中国弯颈霉多糖抗氧化和抑制氧化应激活性研究

白明健; 周颖; 程昊; 边聪; 李名正; 李林; 张春晶

doi:10.13386/j.issn1002-0306.2023030199

中国弯颈霉多糖抗氧化和抑制氧化应激活性研究

Antioxidant and Oxidative Stress Inhibitory Activities of Tolypocladium sinense Polysaccharide

摘要

摘要: 目的：探究中国弯颈霉菌丝体多糖（Tolypocladium sinense polysaccharide，TSP）的体外抗氧化活性和抑制过氧化氢诱导小鼠胰岛MIN6细胞氧化应激导致的细胞凋亡。方法：采用热水浸提法提取中国弯颈霉菌丝体多糖，随后测定其超氧阴离子自由基（superoxide anion， \mathrmO_2^- ·）、羟自由基（hydroxy radical，·OH）、对1,1-二苯基苦基苯肼自由基（P-1,1-diphenylpicryl phenylhydrazine radical，DPPH·）清除能力；采用200 μmol/L过氧化氢（hydrogen peroxide，H₂O₂）诱导小鼠胰岛MIN6细胞氧化应激，给予高剂量和低剂量TSP（0.625、0.156 mg/mL）进行保护，MTT法测定MIN6细胞生存率；倒置显微镜观察细胞形态；采用试剂盒测定培养基中乳酸脱氢酶（lactate dehydrogenase，LDH）水平，细胞内超氧化物歧化酶（superoxide dismutase，SOD）活性和丙二醛（malondialdehyde，MDA）含量；流式细胞术检测细胞凋亡；Western Blot检测核因子E2相关因子2（nuclear factor E2-related factor2，Nrf-2）和磷酸化c-Jun氨基末端激酶（phosphorylated c-jun N-terminal kinase，pJNK）相对表达量。结果：TSP对于三种自由基具有显著的清除效果；细胞实验结果显示，与过氧化氢模型组相比TSP高低剂量给药组细胞生存率显著上升（P<0.01，P<0.05）；悬浮细胞数量极显著下降（P<0.01）；培养基中LDH极显著含量下降（P<0.01）；细胞凋亡率显著降低（P<0.01，P<0.05）；细胞内SOD水平显著增高（P<0.01，P<0.05），丙二醛含量极显著下降（P<0.01）；Western Blot结果显示相较于模型组，高剂量给药组Nrf-2表达量显著增高（P<0.05），高剂量和低剂量给药组pJNK表达量显著下降（P<0.01，P<0.05）。结论：TSP具有显著的抗氧化活性，能够通过调节Nrf-2和pJNK的表达来减少氧化应激导致的细胞凋亡。

Abstract: Objective: To explore the antioxidant activity of Tolypocladium sinense polysaccharide (TSP) in vitro and its inhibitory effect on apoptosis of mouse islet MIN6 cells which were suffered from oxidative stress-induced by hydrogen peroxide. Methods: The T. sinense polysaccharides was extracted by hot water extraction, and then the scavenging activities of superoxide anion radical ( \mathrmO_2^- ·), hydroxyl radical (·OH) and p-1,1-diphenylpicryl phenyl hydrazine radical (DPPH·) was determined. Oxidative stress of mouse islet MIN6 cells was induced by 200 μmol/L hydrogen peroxide (H₂O₂) and protected by high and low dose TSP (0.625, 0.156 mg/mL). The survival rate of MIN6 cells was determined by MTT method, and the cell morphology was observed by inverted microscope. The lactate dehydrogenase (LDH) level in culture medium, intracellular superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured by kit. Apoptosis was detected by flow cytometry. The relative expressions of nuclear factor E2 related factor 2 (Nrf-2) and phosphorylated c-Jun N-terminal kinase (pJNK) were detected by Western Blot. Results: TSP had significant scavenging effect on three kinds of free radicals, and the results of cell experiment showed that compared with the hydrogen peroxide model group, the cell survival rate of high and low dose TSP group increased significantly (P<0.01, P<0.05), the number of suspension cells decreased significantly (P<0.01), the content of LDH in culture medium decreased significantly (P<0.01), and the rate of apoptosis decreased significantly (P<0.01, P<0.05). The level of intracellular SOD increased (P<0.01, P<0.05) and the content of MDA decreased significantly (P<0.01). The results of Western Blot showed that the expression of Nrf-2 in the high dose group was significantly higher than that in the model group (P<0.05), while the expression of pJNK in the high dose group and low dose group was significantly decreased (P<0.01, P<0.05). Conclusion: TSP has significant antioxidant activity, and can regulate the expression of Nrf-2 and pJNK to reduce apoptosis induced by oxidative stress.