| Objective: This study applies the iTRAQ technology(the isobaric tags for relative and absolute quantification)to analyze the pathogenetic mechanism of mycoplasma pneumonia from the perspective of differential protein,include damage and protection mechanism.Besides,the relationship between the mycoplasma pneumonia and imbalance of ROR?t / Foxp3 as well as the notch signaling pathway is further explored and the “QingfeiTouxie(a prescription of traditional Chinese medicine which was made by my instructor Wang xuefengto take away heat in the patient's lung and expel pathogenic factors)” formula is explored in a bid to provide a new thought pattern for the clinical treatment of mycoplasma pneumonia.Material and Method: 1.The technology of iTRAQ had been used to analyze(the expression levels of)main differential proteins for the mice contracting mycoplasma pneumonia(comparison between the mice infected with mycoplasma pneumonia and the normal ones),and whether(the expression levels of)main differential proteins were pathogenic for the mice with the same infection(comparison between the non-infected mice and the infected ones).The targeted 30 Balb/c mice had been divided into the normal group and the infected group at a ratio of 1:2 at random: hence 10 mice in the normal group and the remaining 20 ones in the infected group.All through this research the infected group was given 100 ul nasal drip of MP strain per mouse for the three consecutive days,and in the meanwhile the normal group was given 100 ul nasal drip of 0.9% normal saline per mouse for the same three consecutive days as well.Lung tissues of mice from both groups were taken out after the seven-day medical observation.Moreover,the infected group was further divided into the pneumonia-infected group and the non-pneumonia-infected group depending on the pathological observation results.Samples of the pneumonia-infected group,the normal group and the non-pneumonia-infected group were respectively detected with the methods of iTRAQ proteomics to screen differentially expressed proteins to be further handled for the bioinformatics analysis.2.The targeted 144 SPF-level BABL/c mice were randomly divided into the six groups comprised of the normal group,the model group,the west medicine group,the heavy-dose traditional Chinese medicine group,the medium-dose traditional Chinese medicine group,and the light-dose traditional Chinese medicine group,hence 20 mice in each group.Except the normal group,the other five groups were MP-modeled(to cause pathological symptoms)and next given gavage administration for taking sample issues on the 3rd day,7th day,10 th day and 14 th day after drug injection.Then,the level of IL10 and the level of IL17 in the serum of mice were measured with the methods of Elisa;the pathological sections of mice's lung tissues were observed under a microscope;the level of Foxp3 protein and the level of ROR?t protein were detected with the methods of immunohistochemistry;the expression levels of Notch 1 protein,Notch 2 protein and Notch 3 protein were checked with the methods of Western blot,while the levels of Notch 1 mRNA,Notch 2 mRNA,Notch 3 mRNA,Foxp3 mRNA and ROR?t mRNA were measured with the methods of qPCR.Results: 1.After comparing the pneumonia-infected group and the normal group,this study had identified 141 proteins with quantitative information,including the 85 up-regulated proteins and the 56 down-regulated proteins.Among them,the top ten differential proteins consists of Sptan1,Myh6,Col1a1,Fasn,Col6a3,Iqgap1,Col1a2,Ttn,Limch1 and Ncl that were mainly involved in activities such as body immunity,fatty acid synthesis,carbohydrate metabolism,nucleotide binding,actin binding,bindings of calcium and other metallic ions,actin binding and regulating,and oxidative metabolism.For a more robust research,the Go analysis carried out by this paper reveals that all these differential proteins were mainly engaged in the following engineering processes: immune system processing,development process,anatomical structure,metabolism process of auxiliary factors,multicellular biological development,anatomical structure morphogenesis,response to organic substances,system development,circulatory system development and animal organ development,of which GO:0048731 system development proved the most significant.In terms of molecular function,these differential proteins were mainly playing a role in activities of structural molecular,binding,extracellular matrix structure,protein binding,enzyme binding,binding of the same proteins,self-binding of proteins,S100 protein binding,and binding of platelet-derived growth factors,of which GO:0005515 protein binding turns out the most notable.As far as cell composition was concerned,these differential proteins were mainly making contributions to the compositions of extracellular region,extracellular space,extracellular component,voxel