Research On The Anti-osteoporosis Molecular Mechanisms Of Tonifying Kidney And Activating Blood Formulas Based On Multi-omics

Objective: This preliminary study aims to explore the molecular mechanism of Tonifying Kidney and Activating Blood Formulas(TKABF)in treating postmenopausal osteoporosis(PMOP)using metabolomics,proteomics and genomics.Methods:(1)Three months old female Sprague-Dawley(SD)rats underwent sham operation(Sham)or bilateral ovariectomy(OVX).Bone mineral density(BMD)(mg/cc)was tested at 12 weeks after ovariectomy using a dual-energy X-ray absorptiometer to determine the success of ovariectomized OP model.Bone marrow stromal cells(r BMSCs)were isolated from the shamoperated(NBM)and osteoporotic rats(OBM);Alkaline phosphatase(ALP)measurement was performed to evaluate containing serum of TKABF(cs TKABF)in secretion promotion of r BMSCs in NBM and OBM groups.(2)Three months old female SD rats were randomly divided into TKABF and Control Groups(n=6 in each group).TKABF group rats were dealt with 1.5m L formulas with the dosage of 1.035g/kg·d;Control group rats were given the same volume of distilled water.Daily treatments were given daily via oral gavage for twelve days.Following sacrifice,plasma was collected for metabolite analysis using LC-MS.(3)NBM were dealt with containing serum of TKABF(cs TKABF)for 24 hours.Total proteins were collected for proteome analysis using i TRAQ,through which we can obtain data of different proteins.(4)Three months old female SD rats were randomly divided into sham-operated(Sham)and ovariectomized control(OVX)group.OVX+TKABF group rats were dealt with 3m L formulas with a dosage of 1.035g/kg·d,and OVX and Sham group rats were given 3m L distilled water.Daily treatments were given daily via oral gavages for twelve weeks.Following sacrifice,serum was collected for proteome analysis using i TRAQ,through which we can obtain data of different proteins.(5)OBM were dealt with containing serum of TKABF(cs TKABF)for 24 hours.Total RNA was collected for RNA-seq using illumina Hiseq 4000 platform,through which we can obtain data of different mRNAs.Results:(1)CD29(99.93% and 95.79%)and CD90(99.70% and 99.55%)were positively expressed,while CD45(1.70% and 1.46%)and CD80(4.45% and 0.69%)were negatively expressed in P3 r BMSCs in both NBM and OBM group.The proportions of G0/G1 were 87% and 85.4% in P3 r BMSCs in NBM and OBM group,respectively.Growth curve of both NBM and OBM groups appeared S type.Results of Gomori,Alizarin,oil red O and toluidine blue stain of both NBM and OBM r BMSCs appeared positive.Compared with Sham group,the BMD of right femur,right humerus,left proximal femur and distal as well as L4-5 sites in OVX group were significantly decreased(P?0.01).After twelve weeks' treatment with TKABF,the right femur,right humerus,left proximal femur and distal BMD of OVX + TKABF group were significantly higher than those in OVX group(P?0.01).In comparison with Control group,the ALP secretion activity was significantly increased in cs TKABF+NBM and cs TKABF+OBM group(P?0.01).(2)Metabolite analysis using LC-MS found that TKABF up-regulated 10 and down-regulated 35 plasma metabolites(P<0.05)in comparison with Control group,which were involved in twenty-two metabolic pathways.(3)Proteome analysis using i TRAQ found that cs TKABF up-regulated 17 and down-regulated 26 proteins including Zfyve16(R=0.75,P=0.008)and ZIP1(R=0.70,P=0.001)proteins in comparison with NBM group,which were involved in 22 cellular signaling pathways.(4)There were 17 proteins met the following criteria(Difference between OVX and Sham groups was significant(P<0.05),and difference between OVX+TKABF and OVX groups was also significant(P<0.05),but difference between OVX+TKABF and Sham group was not significant),among them 9 are function-known proteins,which were((OVX+TKABF vs OVX)P <0.05,R=Ratio,R>1 was up-regulated and R<1 was down-regulated)LIFR(R=2.32,P<0.001)?CEH(R=1.92,P=0.003)? FGL2(R=0.47,P=0.012)? TSP-1(R=0.39,P=0.001)?CXCC/RTCK1(R=0.32,P<0.001)?PPlase(R=0.32,P<0.001)?PF4(R=0.29,P<0.001)?KIF(R=0.26,P<0.001)?CCDC60(R=0.24,P<0.001).The other 8 proteins are functionunknown ones.