摘要
目的充分挖掘路路通活性聚糖,评价其生物学功能。方法采用水提醇沉法提取粗聚糖,并经大孔吸附树脂、DEAE-52纤维素阴离子交换层析及G-150凝胶过滤层析进行分离纯化;通过系列光谱、色谱与化学方法对其结构进行初步表征;采用体外自由基清除法评价了其抗氧化活性;通过在人肝星状细胞系(LX2)中测定细胞活性确定了其安全浓度;利用TGF-β诱导的LX2细胞活化模型,评价了其抗肝纤维化活性;利用阿霉素诱导的大鼠心肌细胞系H9c2(2-1)损伤模型,评估了其心肌细胞保护作用。结果从路路通中分离纯化得到一种中性低聚糖LfPs2-2,平均分子量为1.3×10^(3)Da。单糖组成分析显示,LfPs2-2由甘露糖、鼠李糖和葡萄糖以摩尔比30.55∶57.12∶11.93构成。甲基化分析表明,其主链主要由→4)-Manp-(1→、→6)-Glcp-(1→和→2,3,4)-Manp-(1→等残基连接而成,其中→2,3,4)-Manp-(1→为关键结构单元。形貌观察表明,LfPs2-2)呈片状聚集,表面具有褶皱与团簇颗粒。安全性评价显示,该低聚糖(≤100μg/mL)对细胞无显著毒性,对细胞增殖活性无明显抑制。在功能评价方面,LfPs2-2展现出中等强度的体外抗氧化活性:4.00 mg/mL浓度下,清除率为(45.54±4.66)%。更重要的是,LfPs2-2表现出显著的抗肝纤维化活性。在TGF-β诱导的LX-2细胞活化模型中,LfPs2-2能显著的下调肝纤维化关键标志物COL-1和α-SMA的表达水平(P<0.01)。机制研究表明,LfPs2-2通过靶向抑制胶原蛋白脯氨酰4-羟化酶1(C-P4H1)的表达,干扰了胶原蛋白的羟基化修饰与后续成熟过程。此外,LfPs2-2还能有效减轻阿霉素诱导的心肌细胞毒性。结论本研究从路路通中成功分离鉴定出一种具有高纯度与特定结构的低聚糖LfPs2-2,并证实其是一种很有潜力的天然活性物质,为路路通的高值化利用提供了科学依据,尤其在开发新型心肌细胞保护剂和抗肝纤维化药物方面展现出重要的应用前景。
Objective To isolate and purify active glycans from the syncarp(fruit cluster)of Liquidambar formosana(Lulutong),and to evaluate their biological activities.Methods Crude glycans were obtained by water extraction followed by alcohol precipitation.The extract was then purified using sequential chromatographic steps,including macroporous adsorption resin,DEAE-52 cellulose anion-exchange chromatography,and G-150 gel filtration chromatography.The primary structure of the purified glycan was characterized by combined spectral,chromatographic,and chemical analyses.Antioxidant capacity was assessed in vitro by free radical scavenging assays.A safe concentration range was established by measuring viability of the human hepatic stellate cell(HSC)line LX-2.Anti-fibrotic activity was examined in a TGF-β-induced LX-2 activation model.Cardioprotective effects were evaluated in a doxorubicin-induced injury model using the rat cardiomyocyte cell line H9c2(2-1).Results A neutral oligosaccharide,designated LfPs2-2,with an average molecular weight of 1.3×10^(3)Da was isolated.Monosaccharide composition analysis showed that LfPs2-2 is composed of mannose,rhamnose,and glucose in a molar ratio of 30.55∶57.12∶11.93.Methylation analysis indicated that the backbone is mainly formed by→4)-Man p-(1→,→6)-Glc p-(1→,and→2,3,4)-Man p-(1→linkages,with the→2,3,4)-Man p-(1→residue as a key structural motif.Morphological examination revealed that LfPs2-2 assembles into flaky aggregates with a wrinkled,clustered surface.In cytotoxicity assays,LfPs2-2(≤100μg/mL)showed no significant toxicity or inhibition of cell proliferation.Functionally,LfPs2-2 exhibited moderate in vitro antioxidant activity,reaching a scavenging rate of(45.54±4.66)%at 4.00 mg/mL.Notably,in a TGF-β-induced HSC activation model,LfPs2-2 produced strong anti-fibrotic effects,significantly downregulating COL-1 andα-SMA expression(P<0.01).Mechanistic studies indicated that LfPs2-2 exerted its anti-hepatic fibrotic activity,in part,by targeting the expression of collagen prolyl 4-hydroxylase 1(C-P4H1).This inhibition disrupted the hydroxylation modification and subsequent maturation of collagen.Additionally,LfPs2-2 alleviated doxorubicin-induced cardiomyocyte toxicity,indicating potential cardioprotective properties.Conclusion This study successfully isolated and characterized LfPs2-2,an oligosaccharide from Lulutong,confirming its potential as a bioactive natural compound.These findings provide a scientific foundation for the high-value utilization of Lulutong,particularly in the development of novel cardioprotective and anti-fibrotic therapeutics.
作者
李坤梅
史舸捷
张蓓
丁加锐
刘云
李颖燕
潘峰
Li Kunmei;Shi Gejie;Zhang Bei;Ding Jiarui;Liu Yun;Li Yingyan;Pan Feng(The Key Lab of Guizhou Provincial Department of Education for Medical Prevention and Treatment of Tumor,Zunyi Medical University,Zunyi Guizhou 563099,China;Life Sciences Institute,Zunyi Medical University,Zunyi Guizhou 563099,China;School of Basic Medical Science,Zunyi Medical University,Zunyi Guizhou 563099,China;Center of Forensic Expertise,The Affiliated hospital of Zunyi Medical University,Zunyi Guizhou 563003,China)
出处
《合肥医科大学学报》
2026年第2期107-120,共14页
Journal of Zunyi Medical University
基金
国家自然科学基金资助项目(NO:82260850)
贵州省基础研究项目[NO:黔科合基础-ZK(2023)一般524]
合肥医科大学大学生创新创业训练计划项目(NO:S202510661728)。
关键词
路路通低聚糖
结构表征
抗氧化
抗肝纤维化
心肌保护
Lulutong oligosaccharide
structural characterization
antioxidant activity
anti-hepatic fibrosis
cardioprotection
