| Objective: To explore the best active components combination of herbal pair Achyranthis bidentatae and Eucommia ulmoides for preventing glucocorticoid-induced osteoporosis(GIOP)zebrafish model by orthogonal design.To explore the mechanism of β-Ecdysone(βEcd)and Pinoresinol diglucoside(PDG)combination relieves GIOP via observing oxidative stress signaling pathway related molecules expressions in excess glucocorticoids.To provide the basic experimental basis for the intervention of herbal pair Achyranthis Bidentatae and Eucommia ulmoides and their active components on GIOP.Methods: 1.5dpf WT zebrafishes were divided into control group,10μM,1μM,and 0.1μM Dexamethasone(Dex)groups.After 96 h of drug administration,the zebrafish juveniles were analyzed by alizarin red staining using Area and Sum Brightness parameters,and the optimal concentration of Dex was selected for GIOP model.2.Based on the model establishment,the alizarin red staining method was used to observe the protective effects of different concentrations(10,1,0.1 μM)of alendronate as positive control and the main active components of Achyranthes and Eucommia ulmoides on the GIOP zebrafish model.And the optimal concentrations were selected.3.βEcd(Achyranthes bidentata active ingredient)was as loading factor,and PDG、CGA、GA、AU(Eucommia ulmoides Oliv.active ingredient)were as influencing factors using orthogonal design test.Through alizarin red staining method and alkaline phosphatase assay,the best combination of Achyranthes bidentata and Eucommia ulmoides active ingredients was screened out.4.PDG and βEcd-PDG were used to intervene in the GIOP zebrafish model.Alkaline phosphatase activity and Western Blot were used to detect the expression levels of oxidative stress factors such as Ask1、JNK、Akt、FOXO3a,and apoptosis-related factors such as Bax and Bcl-2.Results: 1.10μM Dex could decrease the zebrafish’s Area and Sum Brightness significantly(P<0.01).2.10μM or 1μM βEcd,and 10μM or 1μM PDG、CGA、GA、AU could effectively improve the reduction of Area and Sum Brightness in the zebrafish spine caused by high dose of Dex(P<0.05).3.Through orthogonal design statistical method,the optimal combination of βEcd and PDG was obtained(P<0.05).4.The ALP assay results showed that Dex decreased the ALP activity in the GIOP zebrafish model(P<0.05).Compared with the model group,10,1 μM PDG increased the ALP activity(P < 0.05).In the GIOP zebrafish model with βEcd-PDG,βEcd-PDG increased the ALP activity(P<0.05),and the combination function was better than only prevented with PDG.5.The Western Blot results showed that 10μM Dex increased the expression of P-Ask1,JNK and decreased the expression of P-Akt-473,P-FOXO3 a,and P-JNK in the GIOP zebrafish model.The expression of Bax was significantly increased and the expression of anti-apoptotic factor Bcl-2 was significantly decreased(P<0.05).Compared with the model group,1μM PDG decreased the expression of JNK and increased P-Akt-473,P-FOXO3 a,P-JNK expressions(P<0.05);βEcd-PDG combination decreased the expression of P-Ask1,Bax and Caspase3,increased the expression of P-FOXO3 a,P-JNK(P<0.05).Conclusion: 1.The active components of Achyranthes and Eucommia ulmoides could increase the bone mass and bone density of the zebrafish GIOP model spine induced by high dose of Dex.2.Based on orthogonal design method,the βEcd-PDG combination(Achyranthes bidentata,Eucommia ulmoides active ingredients)was the most effective combination to relieve GIOP in zebrafish model.3.High dose of Dex decreased ALP activity,while PDG and βEcd-PDG combination increased the ALP activity.The effect of βEcd-PDG combination was better than PDG.4.High dose of Dex could activate the oxidative stress signaling pathway in GIOP zebrafish model.The βEcd-PDG combination promoted bone formation by inhibiting oxidative stress and inhibiting apoptosis,and the effects were better than PDG. |
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