| BackgroundOsteoporosis is one of the most common diseases in the world.With the progress of aging and the prolongation of human lifespan,as well as factors such as environmental factors and drug interventions,the incidence of osteoporosis and the number of patients has gradually increased,resulting in heavy socioeconomic burden and public health threat.Among them,glucocorticoid-induced osteoporosis(GIO)is the second largest cause of secondary osteoporosis.With the development of hormone therapy and hormone abuse,the incidence of GIO is increasing year by year.GIO progresses rapidly with serious consequences,and is not affected by a certain age,thus causing a more serious social and public health burden,which greatly affecting the quality of life of patients.There is no term osteoporosis in Chinese medicine literature,but in the records of traditional Chinese medicine books in all dynasties,the symptoms of bone atrophy,bone paralysis,bone pain and other related diseases are similar to osteoporosis.In general,bone atrophy is the most suitable Modern medicine diagnoses osteoporosis,and kidney deficiency is the main mechanism of its pathogenesis.In the published articles and studies,the relationship between TCM constitution and osteoporosis has been further explored.Among them,yang deficiency constitution is most closely related to osteoporosis,followed by qi deficiency constitution and blood stasis constitution.From the point of view of physical fitness for clinical guidance,kidney-invigorating and yang-boosting drugs have always been the focus of the treatment of osteoporosis.Traditional Chinese medicine has a long history and rich experience in the treatment of osteoporosis,and current research has been striving to improve its efficacy and elucidate the mechanism.ObjectivesIn this study,Eucommia ulmoides,Cuscuta,and Drynaria(E.C.D)were selected as medicinal strings,all of which are recommended for yang-deficiency constitution.Its experimental efficacy and mechanism are unclear.We are aimed to explore the treatment efficacy of GIO and the mechanism of E.C.D.Methods and ResultsIn this experiment,GIO rats were injected with glucocorticoid(GC),and E.C.D extract(low dose,medium dose,and high dose)was administered by gavage at the same time.After 8 weeks of administration,rat serum,24-hour urine,and femurs of two sides were collected.Calcium and phosphorus levels in both urine and serum,bone activity-related cytokine level of serum,femoral slice histology staining,femoral biomechanical function test,micro-CT scan and other related tests were performed.After GC inducing GIO,the body weight of GIO rats(GC group)decreased significantly.The 24-hour urinary calcium and phosphorus loss increased significantly compared with Vehicle group,and the serum calcium and phosphorus levels decreased in GC group.E.C.D gavage treatment can alleviate the weight loss of the rats,reduce the 24-hour urinary calcium and phosphorus loss,and improve serum calcium and phosphorus level.Among them,the effect of the high-dose E.C.D group was significantly better than that of the low-dose group.The detection of serum-related protein levels in GIO rats showed that GC modeling led to increased serum expression of osteoclast activity-related proteins,such as RANKL,CTX,while the osteoblast related protein like OPG was inhibited.The E.C.D administration inhibited thCe expression of RANKL and increased the level of serum OPG.At the same time,H&E staining and femoral TRAP staining showed that E.C.D could improve the infiltration of bone fragments and fat in rats and reduce the number and activity of osteoclasts.Bone biomechanical function tests,and micro-CT scan results showed that E.C.D could significantly improve trabecular bone continuity,bone structural integrity,bone mineral density,bone volume,and biomechanical function in experimental GIO rats.And these functions had obvious dose dependence.As the dose increased,the therapeutic effect was more obvious.Based on the above experimental results,E.C.D has a clear therapeutic effect on the GIO of experimental rats,and the therapeutic effect is enhanced with the increase of the dose,which has an obvious dose-dependence.To further verify the effect of E.C.D on GIO and the molecular mechanism,a series of experiments were designed in vitro.In the process of bone metabolism,the balance