Antagonistic Effect And Mechanism Of Tuina On Hippocampal Inflammation In Rats With Cerebral Palsy

Objectives To observe the effect of Tuina on the expression of TNF-? and IL-10 protein and the level of DNA methylation in the hippocampus of cerebral palsy?CP?rats,and explore the antagonistic effect and mechanism of Tuina on hippocampal inflammation in rats with cerebral palsy.Methods Thirty-nine male juvenile rats meet the research standard were slected from ninety-one neonatal rats and randomly divided into three groups: experimental group?n=13?,model control group?n=13?and blank control group?n=13?.The model group and the experimental group were treated with left common carotid artery ligation combined with hypoxia 3.5h to prepare cerebral palsy model,and the blank control group was not treated.After the second day of modeling?ie,Postnatal 5 days,P5?,the experimental group began with early intervention with Tuina,1 time / day,15 min /time,for 4 weeks,blank control group and model control Group without any intervention.Three groups maintained the same temperature,humidity environment and feeding method,the same time point for animal behavior detection.Morris Water Maze?MWM?and Step-down Test?ST?were commenced respectively on P26 and P31 days to observe their learning and memory abilities.All the junenile rats were killed to take hippocampus tissue,7 rats were randomly selected in each group to detect the protein expression levels of TNF-? and IL-10 in the hippocampus with Western Blot.The remaining 6 rats in each group were randomly divided into two groups?n = 3?,and the DNA methylation levels of TNF-? and IL-10 in hippocampus were respectively detected by Sequenom mass spectrometry and Pyrophosphate sequencing.Results1.Morris Water Maze experiment showed : Compared with the blank control group,the latent period of the experimental group was longer,the difference was statistically significant?P<0.05?,the latent period was shorter in the experimental group than in the model control group,but the difference was not statistically significant?P>0.05?.The number of experimental group crossing the platform was higher than the model group,the difference was statistically significant?P<0.05?,compared with the blank control group,there was no significant difference between the experimental group and the blank control group?P>0.05?.Compared with the blank control group,the number of crossing the platform of the model group was less,the difference was statistically significant?P<0.05?.The results showed that the learning and memory ability of the experimental group was better than that of the model control group,and the blank control group was better than the experimental group and the model control group.2.Step-down test showed: The latent time of the experimental group was longer than that of the model control group,shorter than the blank control group,and the difference was not statistically significant?P>0.05?.Compared with the blank control group,the latent period of the model control group was significantly shorter,the difference was statistically significant?P<0.05?.Comparison of three groups of jump times of platform: blank control group<experimental group<model control group.The number of model control group jumping off the patform was less than that of experimental group,the difference was statistically significant?P<0.05?,compared to the blank control group,the times of experimental group was more,the difference was statistically significant?P<0.05?;model group jumped off the platform times was higher than that of the control group and the experimental group,the difference was statistically significant?P<0.05?.The results showed that the learning and memory ability of the experimental group was better than that of the model control group than the blank control group.3.Western Blot showed: the expression of IL-10 protein in the blank control group was higher than that in the model group,and the difference was statistically significant?P<0.05?.The expression level of IL-10 protein in experimental group was lower than that in blank control group,there was no significant difference between the two groups?P>0.05?.The result showed that the expression level of IL-10 protein in experimental group was higher than that in model control group,but lower thna that in control group.The expression level of TNF-? protein in the experimental group was higher than that in the blank control group,the difference was not statistically significant?P>0.05?.Compared with the blank control group,the expression level of TNF-?protein in the model group was higher,the difference was statistically significant?P<0.05?.The results showed that the expression level of TNF-? protein in the experimental group was lower than that in the model control group but higher than in the blank control group.4.The results of Sequenom mass spectrometry: in the promoter region of IL-10 gene,there was no significant difference between the methylation rate of 4 Cp G sites in the three groups?P>0.05?.The IL-10 gene methylation rate of model control group,slightly higher than the blank control group and experimental group,no significant difference?P>0.05?.The results showed that there was no significant difference in the level of IL-10 DNA methylation between the three groups.In the TNF-? gene promoter region,the Cp G₈,9 relative methylation rate of experimental group was higher than the model group but lower than the control group,there were statistically significant differences between the three groups?P<0.05?;the Cp G₃,Cp G₈,9 and Cp G₁4 relative methylation rate of the model control group was lower than the blank control group,the difference was statistically significant?P<0.05?.The results showed that there were differences in the levels of TNF-? DNA methylation between the three groups.5.The results of Pyrophosphate sequencing: there was no significant difference in DNA methylation frequency between the experimental group,the blank control group and the model control group at the four Cp G sites in the promoter region of the inflammatory factor IL-10?P> 0.05?.The results showed that there was no significant difference in the level of IL-10 DNA methylation between the three groups.There was significant difference in DNA methylation frequency between the experimental group,the blank control group and the model control group at the 8 Cp G sites in the promoter region of the inflammatory factor TNF-??P <0.05?.Compared with the blank control group,the methylation frequency of Cp G₁,Cp G₂,Cp G3,Cp G₄,Cp G₅,Cp G₆ and Cp G₇ in the experimental group and the model controlgroup were significantly lower than those in the blank control group?P <0.05?.The methylation frequency of Cp G₁,Cp G₂,Cp G₄,Cp G₆ and Cp G₇ in the experimental group was significantly higher than that in the model control group?P<0.05?.In Cp G₈,the methylation frequency of the experimental group and the model control group was significantly higher than that of the blank control group?P<0.05?.The results showed that the level of TNF-? DNA methylation in the experimental group was higher than that in the model control group,but lower than that in the control group.Conclusions1.Tuina can improve the ability of learning and memory in cerebral palsy neonatal rats.2.Tuina can reduce the proinflammatory cytokine TNF-? expression level in the hippocampus of cerebral palsy rats,increase anti-inflammatory cytokine IL-10 protein expression level,reconstruct the relative stability of inflammation,3.The mechanism of Tuina's effect of regulating the expression of inflammatory cytokines TNF-?and IL-10 in hippocampus of young rats with cerebral palsy may be related to the regulation of DNA methylation of inflammatory cytokines TNF-? and IL-10,at the same time,the change of IL-10 protein expression may be related to the regulation of other factors.