Ursolic Acid Modulates A7r5 Cell Proliferation Via SFRP4/Wnt/β-catenin/FoxO3a/p27 Signaling Pathway

Aim: This study investigated the role of tumor suppressor gene SFRP4 in the rat hyperlipidemia model and the inhibitory effects of ursolic acid(UA) in angiotensin II induced A7r5 cell proliferation. SFRP4/Wnt/β-catenin/Fox O3a/p27 signaling pathway was investigated as the potential mechanism in such effects of UA. Methods: The hyperlipidemia model in SD rats was established, and then immunohistochemistry was used to assess the expression level of SFRP4 in thoracic aorta wall. The A7r5 cell proliferation model was established with induction of 0.1 μM Angiotensin II(Ang II). To investigate the effect of UA, A7r5 cells were exposed to 0.1 μM Ang II for 24 hour, with or without UA pretreatment(5 μM,10 μM,20 μM)for 12 hour. Normal A7r5 cells were kept as control. After treatments, CCK-8 assay was performed to measure cell viability;Real-time PCR and western blotting were used to investigate the m RNA and protein expression levels of SFRP4, β-catenin, Fox O3 a and p27, respectively. Additionally, the intracellular location of β-catenin was observed with immunofluorescence. For the first time, RNA interference was applied to confirm the signaling pathway involved in A7r5 proliferation and the molecular target of UA. A7r5 cells were assigned into Ang II model group, model+si-SFRP4 group, model+WNT inhibitor XAV939(5 μM, 4 hour pretreatment) group, UA group(20 μM), UA+ si-SFRP4 group and UA+ Wnt activator Li Cl(500 μM, 4 hour pretreatment) group. The interference efficacy and target protein expression levels were assessed with western blotting. Statistics were performed with SPSS 17.0. Results: In the animal study, immunohistochemistry results demonstrated the presence of SFRP4 in rat aorta wall, and the expression level(as measured with optic density) in control group was significantly higher than that in model group(P<0.05). In the in vitro study, CCK-8 results indicated that 0.1 μM Ang II treatment for 24 hour significantly increased cell proliferation rate, while pretreatment with 5, 10 or 20 μM UA for 12 hour effectively inhibited the proliferation(P<0.05). Meanwhile, si-SFRP co-treatment further elevated the proliferation rate in both Ang II model group and UA group relative to their counterpart without si-SFRP(P<0.05), suggesting a negative modulation of Ang II induced proliferation by SFRP4, which also contributes to the anti-proliferation effects of UA. On the other hand, real time PCR and western blottingrevealed decreased expression levels the SFRP4, Fox O3 a and p27, increased phosphorylation of Fox O3 a and expression level of β-catenin(P<0.05). These facts,along with the increased nuclear expression of β-catenin observed with immunofluorescence, indicated the activation of Wnt/β-catenin following Ang II exposure.si-SFRP4 treatment silenced the expression of SFRP4 protein and increased the expression level of β-catenin(P<0.05), suggesting the negative modulation on β-catenin by SFRP4. Furthermore, Ang II model + XAV939 treatment decreased the expression ofβ-catenin and p-Fox O3 a protein while increased that of Fox O3 a and p27 relative to Ang II model group(P<0.01), suggesting that β-catenin was located on the upstream of Fox O3 a and P27. In summary, SFRP4/Wnt/β-catenin/Fox O3a/p27 signaling pathway plays an important role in A7r5 cell proliferation. Regarding to UA pretreatment, the m RNA and protein expression levels of SFRP4, Fox O3 a and p27 were remarkably increased, while nuclear β-catenin and p-Fox O3 a expression levels were effectively decreased relative to Ang II model group(P<0.05), suggesting UA’s inhibition of Wnt pathway and activation of downstream transcription factors. Following silence of SFRP4,the expression of β-catenin was significantly elevated(P<0.05), suggesting the involvement of SFRP4 upregulation in UA induced Wnt inhibition. Co-treatment of UA and Li Cl reversed UA’s modulation to downstream proteins Fox O3 a and p27, suggesting that UA’s effect on Fox O3a/p27 is mediated via SFRP4/β-catenin. Conclusions: SFRP4 was confirmed as a new drug target modulating A7r5 cell proliferation, which negatively regulates cell proliferation. UA modulates A7r5 cell proliferation via SFRP4/Wnt/β-catenin/Fox O3a/p27 signaling pathway.