鞘氨醇单胞菌胞外多糖对油酸诱导的IR-HepG2细胞糖脂代谢紊乱的影响

Effects of extracellular polysaccharides from Sphingomonas echinoides on oleic acid-induced glucose and lipid metabolism disorders in IR-HepG2 cells

摘要:

目的 以鞘氨醇单胞菌(Sphingomonas echinoides) 1K04342为研究对象，提取、分离纯化其胞外多糖(extracellular polysaccharide, EPS)，并探究纯化后的多糖对油酸(oleic acid, OA)诱导人肝癌细胞(HepG2)糖脂代谢紊乱的改善作用及机制。方法 选取超声功率、超声时间和培养时间3个因素进行响应面试验设计，确定超声辅助法提取S. echinoides 1K04342胞外多糖的最佳工艺条件；对提取后的粗EPS进行脱蛋白处理，随后通过DEAE-琼脂糖凝胶FF (DEAE-Sepharose Fast Flow)阴离子柱分离，研究分离纯化后的EPS对HepG2细胞增殖活性、葡萄糖消耗率、α-葡萄糖苷酶和胰脂肪酶抑制率的影响，筛选出能减轻OA诱导胰岛素抵抗-HepG2 (insulin resistance-HepG2, IR-HepG2)细胞糖脂代谢紊乱的最佳活性组分，并对所得组分进行理化分析和进一步活性分析。结果 EPS的最佳提取工艺为：超声功率59.39 W；超声时间42.08 s，培养时间9.24 h，该条件下EPS的理论提取量为2.13 g/L。根据实际试验条件，将提取参数调整为超声功率60 W，超声时间42 s，培养时间9.2 h，在此条件下进行试验得到EPS的提取量为2.10 g/L，与理论提取量(2.13 g/L)差异不显著。经过活性筛选得到降低IR-HepG2细胞糖脂代谢紊乱的最佳组分是EPS-2。同时，对EPS-2进行理化分析，EPS-2的总糖含量为74.3%，单糖组成包括岩藻糖、鼠李糖、阿拉伯糖、半乳糖、葡萄糖和甘露糖。EPS-2减轻了OA诱导的IR-HepG2细胞糖脂代谢紊乱。EPS-2能够逆转OA对HepG2细胞活力所产生的负面作用。EPS-2可能通过激活IR-HepG2中IRS-1/PI3K/AKT信号通路，上调下游GSK3β和FoxO1蛋白表达，下调PEPCK和G6Pase mRNA和蛋白表达，进而缓解糖代谢紊乱。EPS-2可能通过激活IR-HepG2中AMPK/ACC1/SREBP-1C信号通路降低甘油三酯(triacylglycerol, TG)、总胆固醇(total cholesterol, TC)和低密度脂蛋白(low-density lipoprotein, LDL)水平，提高高密度脂蛋白(high-density lipoprotein, HDL)水平，进而缓解OA诱导的脂质代谢紊乱。结论 S. echinoides 1K04342胞外多糖EPS-2在IR-HepG2细胞中能有效改善糖脂代谢紊乱。

Abstract:

Objective To extract and purify extracellular polysaccharides (EPS) from Sphingomonas echinoides 1K04342 and investigate the ameliorating effects and mechanisms of the purified EPS on glucose and lipid metabolism disorders induced by oleic acid (OA) in human hepatoma cells (HepG2).Methods A response surface methodology was employed to optimize the ultrasound power, ultrasound time, and incubation time for maximizing the EPS yield of S. echinoides 1K04342 from ultrasound-assisted extraction. The crude EPS underwent deproteinization and was further purified through a DEAE-Sepharose Fast Flow anion exchange column. The effects of the purified EPS on the proliferation, glucose uptake, and α-glucosidase inhibition and pancreatic lipase inhibition rates in HepG2 cells were evaluated. The most effective EPS component for mitigating OA-induced glucose and lipid metabolism disorders in IR-HepG2 cells was selected for in-depth physicochemical and bioactivity analyses.Results The optimal EPS extraction parameters were as follows: ultrasound power at 59.39 W, extraction for 42.08 s, and incubation for 9.24 h, under which the estimated yield of EPS reached 2.13 g/L. Under the adjusted parameters (60 W, 42 s, and 9.2 h), the EPS yield was 2.10 g/L, which was statistically consistent with the theoretical value (2.13 g/L). The optimal component for ameliorating glucose and lipid metabolism disorders in IR-HepG2 cells was EPS-2, which showed the total sugar content of 74.3%, with a monosaccharide profile comprising fucose, rhamnose, arabinose, galactose, glucose, and mannose. EPS-2 alleviated OA-induced glucose and lipid metabolic disorders in IR-HepG2 cells. EPS-2 reversed OA-induced decreases in cell viability. EPS-2 may activate the IRS-1/PI3K/AKT pathway, up-regulating the protein levels of GSK3β and FoxO1 and down-regulating the mRNA and protein levels of PEPCK and G6Pase, thereby mitigating glucose metabolism disorders. EPS-2 may regulate OA-induced lipid metabolism disorders by activating the AMPK/ACC1/SREBP-1C pathway, lowering the triacylglycerol (TG), total cholesterol (TC), and low-density lipoprotein (LDL) levels, and increasing the high-density lipoprotein (HDL) level.Conclusion EPS-2 derived from S. echinoides 1K04342 could effectively ameliorate glucose and lipid metabolism disorders in IR-HepG2 cells.