(5)There were twenty-one mRNAs met the following criteria(Difference between OBM and NBM groups was significant(P<0.05),and difference between cs TKABF+OBM and OBM groups was also significant(P<0.05),but difference between Cs TKABF+OBM and NBM groups was not significant(P>0.05)),among them 13 ones are function-known mRNAs,which were(FC=Fold Change,“+”was up-regulated,“-”was down-regulated;P<0.05)Brd1(FC =4.934,P=7.71E-31)?Zfp219(FC =2.649,P=6.72E-8)?Ahdc1(FC =2.020,P=3.68E-05)?Adra2a(FC =2.001,P=4.30E-5)?Uspl1(FC =1.882,P=0.000106)?Gatad2b(FC =1.572,P=0.001258)?Rfc1(FC =1.564,P=0.001069)?Hspb6(FC =1.38,P=0.00035)? Gcc2(FC=-3.056,P=2.92E-10)?Egr2(FC =-3.055,P=2.91E-13)?Col7a1(FC=-2.331,P=2.49E-06)?Jun(FC=-1.672,P=4.57E-75)and Hs6st2(FC=-1.618,P=0.000699).The other 8 ones are function-unknown mRNAs.With a significant difference between Cs TKABF+OBM and OBM group,orthologues which can encode zinc finger domain and its related gene included: 5 ones containing KRAB zinc finger domain gene,1 with CXXC zinc finger domain gene,1 with BTT zinc finger domain gene,2 SLC39A81,1 SLC39A1 as well as Zinc related genes: 1 MED15,1 KLF2 and 1 TRAF3.Conclusion:(1)TKABF up-regulating plasma metabolite sphinganine level may be related to regulation of sphingolipid signaling pathway of cs TKABF to normal r BMSCs;TKABF massively downregulating plasma metabolite Lyso PC and Lyso PE level may be related to the activation of phospholipase D(PLD)signaling of cs TKABF to normal r BMSCs.TKABF can downregulate plasma histidine levels in normal rats.(2)The up-regulation of CCDC60(containing coiled domain)may be a potential target of renal deficiency type OP model.TKABF can improve BMD in ovariectomized OP rats by downregulating CCDC60 expression.The up-regulation of Gcc2(containing coiled domain)mRNA expression may be one of the characteristics of ovariectomized r BMSCs,and cs TKABF can promote osteoblastic differentiation of ovariectomized r BMSCs by decreasing the level of Gcc2.The Coiled-coil domain may be an important target for prevention and treatment of TKABF to PMOP.(3)cs TKABF can regulate osteoblastic differentiation of normal r BMSCs by down-regulating the zinc transporter protein ZIP1(encoded by SLC30A1 gene)and Zfyve16(FYVE zinc finger)level.(4)The down-regulation of Zfp219(encoding C2H2 zinc finger domain)and Gartad2b(encoding GATA zinc finger domain)mRNAs may be one of the characteristics of ovariectomized r BMSCs,while cs TKABF can up-regulate which to promote osteoblastic differentiation of ovariectomized r BMSCs.The up-regulation of Egr2 mRNA expression may be another characteristic of ovariectomized r BMSCs,whereas cs TKABF can down-regulate which to promote osteoblastic differentiation of ovariectomized r BMSCs.(5)cs TKABF can promote osteoblastic differentiation of ovariectomized r BMSCs by regulating gene orthologues: 5 with KRAB zinc finger domain,1 with CXXC zinc finger domain,1 with BTB zinc finger domain,1 with SLC39A81,1with SLC39A1 and Zinc related orthologues genes MED15,1 KLF2 and 1 TRAF3.The zinc finger domain may be another important target of prevention and treatment of TKABF to PMOP.(6)TKABF can specifically regulate proteins and genes which contain zinc finger domain through down-regulated the level of plasma histidine,and this special regulation may be also involved in the plasma sphingosine up-regulation induced sphingolipid signaling pathway.The possible mechanism may be that TKABF consists of a large number of zinc-rich herbal drug(Tonifying Kidney Chinese Herbal).In sum,TKABF can specifically regulate proteins and genes that contain coiled-coil and zinc finger domain to prevent and treat PMOP.