Urine Proteomics Study Of Yishen Jianpi Yangxue Tongli Recipe In The Treatment Of Polycystic Ovary Syndrome

Polycystic ovary syndrome(PCOS)is a kind of complex,polygenetic endocrine disorders metabolic disease.Recent clinical manifestations of PCOS are scanty menstruation or amenorrhea,chronic anovulation,infertility,hairy and acne,etc.Long term complications were hypertension,diabetes,hyperlipidemia,endometrial carcinoma,etc.Modern Chinese medicine believes that PCOS is a Chinese medicine disease which is phlegm dampness type infertility or amenorrhea.The pathogenesis is phlegm in the uterus.We have studied the Yishen Jianpi Yangxue Tongli Fang(Plantago,dodder,eucommia,coix seed,semen Benincasae etc.)has a therapeutic effect on clinical symptoms and biochemical index of PCOS.But it is still lack clinical study on therapeutic mechanism.Proteomics is a new way to research the protein in recent years,which can display the expression of protein on a large scale and high throughput.But because of the high operating costs and high level of technical requirements,proteomics are hardly applicated in the field of clinical application.Firstly we confirmed the method of extracting urine protein.Secondly,we treatment patients who have PCOS with Yishen Jianpi Yangxue Tongli Fang.Thirdly we use the methods of Mass spectrometry and urinary proteomics study the urine protein expression before and after the treatment of Yishen Jianpi Yangxue Tongli Fang.Fourthly discuss the pathway and functional proteins of Yishen Jianpi Yangxue Tongli Fang in the treatment of polycystic ovary syndrome.Provide exact scientific research foundation and experimental basis for Clinical therapeutic applications.The purpose of this study is to establishment urine protein mass spectrometry and screening differential proteins that treated with Yishen Jianpi Yangxue Tongli Fang before and after PCOS treatment.Use the precipitation method of Ethanol extracted urine protein.Determine the concentration of protein by BCA method.Each group included 10 samples,Reductive alkylation,trypsin digestion,LC-18SPE column desalting,etc.Identified Urine proteins were by liquid chromatography mass spectrometry/mass spectrometry.Confirmed acquisition 2300 proteins(Mascot ion score>10,peptide FDR<0.5%)and 7 pathways involved in differential protein between three groups through identification of proteins by mass spectrometry,database searching and data analysis.Three groups of samples by T-test(p<0.05),the number of differentially expressed proteins were 119,154 and 46,respectively.It can be seen that there is a significant difference in protein expression before and after administration.The results show that drug therapy has a significant effect on the treatment of PCOS.There were no significant differences between 6 and 12 months administration.The results showed that the treatment effect was not significantly improved with the prolongation of drug intervention time.So Yishen Jianpi Yangxue Tongli Fang in the treatment of six months was significantly effective,carrageenan-derived to give treatment,the efficacy of maintenance but did not improve.Objective:We have studied the Yishen Jianpi Yangxue Tongli Fang has a therapeutic effect on polycystic ovary syndrome.In order to study improve the efficacy of PCOS mechanism before and after the treatment of Yishen Jianpi Yangxue Tongli Fang,we study the difference of urine protein expression profile on the level of urinary protein.1.Investigate effects of different methods to extract urine protein from urine.2.Use the methods of Mass spectrometry and urinary proteomics study the urine protein expression before and after the treaatment of Yishen Jianpi Yangxue Tongli Fang.3.Discuss the pathway and functional proteins of Yishen Jianpi Yangxue Tongli Fang in the treatment of polycystic ovary syndromeMethods:1 Use the precipitation method of Ethanol,acetone and TCA extracte urine protein,observation urine protein electrophoresis through SDS-PAGE electrophoresis.2 Test qualitative and quantitative of sample use LC separate and MALDI-TOF/TOF mass spectrometry.Result:1.Extracte urine protein method of Ethanol precipitation have significant advantages compare with the method of acetone and TCA precipitation protein recovery and settling time the longer protein recovery the higher.2.Most electrophoretic bands are similar in the three groups of samples through SDS-PAGE electrophoresis.But still have differential electrophoretic bands.The electrophoretic pattern is clear and suitable for mass spectrometry.3.Confirmed acquisition 2300 proteins through identification of proteins by mass spectrometry,database searching and data analysis.There are 7 pathways involved in differential protein between three groups.Three groups of samples by T testT-test,the number of differentially expressed proteins were 119,154 and 46,respectively.Conclusion:1.Extracte urine protein method of Ethanol precipitation have significant advantages compare with the method of acetone and TCA precipitation protein recovery and settling more than 1 hours protein electrophoretic pattern is the most clear.2.Drug intervention effect more obvious after 6 months of administration than medication for 12 months.After 12 months of administration,the change of the stimulatory protein gradually decreased and have callback trend.There was no significant difference between two groups of patients in the treatment group for 6 months and the medication for 12 months.3.The main function of differential protein is secreted protein and the main form of extracellular secretion exists outside the cell.The differentially expressed proteins were mainly related to 7 pathways.PI3K-Akt signaling pathway is the most sigjnificant and prominent which is related to insulin resistance,PI3K-Akt signaling pathway is the main mechanism of PCOS have IR.