| ObjectiveTo discuss the mechanism of BuShen HuoXue Decoction(BSHXD)on promoting the bone healing,based on the research progress of the current literatures,and clinical observation of efficacy of BSHXD in patients with patellar fractures diagnosed as deficiency of kidney and blood stasis type,and cell experiment on the molecular mechanism of MSCs migration in the bone healing.It thereby provides new thoughts and pharmacological targets for the bone frcture repair and bone remodelling by TCM therapy and enriches the theory of TCM on kidney governs bone and to grow marrow.activating blood and removing blood stasis and to give birth to the new.Methods1.Literature researchLiteratures about the the progress of treatment on patellar fractures were to summarize,especially to analyse the advantages and disadvantage of surgical internal fixations.And the relationship of BMSCs migration,the bone healing and BuShen HuoXue formula was to analyse,depending upon the clinical reports on the TCM therapy for the bone healing,and also the molecular mechanism of inner microenvironment(chemokine,signaling pathway).2.Clinical research60 elderly patients with patellar fractures after k-wire tension band internal fixation were randomly selected to the treatment group and the control group according to the random number table.33 patients in the treatment group continuously took BSHXD for 2 weeks after operation,and patients in the control group did not take the decoction,and took routinely treatment same as that in the treatment group.The pain scores in the two groups were recorded trough VAS methods before and after the treatment.And the patients regularly took X-ray examination to confirm bone healing time.The function of the knee joint in the two groups was to assess through Bostman scoring method after the bone healing.3.Experimental research(1).Effect of the extracts from BSHXD on promoting rat BMSCs migration in vitro and the expression of CCR2 proteinThe petroleum ether extract,ethyl acetate extract,ethyl alcohol extract and the aqueous extract from BSHXD were extracted through Soxhlet’ s extraction respectively.The bone marrow adherent method was used to isolate and culture BMSCs,and P3 cells were used in the experiments.The Transwell assay was used to examined the migration of BMSCs treated with the four extracts from BSHXD with 10,50,100μg/mL.And the expression of CCR2 protein in BMSCs treated with the active components was examined by Western blot and immunocytochemistry.(2).Effect of the active components from BSHXD on the expression of Wnt5a in rat BMSCsThe expression of Wnt5a in BMSCs treated with the active components from BSHXD with 10,50,100μg/mL was detected by Western blot and the RT-qPCR was used to detect the mRNA expression of Wnt5a in the groups.(3).Research on the migration activity of BMSCs with Wnt5a gene silencingRT-qPCR was used to detect the mRNA expression of Wnt5a in BMSCs transfected with Wnt5a-RNAi lentiviral vectors and negative control viral vectors and then the expression quantity of green fluorescence in each group was observed by microscope.The expression of Wnt5a protein was examined by Western blot and the migrated numbers of BMSCs were observed and calculated by Transwell assay.Results1.Literature researchThe methods of internal fixation have advantages and disadvantages for patellar fractures,but they all can totally recover the function of patella,and with adjuvant therapy of BuShen HuoXue formula,it could shortern bone healing time.Meanwhile it’ s benefit to master the law of molecular mechanisms in the bone healing process for seek the pharmacological targets for the bone fracture repair and bone remodelling.The therapeutic targets of BuShen HuoXue formula maybe relate to the migration of BMSCs,and many chemokines and signaling pathways involved in that.It maybe regulates BMSCs migration via MCP-1/CCR2 axis and Wnt5a signaling pathway,and it provides new outlook to treat the fracture with BSHXD.2.Clinical researchVAS Score:Compaired with that before treatment in the same group,the scores after treatment in the two group both reduced,with statistical significance(P<0.01).Howerver,compaired with the scores after treatment in the control group,there was no significant difference in the treatment group,the same as the difference vaslue before and after treatment between the two groups.(P>0.05).The time of bone healing:Compaired with that in the control,the time of bone healing in the treatment group had been shortened,with statistical significance(P<0.01).Bostman Scoring:that in the treatment group was 28.21 ± 1.19,while that in the control group was 26.22±1.15,there was significantly stastistical difference between the two group(P<0.01).3.Experimental researchThe petroleum ether extract,ethyl acetate extract,ethyl alcohol extract and the aqueous extract from BSHXD could promote rat BMSCs migration in vitro,and the three concentrations of the petroleum ether extract all had statistical difference compaired with the control group,and the 100μg/mL was the active concentration(P<0.01).Western blot revealed that the petroleum ether extract promoted the expression of CCR2 protein,there was statistical difference compaired with the control group,the 100μg/mL was also the active concentration,and the same as that by immunocytochemistry.Western blot and RT-qPCR both showed that the petroleum ether extract could promote the expression of Wnt5a in rat BMSCs.RT-qPCR suggested that the three Wnt5a-RNAi lentiviral vectors could suppress the mRNA expression of Wnt5a,and the transfection efficiency of that was about 70%by microscope.It indicated that the expression of Wnt5a protein in the Wnt5a-RNAi group declined,with statistical significance comparied with the control group and the negative control group(P<0.001).And the migrated numbers of BMSCs in the Wnt5a-RNAi group declined in the Transwell assayConclusion1.There is close relationship between BuShen HuoXue formula and the bone healing.It’ s important to analyze the molecular mechanisms involved in BMSCs migration,especially chemokines and signaling pathways,for the therapeutic targets on the bone healing.2.It could shorten the healing time of patients with patellar fractures after operation and improve the function of knee joint after operation treated with BSHXD.3.The petroleum ether extract from BSHXD is the active components involved in BMSCs migration in vitro,and its mechanisms relate to stimulating MCP-1/CCR2 asix and upregulating the expression of Wnt5a.Besides,there is close relationship between Wnt5a signaling pathway and BMSCs migration preliminarily. |
PDF Full Text Request