Study On The Pharmacodynamics Of Anti-Osteoporosis Effect Of Eucommia Cortex And The Network Pharmacology Analysis Of Its Mechanism

Osteoporosis(OP)is a systemic bone metabolism disease,mainly manifested as low bone mass and/or bone tissue microstructure damage,which easily leads to increased bone fragility and fractures.It is likely to occur in the middle-aged and elderly people.With the aging of the population,the incidence of OP is increasing year by year,which seriously affects the quality of life of patients,brings a heavy economic burden to society,as well as becomes a worldwide healthy problem that needs to be solved urgently.Due to the complex pathogenesis and numerous pathogenic factors of OP,including endocrine,mechanical load,nutrition and so on,modern medical treatments of OP mainly start from the perspective of inhibiting bone resorption and promoting bone formation.However,related studies have confirmed that although bone resorption inhibitors and bone formation-promoting drugs can prevent and treat OP,their side effects are serious and there are greater hidden dangers in the safety of clinical medication.According to the manifestation of OP,it belongs to the categories of "bone dysfunction","bone dryness" and "bone impediment" in traditional Chinese medicine(TCM).Its etiology and pathogenesis are mainly the deficiency of kidney essence and the loss of nutrition in the marrow sea,which leads to the loss of bones.Clinical practice has proved that TCM has a good effect in treating OP.Among them,the anti-osteoporosis effect of Chinese herbals for invigorating the kidney yang is extremely prominent.Therefore,this study combined literature researches and clinical practice,selecting the representative kidney-yang-invigorating Chinese herbal-Eucommia cortex as the research object.Through establishing castrated osteoporotic rat models and cell models,the efficacy of Eucommia Cortex on the treatment of OP was confirmed from the overall animal level and in vitro,and the MC3T3-E1 cells proliferation phenotype,which is anti-serum starvation,was screened by drug-containing serum of Eucommia Cortex.In addition,this study used network pharmacology technology,further analyzed and revealed the potential mechanism of the anti-osteoporotic function of Eucommia Cortex.Part one:Study on Animal Pharmacodynamics of Eucommia for OsteoporosisObjective:Establishing a castrated osteoporotic rat model,observing and confirming the efficacy of Eucommia cortex in treating osteoporosis.Methods:Forty-eight healthy male Wistar rats were randomly divided into:control group(C),model group(M),active control group(AC),Eucommia cortex low-dose group(ECL),Eucommia cortex middle-dose group(ECM),Eucommia cortex high-dose group(ECH),intragastric administration to 12 weeks after modeling.Using testing methods including biochemical,dual-energy X-ray absorptiometry,Micro-computed tomography as well as three-point bending test,serum calcium,phosphorus,alkaline phosphatase,bone mineral density,bone tissue morphology and bone biomechanics as evaluation indexes,counting and analyzing the experimental data to explore the efficacy of Eucommia cortex in the treatment of OP.Results:(1)Blood biochemical indexes:Compared with group C,serum ALP,Ca and P contents in group M were significantly decreased(P<0.05);Compared with group M,serum ALP,Ca and P contents in group AC,group ECM and group ECH were significantly increased,and serum Ca and P contents in group ECL were significantly increased(P<0.05).Compared with group AC,serum Ca content of group ECL,ECM and ECH was decreased(P<0.05),serum ALP content had no significant difference(P>0.05),while serum P content of group ECH was significantly increased(P<0.05).There was no significant difference in serum ALP,Ca and P contents between ECL,ECM and ECH groups(P>0.05).(2)Bone mineral density:Compared with group C,femoral BMD in group M was significantly decreased(P<0.05);Compared with group M,femoral BMD of group AC,ECM and ECH was significantly increased(P<0.05);Compared with group AC,BMD of femur in ECM and ECH groups was significantly increased(P<0.05);There was no significant difference in femoral BMD among ECL,ECM and ECH groups(P>0.05).(3)Bone morphological indexes:Compared with group C,BV/TV,Tb.Th and Tb.N of femur in group M were significantly decreased,while Tb.Sp and SMI were significantly increased(P<0.05).Compared with group M,BV/TV of femur in group AC and ECL was significantly increased,Tb.Th of femur in group ECL,ECM and ECH was significantly increased,Tb.N of femur of group AC and ECL was significantly increased,Tb.Sp of femur in group AC and ECL was significantly decreased,and SMI of group ECL was significantly decreased(P<0.05).Compared with group AC,BV/TV of femur of group ECM and ECH was significantly decreased,Tb.Th of femur in group ECL,ECM and ECH was significantly increased,Tb.N of femur of group ECL,ECM and ECH was significantly decreased,while Tb.Sp of femur in group ECL,ECM and ECH was significantly increased(P<0.05).There was no significant difference in BV/TV,Tb.Th,Tb.N,Tb.Sp and AMI between ECL,ECM and ECH groups(P>0.05).(4)Bone biomechanical indexes:Compared with group C,the maximum load of femur of rats in group M was significantly decreased,and the elastic modulus of femur was significantly increased(P<0.05);Compared with the group M,the maximum load of the femur in group ECH was significantly increased,the bending strength of femur in group ECL was significantly increased,and the elastic modulus of femur of the group ECM and ECH were significantly increased(P<0.05).Compared with group AC,the flexural strength of femur in group ECM and ECH was significantly increased,the elastic modulus of femur of group ECM was significantly increased(P<0.05).There was no significant difference in the maximum load,flexural strength and elastic modulus of femur among ECL,ECM and ECH groups(P>0.05).Conclusion:(1)Eucommia cortex has a good effect in the treatment of OP,which can effectively regulate bone metabolism indexes,improve BMD and bone tissue microstructure.Eucommia cortex can increase BMD and bone mass by increasing bone volume fraction,alkaline phosphatase,calcium and phosphorus content in serum,increasing the thickness and mmber of trabeculae bone,reducing the separation degree of bone trabeculae.