Studies On Chemical Constituents And Mechanism Of Rehmanniae Radix Preparata For Diabetic Osteoporosis Based On Molecular Docking Strategy

Diabetes,one of the world's major fatal disease,is a metabolic disease characterized by high blood glucose levels,which is caused by the deficiency of insulin secretion or the dysfunction of insulin.Sustained high blood sugar and long-term metabolic disorder will cause the damage of various organs,especially the eyes,kidney,cardiovascular and nervous system,lead to many complications.As a major bone complication in patients with diabetes,Diabetic osteoporosis(DOP)has attracted more and more attention because of its high incidence,morbidity and disability.As a traditional Chinese medicine,Rehmannia glutinosa Libosch has been used for more than 2000 years for defervesce,promoting blood circulation,and treating Yin deficiency syndrome.Rehmanniae Radix Preparata(RR),prepared from the dry rhizome of Rehmannia glutinosa Libosch,has the function of nourishing yin and tonifying the kidney essence.A large number of clinical and pharmacological experiments show that RR has the effect of anti-tumor,anti stress,anti coagulation,and can reduce blood glucose,reduce bone loss,suggesting that RR can be used for the treatment of diabetes and osteoporosis.The paper studies the therapeutic effects of RR on diabetic osteoporotic rats,and definite the anti-DOP active ingredients of Rehmanniae Radix Preparata.The diabetic osteoporotic rats induced by high sugar and high fat diet combined with streptozotocin(STZ)was applied to evaluate the anti-DOP of RR.In additional,the molecular docking technique was used to predict the anti-DOP targets and the active ingredients of RR.In order to initially determine the chemical constituents for the anti-DOP activity of RR,the effect of active components on MC3T3-E1 osteoblasts damaged by high glucose was observed.1 In vivo anti-diabetic osteoporotic evaluation of Rehmanniae Radix PreparataThe anti-diabetic osteoporotic effects of Rehmanniae Radix Preparata were inverstigated in rats model induced by high sugar,high fat diet combined with injection of STZ.The blood glucose,index of serum and urine was determined by using assay kit.The BMD and bone histomorphology was measured by Micro CT analysis.Compared with control rats,the blood glucose and the trabecular bone gap of region area was significantly increased,and the number of trabecular bone decreased significantly,while the activity of alkaline phosphatase(ALP)and tartrate resistant acid phosphatase(TRAP),and the contents of osteocalcin(OCN)and DPD was remarkably elevated in the modern group.Rehmanniae Radix Preparata and its combination with metformin can significantly increased the activity of ALP and the contents of OCN in serum,and decreased remarkably DPD in urine,while had no effect on the content of TRAP in serum.Moreover,BMD,TMC,BVF was significantly increased by metformin,RR and its combination,and Tb.Sp was reduced obviously.These results suggest that RR might be used as a therapeutic agent for the treatment of diabetic osteoporosis.2 Prediction of the anti-DOP targets and active constituents by using a molecular docking StrategyThe DOP tartets protein database(BMP2?Carbonic anhydrases II?Cathepsin K??-catenin?ERK5?GSK-3??IGF-1R?JNK?PPAR-? and chemical components database of RR were built.Molecular docking technique was used to dock the chemical components with the targets protein.The results indicated that 44 ingredients can be docked with the targets protein,while iridoid glycosides and phenylethanoid glycosides shown strong bindin effect with GSK-3?,IGF-1R and PPAR-?.Western blot method was used to validate the binding properties of catalpol,acteoside and echinacoside with GSK-3?,IGF-1R and PPAR-?.The result showed that three constituents up-regulated the expression of IGF-1R,while down-regulated the expression of PPAR-? and GSK-3?.3 Investigation of effects and mechanism of active compounds from Rehmanniae Radix Preparata on osteoblast injuried with high glucoseSix compounds from Rehmanniae Radix Preparata were evaluated for the effect and mechanism of anti-DOP in MC3T3-E1 osteoblasts damaged by high glucose.Catalpol and acteoside can increase the expression of BMP2,Runx2 and Osteriex,thus increase the activity of osteoblast alkaline phosphatase and bone matrix mineralization.They can also stimulate the production of IGF,increase the expression of IGF-1R,and enhance the phosphorylation of PI3 K and the expression of mTOR.Catalpol and acteoside can inhibit the expression of GSK3b,increase the accumulation of b-catenin in the nucleus which can activate the transcription of target genes and regulate the proliferation and differentiation of osteoblasts.