| Purpose: Gastrointestinal heat retention (GHR) refers to the invisible heat or tangible heat stagnation in the gastrointestinal tract, causing gastrointestinal heat in the qi level, which is mostly caused by food stagnation. GHR is commonly seen in children,as an important factor affecting the occurrence and transmission of pediatric pneumonia. Based on the theory of simultaneous treatment of lung and stomach, the clinical effect in the treatment of pediatric pneumonia can be enhanced. Yinlai Decoction, formulated by the principle of simultaneous treatment of lung and stomach, is composed of Flos Lonicerae, Semen Raphani, Fructus Forsythiae, Radix Scutellariae, Herba Houttuyniae,Radix Peucedani and Fructus Trichosanthis. It can clear the heat of the lung meridian and remove the stagnation of the gastrointestinal tract at the same time. The formula has been applied in clinical treatment of pediatric pneumonia with GHR, and it has remarkable curative effect.Pediatric pneumonia,a common respiratory disease,refers to lung inflammation caused by bacteria, virus, mycoplasma, chlamydia etc. Individuals with pediatric pneumonia typically show fever,cough,polypnea,moist rale and so on. Therefore its intervention and therapy can refer to the principle and method of the pediatric pneumonia in Traditional Chinese Medicine.Lipopolysaccharide(LPS), the major components of Gram-negative bacterial endotoxin, can mediate inflammatory responses. LPS of different bacteria can mediate analogous inflammatory responses because of hypotoxicity, therefore, it can be applied into the establishment of nonspecific pneumonia model.Previous experimental study succeed in duplicating animal model with GHR, fed on high protein and caloric diet. The study showed that GHR can increase the lung index and aggravate its damage, while Yinlai Decoction can obviously reduce the lung injury of the mice model with GHR combined pneumonia. We investigated the pathogenesis of GHR from the aspects inflammation, immunity and intestinal mucosa barrier. However the current study on the mechanism of the GHR causing pediatric pneumonia and Yinlai Decoction lack systematicness .Based on previous study, this project intends to observe the proteins expression characteristics of pneumonia with GHR group and Yinlai Decoction treatment group by isobaric tags for relative and absolute quantitation (iTRAQ). The project aims to reveal the material basis of the occurrence and transmission of the pediatric pneumonia from the point of GHR, to explore the mechanism of Yinlai Decoction in the treatment of pediatric pneumonia with GHR, so as to provide a reliable experiment basis for prevention and treatment of pediatric pneumonia.Methods: The murine pneumonia model (M1 group) was established by LPS aerosol inhalation. Compared with the murine pneumonia model, the murine pneumonia with GHR model (M2 group) was fed on high protein and caloric diet. The treatment group (Y group)was the murine pneumonia with GHR model intervened by Yinlai Decoction. The spleen index,thymus index and lung index of the three groups were estimated and the pathological changes of the lung tissue were observed under microscopy firstly. Then the protein expression in lung tissue of M1 group and M2 group was compared with the normal group by iTRAQ, and the differential protein expression was further studied between M2 group and M1 group, Y group and M2 group.Results:1.Changes in the lung index, spleen index and thymus indexCompared with normal group, the lung index of M1 group and M2 group increased significantly (p<0.05). Compared with M1 group, the lung index of M2 group increased significantly (p<0.05). The lung index of Y group was lower than M2 group, however there was no significant difference(p=0.458).Compared with normal group, the spleen index of M2 group increased significantly(p<0.05). The spleen index of M1 group was higher than normal group, but there was no significant difference(p=0.39). The spleen index of M2 group was higher than M1 group, but the differences were very small(p=0.204). Compared with M2 group, the spleen index of Y group increased significantly (p<0.05).Compared with normal group, the thymus index of M1 group and M2 group were decreased,but the differences were very small(p>0.05). Compared with M1 group, the thymus index of M2 group decreased, but the differences were very small(p>0.05). Compared with M2 group, the thymus index of Y group increased, but the differences were very small(p>0.05).2.Pathological changes of the lung tissueThe lung injury of M1 group was mainly featured with destroyed alveoli, thickened alveoli septum, fusion of pulmonary alveoli and inflammatory cell infiltration. Compared with M1 group, the lung injury of M2 group was more serious with lung consolidation and a large amount of inflammatory cell infiltration. Compared with M2 group, the pathological changes of lung tissue in Y group were reduced with pulmonary septal thickness and interstitial inflammatory infiltration