(2)Eucommia cortex has a certain effect on bone biomechanics.It can improve bone strength by improving the maximum load,bending strength and elastic modulus.(3)Different doses of Eucommia have different regulatory effects on different indexes.Low dose of Eucommia cortex tends to improve bone microstructure,while medium and high doses of Eucommia cortex tend to improve BMD and bone biomechanics.Part two:Study on the cytopharmacodynamics of Eucommia Cortex in the treatment of osteoporosisObjective:To establish the MC3T3-E1 cells model in vitro,observing the state of cell proliferation after the intervention of drug-containing serum of Eucommia cortex,and exploring the effect of drug-containing serum of Eucommia cortex on OB cells.Method:The cell models in vitro were established by processing MC3T3-E1 cells with hypoxia and serum starvation,and the MC3T3-E1 cell models were divided into control group(C),blank serum group(SC),and Eucommia low-dose drug-containing serum group(S-ECL),Eucommia medium-dose drug-containing serum group(S-ECM),and Eucommia high-dose drag-containing serum group(S-ECH).Respectively,used different concentrations of blank serum and Eucommia drug-containing serum to intervene for 48h,using CCK-8 detecting methods,observed the cell proliferation of each group,and explored the effects of Eucommia cortex drug-containing serum on the OB cells model.Results:(1)Cell model results:The results of 24h,48h and 72h were basically the same:Under normoxia,the viability of cells in the 2000 and 5000 groups increased significantly with the increase of FBS concentration(P<0.05);compared with the group 2000,the cell viability of group 5000 was significantly increased under the condition of 0%,1%,2%,5%,and 10%FBS concentration(P<0.05).(2)Cell phenotype results:10%concentration of drug-containing serum:In the absence of FBS,compared with group C,the cell viability of group SC was significantly reduced(P<0.05).Compared with group SC,the cell viability of group S-ECM and S-ECH was significantly increased(P<0.05).The results of 20%and 40%concentration drug-containing serum of Eucommia were basically the same:Under the condition of absence of FBS,compared with group C,the cell viability of group SC was significantly reduced(P<0.05).Compared with group SC,the cell viability of group S-ECL,S-ECM and S-ECH was significantly increased(P<0.05).Conclusions:(1)Under certain conditions,hypoxia and serum starvation conditions can significantly reduce the viability of MC3T3-E1.Considering comprehensively,the optimal cell concentration for establishing a hypoxic cell model is 5*10^4 cells/mL,and the optimal intervening time is 48h.The optimal cell concentration for establishing a serum starved cell model is 5*10^4 cells/mL(5000 group),the best intervening time is 48h,and the best serum starvation concentration is 0%FBS.(2)In cell experiments in vitro,blank serum at 10%,20%and 40%concentration has a killing effect on MC3T3-E1 cells,which shows a certain concentration-dependent.The drug-containing serum of Eucommia cortex of low,medium and high dose groups all have proliferation-promoting effects on MC3T3-E1 cells lineage at 10%,20%and 40%concentration.Among them,the drug-containing serum of Eucommia cortex at a concentration of 20%had the best proliferation-promoting effect on MC3T3-E1 cells linage,when it intervenes for 48 hours.Part three:Study on the anti-osteoporotic mechanism of Eucommia Cortex via network pharmacology.Subjective:To screen the active ingredients of Eucommia Cortex in the treatment of OP by using network pharmacology methods,predicting its targets,and exploring its potential molecular mechanisms.Methods:Using the Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP)and the Traditional Chinese Medicine Information Database(TCMID)to collect the effective active ingredients and its targets of Eucommia Cortex.Collecting OP disease targets from GeneCards,DisGeNET,OMIM and other databases to construct protein-protein interaction network and"drug-active ingredient-target" network,and using DAVID database to analyze the relevant biological functions and signal pathways of key targets.Results:After the data collection,28 effective active ingredients of Eucommia cortex,213 target proteins,and 1725 OP disease-related target proteins were obtained.After target mapping,90 common target proteins of drugs and diseases were obtained,which ranked top after enrichment analysis.Six of the 20 pathways are closely related to OP,namely the AGE-RAGE signaling pathway,the tumor necrosis factor signaling pathway,the C-type lectin receptor signaling pathway,the interleukin 17 signaling pathway,the Toll-like receptor signaling pathway,and the NF-κB signaling pathway.Conclusions:Network pharmacology studies have shown that a variety of active ingredients in Eucommia Cortex can exert anti-osteoporotic effects,and can affect a variety of biological processes and multiple pathways through multiple targets;the enrichment results suggest that Eucommia Cortex may regulate AGE-RAGE,TNF,C-type lectin receptor,IL-17,TLR,NF-κB and other signaling pathways affect the functions of osteoblasts and osteoclasts,and ultimately achieve anti-osteoporosis effects.