Bioinformatics Analysis And Comparative Study Of Testicular Proteins In BPA Exposure And Kidney Deficiency Mice

How to preventand treat theenvironmental estrogens(EEs)Bisphenol-A(BPA)-induced male reproductive disorder has become a hot issue all around the world.BPA exposure and kidney infertility are similar in the aspects of gonadal axis morphology and function,behavioral changes,GPER in testis expression and efficacy of kidney-invigorating Chinese medicine,therefore,we intend to use i TRAQ technology and bioinformatics analysis methods to identify the mouse testis differential proteins,GO and COG annotation and enrichment and network analysis were used to compare the relationship between different proteins,especially reproductive proteins.Provide a reliable experimental basis forsearching better prevention and treatment of BPA-induced reproductive disorders.Part one: Identification and Annotation of Differentially Expressed Proteins in BPA-exposed and Kidney-deficient Mouse TestisObjective: Using i TRAQ and bioinformatics analysis techniques,testis differentially expressed proteins in BPA-exposed and kidney-deficient mice were annotated with GO and COG.Provide a foundation for further study on the correlation of reproductive differential proteins in mice testis of kidney yang deficiency and yin deficiency.Methods: 1.Model preparation and grouping: 40 mice were randomly divided into 4 groups,10 of each group.Normal control group(control): intraperitoneal injection of saline 0.2ml,1 time /d,continuous 7d;Kidney Yang deficiency group(P1): intraperitoneal injection of hydrocortisone 25 mg/kg·d,continuous 10d;Kidney Yin deficiency group(P2): intraperitoneal injection of hydrocortisone 50 mg/kg·d,continuous 7d;BPA group(P3): intraperitoneal injection of BPA solution 100 mg/kg·d,continuous 7d.Cervical dislocation of the mouse,the testicular tissue,reserve.2.Testicular HE staining: preparation of testicular tissue paraffin section.The slices were dewaxed by xylene,dehydrated,and stained with hematoxylin solution and eosin,dehydrated.Xylene transparent,sealed.3.ITRAQ analysis: The extracted testicular protein was treated with reductive alkylation,the protein was fully digested,and the protein concentration was determined using the Brandford method.Polyacrylamide gel was used for electrophoretic detection.Equal amounts protein Tripsin each sample digestion,marker peptides with i TRAQ reagents and mix markup amount for the peptides,and a strong cation exchange chromatography separation,liquid tandem mass spectrometry analysis,get the original mass spectrum data.4.Mass spectrometry analysis and data processing: The original mass spectrometry data were converted into MGF format files by the corresponding tools,the protein identification software Mascot was used to search the corresponding database for identifying proteins.The data is judged by a certain screening threshold,and the authentic differential protein identification result is obtained,and GO and COG annotationswere carried out.5.Statistical method: the spectral data was searched by Mascot 2.3.02,and the database was uniprot-mus musculus.The Confidence Interval,which was used in the identification protein,was 95%,and at least one peptide fragment was more than95% matched with the peptide segment.According to the protein abundance level,when the difference multiple was more than 1.2 times,and the P < 0.05 was statistically tested,it was considered as the difference protein.Results: 1.Testicular HE staining: BPA,kidney yang deficiency and kidney yin deficiency group in mouse testis were appeared different degree of seminiferous tube hollow tubular lumen,wall damage,thin basement membrane,the continuity of the basement membrane was damaged,thecells were disorderly arranged,the number of seminiferous epithelial cells decreased,reduced cell structure clearly,the cavity to reduce the number of sperm,the mesenchymal cells were not evenly distributed..2.Testicular protein identification: Totally 393967 spectrums were generated,23034 peptides and 5803 proteins were identified.And the reproducibility is good,the degree of variation is low,and the credibility is high.3.Testicular protein quantitative analysis: 87 differentially expressed proteins were obtained in the kidney Yang deficiency group,and the expression was increased by 27,and 60 were down-regulated.There were 177 differentially expressed proteins in the kidney Yin deficiency group,84 were up-regulated and 93 were down-regulated.There were 124 differentially expressed proteins in the BPA group,45 were up-regulated and 79 were down-regulated.4.GO annotation for all identified proteins: Proteinswere involved in 23 biological processes,16 cellullar components and 17 molecular functions.The top four cellular components were cell,organelle,membrane,and