Aucubin improved bone recovery and reduced bone resorption in Col10α1: mGFP/ Rankl:HSE:CFP medaka. A Col10α1: nlGFP expression in 14 dpf transgenic medaka larvae after heat-shock-induced RANKL expression and aucubin (25 and 50 μM) treatments. B ALC staining of the mineralized bone matrix in 14 dpf Col10α1: mGFP/Rankl:HSE:CFP medaka, 5 days after heat-shock-induced RANKL expression and aucubin (25 and 50 μM) treatments. C A merge of A and B. D Fixed bone staining with Alizarin Red in 14 dpf Col10α1: mGFP/Rankl:HSE:CFP medaka larvae after RANKL induction and aucubin (25 and 50 μM) treatments

Aucubin reduced VRI-induced vascular insufficiency in zebrafish. A 24 hpf Tg(fli-1a:EGFP)^y1 zebrafish embryos were pre-treated with 500 ng/mL VRI for 3 h, rinsed, and immersed in various concentrations of aucubin (5–20 µM) for 24 h. The expression of EGFP in ISVs of zebrafish embryos. B Quantitative analysis showing the concentration-dependent effects of aucubin on the recovery of ISVs. Data are presented as means ± SD for three independent trials. ^##p < 0.01 versus the control group; **p < 0.01 versus the group treated with VRI alone

Effects of aucubin on VRI-induced down regulation of several pro-angiogenic factors mRNA expression in zebrafish. Total RNAs from different groups of zebrafish pre-treated with VRI (500 ng/mL) for 3 h, and then treated with different concentrations of Aucubin (5, 10 and 20 μM) after VRI withdrawal, were isolated and reverse transcribed to cDNA. The mRNA levels of flt-1 (A), kdr (B), kdrl (C), vegfaa (D), ang-1 (E), ang-2 (F), tie1 (G) and tie2 (H) were tested by RT-PCR. Data are expressed as the mean ± SD, ^##p < 0.01 versus the control group; *p < 0.05 and **p < 0.01 versus the group treated with VRI alone

Effects of aucubin on proliferation in HUVECs. HUVECs were treated with different concentrations of aucubin (6.25–100 µM). A Cell viability was measured by CCK8 assays. B The xCELLigence Real-time cell analysis (RTCA) system showed the data of cell index curves of HUVECs reflected their proliferation in real-time mode. The cell index after aucubin administration at 20 h (C) and 48 h (D) were analyzed. Data are presented as the percentage of the control group (mean ± SD of three or six independent trials). **p < 0.01 versus the control group

Effects of aucubin on the expression of proteins associated with the regulation of angiogenesis. HUVECs were starved in low serum media (0.5% FBS) for 3 h, then treated with VEGF (50 ng/mL, positive control) and different concentrations of aucubin (0–50 µM) for 2 h. A Several key proteins associated with signaling pathways of proliferation and survival in angiogenesis were determined by Western blotting. B–G Expression levels of p-Akt, p-MEK1/2, p-ERK1/2, p-mTOR, p-FAK and p-Src, respectively. Data are presented as the percentage of the control group (mean ± SD of three independent trials). *p < 0.05 and **p < 0.01 versus the control group

Aucubin promoted proliferation, migration and tube formation in su5416-injured HUVECs. A Cell viability was measured by CCK8 assays. B The xCELLigence RTCA system showed the data of cell index curves of HUVECs reflected their proliferation in real-time mode. C The healing area of the wound at the time points of 0 h and 12 h were observed microscopically and calculated by Image J software. Effects of aucubin on HUVECs migration was investigated by wound healing. D The migration rate was determined after treatment. E Effects of aucubin on tube formation in su5416-damaged HUVECs was determined. F Graph showing the quantitative analysis of tube length in the control group, su5416 group, and aucubin groups. Data are shown as mean ± SD of at least three independent experiments. ^##P < 0.01 vs. untreated control. *P < 0.05 and **P < 0.01 vs. su5416

Aucubin promoted angiogenesis by regulating the VEGFR2/MEK/ERK signaling pathway. A The protein expression levels of p-ERK1/2, p-MEK1/2, p-VEGFR2, Bax and Bcl-2 in su5416-injured HUVECs after aucubin treatment (5, 10, and 20 μM) for 48 h were investigated by western blot. B–D Quantitative analysis of protein expression ratios of p-VEGFR2/VEGFR2, p-MEK1/2/MEK1/2 and p-ERK1/2/ERK1/2, respectively. Quantitative analysis of protein levels of Bcl-2 (E), Bax (F) and Bcl-2/Bax expression ratio (G) in HUVECs. (H) Cells were treated with PD98059. MEK inhibitor reversed the protective actions of aucubin on cell viability. Data are shown as mean ± SD of three independent experiments. ^#P < 0.05 and ^##P < 0.01 vs. untreated control. *P < 0.05 and **P < 0.01 vs. su5416. ^$$P < 0.01 vs. su5416 + aucubin group

Scheme summarizing the dual effect of aucubin on promoting VEGFR2 mediated angiogenesis and reducing RANKL-induced bone resorption which may be beneficial to its treatment of osteoporosis