The Mechanism Of Osteoking For Promoting Bone Formation In OPF Rats

Background:Osteoporosis(OP)is caused by a variety of reasons which lead to the decrease of bone mineral density(BMD)and bone quality.Meanwhile,OP damages bone microstructure and increases osteopsathyrosis.Thus a systemic bone disease easily causes osteoporotic fracture(OPF).OPF is a major complication of OP which affects one in five men and every woman over 50 years old.Drugs for OP are divided into two broad categories:One that reduces bone resorption by inhibiting osteoclast activity,represented by diphosphonates;The other one increases bone formation by enhancing osteoblastic activity,represented by parathyroid hormone(PTH)or its analogues.Currently,the two types of drugs for OP are used in clinic.Either anti-resorption drugs or bone-forming drugs has limitations.As a traditional Chinese medicine(TCM)of Yi nationality in Yunnan,osteoking has a long history for bone diseases.In clinic,osteoking has a positive effect on treatment for lumbar disc herniation.Previous studies showed that osteoking healed femoral head necrosis in rabbit model by enhancing the expressions of vascular endothelial growth factor,core binding factor la,and bone morphogenetic protein-2.Studies found that osteoking improved BMD by reducing the expression of dickkopf-1 in rabbits’ OP model.In tree shrews’ OP model,osteoking enhanced the expressions of phosphoinositide 3-kinase/protein kinase B/glycogen synthetase kinase-3 kinase,bone morphogenetic protein-7 and(3-catenin;Osteoking promoted the proliferation of osteoblasts by enhancing the expression of Smads.Osteoking belongs to TCM and there are new methods to study TCM.Network pharmacology provides the relative targets of the TCM components.Proteomics has simplified the study of TCM and demonstrates the relative targets in disease-animal model.Bioinformatics analyzes the results of proteomics and network pharmacology.The relative targets and pathways of osteoking for OP/OPF can be obtained,including target name,molecular function,cell location,biological function and enriched pathway,etc.Objective:To study the mechanisms of osteoking for promoting bone formation in OPF rats.Based on network pharmacology and proteomics,the relative targets of osteoking for promoting bone formation could be obtained.Further experiments should be used to verify the mechanisms of osteoking and provide theories for preventing and treating OP/OPF.Methods:1.The treatment of osteoking in OPF rats:(1)The OPF rats were duplicated and divided into the model control group(intragastric.0.59 ml/kg/2d 0.9%NaCl),the positive drug control group(subcutaneous.0.33 μg/kg/2d rhPTH 1-34),and the osteoking group(intragastric.0.59 ml/kg/2d osteking),respectively.A total of 54 OPF rats were randomly assigned to the three groups and 18 OPF rats in each group.The dosage of drugs used in OPF rats was converted according to the clinical dosage of drugs,the dosage conversion coefficient of drugs and the error coefficient of drugs for human-rat.(2)According to the clinical treatment,three time points of 4 weeks,8 weeks and 12 weeks were set up after treatment with osteoking,and the related indexes were measured as follows:①Weighing.② Dual-energy X-ray analysis was used to measure BMD and BMC.③ Diagnostic X-ray was used to show the callus.(3)After 4,8,and 12 weeks of treatment with osteoking,6 OPF rats in each group were sacrificed and the serum was collected.The BALP,PINP,TRACP-5β,and CTX-Ⅱ of the serum were measured by ELISA.(4)The measurements were performed after 12 weeks of treatment with osteoking as follows:① Micro computed tomography(Micro-CT)was used to measure the indexes of right femur,including bone volume fraction,bone surface area/volume ratio,bone trabecular number,bone trabecular thickness,bone trabecular separation degree,and structural model index.The callus of right femur fracture was measured by high voltage Micro-CT in gray scale.② After decalcification of the third lumbar,hematoxylin-eosin staining was used to show the bone trabecular structure under the microscope.③After decalcification of the third lumbar,alizarin red staining was used to show the mineralization of bone trabeculae under the microscope,and Image J was used to analyze.④ After decalcification of the third lumbar,immunohistochemical staining was used to show the relative target under the microscope,and Image J was used to analyze.2.Network pharmacology was used to obtain the components of osteoking and the relative targets;The relative targets of OP were obtained through GEO(Gene expression omnibus)database;Based on Cyto Scape analysis,the intersections between the relative targets of osteoking and OP were obtained and showed.The potential targets of osteoking for promoting bone formation were obtained.3.After 12 weeks of treatment with osteoking,the right femoral head in the model control group and the osteoking group were collected for proteomics analysis.The above results were analyzed by bioinformatics,including GO database and KEGG database.The different expressed proteins of osteoking in OPF rats were obtained.The molecular function,cell location,biological function,and enriched pathway of relative targets were obtained,respectively.4.The relative targets of different expressed targets were obtained through the analysis of String database.5.rBMSC were isolated and cultured.Four groups were set up as the control group,the inhibitor group(relative target’s inhibitor of osteoking),the osteoking group and the osteoking+inhibitor group.After 4 weeks of culture,alizarin red staining was performed to show the mineralization of osteoblasts from the differentiation of rBMSC.The OD absorbance of alizarin red staining was measured by microplate reader on 560 nm.6.RT-qPCR and WB methods were used to measure the mRNA and protein of relative targets.Results:1.Osteoking increased the BMD and improved fracture in OPF rats:(1)After 12 weeks of treatment with osteoking,the weights of OPF rats in each group was ranked in descending order as the positive drug control group>the osteoking group>the model control group,with statistically significant difference(P<0.05).