The Mechanisms Of Zuoguiwan On Bone Metabolism By Regulating Orexin-A And Its Receptors

Objective:To explore the molecular mechanism of Zuoguiwan(ZGW)in the treatment of postmenopausal osteoporosis through the regulation of Orexin-A and receptor 1(OX1R)and receptor 2(OX2R).Method:Experiment 1:Making ZGW Freeze-dried PowderA 200 mg/ml ZGW extract was prepared with 50%methanol solution.The chemical composition of ZGW extract was analyzed by high performance liquid chromatography-mass spectrometry(UHPLC-MS/MS).The compounds in ZGW were compared with standard substances and the content was calculated.Experiment 2:Establishment of postmenopausal osteoporosis rat model by ovariectomy.Fifty 4-month-old female SPF SD rats were randomly divided into model group(OVX),sham group(Sham),estradiol group(50 ?g/kg/d,OVX-E2),low-dose ZGW group(2.3 g/kg/d,OVX-ZGW(L))and high-dose ZGW group(4.6 g/kg/d,OVX-ZGW(H)).Serum bone turnover markers were detected by enzyme-linked immunosorbent assay(ELISA);Histopathological changes of distal femur were observed by HE staining;Bone mineral density and trabecular microstructure of distal femur were measured and analyzed by CT;The mRNA and protein expressions of Orexin A(Orexin-A),Orexin receptor 1(OX1R)and Orexin receptor 1(OX2R)in hippocampus and tibia,osteoprotegerin(OPG)and nuclear factor kappa B receptor activating factor ligand(RANKL)in tibia were detected by real-time fluorescence quantitative PCR(qPCR)and Western blotting.Experiment 3:Effect of ZGW-containing serum(ZC)on differentiation of bone marrow mesenchymal stem cells(BMSCs)into osteoblastsThe 2.5%containing serum of ZGW were prepared.The control group,ZC group,osteogenic induction fluid group(OS)and 2.5%Zuoguiwan serum-osteogenic induction fluid group(ZCS)were set up.The proliferation of BMSCs was observed by MTT assay and the differentiation of BMSCs into osteoblasts(OB)was detected by Alkaline Phosphatase Calcium cobalt staining.The content of AKP in cell culture medium was detected by alkaline phosphatase AKP.The mRNA and protein levels of Orexin-A,OX1R,OX2R,RUNX2,OPG and RANKL were detected by qPCR and Western blot.Experiment 4:Effect of antagonists on the differentiation of BMSCs into osteoblasts by ZCThe toxic effects of OX1R antagonist,OX2R antagonist and OX1R/OX2R dual antagonist on BMSCs at the concentrations of 200?mol/L,100 ?mol/L,10?mol/L,1 ?mol/L and 100 nmol/L were detected by MTT assay.Control,ZC,ZCS,ZCS-10?mol/L OX1R antagonist group(OX1R10),ZCS-1 ?mol/L OX1R antagonist group(OX1R1),ZCS-100 nmol/OXIR antagonist group(OX1R01),ZCS-10 ?mol/L OX1R antagonist group(OX2R10),ZCS-1?mol/L OX2R antagonist group(OX2R1),ZCS-100 nmol/OX2R antagonist group(OX2R01),ZCS-10?mol/L OX1R/OX2R antagonist group(OX1R2R10),ZCS-1 p.mol/L OX1R/OX2R antagonist group(OX1R2R1),ZCS-100 nmol/L OX1R/OX2R antagonist group(OX1R2R01).Alkaline phosphatase AKP assay was used to detect the content of AKP in cell culture medium.Alkaline Phosphatase Calcium cobalt staining was used to detect the differentiation of BMSCs into osteoblasts.Western blot was used to detect the changes of RUNX2,RANKL and OPG proteins in BMSCs.Result:1.11 compounds in ZGW extract were detected by UHPLC-MS/MS,including Polysaccharide?Monod glycoside?Hyperin?Quercetin?Daucosterol?Ecdysteron?Rutin?Kaempferol hyperoside?Eatalpol?Diosgenin and Sweroside,with the contents were 0.036‰?0.06‰?0.041‰?0.014‰?0.081‰?0.092‰?0.066‰?0.013‰?0.066‰?0.046‰ and 0.056‰,respectively.2.Compared with Sham group,two months after ovariectomy,there were no statistical significance of the weight among all groups(P<0.05).The serum levels of bone resorption markers collagen type I cross-linked carboxyl terminal peptide(?-CTX)and tartrate alkaline phosphatase b(TRAP5b)were increased markedly(P<0.01),while the levels of bone formation markers serum bone alkaline phosphatase(BALP)and osteoprotegerin(OPG)were decreased significantly(P<0.01).The BMD level in all groups were decreased notablely(P<0.01).The femora showed significantly thinned trabeculae and remarkably fractures,incomplete trabecular structure,disordered trabecular arrangement,large amount of empty bone lacunae compared with the Sham group.The strength of femur weakens and the micro structure of femur is severely damaged.In addition,the mRNA and protein levels of OX1R in hippocampus and OX2R and RANKL in tibia of OVX group were significantly higher than those of Sham group(P<0.05,P<0.01),while the mRNA and protein levels of Orexin-A,OPG,OX2R in tibia and Orexin-A,OX2R in brain were significantly lower(P<0.05,P<0.01).Compared with OVX group,OVX-ZGW(L)and OVX-ZGW(H)had no significant difference on body weight of rats after 1 month of administration(P>0.05).However,after 3 months of administration,the weight of rats in OVX-ZGW(L),OVX-ZGW(H)and OVX-E2 groups were decreased significantly(P<0.01).Meanwhile,ZGW significantly could increase the levels of bone formation markers of serum BALP and OPG in ovariectomized rats(P<0.01),inhibite the levels of ?