macromolecular complex.The molecular functionswere binding,catalytic activity,enzyme regulation activity,and structural molecular activity.The biological processes were cellular process,metabolic process,single-organism process,biological regulation,etc.;592 reproductive proteins,554 reproductive process proteins,which are closely related to spermatogenesis,are screened from it.5.COG annotation for all identified proteins: Ranked in the top four were overall functional prediction,posttranslational modifications,protein turnover,chaperones,replication,recombination and repair,transcription,translation,ribosome structure.Conclusion: Both BPA exposure and different doses of hydrocortisone induced kidney Yang deficiency and kidney Yin deficiency model mice induced testicular morphological changes.The results of proteomics also preliminarily confirmed the abnormal expression of testis proteins in the model mice.592 reproductive proteins,554 reproductive process proteins,which are closely related to spermatogenesis,are screened.Part two:Comparative Study on Reproductive-related Proteins of Testis in Mice with Kidney Yang Deficiency and Kidney Yin DeficiencyObjective: Using GO enrichment analysis and network interaction cluster analysis of the testis differential proteins in kidney-yang deficiency and kidney-deficiency mice,the relationship between two groups of differential proteins and reproductive-related differential proteins was explored.Provide key data for further investigation of the correlation between BPA exposure and differential protein expression in testes of kidney deficiency miceMethods: 1.Testis differential protein GO enrichment analysis: the testis differential proteins in the kidney Yang deficiency group and the normal group,the kidney Yang deficiency group and the normal group,the kidney Yin deficiency group and the kidney Yang deficiency group were presented to the Gene Ontology database(http://www.geneontology.org/)each term map,calculating the number of proteins per term,and then appling the hypergeometric test to find the GO term that significantly enriched in the differential protein.With p < 0.05 as the threshold,the GOterm that meets this condition was defined as GOterm which is significantly enriched in the differential protein.Differential proteins closely related to reproduction were screened by comparison of differential proteins that significantly enriched in reproductive or reproductive processes 2.Testis differential protein network analysis: The interaction of mouse testicular differential proteins in kidney yang deficiency group and normal group,kidney yin deficiency group and normal group,kidney yin deficiency group and kidney yang deficiency group was predicted by using STRING10.5(http://www.string-db.org/)database,the network diagram of differential protein interaction was drawn at the same time.When the comprehensive confidence ? 0.15,the data analysis mode of the STRING database is selected to construct the network diagram of protein interaction.Choosing comprehensive confidence acuity 0.7 high confidence value(STRING database by default),at the same time with the help of a STRING of Markov Clustering(MCL)method,analyzing the topology of protein interaction networks,nodal proteins of each group were found.The key proteins in the reproductive related differentially expressed proteins were found by the analysis of the reproductive related differential proteins screened from the GO enrichment function,and the comprehensive confidence was greater than 0.7 and MCL was 3.3.Statistical methods: in the enrichment analysis,the P value of the hypergeometric test was < 0.05,indicating that the difference protein was significantly enriched in this item.Select the comprehensive confidence to be greater than 0.15 to construct the differential protein interaction network.The difference protein network module was constructed by selecting the data analysis mode of the STRING database with the combined confidence value of 0.7 and MCL 3.Results: 1 Comparison of testis differential protein between mice with kidney-yang deficiency and normal ? 