decreased.3.The protein expression characteristics of pneumoniaThis project identified 2626 reliable proteins, with peptides at least 1, which repeated in the three experiments.By the standards of p<0.05 and at least 1.5-fold change in FC,the differential proteins were screened.Compared with normal group, there were 344 differential expression proteins in the lung tissue of M1 group, with 224 up-regulated proteins expression and 120 down-regulated proteins expression. It involved 1287 biological processes, 197 kinds of cellular components,234 molecular functions and 26 KEGG pathways. The biological processes, which were quite different in two groups, were related to the immunoreaction, which involved the response to organic substance, response to cytokine, cellular response to chemical stimulus, response to chemical, cellular response to cytokine stimulus, response to external stimulus, defense response and immune system process. The related KEGG pathways included ECM-receptor interaction, leukocyte transendothelial migration, Fc gamma R-mediated phagocytosis,complement and coagulation cascades,antigen processing,presentation and so on. Some of the KEGG pathways participated in the ribosome synthesis,aminoacyl-tRNA biosynthesis,protein processing in endoplasmic reticulum, glycolysis, gluconeogenesis, pentose phosphate pathway etc.Compared with normal group, there were 521 differential expression proteins in the lung tissue of M2 group, with 273 up-regulated proteins expression and 248 down-regulated proteins expression. It involved 1158 biological processes, 242 kinds of cellular components,234 molecular functions and 39 KEGG pathways. The biological processes,quite different in two groups, covered biological adhesion, cell adhesion, response to organic substance,response to chemical, immune system process, defense response, response to cytokine,organonitrogen compound metabolic process, peptide metabolic process, gas transport, small molecule metabolic process, oxidation-reduction process and so on. Most of the related KEGG pathways were closely associated with ECM-receptor interaction,focal adhesion,bacterial invasion of epithelial cells, leukocyte transendothelial migration, phagosome and so on. Some of the related KEGG pathways in the M2 group were in relation to ribosome synthesis, carbon metabolism and Citrate cycle.The lung tissue of M2 group had 164 differential proteins in comparison with the M1 group. Among the 164 differential proteins, there were 14 up-regulated proteins expression and 150 down-regulated proteins expression. It involved 588 biological processes,131 kinds of cellular components, 144 molecular functions and 20 KEGG pathways. The biological processes were related to small molecule metabolic process, gas transport, purine ribonucleotide metabolic process, ribonucleotide metabolic process, ribose phosphate metabolic process and so on. Some of them were involved in cell adhesion and biological adhesion. Most related KEGG pathways were bound up with oxidative phosphorylation,citrate cycle, butanoate metabolism, valine degradation, leucine degradation, isoleucine degradation, ribosome and so on. Besides that they were related with the immunoreaction and vascular smooth muscle contraction to some extent.The lung tissue of Y group had 87 differential proteins in comparison with the M2 group. Among those, there were 73 up-regulated proteins expression and 14 down-regulated proteins expression. It involved 403 biological processes,112 kinds of cellular components,114 molecular functions and 21 KEGG pathways. The biological processes were related to citrate metabolic process, cellular respiration, tricarboxylic acid metabolic process,ricarboxylic acid cycle, energy derivation by oxidation of organic compounds, generation of precursor metabolites and energy etc. So were the related KEGG pathways. They were bound up with citrate cycle ,oxidative phosphorylation, fatty acid metabolism, tryptophan metabolism, lysine degradation, valine degradation, leucine degradation ,isoleucine degradation and ribosome.Conclusion:1.GHR can aggravate the lung inflammatory injury of the murine pneumonia model established by LPS aerosol inhalation, while Yinlai Decoction can reduce the injury.2.LPS atomization inhalation can not only cause local inflammatory reaction of lung by provoking the inflammatory and immunological responses, but also affect protein synthesis and metabolism of carbohydrate.3.We suppose GHR aggravate the lung inflammatory injury by affecing the material and energy metabolism which may weaken immune function.4.We suppose Yinlai Decoction reduce the lung injury by adjusting the material and energy metabolism of the murine pneumonia with GHR model.5.The change of the citrate cycle probably play an important role in the processes of GHR affecting the occurrence and transmission of pediatric pneumonia. Yinlai Decoction may take effect by regulating the citrate cycle. |
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