projection,cell projection of plasma membrane binding,cytoplasmic part,(intracellular)vacuole,dissolved vacuole,lysosome,with Go: 0005737 cytoplasmic being the most remarkable.The analysis through scatter diagram demonstrates that the largest Go items were L-glutamine,aldosterone,low-density lipoprotein,endodermal cells,endodermal ossification,hydrogen peroxide biosynthesis,negative regulation of oxidative stress,myocardial fiber development,glycogen metabolism,antibiotic biosynthesis,cellular polysaccharide catabolism and polysaccharide catabolism.In the meantime,the KEGG pathway analysis indicated that those with the most significant enrichment contain pentose phosphate pathway,the formations of lysosome,dorsal ventral axis,protein digestion and absorption,amino acid biosynthesis,and interaction between proteasome and ECM receptor.Besides,a further analysis of interactions between proteins indicates that Cdc42,Cyba,Cybb,Syk,Ptpn6,Rasgrp2,Hcls1,Arhgdib,Lcp1,Actr2,Myh6,l1gap1,Arpc2,Pfn1,Ttn,Ldb3,Actn2,Serpina3 k,Myl7,Ppp1r12 c,Cdc42bpg,Psmb8,Psmd11,Psma2,Psme1,Col5a1,Col5a2,Col1a2,Col1a1,Col6a2,Col6 a and Col6a1 were in the critical positions,so they could be the vital proteins for the incidence of mycoplasma pneumonia.This study had also conducted a modularization analysis of complex networks,putting forward the four major modules as follows: the module one was mainly engaged in the metabolism of organic substances and cells,the module two was mainly responsible for the reaction to oxygen-containing compounds and the occurrence of anatomical structure and morphology,the module three was mainly associated with protein and ion binding,supramolecular polymer,actin cytoskeleton,circulatory system development,nitrogen metabolism,and the module four was mainly related to cell communication and signal transmission.2.After comparing the non-pneumonia-infected group and the pneumonia-infected group,this study had recognized the 5 up-regulated proteins and the 28 down-regulated proteins.The top ten differential proteins were all down-regulated,consisting of Fasn,Cavin1,Anxa2,Hnrnpm,Cbr2,A1dh2,Tagln2,Inmt,Mthfd1,Actc1,Tufm,Inmt,Mthfd1,Actc1,Tufm,Pgd,Psmd11,Capn2,Arpc2,Ddost,Gstt1,Cct6 a,Cox4il and Ivd that were mainly involved in activities such as body immunity,fatty acid synthesis,lung metabolism,actin binding,catalysis.And at the same time the Go analysis carried out by this paper reflects that all these differential proteins were mainly engaged in the following engineering processes: cofactor metabolism,redox process,organic matter metabolism,of which Go: 0051186 cofactor metabolism process turns out the most significant.In the light of molecular function,these differential proteins were mainly playing a part in activities of catalytic activity,oxidoreductase activity,small molecule binding,nucleotide binding,ion binding,anion binding,coenzyme binding,nucleoside phosphate binding,cell adhesion molecule binding,actin binding,actin silk binding,lipid binding,ion binding,anion binding,protein-containing complex binding,of which Go: 0016491 oxidoreductase activity turns out the most notable.As far as cell composition was concerned,these differential proteins were mainly making contributions to the compositions of cell ventral surface,podocyte loop,actin filament branch point,cytoplasm,cytoplasmic part,intracellular part,intracellular,mitochondria,organelle,membrane-bound organelle,intracellular organelle,with GO: 0061835 cells ventral being the most remarkable.In the meanwhile,the KEGG pathway analysis suggested that those with the most significant enrichment contain dorsal ventral axis formation,Notch signaling pathway,tryptophan metabolism,arginine and proline metabolism,valine,leucine and isoleucine degradation,inflammatory bowel disease,glyceride metabolism,glutathione peptide metabolism,endocrine resistance,P450 xenobiotics metabolization through cytochrome,thyroid hormone signaling pathway,endoplasmic reticulum protein processing,and metabolic pathways.In addition,another analysis of interactions between proteins revealed that Gm17087,Actc1,Arpc2,Tagln2,Anxa2,Serpina3 g,Hsph1,Cct6 a,Notch2,Notch3,Notch1,Pard3,Foxp3,ROR?t,Serpinb6 b,Clca3,Pard3,Ndufv3,Cox4i1,Ddost,Hnrnpm,Nxf1,Pgd,Fasn,Gpam,and Fabp4 were in the crucial positions,so they could be the vital proteins for the absence of pneumonia despite mycoplasma infection.This study had also done a modularization analysis of complex networks,figuring out the four major modules as follows: the module one is mainly engaged in immune regulation,the module two was mainly responsible for actin binding,the module three is mainly associated with fatty acid synthesis,transformation and catalysis,and the module four was mainly related to induction of signal events and induction of inflammatory factors.3.From the mice infected with MP,this study can see through the microscope that congestion