| Spleen deficiency syndrome is veterinary clinical common disease and frequently-occurring disease.The main symptoms are weight loss,fatigue,lethargy,fatigue and indigestion and so on?Traditional Chinese veterinarians have its good therapeutic effect for this disease,but the proteomics and metabonomics study of spleen deficient is very less and the material foundation of modern science is not very clear.Si-jun-zi-Decoction(SJZD),a classic traditional Chinese medicine(TCM)composed of Codonopsis pilosula(Franch.)Nannf,Atractylodes macrocephala,Poria cocos(Schw.)Wolf,and Glycyrrhiza uralensis Fisch,has been demonstrated to have therapeutic effects for spleen deficiency syndrome.However,there are very few reports about the detailed therapeutic mechanisms and the pesticide effect material foundation of SJZD on spleen deficiency syndrome using a modern scientific strategy.In this study,the spleen deficiency syndrome rat model was built with reserpine injection method.At first,the intervention effect of SJZD on spleen deficiency syndrome was preliminary evaluation through pharmacodynamics study.By comparing the differences proteins of liver tissues by Label free proteomics techniques and metabolites in serum and liver tissues by Liquid chromatography-quadrupole-time of flight-mass spectrometry(LC-Q-TOF-MS)metabolomic techniques,the different express protein and small molecule metabolites of spleen deficiency syndrome and SJZD intervention spleen deficiency syndrome were screened.At last,the different express protein and small molecule metabolites were analyzed with bioinformatics analysis method,the potential target protein and biomarker of spleen deficiency syndrome and SJZD intervention spleen deficiency syndrome were excavated.At the same time,the corresponding metabolic pathways and biological molecular interaction network was established.We hope to probe the mechanisms of SJZD intervention spleen deficiency syndrome from the aspect of proteomics and metabonomics and to provide a scientific basis for the clinical diagnosis and treatment of spleen deficiency syndrome.The main content and results were as follows:1.The pharmacodynamics study of SJZD on spleen deficiency syndrome rats 40 healthy adult male SD rats were randomly divided into the nature control group(NC group),the model group(M group),the Si-jun-zi-decoction intervention group(SJZD group)and the position control group(PC group).The spleen deficiency syndrome rat model was built with reserpine injection method.Evaluating indicators included clinical signs,viscera index,the physiological and biochemical index,praxiology index,stomach,duodenum,spleen and liver pathological alteration.The results indicated that SJZD could improve the clinical symptoms of spleen deficiency syndrome rats.amylase(AMY),lipase(LPS),pepsase,gastrin,ghrelin,succinodehydrogenase(SDH),isocitrate dehydrogenase(ICD)activity and content in serum and pepsase,gastrin and ghrelin content in stomach and SDH,ICD,Na+K+-ATPase and Ca2+Mg2+-ATPase activity in liver were significantly decreased in the M group compared with the NC group rats(P<0.05).However,the content of lactic dehydrogenase(LDH)in serum and in liver were significantly increased in the M group compared with the NC group(P<0.05).Rats in the PC groups and the SJZD groups had increased or decreased levels of above parameters compared with the M group(P<0.05).Praxiology results indicated that compared with the NC group,the horizontal crawl lattice and vertical number of the M group rats significantly decreased(P<0.01).The the horizontal crawl lattice and vertical number of the SJZD group and the PC group rats significantly increased(P<0.01)compared with the M group.Pathological examination showed that compared with the NC group,the stomach,duodenum,spleen and liver of the M group rats had different degree of pathological changes.Compared with the M group,the SJZD group and PC group rats pathological change in stomach,duodenum,spleen and liver significantly tended to normal.The above results hinted that SJZD possesses protective effects on rats with spleen deficiency syndrome.2.Liver proteomics analysis Label free proteomics technology was employed to identify differentially expressed protein.The differentially expressed protein of spleen deficiency syndrome and spleen deficiency syndrome intervened with SJZD were screened out according to the change fold >1.5(<0.67).At last,the differentially expressed protein was analyzed with Gene Ontology(GO)annotation and enrichment?Kyoto Encyclopedia of Gene and Genomes(KEGG)annotation and enrichment and protein interaction network analysis?The M group vs the NC group,the results showed that 267 differentially expressed proteins(125 proteins up-regulated,142 proteins down-regulated)as the differential expression protein of spleen deficiency syndrome were screened.The M group vs the SJZD group,247 differentially expressed proteins(81 proteins