| BackgroundThe clinical antipyretic effect of pediatric tuina is remarkable,but the mechanism is unclear,which not only affects the clinical application and promotion of pediatric tuina,but also is not conducive to the inheritance and development of pediatric tuina.Therefore,exploring the antipyretic mechanism of pediatric tuina has become an urgent scientific problem to be solved.ObjectiveThis study aimed to clarify the antipyretic mechanism of pediatric tuina and to provide scientific basis for clinical pediatric tuina in the treatment of fever.The antipyretic efficacy of six antipyretic manipulations on lipopolysaccharide(LPS)febrile young rabbits was explored by efficacy evaluation,so as to clarify the evidence of antipyretic effect of pediatric tuina.The effect of six antipyretic manipulations on hypothalamic metabolites was explored by liquid chromatography-mass spectrometry(LC-MS)untargeted metabolomics,so as to clarify the antipyretic mechanism of pediatric tuina from the metabolite level.The effect of six antipyretic manipulations on hypothalamic proteins was explored by data independent acquisition(DIA)quantitative proteomics,so as to clarify the antipyretic mechanism of pediatric tuina from the protein level.MethodsThis study was divided into three experiments.The antipyretic efficacy of six antipyretic manipulations was studied in Experiment 1,the effect of six antipyretic manipulations on hypothalamic metabolites was studied in Experiment 2,and the effect of six antipyretic manipulations on hypothalamic proteins was studied in Experiment 3.Two-month old New Zealand young rabbits were randomly assigned into three groups:the normal group,the model group,and the tuina group.Both the model group and the tuina group were injected intravenously with 0.5 μg/kg LPS,the normal group was injected with saline.Six antipyretic manipualtions(pushing Tianmen,pushing Kangong,kneading Taiyang,kneading Erhougaogu,clearing Tianheshui,and pushing Jizhu)were administered in the tuina group 1 h after the LPS injection.1.The maximum rising height(ΔTmax)of anal temperature and the total response index(TRI)were calculated by tracing the change curve of anal temperature 6 h after the LPS injection,to evaluate the antipyretic effect of six antipyretic manipulations on LPS febrile young rabbits.Then the young rabbits were divided into low-fever group(increased by 0.6～1℃),medium-fever group(increased by 1～1.5℃),and high-fever group(increased by more than 1.5℃)according to the rising height of anal temperature 1 h after modeling.To evaluate the antipyretic effect of six antipyretic manipulations on LPS febrile young rabbits with different heat degrees by the change of anal temperature,ΔTmax,and TRI,preliminarily determining the appropriate heat degree of six antipyretic manipulations.2.The differential metabolites in the hypothalamus of young rabbits were screened by LC-MS untargeted metabolomics,bioinformatics analyses such as HMDB and KEGG were carried out,thus to clarify the antipyretic mechanism of pediatric tuina from the metabolite level.3.The differential proteins in the hypothalamus of young rabbits were screened by DIA quantitative proteomics,bioinformatics analyses such as GO and KEGG were carried out,and the relevant proteins were verified by WB,thus to clarify the antipyretic mechanism of pediatric tuina from the protein level.Results1.The results of efficacy evaluation1.1 Antipyretic effect of six antipyretic manipulations on LPS febrile young rabbits① The results of anal temperature change showed that the anal temperature in the tuina group decreased compared with the model group 2～6 h after modeling(P<0.05),the anal temperature in the tuina group increased compared with the normal group 2～6 h after modeling(P<0.05).② The results of ΔTmax showed that ΔTmax in the model group and the tuina were higher than those in the normal group(P<0.01),ΔTmax in the tuina group was lower than in the model group(P<0.01).③The results of TRI showed that the 2～6 h TRI in the model group and the tuina group were higher than those in the normal group(P<0.01),the 2～6 h TRI in the tuina group were lower than that in the model group(P<0.01).1.2 Antipyretic effect of six antipyretic manipulations on LPS febrile young rabbits with different heat degreesLow fever:① The results of anal temperature showed that the anal temperature in the lowfever tuina group decreased compared with the low-fever model group 2～6 h after modeling(P<0.05).There was no difference between the low-fever tuina group and the normal group 2 h,3 h,and 6 h after modeling(P>0.05).②The results of ΔTmax showed that ΔTmax in the lowfever model group and the low-fever tuina were higher than those in the normal group(P<0.01),ΔTmax in the low-fever tuina group was lower than in the low-fever model group(P<0.01).③The results of TRI showed that the 2～6 h TRI in the low-fever model group and the low-fever tuina group were higher than those in the normal group(P<0.01),the 2～6 h TRI in the low-fever tuina group were lower than that in the low-fever model group(P<0.01).Medium fever:① The results of anal temperature showed that the anal temperature in the medium-fever tuina group decreased compared with the medium-fever model group 2～5 h after modeling(P<0.01).