| Cerebral palsy(CP)is a syndrome caused by non-progressive brain injury and developmental disorders from pregnancy to infancy.It mainly manifests as movement disorders and postural abnormalities,and is often accompanied by sensation,perceptual,cognitive,and behavioral disorders,as well as epilepsy and secondary musculoskeletal problems.In recent years,with the development of perinatology and medical technology,the incidence of CP has shown an increasing trend,and CP has become the main disease that causes children’s disability.Therefore,CP not only seriously damages the physical and mental health of infants and young children,but also brings a heavy burden to the family and society.It is currently believed that the occurrence of CP is caused by a variety of factors during perinatal period,among which ischemia-hypoxia,infection and inflammation,premature/low birth weight infants and neonatal jaundice are the main high-risk factors.The main pathological change of CP is the damage of white matter,which is often accompanied by impairment of cognitive function.In the process of CP caused by ischemia-hypoxia,infection and inflammation,on the one hand,it will activate the microglia in the brain.On the other hand,due to the damage of blood-brain barrier,the peripheral neutrophils and macrophages will enter the brain through the blood-brain barrier.These innate immune cells of the body will produce an inflammatory response.There are damage-associated molecular patterns(DAMP)on the surface of damaged brain cells after CP,which can be recognized by immune cells containing pattern recognition receptors(PRR)to initiate an immune response.NOD-like receptor family(nucleotide-binding oligomerization domain like receptor,NLR)is one of the pattern recognition receptors,among which NLRP3 is a molecule that has attracted much attention in the NLRs family in recent years.When NLRP3 is activated,it can form an NLRP3 inflammatory complex with the adaptor protein ASC(apoptosis-associated speck-like protein containing)and pro-caspase-1 to initiate pyroptosis.Pyrolysis is a form of cell death that depends on Caspase.Pyroptosis can activate Caspase-1 by shearing inactive pro-caspase-1,which activates gasdermin D(GSDMD)and promotes the production of IL-1β and IL-18,which ultimately triggers cell expansion,rupture,and releasing of cell content leading to local inflammation.Therefore,pyroptosis belongs to cell inflammatory necrosis,which is involved in the pathophysiological process of a variety of diseases such as hypoxic-ischemic brain injury and cerebral hemorrhage.In this study,by replicating an animal model of hypoxic-ischemic encephalopathy in neonatal rats,we observed the role of pyroptosis in the pathogenesis of CP.At the same time,spinal Tuina was given to young rats with ischemic-hypoxic brain injury to observe the effect of spinal Tuina on improving the learning and memory impairment of CP young rats,and to explore the ameliorative mechanism of spinal Tuina on cognitive function impairment after CP from the perspective of pyroptosis.Purposes: Spinal Tuina was given on an animal model of CP to observe that Tuina inhibited NLRP3 in the hippocampus of young rats with CP and that the pyroptosis induced by spinal Tuina can improve the cognitive function impairment of CP young rats.Methods: Thirty male SD newborn rats were randomly divided into: Sham group,Model group and Tuina group,with 10 rats in each group.The young rats in the model group and Tuina group were ligated and coagulated on the right common carotid artery on the 7th day after birth,and were treated with hypoxia(8%O2 and 92%N2)for 2.5 hours after the operation.Rats in the sham group were only given a neck incision and separation of the right common carotid artery on the 7th day after birth.All the pups were returned to the mothers for feeding after the operation.The young rats in the Tuina group were given spinal Tuina once a day after the operation from the 2th day to the 49 th day after birth.The pups of each group were weighed every two days from the 3th day to the 49 th day after birth;the balance beam test was carried out from the 40 th day to the 43 th day after birth;the light-dark box test was carried out from the 44 th day to the 45 th day after birth;Morris water maze test was performed from the 46 th day to the 50 th day after birth.All experimental pups were sacraficed on the 50 th day after birth.Immunohistochemistry was used to detect the expression of NLRP3 in the hippocampus of the pups,and Western blot was used to detect changes in the protein expression of NLRP3,Caspase-1,cleaved-Caspase-1,GSDMD,IL-1β and IL-18 in the hippocampus.Results:1.The body weights of the pups in model group and Tuina group on the 7,9,11,13,15,and 25 days after birth were significantly lower than those in the sham operation group(the corresponding time points were all p<0.05),and after birth at 27,31,33,35,37,39,41,43,45,47,49 days,the weight of the pups in model group was still significantly lower than that of sham operation group(the corresponding time points were all p<0.05),while the body weight of the pups in Tuina group was significantly higher than that of the model group(the corresponding time points were p<0.05),but the weight of the young rats in Tuina group was not significantly different from that of sham operation group(the corresponding time points were p>0.05).2.In