Abstract：
The influence of SGLT-1 on perivascular preadipocytes (PVPACs) and vascular remodeling is not well understood. This study aimed to elucidate the role and mechanism of SGLT-1-mediated PVPACs bioactivity. PVPACs were cultured in vitro and applied ex vivo to the carotid arteries of mice using a lentivirus-based thermosensitive in situ gel (TISG). The groups were treated with Lv-SGLT1 (lentiviral vector, overexpression), Lv-siSGLT1 (RNA interference, knockdown), or specific signaling pathway inhibitors. Assays were conducted to assess changes in cell proliferation, apoptosis, glucose uptake, adipogenic differentiation, and vascular remodeling in the PVPACs. Protein expression was analyzed by Western blotting, immunocytochemistry, and/or immunohistochemistry. The methyl thiazolyl tetrazolium (MTT) assay and Hoechst 33342 staining indicated that SGLT-1 overexpression significantly promoted PVPACs proliferation and inhibited apoptosis in vitro. Conversely, SGLT-1 knockdown exerted the opposite effect. Oil Red O staining revealed that SGLT-1 overexpression facilitated adipogenic differentiation, while its inhibition mitigated these effects. 3H-labeled glucose uptake experiments demonstrated that SGLT-1 overexpression enhanced glucose uptake by PVPACs, whereas RNA interference-mediated SGLT-1 inhibition had no significant effect on glucose uptake. Moreover, RT-qPCR, Western blotting, and immunofluorescence analyses revealed that SGLT-1 overexpression upregulated FABP4 and VEGF-A levels and activated the Akt/mTOR/p70S6K signaling pathway, whereas SGLT-1 knockdown produced the opposite effects. In vivo studies corroborated these findings and indicated that SGLT-1 overexpression facilitated carotid artery remodeling. Our study demonstrates that SGLT-1 activation of the Akt/mTOR/p70S6K signaling pathway promotes PVPACs proliferation, adipogenesis, glucose uptake, glucolipid metabolism, and vascular remodeling.NEW & NOTEWORTHY SGLT-1 is expressed in PVPACs and can affect preadipocyte glucolipid metabolism and vascular remodeling. SGLT-1 promotes the biofunctions of PVPACs mediated by Akt/mTOR/p70S6K signaling pathway. Compared with caudal vein or intraperitoneal injection, the external application of lentivirus-based thermal gel around the carotid artery is an innovative attempt at vascular remodeling model, it may effectively avoid the transfection of lentiviral vector into the whole body of mice and the adverse effect on experimental results.
摘要：
SGLT-1对血管周围前脂肪细胞（PVPAC）和血管重塑的影响尚不清楚。本研究旨在阐明SGLT-1介导的PVPACs生物活性的作用和机制。在体外培养PVPAC,并使用基于慢病毒的热敏原位凝胶(TISG)将其离体应用于小鼠的颈动脉。各组用Lv-SGLT1(慢病毒载体，过表达)，Lv-siSGLT1(RNA干扰，击倒)，或特异性信号通路抑制剂。进行测定以评估细胞增殖的变化，凋亡，葡萄糖摄取，成脂分化，和PVPAC中的血管重塑。蛋白质印迹分析蛋白质表达，免疫细胞化学,和/或免疫组织化学。甲基噻唑基四唑（MTT）测定和Hoechst33342染色表明，SGLT-1过表达在体外显着促进PVPACs增殖并抑制凋亡。相反，SGLT-1敲低产生相反的效果。油红O染色显示SGLT-1过表达促进成脂分化,而它的抑制作用减轻了这些影响。3H标记的葡萄糖摄取实验表明，SGLT-1过表达增强了PVPAC的葡萄糖摄取，而RNA干扰介导的SGLT-1抑制对葡萄糖摄取没有显著影响。此外，RT-qPCR,西方印迹,和免疫荧光分析显示SGLT-1过表达上调FABP4和VEGF-A水平并激活Akt/mTOR/p70S6K信号通路,而SGLT-1敲低产生相反的效果。体内研究证实了这些发现，并表明SGLT-1过表达促进了颈动脉重塑。我们的研究表明，SGLT-1激活Akt/mTOR/p70S6K信号通路促进PVPACs增殖，脂肪生成,葡萄糖摄取，糖脂代谢，和血管重塑。
