Effect Of Erxian Decoction On Senile Osteoporosis And Its Network Pharmacology

Objective: Er-xian Decoction(EXD)is made up by the proportion of Epimedium brevicornu Maxim.Curculigo orchioides Gaertn.Morinda officinalis How.Cupressus funebris Endl.Angelica sinensis(Oliv.)Diels and Anemarrhena asphodeloides Bunge.At present,it is widely used in the treatment of osteoporosis.This work aims at identifying the effect of Er-xian Decoction on the microstructure and behavior of senile osteoporosis bone,and clarifying the protective effect of EXD on oxidative injury of osteoblasts,and analyzing the mechanism of EXD on senile osteoporosis via network pharmacology.Method: The effect of Er-xian Decoction on the serum biochemical indexes of senile osteoporosis rats was observed by aging osteoporosis model injected with D-galactose(D-Gal)injection;Measurement of bone mineral density and bone histomorphology were measured by Micro-CT;Using the Morris water maze test method to locate the navigation and the space exploration experiment,the difference of the swimming data among each group of rats was analyzed;Primary osteoblasts were isolated from the calvaria of neonate 24-hour Wistar rats.The effects of EXD extract and its active components on the proliferation of(MTT),active oxygen cluster(ROS)and apoptosis of oxidized osteoblasts were observed;Western blot method was used to observe the effect of EXD on osteoblast bone morphogenetic protein(Runx2)after oxidative injury;Cytoscape software and target database were applied to predict the target of anti-aging osteoporosis of EXD,and the main KEGG pathway was enriched.The Western blot method was utilized to verify the regulation of EXD on the key targets such as p-Akt,p-mTOR,Beclin1 and so on.Results:Compared with the Control group,the serum GSH-Px and ALP level decreased,the MDA,DPD and IL-6 level increased in the model group,the bone mineral density decreased significantly,the bone mass was lost seriously,the latency time of locating navigation was prolonged,and the space exploration ability was decreased in themodel group;Compared with the model group,the serum GSH-Px and ALP level raised,the MDA,DPD and IL-6 level declined in the experimental group,the bone resorption rate of the experimental group was declined,the bone mineral density of the femur was raised,the bone microstructure was significantly improved,the time of the navigation latent period was shortened,and the space exploration ability was significantly improved;The extract of EXD and active component of EXD(Ferulic acid,Curculigoside,Icarlin,Timosaponin B ?,Monotropein and Berberine)have significant protective effects on proliferation of oxidative-injured osteoblast model.At the same time,the level of ROS was decreased and the ability of anti-apoptosis and anti-oxidation of osteoblasts was increased.Moreover,the effective components of EXD(Ferulic acid,Curculigoside,Icarlin,Timosaponin B ?,Monotropein and Berberine)could increase the expression of Runx2 protein in oxidative-injured osteoblasts;The results of the network pharmacology show that the biological process involved in the anti-aging osteoporosis target of EXD is related to the oxidative stress pathway,and the results show that the drug effect of EXD can lower the expression of p-Akt,p-mTOR protein in the osteoblast and increase the protein expression of Beclin1.We can also draw a conclusion that EXD can reduce the phosphorylation of Akt and mTOR by inhibiting Akt/mTOR signaling pathway,and increase the autophagy of the cells and inhibit the oxidative damage of the osteoblast.Conclusions:(1)Er-xian Decoction can effectively inhibit the level of oxidative stress in senile osteoporosis rats,increase the bone density,and enhance the exercise and learning ability of the aging rats.(2)The effect of Er-xian Decoction and its effective components on the proliferation of oxidative damaged osteoblast,ROS level,anti-apoptotic ability and the protein expression of Runx2 were significantly improved.(3)The results of the pharmacological analysis of the network and the experimental verification demonstrate that the mechanism of the anti-aging osteoporosis of the Er-xian Decoction may be related to the regulation of the Akt/mTOR signaling pathway,promote the autophagy reaction of the cells,and improve the anti-oxidation and anti-apoptotic abilities of the osteoblast.