up-regulated,166 proteins down-regulated)as the differential expression protein of Si-jun-zi decoction intervened with spleen deficiency syndrome were screened.GO enrichment analysis showed the differential expression protein of spleen deficiency syndrome mainly distributed in cytoplasm,cell membrane and extracellular region and the main function of the proteins was the binding activity and enzyme activity and these proteins were involved in various metabolic processes.GO enrichment analysis showed the differential expression protein of Si-jun-zi decoction intervened with spleen deficiency syndrome mainly distributed in cytoplasm,cell membrane and extracellular region and the main function of the proteins was the activity of enzyme catalysis,transformation and transfer and these proteins were involved in various metabolic processes.With comparative analysis the target pathways involved the differential expression protein of spleen deficiency syndrome and the target pathways involved the differential expression protein of Si-jun-zi decoction intervened with spleen deficiency syndrome by KEGG enrichment analysis revealed the mechanism of SJZD intervened with spleen deficiency syndrome was mainly through regulation 4 pathways(bile secretion,pyruvate metabolism,cysteine and methionine metabolism,fatty acid metabolism)to achieve.The differential expression protein interaction networks analysis revealed that the proteins were involved in multiple biological function and metabolic pathway which consistent with the results of GO annotation and KEGG annotation.3.The metabolomics research of plasma and liver tissue LC-Q-TOF/MS based metabolomics approach was employed to investigate the metabolic profiles of serum and liver tissue extracts from the NC,M,SJZD and PC group rats.Pattern recognition method was used to analyze the data and to find the differences metabolites of serum and liver tissue.The potential biomarker was analyzed with correlation analysis,heatmaps analysis,metabolic pathway and metabolite interaction network analysis.The results indicated that the metabolic profiles of the M group and the NC group could be completely separated,and the SJZD group and the PC group showed a trend to near the NC group.According to VIP>1.0 and P<0.05,a total of 66(20 in plasma,46 in liver tissue)spleen deficiency syndrome and 81(34 in plasma,47 in liver tissue)spleen deficiency syndrome intervened with SJZD potential biomarkers were identified.The metabolic pathways of potential biomarkers were built the Met PA metabolic pathway analysis software.The pathways were selected as the target pathways according to impact value>0.10.With comparative analysis the target pathways involved the potential biomarkers of spleen deficiency syndrome and the target pathways involved the potential biomarkers of Si-jun-zi decoction intervened with spleen deficiency syndrome revealed the mechanism of SJZD intervened with spleen deficiency syndrome was mainly through regulation 8 pathways(glycerophospholipid metabolism,fatty acid elongation in mitochondria,Synthesis and degradation of ketone bodies,fatty acid metabolism,sphingolipid metabolism,terpenoid backbone biosynthesis,valine,leucine and isoleucine degradation,butanoate metabolism)to achieve.At last,the potential biomarkers interaction networks were constructed using cytoscape metabolite interaction network analysis software and 3(Acetyl-Co A,3-hydroxy-3-methylglutaryl-Co A,Hexanoyl-Co A)potential biomarkers of strong correlation with spleen deficiency syndrome intervened with SJZD were selected.In conclusion,SJZD can relieve sundry symptom of spleen deficiency syndrome rats to different degree.SJZD can improve the imbalance of physiological and biochemical indexes and the pathological changes of tissues and organs.The proteomics and the metabolomics results revealed that the intervention mechanism of SJZD on spleen deficiency syndrome was mainly through regulation bile secretion,pyruvate metabolism,cysteine and methionine metabolism,fatty acid metabolism pathways and glycerophospholipid metabolism,fatty acid elongation in mitochondria,synthesis and degradation of ketone bodies,fatty acid metabolism,sphingolipid metabolism,terpenoid backbone biosynthesis,valine,leucine and isoleucine degradation,butanoate metabolism and so on multi target intervention on spleen deficiency syndrome.Proteomics and metabolomics analysis also reveals SJZD intervention on spleen deficiency syndrome mainly related to metabolism of main nutrients such as lipid metabolism,protein and amino acid metabolism,sugar metabolism and so on.At the same time,the fatty acid metabolism pathway was screened as the main target metabolism pathway of SJZD intervention on spleen deficiency syndrome.The study provides a theoretical basis for revealing the the essence of spleen deficiency syndrome and intervention mechanism of SJZD on spleen deficiency syndrome. |
PDF Full Text Request