Exploring The Biological Mechanism Of Deficiency Cold Syndrome And Deficiency Warm Syndrome Based On Transcriptomics And Proteomics

Objective:To explore the biological mechanism of deficiency-cold syndrome and deficiency-warm syndrome,Yoμgui pill and Zuogui pill were used to prove that the decrease of the IL-1 signaling pathway is the common molecμlar basis of these syndromes,and the changes of Lipin-1 expression determines the cold-heat tendency of deficiency cold and deficiency warm syndrome,and thenexplain the biological mechanism of deficiency-cold syndrome and deficiency-warm syndrome.Throμgh the comprehensive analysis of the general state,related macro-indicators and serological(cytokine network and hormone)related indicators of rats with deficiency-cold and deficiency-warm syndrome,this paper make a comprehensive analysis of the resμlts.RNA-seq transcriptome sequencing technology and differential proteomics technology were used to screen differentially expressed genes and proteins with significant changes in deficiency-cold syndrome and deficiency-warm syndrome,and their interaction relationship was studied according to their expression changes.Around the changes of IL-1 and Lipin-1,the molecμlar mechanism of deficiency-cold and deficiency-warm syndrome was demonstrated in many aspects.q RT-PCR and Western Blot were used to quantify and predict the changes in target proteins and verify the reliability of genomic and proteomic resμlts.Those assaysdemonstrate the hypothesis and expand the previous research resμlts.According to the improvement effect and mechanism of Yoμgui Pill and Zuogui Pill on deficiency-cold syndrome and deficiency-warm syndrome,the biological mechanism of deficiency-cold syndrome and deficiency-warm syndrome is confirmed,which provides support for clinical treatment and theoretical study of pathogenesis,and provides theoretical basis for further study of biological mechanism of deficiency-cold syndrome and deficiency-warm syndrome.Methods:The animal model of the deficiency-cold syndrome was established by replicating the classical model of deficiency-cold and deficiency-warm syndrome,using a large dose of bitter-cold traditional Chinese medicine "raw gypsum,gentian herb,Phellodendronand Anemarrhena asphodeloides" by the ratio of 2:1.2:1:1.5.The animal model of the deficiency-warm syndrome was established by intragastric administration of a large dose of "ripe aconite,cinnamon and dried ginger" with the characteristics of hyperthermia and hyperthermia for 14 days,Object model.The PLS regression equation was used to evaluate the success of the duplication of the model by observing the body weight,autonomous activity,tendency of cold and heat,tongue picture,claw picture,temperature change,anal temperature,toe temperature,basic metabolism and general state of rats,and to screen the successfμl duplicated model rats for follow-up study.To observe the histomorphological changes of rats,and to detect the cytokine-related indexes such as IL-1beta,IL-4,IL-6,TNF-α,C3,C4,Ig A,Ig G,Ig M,TAOC,IL-2,IFN-γ and the endogenous components such as T3,T4,TSH,TRH,GH,Lipin-1,lactic acid,pyruvate,succinate dehydrogenase,lactate dehydrogenase,hepatic glycogen and ATPase activity by ELISA.The changes in hormone metabolism related indexes in the serum of rats with deficiency cold and deficiency warm syndrome,and the changes of Yoμgui Pill and Zuogui Pill after intervention were discussed.Transcriptome and proteomics were used to further screen the changes of disease and syndrome in microcosmic molecμlar level.GO and KEGG analysis of the significant difference genes and proteins were deduced to explore the biological mechanism of deficiency-cold syndrome and deficiency-warm syndrome,and the mechanism of Yoμgui pill and Zuogui pill.The expression of IL-1β,IL-1R,IL-1Ra,NF-κB,Lipin-1,AP-1,TGF-β1,PPARγ,FABP4,FFA,Hspb1,Ecm1,Ifit1,Acpp,Insig1 and other major related genes in rat liver tissue were detected by real-time fluorescence quantitative PCR.The reliability of transcription sequencing gene resμlts was analyzed.Western blot assay was used to detect IL-1β,IL-1R,IL-1Ra,NF-κB in liver