Study On The Prevention Of Postmenopausal Osteoporosis By Cornuside Through Antioxidant Stress

Objective:PMOP is a common bone metabolic disease in postmenopausal women.Iron accumulation is an independent risk factor of PMOP.At present,drug treatment of PMOP is not good enough with some drawbacks.Traditional Chinese medicine has some outstanding advantages such as natural,low toxicity and has great potential in the prevention of PMOP.However,the lack of studies on the mechanism of relevant traditional Chinese medicine ingredients limits the development of traditional Chinese medicine in the treatment of PMOP.Cornus is a traditional Chinese medicine for tonifying kidney and strengthening bone,which exists in the prescription for strengthening bone,and has great potential in the prevention of PMOP.The potential mechanisms in the prevention of PMOP were screened by network pharmacology so as to provide ideas for the treatment of PMOP with traditional Chinese medicine.Methods:(1)To screen the potential role of Cornus in the treatment of PMOP through network pharmacology.(2)Taking human osteoblast cell line h FOB1.19 as the object,CCK-8 assay was used to detect the cell activity after intervention with Cornuside to determine the subsequent experimental concentration and using FAC to establish the iron-overloaded osteoblast model.(3)Using ALP staining and alizarin red staining to detect the effect of Cornuside on the activity and mineralization of iron-overloaded osteoblast model.(4)Using Annexin V-FITC kit to detect the effect of Cornuside on apoptosis of iron-overloaded osteoblast model.(5)Using flow cytometry and fluorescence method to detect the effects of Cornuside on oxidative stress related markers of iron-overloaded osteoblast model.(6)Using Western blot assay to detect the effects of Cornuside on the expression of osteogenic associated proteins of iron-overloaded osteoblast model.Results:(1)A total of 30 effective targets of Cornus in the treatment of PMOP were screened out.Among them,PTGS2、PTGS1、PRKACA、PGR and ADRB2 could play an important role,and the regulation of lipolysis in adipocytes,the TNF,p53 and Calcium signaling pathway,involved in many biological functions such as cell differentiation apoptosis and antioxidant stress,may be their potential mechanism.(2)The result of CCK-8 assay showed that concentration between 0-20 μM of Cornuside had a positive effect on osteoblast activity and concentration more than 20 μM had a negative effect.Thus,we used 20μM as the experimental concentration in the following experiment.(3)The results of ALP staining and ARS staining showed that FAC could reduce the staining and Cornuside could improve the staining,suggesting that Cornuside can improve the activity and mineralization ability of iron-overloaded osteoblast model.(4)The results showed that FAC could increase apoptotic rate and Cornuside had an inhibiting effect,suggesting that Cornuside can reduce the apoptosis of iron-overloaded osteoblast model.(5)The results of flow cytometry and fluorescence method showed that FAC could increase the level of ROS and reduce the level of SOD,while Cornuside had an opposite effect,suggesting that Cornuside can alleviate the Oxidative stress level of iron-overloaded osteoblast model.(6)The results of Western blot assay showed that FAC could reduce the expression of osteogenic associated proteins and Cornuside could improve these expressions,suggesting that Cornuside could improve the osteogenic activity of iron-overloaded osteoblast model.Conclusions:Cornuside is a potential component of traditional Chinese medicine Cornus in the prevention of PMOP,which can promote the activity and mineralization ability,up-regulate the expression of osteoblast-related proteins and reduce the oxidative stress level and apoptosis of iron-overloaded osteoblast model.