| ObjectiveThe goal of this study was to observe the effect of Tuina on the differentiation of oligodendrocyte precursor cells in the white matter of cerebral palsy rats,to analyze its relationship with Notch1/Hes5 signaling pathway,to reveal the possible mechanism of Tuina promoting white matter repair in the treatment of cerebral palsy.MethodsExperiment 1:The 52 Sprague Dawley pups born naturally were strictly selected on postnatal day 3(P3).The remaining 50 newborn pups weighing about 9g were randomly divided into the Normal group(n=12),the Sham group(n=12),and the Model group(n=26).The Model group was prepared by left common carotid artery fusion combined with hypoxia.The model was verified successfully by righting reflex test on the second day after the model was made(P4).Then 24 young rats in the model group were randomly divided into CP group(n=12)and Tuina group(n=12).No operation was performed in the Normal group.The Sham group only cut the skin on the left neck,then separated the carotid artery and the vagus nerve without melting and hypoxia.On the third day after modeling(P5),Tuina intervention was performed on the Tuina group.Each young rat was interfered with for 15 minutes,once a day,6 days a week,for a total of 8 weeks.The other three groups of young rats and female rats were fed separately in cages without intervention.The body weight of the four groups of rats was monitored and recorded at a fixed time point.After 8 weeks of intervention(P61),the movement and balance functions of the four groups of young rats were tested by the Foot-Fault test and Roat-rod test.Subsequently,the four groups of young rats were sacrificed and white matter was collected.The myelin sheath of white matter was detected by Luxol Fast Blue staining and transmission electron microscopy.The location and expression of MBP were detected by immunofluorescence and Western Blot,and the expressions of Notch1 and Hes5 proteins were detected by Western Blot.Experiment 2:Another 36 young rats were selected and randomly divided into the Saline group(n=9),Saline + Tuian group(n=9),DAPT group(n=9),and DAPT+ Tuina group(n=9).The Saline group and the Saline + Tuina group were intraperitoneally injected with 10mg/kg/ piece of normal saline on the first day after modeling(P4),once a day for 6consecutive days;The DAPT group and DAPT+ Tuina group were injected with DAPT in the same dosage,way and time course;The Saline + Tuina group and DAPT+ Tuina group received the same Tuina intervention on the second day after modeling(P5),while the other three groups of young rats and female rats were fed separately in cages without intervention.The four groups of young rats were sacrificed after the intervention(P61),and white matter was collected.The myelin sheath of white matter was detected by transmission electron microscopy.The localization and expression of PDGFR-α and MBP were detected by immunofluorescence and Western Blot.Results1.The weight of infant cerebral palsy model rats decreased obviously,Tuina accelerated the weight increase of infant cerebral palsy rats,and with the extension of time,the degree of weight increase was more significant(P<0.001).2.After Tuina,the F-F score and Roat-rod score of cerebral palsy rats were significantly increased,and cerebral palsy rats’ motor function and balance function was significantly enhanced(P < 0.001).3.The Notch1/Hes5 pathway was over-activated in cerebral palsy rats,and the Notch1/Hes5 pathway was inhibited after Tuina,the expression levels of Notch1 and Hes5 proteins were significantly decreased(P<0.001).4.After Tuina,the proportion of myelin sheath,the number of myelinated nerve fibers(P < 0.05),and the thickness of myelin sheath were significantly increased,and the phenomenon of loose myelin structure,irregular shape,and turbidity of cytoplasm was significantly improved(P < 0.001).The number of myelinated nerve fibers and the thickness of the myelin sheath were significantly increased(P < 0.05),and the structure of the myelin sheath was improved(P < 0.001),which was consistent with the effect of Tuina under the condition of DAPT inhibitor.5.After Tuina,The mean fluorescence intensity of MBP in cerebral palsy rats was slightly increased after the Tuina,but the difference was not statistically significant(P >0.05).The protein expression of MBP was significantly increased(P < 0.0001).The Notch1/Hes5 pathway was inhibited after Tuina,and the expression levels of Notch1 and Hes5 proteins were significantly decreased(P < 0.001).The average fluorescence intensity(P < 0.001),and protein expression of PDGFR-α in cerebral palsy rats were significantly decreased(P < 0.05).,consistent with Tuina’s effect The average fluorescence intensity(P< 0.01)and protein expression of MBP were significantly increased(P < 0.001),which was consiconsistent with Tuina’s effectibitor the condition.Conclusion1.Tuina can promote the growth and development of cerebral palsy rats,improve its movement and balance function,promote oligodendrocyte myelination in the cerebral white matter of cerebral palsy rats,and repair its white matter injury.2.Tuina promotes white matter repair in young cerebral palsy rats,and the possible mechanism is to enhance the differentiation and maturation of oligodendrocyte precursor cells by inhibiting Notch1/Hes5 signaling pathway. |
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