② The results of ΔTmax showed that Δ Tmax in the medium-fever model group and medium-fever tuina group were higher than those in the normal group(P<0.01),ΔTmax in the medium-fever tuina group was lower than in the medium-fever model group(P<0.01).③ The results of TRI showed that the 2～6h TRI in the medium-fever model group and the medium-fever tuina group were higher than those in the normal group(P<0.01),the 2～6 h TRI in the medium-fever tuina group were lower than that in the medium-fever model group(P<0.01).High fever:① The results of anal temperature showed that there was no difference between the high-fever tuina group and the high-fever model group 2 h,4 h,and 6 h after modeling(P>0.05),the anal temperature in the high-fever tuina group decreased compared with the high-fever model group 3 h and 5 h after modeling(P<0.05),and increased compared with the normal group 2～6 h after modeling(P<0.01).② The results of ΔTmax showed thatΔTmax in the high-fever model group and high-fever tuina group were higher than those in the normal group(P<0.01),ΔTmax in the high-fever tuina group was lower than in the high-fever model group(P<0.05).③The results of TRI showed that the 2～6 h TRI in the high-fever model group and the high-fever tuina group were higher than those in the normal group(P<0.01),the 3～6 h TRI in the high-fever tuina group were lower than that in the high-fever model group(P<0.05).2.Effects of six antipyretic manipulations on hypothalamic metabolites in LPS febrile young rabbitsMetabolomics results:① There were 95 differential metabolites in the tuina group vs.the model group,enriching 49 pathways,among which sphingolipid metabolic pathway was the most relevant.② There were 38 common differential metabolites between the model group vs.the normal group and the tuina group vs.the model group,of which 10 differential metabolites showed reversal trends,including N-Acetyl-L-glutamic acid,N-Acetyl-L-glutamate,Ecgonine,Topaquinone,Labienoxime,N-Acetylisoputreanine,1-(11Z,14Z-eicosadienoyl)-glycero-3phosphate,3-hydroxy-3-methyl-Glutaric acid,GDP-D-mannose,Peonidin 3-rhamnoside.③There were 28 unique differential metabolites in the tuina group vs.the model group,such as L-glutamate,oxidized glutathione,indoxylsulfuric acid,adenosine,and cytidine.3.Effects of six antipyretic manipulations on hypothalamic proteins in LPS febrile young rabbitsProteomics results:① There were 247 differential proteins in the tuina group vs.the model group,enriching 262 pathways.②There were 153 common differential proteins between the model group vs.the normal group and the tuina group vs.the model group,and 152 differential proteins showed reversal trends after tuina intervention,enriching 228 pathways,among which the signal transduction pathways were the most.cGMP-PKG,MAPK,PI3K-Akt,HIF-1,cAMP and other signaling pathways were related to this study.WB results showed that compared with the normal group,there was no significant difference in the contents of S100A4,VIM,SMAD2,NT5E,TRIM32,TRPV2,and STAT5 in the tuina group(P>0.05).Compared with the model group,the contents of S100A4,VIM,SMAD2,and NT5E in the tuina group decreased significantly(P<0.05),and the contents of TRIM32,TRPV2,and STAT5 increased significantly(P<0.01).The results of metabolomics combined with proteomics analysis showed that there were 33 same pathways,including GABAergic synapses,glutamatergic synapses,cholinergic synapses,and other nervous system pathways,tyrosine metabolism,phenylalanine metabolism,purine metabolism,pyrimidine metabolism,and other metabolic pathways were related to this study.ConclusionsThrough the efficacy evaluation,combined with metabolomics and proteomics,the conclusions in this study were drawn as follows:1.The six antipyretic manipulations can effectively reduce the anal temperature,fever intensity,and fever duration of LPS febrile young rabbits.Among them,it had the best antipyretic effect on young rabbits with low fever,which can reduce the anal temperature to the normal level.It had a good antipyretic effect on young rabbits with medium fever,and can reduce the anal temperature,fever intensity,and fever duration of young rabbits with medium fever.It had no obvious antipyretic effect on young rabbits with high fever,it can reduce the fever intensity and fever duration,but can not significantly reduce the anal temperature.2.The antipyretic mechanism of six antipyretic manipulations was related to regulating the expression of differential metabolites,such as N-Acetyl-L-glutamic acid,N-Acetyl-Lglutamate,Ecgonine,Topaquinone,Labienoxime,N-Acetylisoputreanine,1-(11Z,14Zeicosadienoyl)-glycero-3-phosphate,3-hydroxy-3-methyl-Glutaric acid,GDP-D-mannose,Peonidin 3-rhamnoside.3.The antipyretic mechanism of six antipyretic manipulations was related to the regulation of cGMP-PKG,MAPK,PI3K-Akt,HIF-1,cAMP,and other signal transduction pathways.The antipyretic mechanism of six antipyretic manipulations was related to upregulating the expression of TRIM32,TRPV2,STAT5 and downregulating the expression of S100A4,VIM,SMAD2,NT5E.In conclusion,the six antipyretic manipulations had significant antipyretic effect,and its antipyretic mechanism was related to the regulation of differential metabolites and differential protein expression in hypothalamus. |
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