in the tracheal wall and surrounding interstitium,incomplete alveolar wall,a small amount of secretion in the alveoli,thickening of alveolar septum infiltrated by inflammatory cells,and telangiectasia,consistent with the syndromes of pneumonia.In Comparison with the model group,the symptoms of pulmonary inflammation in the west medicine group,the heavy-dose traditional Chinese medicine group,the medium traditional Chinese medicine group were relatively lighter,including mild pulmonary interstitial edema,relatively complete alveolar wall,and only infiltration of a small number of inflammatory cells.There was no significant difference between the light-dose traditional Chinese medicine group and the model group due to relatively incomplete alveolar wall,moderate congestion and edema in the lung tissues,and the bronchioles and alveolar space infiltrated with moderate amounts of inflammatory cells.4.The level of IL-10 and the level of IL-17 in the serum of mice were measured with the methods of Elisa,results from which demonstrated that the level of IL-10 in the serum of mice infected with MP fell dramatically(P<0.05)while the level of IL-17 in the serum of mice infected with MP went up greatly(P<0.05).The following four groups was ranked according to the levels of IL-10 in the serum of mice infected with MP like this: the normal group > the heavy-dose traditional Chinese medicine group >the medium-dose traditional Chinese medicine group>the model group.As regards to the level of IL-10 in the serum of mice infected with MP,there was no significant difference for statistics between the west medicine group and the heavy-dose traditional Chinese medicine group,the medium-dose traditional Chinese medicine group as well as the light-dose traditional Chinese medicine group;in comparison with all those four groups,the model group have lower levels of IL-10.There is no significant difference for statistics between the medium-dose traditional Chinese medicine group and the light-dose traditional Chinese medicine group;in comparison with both groups,the model group had lower levels of IL-10.There was no significant difference for statistics between the light-dose traditional Chinese medicine and the model group.The differences in the levels of IL-10 among all six groups at different times were of no importance for statistics.The following four groups was ranked according to the levels of IL-17 in the serum of mice infected with MP like this: the model group > the medium-dose traditional Chinese medicine > the heavy-dose traditional Chinese medicine > the normal group.There was no significant difference for statistics between the west medicine group and the medium-dose traditional Chinese medicine group;but in comparison with both groups,the light-dose traditional Chinese medicine and the model group have higher levels of IL-17.Moreover,there was no significant difference for statistics between the light-dose traditional Chinese medicine and the model group;however,in comparison with both groups,the medium-dose traditional Chinese medicine,the heavy-dose traditional Chinese medicine and the west medicine group had lower levels of IL-17.The differences in the level of IL-17 among all six groups at different times were valuable for statistics: the levels of IL-17 in the serum of mice from all six groups on the 3rd day and 14 th day were lower than those in the serum of mice from all six groups on the 7th day and 10 th day(P<0.05).5.The level of Foxp3 protein and the level of ROR?t protein in the lung tissues of mice infected with MP were measured with the methods of immunohistochemistry,findings from which indicated that MP infection caused a decrease of Foxp3 protein(P<0.05)and an increase of ROR?t protein(P<0.05),leading to an imbalance of ROR?t/ Foxp3.In the traditional Chinese medicine group,ROR?t protein could be down-regulated while Foxp3 could be up-regulated but in direct proportion to the dosage.There was no significant difference between the west medicine group as well as the medium-and light-dose traditional Chinese medicine groups.6.Notch1,Notch2 increased significantly after MP infection,but Notch3 changed little.The expression levels of Notch 1 protein,Notch 2 protein and Notch 3 protein in the lung tissues of mice were detected with the methods of Western blot,outcome from which suggested that there was a notable increase(P<0.05)in the expression level of Notch 1 protein and the expression level of Notch 2 protein in the lung tissues of mice with MP infection.There was no significant difference for statistics in the expression level of Notch 3 protein.The differences in the expression levels of Notch 1 protein tested and detected at different times were of value for statistics(P<0.05)in direct proportion to the duration of disease.In comparison with the model group,these four groups composed of the heavy-dose traditional