(2)From 4th week to 12th week after treatment with osteoking,the BMD in the osteoking group increased gradually;After 12 weeks of treatment with osteoking,compared with the model control group,the BMD and BMC in the osteoking group increased by(4.5±1.06)%and(20.6±23.67)%(P<0.05),respectively;however the two indexes were lower than the positive drug control group.(3)After 8 weeks and 12 weeks of treatment with osteoking,compared with the model control group,the x-ray results of OPF in the osteoking group showed better callus.After 8 weeks of treatment with osteoking,the positive drug control group showed the callus healing line.After 12 weeks of treatment with osteoking,the osteoking group showed the callus healing line which was later than the positive drug control group and earlier than the model control group.(4)From 4th week to 12th week after treatment with osteoking,the BALP in the osteoking group increased by(49.6±6.13)%and PINP decreased by(80.6±3.26)%(P<0.05);After 12 weeks of treatment with osteoking,compared with the model control group and the positive drug control group,the BALP and PINP in the osteoking group increased(P<0.05);After 12 weeks of treatment with osteoking,TRACP-5β and CTX-II in the osteoking group were lower than the sensitivity of ELISA(0.078 ng/ml).(5)After 12 weeks of treatment with osteoking,compared with the model control group,the ratio of bone volume/surface area,the thickness of trabecular bone and the number of trabecular bone in the osteoking group increased(P<0.05).However,these indexes in the osteoking group were lower than the positive drug control group(P<0.05).The results of high-voltage Micro-CT showed that compared with the model control group,the gray degree of callus in the osteoking group increased(P<0.05).The gray degree of callus in the osteoking group was similar to the positive drug control group(P>0.05).(6)After 12 weeks of treatment with osteoking,hematoxylin-eosin staining showed that compared with the model control group,the bone trabecular structure in the osteoking group was more.Alizarin red staining showed that compared with the model control group,the mineralization in the osteoking group increased(P<0.05).However,the bone trabecular structure and mineralization in the osteoking group were less than the positive drug control group.2.The results of network pharmacology:(1)Osteoking had 7 natural components with a total of 874 active components.According to the OB≧30%and DL≧0.18,a total of 129 components were obtained.(2)The 129 components of osteoking were analyzed by network pharmacology.A total of 1056 relative targets from the 129 components were obtained and 119 unique targets were obtained by deleting duplicates.(3)GO analysis showed that the relative targets of osteoking were focused on the interaction of nerve tissues,the response of cells to drugs,the response to toxic substances,the involvement in synaptic signal transduction,the response to ammonium ion and the involvement in ion balance,etc.(4)The secondary dig and analysis were based on GEO database from GSE35955,GSE35958 and GSE35959 chips.A total of 665 relative targets which related to OP were obtained.(5)The "component-target-disease" visual graph between OP and osteoking was manufactured by Cyto Scape analysis.Three relative targets were obtained,including GABRA2,PLAU and Hsp 90-β.3.The results of proteomics:(1)After 12 weeks of treatment with osteoking,there were 7 up-regulated proteins(Hsp 90-β,Fatty acid-binding protein,etc.)and 14 down-regulated proteins(Mgp,Adipocyte lipid-binding protein,etc.).In addition,the expressions of Keratin(typeⅠ/Ⅱ)were up-regulated and down-regulated.(2)The physiological effects of osteoking were analyzed by GO database,including the involvement in biological body regulation,the stress to external stimuli,the tissue physiological and pathological processes.The functions of osteoking were located in the extracellular matrix and to combine molecular substances,regulate molecular functions,participate in molecular structure activities and catalysis,etc.(3)The mechanism of osteoking for promoting bone formation was related to TGF-βsignal pathway through the analysis of KEGG database.4.Hsp 90-β and Mgp were the important targets of osteoking for promoting bone formation in OPF rats:(1)After 12 weeks of treatment with osteoking,compared with the model control group,the expression of Hsp 90-β in the osteoking group which measured by RT-qPCR and WB increased(P<0.05)in vivo;After 4 weeks of culture,compared with the control group,the expression of Hsp 90-β in the osteoking group which measured by RT-qPCR and WB increased(P<0.05)in vitro.(2)After 4 weeks of culture,compared with the control group and the Hsp 90-βinhibitor group,the mineralization of osteoblast in the osteoking group which measured by alizarin red staining increased(P<0.05);Compared with the osteoking group,the mineralization of osteoblast in the osteoking+Hsp 90-β inhibitor group decreased(P<0.05);Compared with the control group and the Hsp 90-β inhibitor group,the mineralization of osteoblast in the osteoking+Hsp 90-β inhibitor group increased(P<0.05).(3)The relative targets of Mgp were obtained from the String database.The relative targets of Mgp were Bmp-2,Bglap and Sparc.(4)After 12 weeks of treatment with osteoking,compared with the model control group,the expression of Mgp decreased(P<0.05)and the expressions of TGF-β,Bmp-2,Bgalp,Sparc increased(P<0.05)in the osteoking group which were measured by RT-qPCR and WB.Conclusion:1.Osteoking increases the BMD and improves fracture in OPF rats.2.The relative targets between OP and osteoking are GABRA2,PLAU and Hsp 90-β.3.The mechanism of osteoking for promoting bone formation are related to TGF-βsignal pathway.4.Osteoking enhances the expression of Hsp 90-β and inhibits the expression of Mgp for promoting bone formation in OPF rats.