| Background:The clinical effect of Bushen Huoxue Decoction on osteoporosis(OP)of kidney deficiency and blood stasis type is significant,but the mechanism of action is not clear.Previous studies in this study have shown that Bushen Huoxue Decoction can promote the proliferation and osteogenic differentiation of MSCs,and can significantly upregulate the expression of Runx2/Osterix,which may be related to the expression of miR-488.However,it is unclear whether its mechanism of action involves the involvement of extracellular vesicles.Objective1.Observe the effect of Bushen Huoxue Decoction on traditional Chinese medicine syndrome score,bone metabolism,clinical efficacy,bone mineral density(BMD),FRAX score,and its safety evaluation in patients with osteoporosis(OP)of kidney deficiency and blood stasis type.2.Through animal experiments,observe the effects of Bushen Huoxue Decoction on BMD,bone microstructure,and osteogenic related factors in OP rats;Through cell experiments,observe the effects of Bushen Huoxue Decoction on the expression of miR-488 and osteogenic related factors in bone marrow mesenchymal stem cells(MSCs)of OP rats,as well as the secretion of extracellular vesicles.Methods1.Clinical researchA prospective exploratory open study method was used to recruit 49 patients with bone loss or osteoporosis who received outpatient or inpatient treatment at Guangdong Provincial Traditional Chinese Medicine Hospital from September 2021 to December 2022.According to whether the patients took traditional Chinese medicine or not,they were divided into an experimental group of 26 cases and a control group of 23 cases.The experimental group was treated with Bushen Huoxue Decoction on the basis of Western medicine’s anti osteoporosis treatment,while the control group was treated with Western medicine’s anti osteoporosis treatment,namely alendronate sodium tablets,calcitriol capsules,and calcium carbonate D3 tablets,all of which lasted for 3 months.Before and after treatment,traditional Chinese medicine syndrome scores,BMD,bone metabolism tests,efficacy and safety evaluations were conducted.The FRAX tool was used to evaluate the probability of major osteoporosis fractures occurring in patients over the next 10 years.Data analysis was conducted using t-tests,rank sum tests,chi square tests,and repeated measures analysis of variance.2.Experimental research2.1 Animal experiments:The ovariectomy method was used to construct an OP rat model.After modeling,the BMD and bone microstructure of the rats were examined using micro-CT.The serum levels of OPG and RANKL were measured using enzyme-linked immunosorbent assay(ELISA).After the successful establishment of the model,the rats were divided into groups and given Bushen Huoxue Decoction,Alendronate Sodium,and clean distilled water by gavage.After 4 weeks of administration,micro-CT was used to examine the changes in BMD in rats,immunohistochemistry was used to detect the expression of Runx2,Osterix,OPG,and RANKL,and ELISA was used to detect serum OPG and RANKL levels.2.2 Cell experiments:①After 4 weeks of administration to OP rats,bone marrow MSCs were isolated and cultured,and the expression of miR-488,Runx2,and Osterix mRNA was detected using RT-qPCR and Western blot.②Collect,extract,and identify extracellular vesicles from four groups of rat MSCs,then measure the secretion of MSCs using NTA counting method,and detect the expression of miR-488,Runx2,and Osterix in the extracellular vesicles using RT-qPCR and Western blot methods.③Using extracellular vesicles to intervene in MSCs,the proliferation activity of extracellular vesicles on MSCs was detected by CCK8 method,and the expression of miR-488,Runx2,and Osterix in MSCs was detected by RT-qPCR and Western blot.Results1.Clinical researchAfter treatment,the total effective rate of the experimental group was 90.9%,while that of the control group was 84.2%.The experimental group was higher than the control group,but the difference was not statistically significant(P=0.649);The traditional Chinese medicine syndrome scores of the two groups of patients changed with treatment time,showing a downward trend.The experimental group was better than the control group,and the difference was statistically significant(P<0.05);After treatment,the bone metabolism indicators of both groups of patients decreased to varying degrees compared to before treatment,but the experimental group was not better than the control group;After treatment,the serum 25(OH)2VitD3 levels in the two groups of patients increased compared to before treatment(P<0.05),and the experimental group was higher than the control group,but the difference was not statistically significant(P>0.05);The difference in bone density between the two groups of patients after treatment and before treatment was statistically significant(P<0.05),but the experimental group was not superior to the control group(P>0.05);The FRAX scores of both groups of patients decreased significantly after treatment compared to before treatment(P<0.05),but the experimental group was not better than the control group(P>0.05);Before and after treatment,there was no significant change in liver and kidney function in both groups of patients.Both groups of patients did not experience serious adverse drug reactions during the clinical trial.2.Experimental research2.1 Animal experimentsAfter 4 weeks of ovariectomy for modeling,micro-CT testing showed a decrease in BMD,a decrease in the number and thickness of bone trabeculae,sparse and fractured arrangement of bone trabeculae,poor structural integrity,and increased gaps between bone trabeculae in OP rats,indicating successful modeling;After modeling,the serum OPG level of OP rats decreased,while the serum RANKL level increased;After treatment with Bushen Huoxue Decoction,the BMD of OP rats significantly increased,with an increase in the number and thickness of bone trabeculae.The bone trabeculae were thick,plump,and structurally intact,with fewer gaps between them.At the same time,serum OPG levels increased while RANKL levels decreased;Bone immunohistochemistry staining showed that after treatment with Bushen Huoxue Decoction,the expression of Runx2,Osterix,and OPG in osteocytes and osteoblasts of OP rats was strongly positive.RANKL was mainly expressed in osteoclasts of the model group.2.2 Cell experimentsBushen Huoxue Decoction can inhibit the expression of miR-488 in MSCs of OP rats and upregulate the expression of Runx2 and Osterix;Bushen Huoxue Decoction can promote the secretion of extracellular vesicles in MSCs of OP rats,and is superior to the model group and alendronate sodium group;The expression of miR-488 in the exosomes of MSCs in the Bushen Huoxue Decoction group decreased,while the expression of Runx2 and Osterix increased;Exosomes intervention in MSCs can increase their proliferative activity,and the effect is even better than that of alendronate sodium;Meanwhile,after intervention with MSCs exosomes,the expression of miR-488 decreased in MSCs,while the expression of Runx2 and Osterix increased.Conclusion1.Bushen Huoxue Decoction has a certain effect on improving the traditional Chinese medicine syndrome of OP patients with kidney deficiency and blood stasis type,but it needs to be further validated through a large sample size,strict,and clinical randomized controlled trial.2.Bushen Huoxue Decoction can increase BMD and improve bone microstructure in OP rats,possibly by inhibiting the expression of miR-488 in MSCs exosomes and enhancing Runx2 and Osterix transcription and translation,promoting osteogenesis. |
PDF Full Text Request