platform(P<0.05)and the percentage of the platform quadrant distance(P<0.001)of the Massage Group were significantly improved compared with the Model Control Group,indicating that massage can improve the spatial positioning learning and memory ability of cerebral palsy rats.4.The results of HE staining showed that the left hippocampal CA1 region pyramidal cell layer was arranged disorderly of the Model Group,the distance between the cells was significantly widened,the neurons were degenerated and necrotic,and the cell body became significantly smaller,and a small number of nuclei were proliferated with rounded microglial cells;Compared with the Model Group,the cell arrangement in the Massage Group was relatively regular,and neuronal vacuolar degeneration or necrosis was reduced,indicating that massage can improve brain tissue damage caused by ischemia and hypoxia of modeling.5.ELISA experimental results show that:The expression of peripheral blood BDNF of the Model Control Group was lower than the Blank Control Group and the Sham Control Group(P<0.01).The expression of the Massage Group was higher than the Model Group(P<0.05),while lower than the Blank Control Group(P<0.05).The expression of peripheral blood Trk B of the Model Control Group was lower than the Blank Control Group and the Sham Control Group(P<0.01).the expression of the Massage Group was higher than the Model Group(P<0.05),while lower than the Blank Control Group(P<0.05).The results showed that the massage can effectively promote the expression of BDNF and Trk B protein in the peripheral blood of cerebral palsy rats.6.Western Blot experimental results show that:The expression of hippocampal BDNF protein of the Model Control Group was lower than the Blank Control Group and the Sham Control Group(P<0.01).The expression of the Massage Group was higher than the Model Control Group(P<0.01),and without difference compared with the Blank Control Group and the Sham Control Group(P>0.05);The expression of Trk B protein of hippocampus of the Model Control Group was significantly lower than the Blank Control Group and the Sham Control Group(P<0.01).The expression of the Massage Group was significantly higher than that of the Model Control Group(P<0.01),and without difference compared with the Blank Control Group and the Sham Control Group(P>0.05).The results showed that massage therapy can effectively promote the expression of BDNF and Trk B protein in the hippocampus of cerebral palsy rats.7.QPCR results show that:The expression of hippocampal circular RNA mmu＿circ＿0001724 of the Model Control Group was significantly higher than that of the Blank Control Group and the Sham Control Group(P<0.01),and the expression of the Massage Group was lower than that of the Model Control Group(P<0.05),and there was no statistical difference compared with the Blank Control Group and the Sham Control Group(P>0.05).It shows that massage can down-regulate the expression of circular RNA mmu＿circ＿0001724 in hippocampus of cerebral palsy rats.8.Correlation analysis between the expression of hippocampal circular RNA mmu＿circ＿0001724 and the expression of BDNF and Trk B proteins of the four groups rats showed that:The expression of circular RNA mmu＿circ＿0001724 in the hippocampus of the brain was significantly negatively correlated with the expression of BDNF and Trk B protein in the hippocampus(P<0.01).Conclusion:1.The massage can improve the growth and development of cerebral palsy rats.2.The massage can improve the learning and memory ability of cerebral palsy rats.3.The possible mechanism of massage to improve the learning and memory ability of cerebral palsy rats is achieved by down-regulating the expression of circular RNA mmu＿circ＿0001724 in hippocampus,and up-regulating the expression of BDNF and Trk B protein in hippocampus and peripheral blood,and promoting the differentiation and maturation of hippocampal neurons and enhancing synaptic plasticity.This research can provide experimental basis for the clinical application of massage therapy to improve the learning and memory ability for cerebral palsy children.