Chinese medicine group,the medium-dose traditional Chinese medicine group,the light-dose traditional Chinese medicine group and the west medicine group can down-regulate the expression level of Notch 1 protein and the expression level of Notch 2 protein.And the heavy-dose traditional Chinese medicine group had the best effect.7.The expression levels of Notch 1 protein,Notch 2 protein,Notch 3 protein,ROR?t mRNA in the lung tissues of mice infected with MP were detected with the methods of qPCR,results from which showed that there was a marked rise(P<0.05)in the expression level of ROR?t protein in the lung tissues of mice with MP infection and a remarkable decline(P<0.05)in the expression level of Foxp3 protein in the lung tissues of mice with MP infection.The differences in the expression levels of ROR?t mRNA at different times are of no importance for statistics.There were differences in the levels of ROR?t mRNA among the model group,the normal group,the west medicine group,the heavy-dose traditional Chinese medicine and the medium-dose traditional Chinese medicine(P<0.05).There was no valuable difference in the level of ROR?t mRNA for statistics between the model group and the low-dose traditional Chinese medicine.In addition,the differences in the levels of ROR?t mRNA among the remaining groups were of no value.The differences for statistics in the levels of Foxp3 mRNA in the lung tissues of mice at different times were not vital,but there were differences among groups including the following observations: the level of Foxp3 mRNA in the normal group was higher than those of the other groups;the level of Foxp3 mRNA in the heavy-dose traditional Chinese medicine was higher than that of the model group;and the differences for statistics among the remaining groups were of no value.The mice with MP infection had a notable increase in the expression level of Notch 1 protein and the expression level of Notch 2 protein(P<0.05).There was no significant difference for statistics in the expression level of Notch 3 protein.The differences for statistics in the expression levels of Foxp3 mRNA measured at different times were of no importance.Different from the model group,these four groups containing the heavy-dose traditional Chinese medicine group,the medium-dose traditional Chinese medicine group,the light-dose traditional Chinese medicine group,and the west medicine group can down-regulate the expression level of Notch 1 protein and the expression level of Notch 2 protein.And the heavy-dose traditional Chinese medicine group have the best effect.Conclusion: 1.The iTRAQ proteomics research technology can be widely applied to analyze(the expression levels of)differential proteins so as to reveal the disease mechanism and the relevant status.2.The incidence of mycoplasma pneumoniae pneumonia for a large part is closely associated with body immunity,fatty acid synthesis,carbohydrate metabolism,nucleotide binding,actin binding,oxidative metabolism,etc.3.Whether mycoplasma pneumoniae pneumonia causes illness or not in the targeted mice with the same infection heavily depends on body immunity,fatty acid synthesis,lung metabolism,actin binding,catalysis and so on.4.The level of IL10 in the serum of mice infected with MP drops while the level of IL17 in the serum of mice infected with MP rises.Apart from that finding,each dose of the traditional Chinese medicine can regulate the imbalance of IL-17/IL-10 with the effect in direct proportion to dosage.The effect of the west medicine group is similar to those of the medium-dose traditional Chinese medicine as well as the small-dose traditional Chinese medicine.The levels on the 3rd day and 14 th day are lower than those on the 7th day and 10 th day.5.An imbalance of ROR?t/Foxp3 in the mice with MP infection is mainly attributed to a decrease in the level of Foxp3 protein and the level of Foxp3 mRNA as well as an increase in the level of ROR?t protein and the level of ROR?tmRNA.The “QingfeiTouxie(a prescription of traditional Chinese medicine to take away heat in the patient's lung and expel pathogenic factors)” formula could effectively treat mycoplasma pneumonia likely through regulating the imbalance of ROR?t/Foxp3 and Notch pathway,with an effect in direct proportion to dosage regardless of the length of treatment with medicine.6.The “QingfeiTouxie(a prescription of traditional Chinese medicine to take away heat in the patient's lung and expel pathogenic factors)” formula can regulate the expression level of Notch 1 protein as well as the expression level of Notch 2 protein and mainly down-regulate the expression level of Notch 1 protein and the expression level of Notch 2 protein with an effect in direct proportion to dosage regardless of the length of treatment with medicine.The expression level of Notch 3 has not been heavily affected. |
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