-CTX and TRAP5b in serum(P<0.01),increase the number of bone trabeculae,reduce the vacuoles and trabecular segregation.The levels of BMD,bone micro structure were improved bone strength(P<0.01).In addition,ZGW could down-regulate the mRNA and protein levels of OX1R and RANKL in brain and tibia of ovariectomized rats,and up-regulate the mRNA levels of OX2R in the central and bones(P<0.01).Although the mRNA level of Orexin-A was improved in brain(P<0.01),there is no significant effect on the protein expression of Orexin-A(P>0.05).3.Compared with Control,MTT assay showed that ZC had limited effect on the proliferation of BMSCs.There was no significant difference of the AKP content in culture medium and a few nuclei in ZC group were purple-blue after cultured with ZC(P>0.05).The mRNA and protein levels of Orexin-A,OX1R,OX2R,RUNX2,OPG and RANKL were no significant difference in BMSCs cells after cultured with ZC(P>0.05).Compared with OS group,a large number of nuclei in ZCS were purple-blue,and AKP content was significantly increased(P<0.01).The mRNA and protein levels of Orexin-A,OX2R,RUNX2 and OPG in BMSCs were significantly increased(P<0.05,P<0.01),while the expressions of RANKL and OX1R were markedly decreased(P<0.05,P<0.01).4.The minimal concentration ranges of toxicity to BMSCs of OX1R antagonist,OX2R antagonist and OX1R/OX2R dual antagonist were 10 ?mol/L,1 ?mol/L and 100 nmol/L.Compared with ZCS group,the AKP content of OX1R10 group was not significantly higher(P>0.05)at 3 days of incubation,while the AKP content of OX2R10 and OX1R2R10 groups were decreased markedly(P<0.01).Compared with ZCS group,the AKP content in OX1R10 and OX1R2R10 groups were increased significantly(P<0.01),while OX2R10 group was decreased(P<0.05)at 7 days of incubation.Compared with OS group,the staining of Calcobalt staining of alkaline Alkaline Phosphatase Calcium cobalt in BMSCs was significantly deeper after adding ZGW containing serum.Compared with ZCS group,the staining of OX1R10,OX1R01 and OX1R01 groups were more obvious,and the OX1R10 group was more obviou.The staining of OX2R10,OX2R1,OX2R01 groups were significantly lighter,and the OX2R01 group was more obvious.The staining of OX1R2R10,OX1R2R1,OX1R2R01 groups were significantly lighter,and the OX1R2R01 group was more obvious.Compared with ZCS group,the protein expressions of RUNX2 and OPG in OX1R10 group and OX1R1 group were increased significantly,while the protein expressions of RANKL was decreased(P<0.05,P<0.01).However,there were no significant effect of the protein expressions of RUNX2,OPG and RANKL in OX1R01 group(P>0.05).Compared with OX1R10 group,the protein levels of RUNX2 and OPG in OX1R01 group and OX1R01 group were decreased,while the protein expression of RANKLwas increased(P<0.01).Compared with ZCS group,the protein levels of RUNX2 and OPG in OX2R10 group and OX2R1 group were decreased,while the RANKL was increased(P<0.05,P<0.01).There were no significant difference of the protein levels of RUNX2 and OPG in OX2R01 group(P>0.05),while the expression of RANKL protein was increased(P<0.01).Compared with OX2R10 group,the protein levels of RUNX2 and OPG protein in OX2R01 group and OX2R01 group were increased,while the RANKL was decreased(P<0.01).Compared with ZCS group,the protein levels of RUNX2 and OPG in OX1R2R10 group and OX1R2R1 group were decreased,while the level of RANKL protein was increased(P<0.05,P<0.01).There was no significant difference of the protein levels of RUNX2 in OX1R2R01 group(P>0.05),while the protein levels of OPG and RANKL protein were increased(P<0.05,P<0.01).Compared with OX1R2R10 group,the protein levels of RUNX2 and OPG protein in OX1R2R01 group were increased,while the RANKL was decreased(P<0.05,P<0.01).The protein levels of RUNX2 in OX1R2R1 group were increased,while the protein level of RANKL was decreased significantly(P<0.05,P<0.01).However,there was no significant difference of OPG(P>0.05).Conclusions:1.Eleven compounds,including Polysaccharide?Monod glycoside?Hyperin?Quercetin?Daucosterol?Ecdysteron?Rutin?Kaempferol hyperoside?Eatalpol?Diosgenin and Sweroside couls be used as the quality control indicators of ZGW in the treatment of osteoporosis.2.ZGW could regulate the bone resorption and bone formation markers in serum of ovariectomized rats,increase the number of bone trabeculae,reduce the number of bone vacuoles and bone trabecular separation,improve bone mineral density and bone microstructural level,and enhance bone strength.At the same time,ZGW could down-regulate the expressions of OX1R and RANKL in central ovariectomized rats,and up-regulate the expression of Orexin-A and OX2R in central and peripheral bone centers.3.There was no significant effect of ZGW on the proliferation and differentiation of BMSCs into osteoblasts.ZCS combined with OS could significantly increase the induction ability of BMSCs into towards osteoblasts than using OS alone.4.OX1R antagonist could promote the proliferation and differentiation of BMSCs in the direction of osteoblasts by ZCS.OX2R antagonist and OX1R/OX2R dual antagonist could inhibit the proliferation and differentiation of BMSCs in the direction of osteoblasts by ZCS.5.The molecular mechanism of ZGW in improving perimenopausal syndrome,promoting bone formation,inhibiting bone resorption,increasing bone mass and improving bone trabecular microstructure may be related to the regulation of Orexin-A,OX1R and OX2R receptors.