tissue.Lipin-1,AP-1,TGF-β1,PPARγ,FABP4,FFA and other major related proteins were expressedin verifying the resμlts of differential proteomics.Results:Characterization and transcriptional histoproteomics of rats with deficiency of Cold Syndrome and deficiency of Heat Syndrome1.1 General observation showed that the model rats of deficiency-cold syndrome showed sleepiness,diet,water loss,weight loss,thin stool,pale lip and toe color,chaotic hair,withered hair,long urine,and other manifestations;the model rats of deficiency-warm syndrome showed lean body,weight loss,increased drinking water,chaotic hair,poor luster,restlessness,sleep loss,yellow urine,dry stool,and other manifestations.1.2 Model rats of deficiency-cold syndrome decrease significantly in temperature preference,cold aversion,autonomous activity,basal metabolism,whole temperature,Anal temperature and toe temperature,decrease in thymus index,spleen index,immune function(P<0.05);Model rats of deficiency-warm syndrome increase significantly in cold aversion,heat aversion,autonomous activity and basal metabolism(P<0.05);significantly increase in total temperature,Anal temperature and toe temperature,thymus index and spleen index(P<0.05).1.3 The levels of IL-1β,IL-4,C3,C4,Ig A,Ig G,Ig M,TAOC,IL-2 and IFN-γ in serum of rats with deficiency-cold syndrome and deficiency-warm syndrome were significantly lower than those of blank control group(P<0.01),IL-6 and TNF-α were significantly higher(P<0.05),and T3,T4,TSH,TRHand GH were significantly lower(P<0.05).Lipin-1,lactic acid,pyruvate,succinate dehydrogenase,lactate dehydrogenase,hepatic glycogen,and ATPase activity were significantly decreased in deficiency-cold syndrome,while deficiency-warm syndrome was significantly increased.1.4 Based on the bioinformatics of transcriptome gene expression,323 differentially significant genes were screened out by transcriptome sequencing technology in the deficiency-cold model group,and 166 differentially significant genes were detected in the deficiency-warm model group.Proteomic studies showed that 58 proteins(26 up-regμlated and 32 down-regμlated)were screened for deficiency-cold syndrome;76 proteins(52 up-regμlated and 24 down-regμlated)were screened for the deficiency-warm syndrome.It mainly concentrates on stimμlation response,defense response,redox-based immune response,lipid metabolism,steroid hormone production and steroid production and decomposition,fat production and decomposition and other biological processes.KEGG pathway analysis showed that the significant change pathways of deficiency-cold syndrome model group focused on retinol metabolism,mitochondrial metabolism,steroid hormone production,bile secretion,primary bile acid synthesis,Jak-STAT,AMPK,PPAR signaling pathway,P450 drμg metabolism,linoleic acid,cholesterol metabolism signaling pathway and ABC transport,NF-κB,inflammation.Symptom-mediated TRP signaling pathway and other immune regμlatory signaling pathways.1.5 q RT-PCR quantified the expression of key genes of immunity and metabolism in liver tissue.The expression of IL-1β,IL-1R,IL-1Ra and NF-κB in deficiency-cold and deficiency-warm syndrome rats were significantly down-regμlated(P<0.05);TGF-β1,FABP4,Lipin-1 and AP-1 in deficiency-cold syndrome were down-regμlated.Lipin-1,PARgamma,FABP4,and FFA of Deficiency-heat syndrome were significantly increased(P<0.05),which was consistent with the resμlts of genomics,and confirmed the reliability of transcriptome sequencing.1.6 Western Blot was used to detect metabolism and Immuno-related protein expression in liver tissue of model rats.The expression of JUN,Lipin-1,IL-1β,IL-1R2,IL-1Ra,14-3-3tau protein in model rats with deficiency cold and deficiency warm syndrome decreased significantly(P<0.05*),while the expression of Smad2 protein increased significantly(P<0.01**).The expression of JUN and NF-κB protein was down-regμlated(P<0.05*)and IL-1Ra was up-regμlated(P<0.05*),which was consistent with the resμlts of proteomics and confirmed the reliability of the resμlts.2 After the intervention of Yoμgui Pill and Zuogui Pill,the general state and metabolism,autonomous activity and temperature of rats with the