18 differentially expressed proteins closely related to reproduction were screened out,of which Rps19,Rps28 and D1Pas1were up-regulated,down-regulated as Svs2,Svs3 a,Banf1,Sod1,Rplp2,Rps5,Pdzd8,Lipe,Rplp1,Rpl11,Mif,Pebp1,Spata18,Cyp17a1 and Mea1.? Compared with the normal group,there were 32 key differentially expressed proteins in the kidney Yang deficiency group,including Hbb-b2,Hbb-b1,Ephx1,Gsta3,Eef1b2,Rps5,Rplp2,Rps28,Rplp1,Rpl11,Rps19,Ubc,Ubqln2,Histlh3 e,Hist3h2ba,H2 afy,Serpina3k,Gtpbp2,Orm1,Fgb,Glb1,Akr1b8,Apoa4,Apoa1,Serpinc1,Hpx,Svs3,Svs6,Svs4,Svs1,Svs2,Svs5.? There were 8 key proteins in the reproductive related differential protein,including Rplp1,Rplp2,Rpl11,Rps5,Rps28,Rps19,Svs2,Svs3 a.2.Comparison of mouse testis differential proteins between Kidney Yin deficiency group and normal group and the same key proteins as the kidney-yang deficiency group ? There were 34 differentially expressed proteins closely related to reproduction,of which 8 differentially expressed protein expressions were up-regulated,including Nme1,Vim,Calr3,Rps28,Insl3,Anxa1,Kif4,Hsd3b1.26 differentially expressed proteins were down-regulated,including Svs2,Ccin,Dynlrb1 Rpl35,Rplp2,Eqtn,Cabs1,Mif,Adad1,Sod1,Smcp,Izumo1,Bsg,Hspa1 l,Spa17,Mea1,Smrp1,Svs3 a,Banf1,Rpl23,Cabyr,Rpl11,Pebp1,Pacrg,Txndc2,Ace.? Compared with the normal group,there were 74 key differentially expressed proteins in the kidney Yin deficiency group,including Try5,Prss1,Khsrp,Exosc5,Rpl35,Rpl23,Rpl11,Rplp2,Eef1b2,Srp9,Rps15,Rps28,Svs6,Svs3 a,Svs4,Svs1,Svs2,Svs5,Col22a1,Col6a1,Eno3,Tkt,Spa17,Akap3,Cabyr,Fscb,Apoa4,Apoa1,Hpx,Fga,Fgb,Orm1,Lgals1,Anxa2,Anxa1,Gnaq,Dynlrb1,Kif3 b,Kif4,Gnai2,Rapgef3,Zyx,Parva,Mphosph8,Hist1h3 e,H2afy,Rbbp4,lgbp1 b,Ppp4c,Srsf6,Syf2,Hnrnpul1,Lsm3,Snrpc,Snrpb,Gemin7,Ephx1,Gstp1,Adh1,Actr3,Arpc3,Nck1,Dstn,Psma3,Ubc,Ube2v2,Arhgef7,Hspb1,Mcm7,Uchl1,Chmp4 b,Nedd8,Cops3.? There were 7 key proteins in the reproductive related differential protein,rpl2,Rpl11,rp 28,Rpl35,Rpl23,Svs2,and Svs3 a.? The same key difference proteins of the kidney Yang deficiency group were Ephx1,Eef1b2,Rplp2,rpl2,Rpl11,Ubc,Hist1h3 e,H2afy,Orm1,Fgb,Apoa1,Apoa1,Apoa4,Hpx,Svs6,Svs4,Svs2,Svs5.? The same reproductive key difference proteins in the kidney-yang deficiency group were Rplp2,Rpl11,Rps28,Svs2,Svs3 a.Among them,Rps28 was the up-regulation,while Rpl11,Rplp2,Svs2,and Svs3 a were down-regulated.3.Comparison of mouse testis differential proteins between Kidney Yin deficiency group and Kidney Yang deficiency group ? There were 22 differentially expressed proteins closely related to reproduction,of which 10 differentially expressed protein expressions were up-regulated,including Gpx3,Calr3,Star,Nme1,Ncapd2,Vim,Cast,Sod1,Rps4 x,Cygb.12 differentially expressed proteins were down-regulated,including Cabyr,Selenof,Pacs1,BC048507,Ube2 i,Gpx4,Smcp,Ace,Spa17,Ybx3,Odf2,Svs3 a.? There were 37 key difference proteins between kidney Yin deficiency group and kidney Yang deficiency group,including Try5,Prss1,Setx,Scaf4,Cobra1,Ercc3,Snrpb,Cd2bp2,Lsm3,Haus4,Odf2,Ssna1,Dctn3,Dync2li1,Gm20390,Nme1,Ppid,Ttc12,Syne2,Tex12,Ube2 i,Nedd8,Lcp1,Actg1,Arpc3,C4 b,C3,Gnai3,Hbb-b2,Hbb-b1,mt-Atp8,Atp5 j,Gpx1,Gpx3,Gpx4,Atox1,Sod1.? The reproductive related nodal proteins in the kidney Yin deficiency group and the kidney Yang deficiency group were Sod1,Gpx4 and Gpx3,however,Gpx4 and Gpx3 are not differential proteins,and Sod1 is the difference protein of two kinds of kidney deficiency,and the expression of kidney yin deficiency mouse testis is higher than that of kidney yang deficiency,and P<0.001.Conclusion: The up-regulation of Rps28 in testis and the down-regulation of Rpl11,Rplp2,Svs2 and Svs3 a may be the common material basis for the two types of kidney deficiency.However,the expression of Sod1 in the two groups may be the key material basis for distinguishing the two types of kidney deficiency.Part three: A preliminary study on the correlation of mouse testis proteins between BPA exposure group and kidney deficiency groupObjective: Interacting Cluster Analysis was performed on the testicular differential protein in the BPA group and the normal mice,in order to screen BPA-induced mouse testicular key differential proteins.And compare it to the key protein of kidney deficiency testis,in order to find the same differential proteins,to explore the correlation between BPA exposure and kidney deficiency mice in testis proteome.Methods: 1.Mass spectrum analysis and data processing: the same as the first part.2 Testicular differential protein network analysis: the same as the second part.Results: 1 There were 50 key difference proteins in the BPA group compared with the normal group,including Serpina3 k,Orm1,Gclm,Oplah,Cyp17a1,Taldo1,Ldha,H2-Ke6,Hsd3b1,Actrt2,Arpc4,Gapvd1,Ddx21,Nat10,Rps3 a,Rps17,Rpl27,Rpl17,Rps13,Rps19,Eif3 g,Eif3f,Rps9,Rps10,Rps14,Kif4,Rab1,Surf4,Svs6,Svs4,Svs3 a,Svs1,Svs2,Apoa1,Calr,Pdia6,Prdx4,Xpot,Ranbp1,Tnpo1,Gemin7,Snrpb,Lsm3,Hnrnpd,Psma4,Psmd7,Adh5,Aldh1a1,Gstm6,Ephx1.2 BPA exposure was the same the key difference proteins in testicular as kidney Yin deficiency and kidney Yang deficiency mice,including the down-regulated proteins Svs1,Svs2,Svs3 a,Svs4,Svs6,the up-regulated proteins Apoa1,Orm1.Conclusion: BPA was the same as the key differential proteins in testis of mice with kidney yin deficiency and Yang deficiency.The correlationof testicular protein expression between BPA exposure and kidney-deficient mice was initially verified,providing experimental evidence for further research.