Study On The Role And Mechanism Of White Matter CircRNA Mmu＿circ＿0001724 In The Treatment Of Cerebral Palsy In Young Rat

Objective To observe the expression of circ RNAmmu_circ₀001724 in the white matter of young rat after Tuina,and to explore its role and mechanism in the treatment of cerebral palsy in young rats.Method Seventy-two newborn Sprague Dawley（SD）rats were randomly divided into two groups according to the random number table,with 36 rats in each group.One group was randomly divided into normal group（n= 18）and sham group（n= 18）.In the other group,the left common carotid artery ligation combined with hypoxia method was used to prepare the model,and all the models were recorded in P1（P0 on the modeling day and so on,P1,P2,P3...）.The model was verified by turn-over experiment.After 9 mice died or failed in modeling,the remaining 17 mice were randomly divided again into the massage group（n=14）,the model group（n=13）,and the sham operation group.On the day of modeling,the left neck skin was cut open,and the blood vessels were separated without ligation or hypoxia treatment.P1 One mouse died in the massage group and two mice died in the model group.In order to ensure the same number of feeding pups in each group,6 mice were randomly removed from the normal group and the sham operation group,i.e.,the normal group（n= 12）,the sham operation group（n= 12）,the model group（n=11）and the massage group（n=13）.From P0,the body weight of four groups of young rat was recorded at the same time point every day,and the body length and tail length of four groups of young rat were recorded at five time points: P0,P7,P14,P21 and P28.P1 began the Tuina intervention,intervention for 6 consecutive days,rest for a day,intervention for 4consecutive weeks,the other three groups did not Tuina intervention,only in the Tuina group Tuina intervention fasting treatment;P24 Conduct behavioral training for three consecutive days,including balance beam training and gait analysis training;P27Behavioral tests,including balance beam test and gait analysis test,were conducted to evaluate the motor function of four groups of pups.P29,that is,1 day after the end of intervention,the four groups of young rats were sacrificed.The brains of the four groups were removed,and the white matter and cerebral cortex were placed in 1.5m LEP tubes,respectively,and transferred to-80℃ refrigerator through dry ice for preservation.The structural changes of axons were observed by electron microscopy.G value（the ratio of inner and outer diameters of axons）was calculated.The myelination degree of axons in the four groups was compared.The expression level of target circ RNA in brain white matter was detected by q PCR.WB was used to detect the expression level of myelin basic protein（MBP）in the cerebral cortex of young rat.All data were analyzed using IBM SPSS Statistics Version 26.0.0.0.Result 1.Comparison of general conditions of four groups of young rats The body weights of the four groups were compared at P0,P7,P14,P21 and P28 respectively.The results showed that from P7,the average body weights of the sham operation group and normal group were higher than that of the model group.The average body weight of the Tuina group from P21 was higher than that of the model group,and the difference was statistically significant.The body length and tail length of P0,P7,P14,P21 and P28 of the four groups were compared respectively.The results showed that from P7,the average body length and tail length of the sham operation group,normal group and Tuina group were all longer than that of the model group,and the difference was statistically significant.2.Comparison of motor function of four groups of young rats（1）Comparison of the length of support between two hind legs of young rats in four groups（left hind foot vs.right hind foot）The average supporting duration ratio of the two hind limbs in the Tuina group was1.01±0.13,the average supporting duration ratio of the two hind limbs in the model group was 1.17±0.11,the average supporting duration ratio of the sham group was 1.01±0.12,and the average supporting duration ratio of the normal group was 1.04±0.19.The comparison between the Tuina group and the normal group and the sham group showed no statistically significant difference,while the comparison between the Tuina group and the model group showed statistically significant difference（P<0.01）.（2）Comparison of the ratio of the maximum ground contact area between the two hind feet of young rats in four groups（left hind foot vs.right hind foot）The average ratio of the maximum ground touch area of the two hind feet in the Tuina group was 1.01±0.14,the average ratio of the model group was 1.18±0.1,the average ratio of the sham group was 1.00±0.11,and the average ratio of the normal group was1.01±0.17.The comparison between the Tuina group and the normal group and the sham group showed no statistically significant difference,while the comparison between the Tuina group and the model group showed statistically significant difference（P<0.01）.（3）Comparison of the balance function of the four groups The average time to pass the balance beam in the Tuina group was 6.88±0.8,the average time to pass the balance beam in the model group was 9.44±1.76,the average time to pass the balance beam in the sham group was 5.55±0.65 and the average time to pass the balance beam in the normal group was 5.42±0.65.The comparison between the Tuina group and the normal group and the sham group showed statistically significant differences（P<0.01）,and the comparison between the Tuina group and the model group showed statistically significant differences（P<0.05）.3.Comparison of axon myelination levels in four groups The average myelin G value of the Tuina group was 0.76±0.07,the model group was0.81±0.06,the sham group was 0.73±0.08,and the normal group was 0.72±0.06.The comparison between the Tuina group and the normal group and the sham group showed no statistically significant difference,while the comparison between the Tuina group and the model group showed statistically significant difference（P<0.05）.4.Comparison of circ RNAmmu_circ₀001724 expression levels in white matter of four groups The average expression level of circ RNAmmu_circ₀001724 in white matter of the Tuina group was 1.65±0.39,the average expression level of the model group was1.87±0.61,the average expression level of the sham group was 1.35±057,and the average expression level of the normal group was 1.23±0.36.The difference between the Tuina group and the normal group and the sham group was not statistically significant,and the difference between the Tuina group and the model group was not statistically significant.The average expression level of circ RNA in the model group was higher than that in the normal group,and the difference was statistically significant（P<0.05）.5.Comparison of MBP protein expression levels in four groups The mean expression level of MBP protein in the cerebral cortex of the Tuina group was 0.86±0.22,the mean expression level of the model group was 0.41±0.2,the mean expression level of the sham group was 0.83±0.25,and the mean expression level of the normal group was 0.88±0.2.The comparison between the Tuina group and the normal group and the sham group showed no statistically significant difference,while the comparison between the Tuina group and the model group showed statistically significant difference（P<0.01）.Conclusion 1.Tuina can promote the growth and development of infant CP mice,and improve the motor function and balance function of infant CP mice.The experimental results are consistent with the previous experimental results of the research group.2.Tuina can improve the degree of myelination of oligodendrocyte axons and promote the repair of white matter in CP infant rat.3.The mechanism of Tuina promoting white matter repair in CP infant rats may be realized by down-regulating the expression level of circ RNA mmu＿circ＿0001724circ RNA in brain white matter of CP infant rats,up-regulating the expression of MBP protein,and affecting the functional state of mi RNA.