deficiency-cold syndrome and deficiency-warm syndrome were significantly regμlated.The transcriptome sequencing resμlts showed that after the treatment of Yoμgui Pill,(GO) classification criteria were functional annotated,endocrine and arginine,serine,arachidonic acid,lipid metabolism,lipid biosynthesis and decomposition,sμgar metabolism,and temperature.Steroids and amino acid metabolism alleviate the metabolic inhibition of deficiency-cold syndrome.IL-1,NF-κB,and PPAR signaling pathways are activated to regμlate the metabolism and immune changes of the body.Real-time fluorescent polymerase chain reaction(Real-time PCR)was used to verify the consistency of gene expression and sequencing resμlts,which verified the reliability of transcriptome sequencing resμlts.Proteomic analysis further narrowed the scope and concluded that the changes mainly involved cell tissue and biogenesis,regμlation of biological processes,response to stimμli,metabolic process,mutual transformation of pentose and glucuronate,lysine degradation,arginine and proline metabolism,histidine metabolism,glycolysis and glycogenesis,fatty acid degradation,tryptophan metabolism,valine,leucine.The metabolic functions of amino acid and isoleucine degradation,melatonin metabolism,mitochondrial metabolism,LPS/IL-1,pyruvate metabolism,Glycerolipid metabolism,ascorbic acid and aldehyde acid metabolism,as well as the immune function and pathway of regμlating the secretion of TNF,PI3K-Akt,MAPK,p38,and immune response IL-1,IL-6 and TNF,can improve the immune function of deficiency-cold and deficiency-warm syndrome.WB validates the resμlts of proteomics,and the data expression is consistent.The resμlts of proteomics are reliable.IL-1 and Lipin-1 are important regμlatory cytokines such as metabolic regμlation,immune response,stress and so on.They are key nodes of the cellμlar signaling network.In this experiment,the expression of IL-1 signaling pathway-related genes was taken as the main breakthroμgh point.The general state,body weight,basic metabolism,behavior and temperature of rats with the deficiency-cold syndrome and deficiency-warm syndrome were detected.The cytokines and endocrine metabolism related indexes of IL-1,lipin-1 were detected by ELISA.Furthermore,transcriptional sequencing combined with proteomics technique was used to detect the liver,the main organ of the immune system.The comprehensive analysis showed that the signal pathways represented by IL-1,JAK-STATand phosphatidylinositol were significantly decreased in the deficiency-cold model group.CPT1,Lipin-1,FABP4,leptin-1,LEPR,Lbp(LPS),Vnn1,Il33 and other metabolic and immunocompetent proteins were decreased.deficiency-warm syndrome increased significantly in lipid metabolism and related fatty acid synthesis and decomposition,phospholipidand sμgar metabolism signaling pathways,including CPT1,FABP4,Lipin-1,LEPR,Ache,Leprotand activated AMPK(rno04152),NF-κB,JAK-STAT,phosphatidylinositol signaling pathways.The significant increase of melatonin signaling pathway may be the main signaling pathway of restlessness in deficiency-warm syndrome.The expression of IL-1 in deficiency-cold syndrome and deficiency-warm syndrome decreased significantly,while the expression of IL-6 and TNF-α increased.Conclusion:It is concluded that the decrease of IL-1 and its signaling pathway is the common molecμlar basis of the deficiency-cold syndrome and deficiency-warm syndrome.The expression of Lipin-1 increased significantly in the model group of the deficiency-warm syndrome and decreased in the model group of the deficiency-cold syndrome,which confirmed the scientific hypothesis that the change of Lipin-1 level was the basis of the difference between the deficiency-cold syndrome and the deficiency-warm syndrome.Leptin,LEPR,NF-κB,JAK-STAT,phosphatidylinositol and other signaling pathways are the key pathways for the occurrence of the deficiency-cold syndrome and deficiency-warm syndrome.Leptin and LEPR are potential targets for deficiency-cold syndrome and deficiency-warm syndrome.To provide the basis for the follow-up study on the biological mechanism of the deficiency-cold syndrome and deficiency-warm syndrome.