Taohong Siwu Decoction Regulates The Molecular Mechanism Of Mesenchymal Stem Cell Homing In Fracture Healing

The theory of"activating blood circulation and removing blood stasis"in the early stage of fracture is based on the summary of the experience gained from the long-term practice of predecessors,and the mechanism of its promoting fracture healing lacks the further research.Taohong Siwu decoction is the basic,representative and classic recipe of"promoting blood circulation and removing blood stasis".To reveal the molecular mechanism of Taohong Siwu decoction in promoting fracture healing by"activating blood circulation and removing blood stasis"in the early stage of fracture is of great significance.This study will to study the effect and the molecular mechanism of Taohong Siwu decoction on right femur shaft fracture model in rats,which were administered by intragastric administration.It will enrich the connotation of the theory of"activating blood circulation and removing blood stasis"used in the early stage of fracture.Part 1Background:The treatment of osteodiseases in Orthopaedics&Traumatology of TCM is under the guidance of the theoretical system of“Holistic Concept”and“Syndrome Differentiation and Treatment”.Based on the theory of"Three-stage syndrome differentiation and treatment",the damaged bones in early stage caused a qi stagnation of blood and blood stasis,which will be turnover by promoting blood circulation and removing blood stasis.However,the rationality and effectiveness of the theory should be demonstrated for further research.Objective:To establish a rat model of open right femoral shaft fracture with osteotomy and to study the effect of the basic prescription of“promoting blood circulation and removing blood stasis”known as Taohong Siwu Decoction(TSD)on femoral shaft fracture healing with an early intervention.Methods:A rat model of open right femoral shaft fracture with osteotomy was established,and32 rats were randomly divided into four groups:the model group(0ml TSD+1ml distilled water,n=8);low dose group(0.25ml TSD+0.75ml distillation,n=8);medium dose group,(0.5ml TSD+0.5ml distilled water,n=8);high-dose group,(1ml TSD+0ml distilled water,n=8).On the first day after operation,rats were administered by intragastric administration with the concentrated decoction of TSD twice a day for 5days.After 21 days,the specimens from right femur were collected and bone volume fraction BV/TV(%)and bone mineralization density BMD(mg/cc)were analyzed by micro-CT,and the bone matrix and cells were observed by Hematoxylin-Eosin stain(H&E stain).Results:BV/TV(%)in the control group,low-dose group,medium-dose,high-dose group were 51.28%,54.41%,61.98%and 69.81%,respectively.There was no difference between the control group and the low-dose group(P=0.651),and the high-dose group was significantly higher than the medium-dose group(P=0.032),and the medium-dose group was significantly higher than the low-dose group(P=0.041).There was a concentration gradient between the three-dose groups.BMD(mg/cc)in the control group,low-dose group,medium-dose,high-dose group were 500.72 mg/cc,526.6 mg/cc,598.7 mg/cc,678.2 mg/cc,respectively.There was no difference between the control group and the low dose(P=0.671),the low dose was significantly lower than the medium dose(P=0.018),and the medium dose was significantly lower than the high dose(P=0.008).BMD increased significantly in the high dose group.H&E staining(100 x,400 x)showed that there are many chondrocytes in a group and apoptosis chondrocytes in lacunae in the callus.After TSD treatment,callus ossification process is accelerated with chondrocytes apoptosis,osteoblast proliferation,and bone matrix secretion and calcification increase.The effect is obvious in high concentrations.Conclusion:The early intervention of a basic prescription of"activating blood circulation and removing blood stasis",high dose(20g/kg)of TSD could obviously enhance the ability of osteogenesis,promote the volume of callus and increase the density of bone mineralization.Part 2Background:It is difficult to study the pharmaceutical substance and mechanism of action of TCM compound at the cellular and molecular level.However,the modern network pharmacology based on system