Metabolomics Study Of Morindae Officinalis Radix And Rehmanniae Radix Praeparata On Protecting Against Glucocorticoid-induced Osteoporosis

Glucocorticoid-induced osteoporosis(GIOP)is a systemic metabolic osteopathy with low bone mineral density(BMD),low bone quality and high fracture incidence caused by long-term glucocorticoid(GC)administration.Since the high morbidity and disability of GIOP,the research and development of anti-GIOP drugs has become a hot spot of widespread concern.Traditional Chinese medicine(TCM)is effective and safe with little side effects.In addition,TCM considers that kidney is in charge of bone.Thus,tonifying kidney herbs are being recognized and approved in dealing with osteoporosis.Morindae Officinalis Radix(MO)is the dry root of Morinda officinalis How.with the function of nourishing kidney and preventing rheumatism,and has long been used as a traditional herbal medicine for tonifying kidney-yang.Rehmanniae Radix Praeparata(RR)is prepared from the dry rhizome of Rehmannia glutinosa Libosch.,and has been traditionally used for tonifying kidney-yin in China.Ample experiments show that MO,RR and their chemical constituents can prevent post-menopausal osteoporosis and senile osteoporosis,while there is few evidence on GIOP.More importantly,MO is the Chinese herb for tonifying kidney-yang,while RR belongs to tonifying kidney-ying medicine,the similarity or difference between their anti-GIOP mechanisms also need to be elucidated.In this study,dexamethasone(DEX)-treated rats and DEX-injured osteoblasts were selected as research objects,and the in vivo and in vitro pharmacodynamics study as well as UHPLC-Q/TOF-MS-based metabolomics analysis were used to explore the preventing effect of MO and RR on GIOP and their underlying mechanisms.1.Effects of MO and RR on GIOP rats and its metabolomics studyGIOP rats model was established by injecting DEX via the caudal vein.The bone micro-architecture,bone histomorphometry parameters,bone related biochemical markers,metabolic profiles and metabolic pathways were assayed to evaluate the effects of MO and RR on GIOP rats.As a result,MO and RR both significantly improved the micro-architecture of trabecular bone,enhanced the BMD,regulated the expression of bone formation markers alkaline phosphatase(ALP)and bone morphogenetic protein 2(BMP-2),and inhibited the expression of bone resorption markers c-terminal telopeptides of type I collagen(CTX-I)and deoxypyridinoline(DPD).For urine metabolomics study,a total of 27 differential metabolites were detected in DEX group vs.the control group,of which 15 were reversed significantly after MO treatment,and these metabolites wereinvolved in arachidonic acid(AA)metabolism,retrograde endocannabinoid signaling and amino acid metabolism.10 differential metabolites were reversed significantly after RR treatment,and these metabolites mainly participated in steroid hormone biosynthesis and amino acid metabolism.By metabolic pathway analysis,it was further ascertained that MO mainly regulated AA metabolism,while RR majorly intervened steroid hormone biosynthesis.2.Effects of MO and RR on DEX-injured osteoblasts and its metabolomics studyPrimary osteoblasts were prepared from the calvarias of neonate rats and were injured with DEX.The cell proliferation,ALP activity,extracellular matrix mineralization,osteoblast metabolic profiles and metabolic pathways were assayed to evaluate the effects of MO and RR on DEX-injured osteoblasts.As a result,MO,monotropein(Mon),rubiadin-1-methyl ether(Rub),as well as RR,catalpol(Cat)and acteoside(Act)all significantly improved the cell proliferation,differentiation and bone mineralization levels of DEX-injured osteoblasts,and promoted bone formation.For osteoblast metabolomics study,a total of 23 differential metabolites were detected in DEX group vs.the control group.After drugs treatment,MO further induced 7 metabolites changes compared with DEX group,and they were mainly involved in AA metabolism and D-Glutamine and D-glutamate metabolism.RR further induced changes in 11 differential metabolites,which were mainly involved in amino acids metabolism and biosynthesis.3.Effects of MO,RR and their chemical constituents on key proteins expression of metabolic pathwayDEX-injured osteoblasts were used to perform Western blotting analysis,and to verify the regulatory effects of MO,RR and their chemical constituents on key proteins expression of metabolic pathway.As a result,compared with DEX group,MO significantly increased the expression of 5-lipoxygenase(5-LOX)and leukotriene A4 hydrolase(LTA4H),and inhibited the expression of cyclooxygenase 2(Cox-2)and prostaglandin E2 receptor type 3(EP3)of AA metabolism.RR significantly up-regulated the aromatase(CYP19A1)and cytochrome P450 17A1(CYP17A1),and down-regulated the11?-hydroxysteroid dehydrogenase(HSD11B1)of steroid hormone biosynthesis.These results further suggested that MO could prevent bone loss by regulating AA metabolism,while RR could maintain bone homeostasis via affecting steroid hormone biosynthesis.