Differentially Expressed Serum Proteins In Osteoporosis Patients With Yang And Yin Deficiency Constitution Based On TMT

Objective:In this study,we explored the correlation between Yang-deficiency constitution and osteoporosis(OP),Yin-deficiency constitution and OP by meta-analysis.The differential proteins were screened in serum of OP patients with Yang-deficiency constitution and OP patients with Yin-deficiency constitution by Tandem Mess Tags(TMT)combined with mass spectrometry technology.These differential proteins were verified by ELISA method so as to find the specific protein that could treat as biomarkers for OP patients with Yang-deficiency constitution and OP patients with Yin-deficiency constitution,so that it could provide molecular markers for the diagnosis of OP with different constitutions and targets for the development of drugs for the treatment of OP with different constitutions.Methods:1.Keywords related to "constitution" and "osteoporosis" were used to search the databases.Studies related to analysis the relationship between Yang-deficiency and osteoporosis,Yin-deficiency constitution and osteoporosis were screened.Studies were included according to the inclusion and exclusion criteria.The baseline data information and the number of OP incidences in Yang-deficiency constitution,Yin-deficiency constitution and ideal constitution were extracted.The study quality evaluation was conducted by using NOS score table and AHRQ list,Rev Man 5.4 was used to conduct meta-analysis.Funnel plot was applied to evaluate the existence of publication bias.2.32 cases of OP with Yang-deficiency constitution,32 cases of OP with Yin-deficiency and32 cases of non-osteoporosis with ideal constitution were selected from the biological sample bank of Gansu traditional Chinese medicine hospital.In each group,8 samples were selected to make a mixed sample.Low abundance protein was obtained by removing high abundance protein.Then,the low abundance proteins were extracted by SDT cleavage method and enzymolysis,.The obtained peptides were labeled by TMT method,mass spectrometry was used to screen out the differential proteins in the serum of between Yang-deficiency OP and non-osteoporosis with ideal constitution,Yin-deficiency OP and non-osteoporosis with ideal constitution,GO and KEGG databases were used to bioinformatics analysis.3.The differential proteins with important physiological functions were selected to verify.18 cases of OP with Yang-deficiency,OP with Yin-deficiency,OP with ideal constitution and non-osteoporosis with ideal constitution were selected from the serum of biological sample bank.ELISA method was used for verification,so as to find the sensitive differential proteins of OP with Yang-deficiency or OP with Yin-deficiency.Results:1.Five cross-sectional studies and five case-control studies were included,Four of them were high quality studies,six were medium quality studies.Meta-analysis showed that the OR for the risk of OP in Yang-deficiency constitution was 2.62(95% CI: 1.96-3.49,P<0.01),the OR for the risk of OP in Yin-deficiency constitution was 1.77(95% CI: 1.13-2.77,P<0.01).Funnel plot showed that there was no significant publication bias.2.SDS-PAGE showed good protein removal in each sample group after removal of high abundance proteins,the mass spectra showed normal results.Mass spectrometry analysis showed that a total of 757 proteins were identified and quantified in the Yang-deficiency constitution OP group and the ideal constitution non-OP group,of which 9 proteins were significantly differentially expressed in the sera of Yang deficiency OP patients,6 proteins were significantly upregulated including EIF4A2,PLXNB1,GKN1,ERAP2,DKK3,TAGLN3 and 3 proteins significantly down-regulated TMSB4 X,POTEJ and IGHV2-26.Bioinformatics analysis showed that the differential proteins of Yang-deficiency constitution OP have mainly binding and catalytic functions and are mainly involved in biological processes such as metabolic processes,cellular processes and stress responses.Pathway annotation analysis showed that these differential proteins were mainly enriched to the RNA transport pathway.A total of 732 proteins were identified and quantified in the Yin-deficiency constitution OP group compared to the ideal constitution non-OP group.5 proteins were significantly differentially expressed,including 5 up-regulated proteins: RAB7 A,PON1,IGHV1-69-2,IGLV3-21 and IGHV-?-2.No proteins were significantly down-regulated.Bioinformatics analysis showed that the differential proteins of Yin-deficiency OP all have binding and catalytic functions and are involved in biological processes such as metabolic processes,immune processes,bioregulatory processes and stress responses,and pathway annotation analysis showed that these proteins are mainly enriched in the autophagic and mitochondrial autophagic pathways.3.The expression levels of EIF4A2 protein in the Yang-deficiency constitution OP group were significantly up-regulated compared with the Yin-deficiency constitution OP group,the ideal constitution OP group and the ideal constitution non-OP group(P<0.05).There was no significant difference in the expression levels of TAGLN3 and PLXNB1 proteins between the four groups(P>0.05).The expression levels of EIF4A2,TAGLN3 and PLXNB1 proteins were not significantly different between ages and genders(P>0.05).There were significant differences in RAB7 A protein expression levels in the Yin-deficiency constitution OP group compared with the Yang deficiency constitution OP group,the ideal constitution OP group and the ideal constitution non-OP group(P<0.05).In Yin-deficiency OP group,Yang-deficiency OP group and ideal constitution OP group the expression level of PON1 protein were significantly higher than non-OP ideal group(P<0.05).There was no significant difference between Yin-deficiency constitution OP group,Yang-deficiency constitution OP group and ideal constitution OP group(P>0.05)and the expression levels of RAB7 A and PON1 proteins were not significantly different between ages and genders(P>0.05).Conclusions:1.People with Yang-deficiency and Yin-deficiency constitution have higher risk of osteoporosis than ideal constitution.2.Compared with the non-osteoporosis with ideal constitution,there exist differentially expressed proteins in the serum of the OP patients with Yang-deficiency constitution and the OP patients with Yin-deficiency constitution.EIF4A2 may be a potential bioprotein marker in the serum of OP patients with Yang-deficiency constitution,the pathogenesis of Yang-deficiency OP may be related to abnormal RNA transport function.RAB7 A may be a potential bioprotein marker in the serum of OP patients with Yin-deficiency constitution.PON1 is a sensitive differential protein of OP.PLXNB1 and TMSB4 X are not yet indicated as sensitive differential proteins of Yang-deficiency OP or OP.