the balance beam test,pups in sham operation group,model group and Tuina group took(5.10±0.99)seconds,(21.80±15.32)seconds and(7.22±3.22)seconds to cross the balance beam,respectively.Compared with sham operation group,the time for the young rats in model group to cross the balance beam was significantly longer(p<0.05);and compared with model group,the time for the young rats in Tuina group to cross the balance beam was significantly reduced.However,there was no significant difference between the sham operation group and the Tuina group;the number of hindlimb slipping times during the three groups of young rats crossing the balance beam was(0.10±0.32))times,(3.00±1.25)times,and(1.50±1.08)times.Compared with sham operation group,the pups in the model group and the Tuina group had significantly increased hindlimb slipping times through the balance beam(p<0.05 for both groups),but the number of hindlimb slipping of young mice in the Tuina group was significantly lower than that in the model group(p<0.05).3.In the light-dark box test,the number of pups entering the dark box in the sham operation group,model group and Tuina group were(0.90±0.88)times,(6.10±1.66)times and(3.40±1.71)times,respectively.Compared with sham operation group,the number of entering the dark box of pups in model group and Tuina group was significantly higher(p<0.05 for both groups)than sham operation group,while the number of pups entering the dark box in the Tuina group was significantly less than the model group(p<0.05);The latency of the rats entering the dark box for the first time were(133.10±43.16)seconds,(29.30±14.87)seconds,and(58.50±24.04)seconds.Compared with sham operation group,the latency of the young rats in model group and Tuina group was significantly shorter(two The p value of the group is p<0.05),while there was no significant difference between model group and Tuina group.4.In the Morris water maze test,the latency for pups in sham operation group,model group and Tuina group to find the platform was(6.38±2.55)seconds,(34.17±10.16)seconds and(17.82±3.65)seconds,respectively.Compared with sham operation group,the latency of the pups in model group and Tuina group was significantly higher(p<0.05 for both groups),but the latency of the pups in Tuina group was significantly lower than that of model group(p<0.05);The percentages of residence time of sham operation group,model group and Tuina group in the quadrant where the small platform is located are(63.46±12.51)%,(30.27±15.24)% and(43.41±9.21)%,respectively.Compared with sham operation group,the young rats in model group and Tuina group staying in the platform quadrant was significantly reduced(p<0.05 for both groups),but the percentage of residence time in the quadrant of the platform of the young rats in Tuina group was significantly higher than that in the model group(p<0.05).5.The expression of NLRP3 was detected by immunohistochemistry,and the results showed that NLRP3 was mainly expressed in the cytoplasm of cells.Western blot was used to detect the expression changes of NLRP3 in hippocampus.The results showed that the expression of NLRP3 in the hippocampus of young rats in model group was significantly higher than that in sham operation group and Tuina group(p<0.05 for both groups);and the expression of NLRP3 in the hippocampus of young rats in Tuina group was significantly lower than that in model group.Nevertheless,there was no significant difference in the expression of NLRP3 in the hippocampus of young rats betweean Tuina group and sham operation group.6.Western blot was adapted to detect the changes in the expression of Caspase-1 in the hippocampus of three groups of young rats.The results showed that the expression of Caspase-1 in the hippocampus of the young rats of model group and Tuina group was significantly increased compared with sham operation group(p<0.05 for both groups),while there is no significant difference between model group and Tuina group.However,the expression of active cleaved-Caspase-1 in the hippocampus of the three groups of young mice were different from that of Caspase-1.The expression of cleaved-Caspase-1 in the hippocampus of young rats in model group and Tuina group was significantly higher than that in sham operation group(p<0.05 for both groups),but the expression level of cleaved-Caspase-1 in Tuina group was significantly lower than that of model Group(p<0.05).7.The expressions of GSDMD,IL-1β and IL-18 in the hippocampus of young rats in sham operation group,model group and Tuina group showed the same changes after Western blot detection,that is,the expression of GSDMD,IL-1β and IL-18 in the hippocampus of young rats in model group and Tuina group were significantly higher than those in sham operation group(p<0.05 for both groups),while the expressions of GSDMD,IL-1β and IL-18 in Tuina group were significantly lower than those in the model group(p<0.05).Conclusion:1.Tuina can significantly improve the growth and development of young rats with CP,and promote the recovery of motor balance ability of young rats with CP.2.Tuina can significantly improve the cognitive function of young rats with CP.3.The expression of NLRP3 in the hippocampus of young rats with CP was down-regulated after spinal Tuina intervention,leading to the suppression of pyroptosis in hippocampus after CP. |
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