biology and network biology provides a new method for the study of TCM compounds.Objective:We will use network pharmacology to predict the pharmacodynamic basis and target of TSD for promoting femoral shaft fracture healing.Methods:TCMSP database was used to screen the active chemical components and corresponding target genes of TSD,to screen the target genes of femoral shaft fractures by Gene Cards database.All target genes were transformed by Uni Prot database,and protein interaction network were established by String database;A"drug-component-gene-disease"visual network were built by Cytoscape3.6.0.The R(R3.5.3)language package was used to carry out the GO enrichment analysis of the target genes and the KEGG biological pathway enrichment analysis.Results:764 compounds were obtained from TSD.There were 85 active components satisfying OB≥30%and DL≥0.18,among which,there were 44 active compounds with target genes,corresponding to 111 genes.There were 897 genes related to femoral shaft fracture,and 394 disease genes were obtained according to the correlation coefficient≥4.By combining the disease target genes and active compound target genes,28 target genes were obtained,and 14 active compounds were corresponding:beta-sitosterol,baicalein,ellagic acid,(+)-catechin,paeoniflorgenone,perlolyrine,6-hydroxykaempferol,myricanone,perlolyrine,FA,quercetagetin,beta-carotene,4-[(E)-4-(3,5-dimethoxy-4-oxo-1-cyclohexa-2,5-dienylidene)but-2-enylidene]-2,6-dimethoxycyclohexa-2,5-dien-1-one,GA87,(2R,3R)-4-methoxyl-distylin.With nodal degree≥6,18 key target genes were obtained:VEGFA,IL6,TP53,JUN,CTNNB1,TNF,TGFB1,ALB,CASP3,ESR1,MMP9,PPARG,FOS,AR,CAV1,IGF2,MMP2,HIF1A.These genes were mainly enriched in biological functions such as heterodimer activity,transcription factor binding,protease and protein phosphatase binding,and growth factor activity.Mainly moderate the AGE-RAGE signaling pathway in diabetic complication,Proteoglycans in cancer,Fluid shear stress and atherosclerosis,HIF-1signaling pathway and other signaling pathways.Conclusion:the potential 14 major active components,such as paeoniflorgenone and perlolyrine and 18 targets,such as VEGF and HIF-1 and signaling pathways of TSD in the treatment of femoral shaft fracture were obtained through network pharmacology,which will provide research scope for further study of the molecular mechanism of drug action and provide research direction for the follow-up experimental studies.Part 3Background:Mesenchymal stem cell(MSC)are an attractive and promising seed cell for the treatment of various diseases in orthopaedic research in recent years.With the deepening of research,it has been found that MSCs homing also plays an important role in bone formation and the treatment of osteodiseases.MSCs homing is a prerequisite for bone formation.Only after MSCs first homing to the injury and then differentiating into osteoblasts can they participate in bone tissue repair.Objective:We will observe the mobilization of MSCs from bone marrow by TSD and its molecular mechanism.Methods:The rats were divided into the normal group(1ml distilled water,n=8);fracture group(1ml distilled water,n=8);TSD treated group(1ml TSD,n=8).On the 1st day after operation,rats were administered by intragastric administration with TSD twice a day for 5 days.The number of CD45~-CD90~+CD29~+MSCs in peripheral blood was screened by flow cytometry(FCM)with Tri-color marking.The MSCs in peripheral blood were identified as the cells from bone marrow by surface markers,cell morphology,osteogenic differentiation and adipogenic differentiation.The differential expression of 29 cytokines in the early stage of fracture was determined by proteome profiler array.Results:The number of CD45~-CD90~+CD29~+mononuclear cells in peripheral blood after fracture was significantly higher than that of normal rats(P=0.031).Moreover,TSD can promote the increase of CD45~-CD90~+CD29~+mononuclear cells in peripheral blood of fracture rats(P=0.034).After identification of CD45~-CD90~+CD29~+mononuclear cells in peripheral blood and bone marrow MSCs,it was found that they were negatively expressed in CD45,and positively expressed in CD90 and CD29.All of them are in the shape of long shuttle,growing adherently to the culture plate.Both