| ObjetivesThe Modified Danggui Buxue Tablet(MDBT)is a TCM formula that made of Astragalus Radix(AR),Angelica sinensis Radix(ASR)and Epimedii folium(EF).In TCM theory it can "tonifying kidney and invigorating Yang,invigorating Qi and generating blood",so can be used for Postmenopausal osteoporosis(PMOP).PMOP is cause by the estrogen descend after menopause and the hormone replacement therapy(HRT)is the common therapeutic method to prevention and cure the PMOP.After the occurrence of osteoporosis,patients need to take drugs for a long time,which have certain curative effect,but the effect is not satisfactory.In TCM theory the osteoporosis can belong to the category of "Gu-wei",which caused by kidney deficiency and other organs disharmony.The holistic treatment to prevention and cure the PMOP is the main principle of TCM,which will tonifying kidney and spleen to balance Yin-yang and Zang-fu.By the early research we found that MDBT can promote MG-63 cell proliferation and differentiation,increase the influence of estrogen gene expression level in MCF-7 cell.Such result show that MDBT have the potential effect for the prevention and treatment of PMOP and other disease of postmenopausal,but the specific mechanism of action and the actual effect is uncertain.On the other hand,in recent years there are reports show that EF can cause liver injure.The MDBT content abundant of EF so the long-term safety evaluation must be pay attention.This study adopts the method of network pharmacology and animal phannacology,to predict and study the mechanisms of MDBT for the prevention and treatment of PMOP.On the other hand,we used the computer toxicology method and long-term toxicology experiment to evaluate the safety of MDBT.All the result can provide the basis for the clinical application of MDBT and the new drug development.Methods1.Network pharmacological study on MDBTCollect the components of AR,ASR and EF by searching TCMSP database and relevant literature.Set the Oral bioavailability(OB)?30%,Drug-likeness(DL)?0.18 and Caco?-0.4 as the pharmacokinetic screening indexes to screen the active components and predictive target(PT)of MDBT.Through OMIM,PharmGKB,DurgBank,TTD and CTD databases to collect the anti-osteoporosis target(AT).The intersection targets of PT and AT are the directly anti-osteoporosis target(PAT).Then using the STRING database to find out the indirectly anti-osteoporosis target(KT)of MDBT.The union set of PAT and KT is the core target(CT)for MDBT on the prevention and treatment of osteoporosis.Used the DAVID database to conduct the Gene Ontology(GO)enrichment and Pathway enrichment on CT to comprehensively the molecular mechanism and potential pharmacological activity of MDBT.2.Computational toxicological study on MDBTThe potential toxicity of MDBT was predicted and analyzed by software eMolTox and admetSAR.eMolTox can predict the hepatotoxicity and other organ toxicity of compound.The admetSAR is a ADME/T software can predict the interaction between the compound with the Cytochrome P450(CYP450),P-glycoprotein(P-gp)and Renal organic cation transporter(ROCT),which can forecast the toxic ingredient of MDBT.3.The effect of MDBT on the bone of naturally aged female ratsThe estrogen tablets(10.42 g/mL),Yougui pills(0.45g/mL)and the MDBT(0.22g/mL,0.11 g/mL,0.055g/mL)were gave to the 11 months old female rats to study the effect on the prevention and treatment of osteoporosis.After 4-months of administration,detect the BMD of body,lumbar vertebra,bilateral femoral neck,bilateral femur,the trabecular area,trabecular area ratio and trabecular number of lumbar vertebrae.After statistical analysis,the differences between groups were compared4.Long-term safety evaluation of MDBT in naturally aged female ratsThe 11 months old female rats were divided into 4 groups,which administrate distilled water.MDBT(0.88g/m L.0.44g/m L.0.11g/mL)for 6-months.After 6 months administration,drugs were stopped and administrate the same dose distilled water for 1-month.The signs,behavior and activity,daily food intake,water intake and defecation of rats were observed.After 3 and 6 months of administration and 1-month withdrawal,the levels of hematology,blood biochemical indexes,sex hormones and organ indexes were measured.After statistical analysis,the differences between groups were compared.Results1.Network pharmacological study on MDBTThe results showed that the MDBT contained 33 active ingredients and 235 related targets.53 core targets were identified.The important targets of regulating perimenopausal women hormone such as estrogen receptor and androgen receptor were coverd.Through Pathway enrichment analysis,it was found that MDBT could affect bone metabolism through direct and indirect pathways.In addition,the pathways with changes in hormone levels is enriched,which associate with the disease of CVDs,kinds of cancer and other perimenopausal syndrome.2.Computational toxicological study on MDBTMost of the active components of MDBT were predicted can be hepatotoxic.The most toxic enrichment organs was liver.Combined with the P-gp effect analysis,icarintin and 8-prenyl-flavone in EF were identified with high risk of hepatotoxicity.The prediction result indicated that the active compounds had no inhibitory effect on ROCT,suggesting that it had no nephrotoxicity.3.The effect of MDBT on the bone of naturally aged female ratsThe results showed that the BMD,trabecular area,trabecular area rate and number of aging female rats were improved to certain extent after 4-months of MDBT supplementation as compared with the blank group.The results showed that MDBT could alleviate osteoporosis in aging female rats to certain extent and alleviate the degeneration of bone trabecular structure,suggestive of a certain application prospect in the treatment of female perimenopteric osteoporosis.4.Long-term safety evaluation of MDBT in naturally aged female ratsThe results showed that the animals in each group,including the blank group,were generally in good condition,with no abnormal changes in appearance,physical signs,behavior,food intake,water intake,weight gain.etc.The hematological and biochemical examinations of the three dose groups and the control group were within the normal range.Histopathological examination of the main organs in each group showed no obvious abnormality.The above indicators did not change after 1-month withdrawal.The test results showed that MDBT of high,medium and low doses for 6-months had no obvious effect on senile female rats,after 1-month withdrawal no delayed toxicity reaction.The result showed that MDBT application was relatively safe.On the other side,the study also found that the MDBT could increase the testosterone,progesterone and estradiol levels,and reduce the levels of follicle stimulating hormone,luteinizing hormone and prolactin.ConclusionThe results of the network pharmacological study on MDBT showed that MDBT could affect bone formation through multiple targets and multiple biological pathways,providing an explanation for the application of MDBT in postmenopausal osteoporosis.On the other hand,this study also found that MDBT had potential application prospect in improving immunity,tumor diseases,cardiovascular and cerebrovascular diseases,etc.Through computer toxicology study,it was found that MDBT had potential toxicity or enrichment on liver and endocrine system,of which the higher risk ingredients of hepatotoxicity was from EF.Through long-term safety evaluation study,it was found that the clinical application dose of MDBT had no toxic effect on liver and kidney,suggesting that its clinical application had a wide margin of safety.As for the effects on endocrine system,it was found that MDBT could regulate E2,P,FSH,LH and other hormone levels in vivo,suggesting that MDBT might prevent and cure postmenopausal osteoporosis by regulating hormone levels and promoting bone formation. |
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