between osteoclast and osteoblastic activity is critical for bone remodeling and bone-related diseases.In vitro experiments,the Raw264.7 cell line was used for osteoclast differentiation-related experiments,and mouse primary bone marrow mesenchymal stem cells(MSCs)were used for osteoblast-related experiments.The results of CCK8 toxicity experiments showed that E.C.D had no toxic effect on Raw264.7 cells in the range of 1μg/mL-500 μg/mL but could significantly promote the proliferation of Raw264.7 and MSC cells with increasing dose.For further experiments,we selected E.C.D 1 μg/mL as the low dose group,50 μg/mL as the medium dose group,and 200 μg/mL as the high dose group according to the dose gradient CCK8 assay.Raw264.7 was induced to differentiate into osteoclasts in vitro with M-CSF and RANKL,and E.C.D was used for in vitro intervention by different doses.The results were verified by mRNA transcription level,protein expression,as well as morphological observation and immunofluorescence staining.qPCR and Western Blot results showed that E.C.D could inhibit the differentiation of Raw 264.7 cells into osteoclasts and reduce the expression of RANKL,NFATc1,TRAP and other genes in vitro.And as the dose increased,the inhibitory effect became more obvious.The results of TRAP staining and F-actin immunofluorescence staining also showed that E.C.D could inhibit the formation of mature osteoclasts.Further bone resorption experiments showed that E.C.D could ultimately inhibit the bone resorption function of osteoclasts.On the other hand,E.C.D can promote the expression of OPG in bone marrow MSCs of mice.As a cell regulatory protein,OPG can promote osteoblast differentiation and inhibit the formation of osteoclasts.E.C.D extract also increased the expression of Wntl and Runx2 which are related to osteoblast formation in MSCs.This result showed that E.C.D has the potential to promote osteogenic differentiation.Pretreatment of MSCs with E.C.D to obtain conditioned medium(CM)also can inhibit the differentiation of Raw264.7 cells into osteoclasts.This experimental result shows that E.C.D can not only directly regulate the differentiation of MSCs,promote the formation of osteoblasts,but also regulate the paracrine effect of MSCs to inhibit osteoclast differentiation,and exerts an indirect effect.To further explore the molecular biological mechanism behind E.C.D treatment of GIO,we performed high-performance liquid chromatography-mass spectrometry(HPLC)and comprehensive network pharmacology analysis of E.C.D extracts.43 active ingredients in E.C.D extracts were identified by HPLC based on the comparison with literature and databases.Through literature search,database search,and network pharmacology analysis,the potential regulated protein targets by 43 active ingredients contained in E.C.D were identified.We compared 43 targets with the protein targets related to osteoporosis diseases,and finally the E.C.D-mediated potential therapeutic targets for osteoporosis were determined.KEGG enrichment analysis classified the relationship between the targets and the molecular pathways that may be involved in the regulation of osteoporosis.Among them,the PI3K/Akt pathway was anchored by the most protein targets,indicating that the PI3K/Akt signaling pathway may be involved in the regulation of osteoporosis by E.C.D.To investigate the regulation of E.C.D on osteoclast genesis,we further verified the PI3K/Akt pathway based on network pharmacology analysis at the cellular level.After M-CSF/RANKL stimulation,the expression of PI3K/Akt pathway was up-regulated in Raw264.7 cells,which in turn promoted osteoclast differentiation.E.C.D inhibits PI3K,Akt phosphorylation of Raw264.7 cells,thereby inhibiting osteoclast differentiation.Further,we selected PI3k protein inhibitor for robust verification,and found that inhibiting PI3K can inhibit Akt phosphorylation,thereby inhibiting the level of RANKL protein and osteoclast differentiation.ConclusionIn conclusion,E.C.D can inhibit osteoclast differentiation in vivo and in vitro to treat experimental glucocorticoid-induced osteoporosis and can promote the expression of osteogenesis-related genes in MSCs.E.C.D possibly exerts the function of inhibiting osteoclast differentiation by regulating the PI3K/Akt signaling pathway. |
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