can differentiate into osteoblasts and adipocytes.Therefore,peripheral blood CD45~-CD90~+CD29~+cells are bone marrow-derived MSCs.The number of MSCs in peripheral blood of rats with open femoral shaft fractures increased significantly compared with that of rats in the normal group,and there was a significant difference between the two groups in CINC-1(2.84 times).It has a chemotactic effect on neutrophils,and neutrophils can release proteolytic enzyme to inactivate cytokine activity and reduce the mobilization of MSCs.Interestingly,after 5 days of TSD intervention,the number of MSCs in peripheral blood significantly increased compared with that in the non-intervention group.The results of proteome profiler array showed that TSD could up-regulate the levels of CINC-1(1.51 times),Fractalkine(3.75 times),L-selectin(1.82 times)and VEGF(2.08 times)in peripheral blood of fracture rats,and down-regulate the levels of IL-1(1.48 times).Fractalkine and VEGF have been shown to directly induce bone marrow mobilization of MSCs,among which VEGF can also amplify sinusoidal capillaries in bone marrow to promote the mobilization of MSCs.Furthermore,network pharmacology predicted that VEGF was closely related to other target genes.Therefore,we speculated that TSD promoted bone marrow mobilization of MSCs in relation to up-regulation of cinc-1,Fractalkine and VEGF.Conclusion:TSD can promote bone marrow mobilization of MSCs,and its molecular mechanism may be related to up-regulation of peripheral blood CINC-1,Fractalkine and VEGF.Part 4Background:MSCs migration is affected by the change of the external micro-environment and the change of their biological behavior.With the increase of peripheral blood derived MSCs successfully migrating to the fracture site,it can provide sufficient stem cells for bone tissue repair,which plays a very important role in accelerating fracture healing.Objective:We will study the effect of TSD on peripheral blood derived MSCs migration in peripheral blood and its molecular mechanism.Methods:Rat were divided into two groups after operation,TSD untreated group(1ml distilled water,n=12)and TSD treated group(1ml TSD,n=12).On the 1st day after the operation,rats were administered by intragastric administration with TSD twice a day for 5 days.The migration ability of peripheral blood derived MSCs was detected by wound healing assay and cell migration assay.The number of MSCs homing at the callus at the early stage of fracture(5d)was detected by immunohistochemistry(IHC-P).Proteome profiler array was used to detect the expression of cytokines in bone callus.Results:Wound healing assay and cell migration assay showed that TSD could enhance peripheral blood derived MSCs.The expression of CD45~-CD90~+CD29~+on the surface of MSCs was detected by IHC-P,and it was found that CD45~-CD90~+CD29~+cells in bone callus increased significantly after TSD treatment for 5 days compared with TSD untreated group,indicating that TSD can promote the migration of peripheral blood derived MSCs to bone callus.The results of proteome profiler array showed that TSD could promote the up-regulation of CINC-1(2.91 times),CINC-3(1.59 times),LIX(1.5 times),Thymus Chemokine(2.55 times)and down-regulation of TIMP-1(2.98times)in bone callus in fracture rats.CINC-1(CXCL1)has been shown to bind to CXCR1 receptor in MSCs membrane to promote its migration.LIX(CXCL5)has also been shown to promote osteogenic differentiation and migration of MSCs.Thymus Chemokine(CXCL7)can promote MSCs migration by binding to CXCR2.VEGF has been reported to promote the formation of focal adhesion and stress fibers and enhance the migration ability of MSCs.TIMP-1 is an active natural protease inhibitor of MMPs,which can inhibit the degradation of MMP-2,MMP-9 and other proteases,leading to the degradation of vascular endothelial cell basal membrane and bone callus matrix,and can promote the migration of peripheral blood MSCs to the bone callus site.Conclusion:TSD can promote peripheral blood derived MSCs migration to the fracture sit,and its molecular mechanism may be related to enhancing peripheral blood derived MSCs,up-regulating the expression of CINC-1,LIX,Thymus Chemokine and VEGF,and down-regulating the expression of TIMP-1 in bone callus.