Stydy On The Efficacy And Mechanism Of Bushen Huatan Decoction On Phlegm-dampness Pattern Of Polysystic Ovary Syndrome Based On Proteomics

Objective1.The techniques of iTRAQ(isobaric tagging for relative and absolute protein quantification)labeling,together with liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis were used to investigate serum protein changes of Bushen Huatan decoction intervention to phlegm-dampness pattern of PCOS.2.We analysed these differentially expressed proteins to find out the therapeutic mechanism of TCM,as well as providing the basis of proteomics for clinical treatment.Methods:The patients were recruited from March,2016 to December,2016 in clinic of Heilongjiang University of Chinese Medicine.All subjects met the inclusion criteria.All 30 patients were treated with Bushen Huatan decoction for three menstrual cycles.Through the combination of clinical research and proteomics technology,the clinical characteristics,steroid hormones,glucose metabolism,ipid metabolism and the integral of TCM syndrome were measured.Serum before and after treatment from 12 patients were obtained.The serum were immunodepleted,labeled by the iTRAQ technique,and analyzed by LC-MS/MS to identify proteomic changes associated with Bushen Huatan decoction treatment.Results:1.Clinical findings:①After Bushen Huatan decoction intervention three menstrual cycles,Waist circumference,mF-G and incidence of acne decreased significantly(P<0.05),the difference was statistically significant;BMI,Hip circumference,WHR,incidence of acanthosis nigricans and lipid leakage declined(P>0.05).② After Bushen Huatan decoction intervention for three menstrual cycles,the level of SHBG significantly increased as well as the level of FAI significantly decreased(P<0.05),the difference was statistically significant.③ After Bushen Huatan decoction intervention for three menstrual cycles,FAI,FINS,HOMA-IR,TG,apoB/A1 decreased significantly(P<0.05),the difference was statistically significant.④After using Bushen Huatan decoction intervention for three menstrual cycles,the loose stool,palpitations and fatigue symptoms improved significantly,the difference was statistically significant(P<0.05).⑤During the three menstrual cycles,the ovulation rate were 10%,13.33%,and 26.67%,respectively,and the total ovulation rate was about 16.67%.It showed that the ovulation rate increased gradually during the treatment.2.Results of proteomics research:The LC-MS/MS analysis identified an average total of 636 proteins,of which 63 were different.25 proteins were up-regulated and 38 proteins were down-regulated.The major biological processes involved in these differential proteins were cellular process(90.4%),single biological process(87.3%),biological control(84.1%),stress(77.8%),location(66.7%);the major molecular functions were binding activity(76.1%),catalytic activity(2 8.5%),molecular function regulation(12.6%),molecular transduction activity(9.5%),signal transduction activity(7.9%).These 63 proteins mainly located in organelle(87.3)、extracellular region(84.1%)、cellular composition(79.3%).The major signaling pathways involved in these differential proteins including:the process of environmental information,including PI3K-Akt signaling pathway,ECM receptor interaction,cGMP/PKG signaling pathway,AGE-RAGE signaling pathway,JAK-STAT signal transduction pathway,Rap1 signaling pathway,MAPK signaling pathway,etc.Metabolic pathways,such as peanut four acid metabolism and carbon metabolism,glycolysis.Cell behavior,including the role of focal adhesion kinase and lysosomal function,tight junction.Signal pathway in biological systems,such as the complement and coagulation cascades,platelet signal transduction pathway;viral carcinogenesis related signal pathway.We analyzed the pathway enrichment analysis of KEGG pathway proteins,found the mechanism of Bushen Huatan Decoction for treating phlegm dampness type PCOS may be mainly involved in complement and coagulation cascade,Fc-gamma-R mediated phagocytosis and Jak-STAT signaling pathway.The expression of protein products of various hierarchical clustering of 63 differences,have important differences between 10 proteins,mainly of regulation of inflammatory response,including gelsolin and vasodilator-stimulated phosphoprotein;lipid metabolism,including proprotein convertase subtilisin/kexin type 9,apoE2,apoM;sugar metabolism,selenoprotein P,glyceraldehyde-3-phosphate dehydrogenase;oxidative reaction,including Superoxide dismutase;regulation of complement activation,including C1 esterase inhibitor and complement component 1 q subcomponent.Conclusion:1.After using BH decoction intervention for three menstrual cycles,Waist circumference,mF-G,incidence of acne,FAI、FINS、TG、ApoB/A1、HOMA-IR decreased significantly compared to before intervention.The level of SHBG increased significantly.2.The 10 proteins,mainly of regulation of inflammatory response,lipid metabolism,oxidative reaction,sugar metabolism,regulation of complement activation involved.3.The mechanism of Bushen Huatan Decoction in treating PCOS may be mainly involved in complement and coagulation cascade,Fc-gamma-R mediated phagocytosis and Jak-STAT signaling pathway,the findings need to be validated in future studies.4.The serum proteomic method of iTRAQ can effectively and sensitively screen the differential proteins before and after the treatment of TCM compound,and provide a new idea for the study of the therapeutic mechanism of traditional Chinese medicine.