摘要
目的探讨沙海蜇毒素对小鼠单核巨噬细胞白血病细胞RAW264.7的毒性作用及其机制。方法采用含10%胎牛血清、1%青-链霉素的高糖DMEM培养液,在37℃、5%CO_(2)和饱和湿度的培养箱中培养RAW264.7细胞,待细胞贴壁传代后将RAW264.7细胞分为对照组和沙海蜇触手提取物(TE)组。对照组细胞不进行额外处理;TE组细胞使用不同浓度(0~100μg/ml)的沙海蜇TE干预。采用CCK-8法检测2组细胞活力;流式细胞术检测对照组和TE组半抑制浓度(IC_(50))时的细胞凋亡情况;DCFH-DA荧光探针观察对照组和TE组IC_(50)时的细胞活性氧(ROS)水平;分别使用硫代巴比妥酸法、黄嘌呤氧化酶法及紫外吸收法检测对照组和TE组IC_(50)时的细胞丙二醛(MDA)水平及超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性变化;RT-qPCR法检测对照组和TE组IC_(50)时的细胞CXC基序趋化因子10(Cxcl10)、白细胞介素-6(IL-6)、肿瘤坏死因子(TNF-α)mRNA表达水平;Western blotting实验检测对照组和TE组IC_(50)时的细胞p38、p-p38蛋白表达水平。结果TE组细胞活力明显低于对照组,并呈现浓度依赖式下降;TE组细胞IC_(50)为21.9μg/ml,细胞凋亡率[(22.58±0.15)%]明显高于对照组[(4.62±0.05)%],差异有统计学意义(P<0.001)。IC_(50)的TE组细胞ROS、MDA水平、CAT活性明显高于对照组,SOD活性明显低于对照组,差异均有统计学意义(P<0.01或P<0.001)。与对照组比较,IC_(50)的TE组细胞Cxcl10 mRNA相对表达量上升(265.7±94.5)倍,IL-6 mRNA相对表达量上升(342.9±94.5)倍,TNF-αmRNA相对表达量上升(26.2±5.5)倍,差异均有统计学意义(均P<0.001)。与对照组比较,IC_(50)的TE组细胞p38蛋白磷酸化水平明显升高,差异有统计学意义(P<0.01)。结论沙海蜇毒素通过调控p38过度磷酸化诱导RAW264.7细胞氧化应激、上调促炎因子水平,进而导致细胞凋亡。
Objective:To investigate the toxic effects and mechanisms of Nemopilema nomurai toxin on mouse monocyte macrophage leukemia RAW264.7 cells.Methods:The RAW264.7 cells were cultured in high-glucose Dulbecco’s modified Eagle medium(DMEM)supplemented with 10%fetal bovine serum and 1%penicillin-streptomycin in an incubator at 37℃,5%CO 2,and saturated humidity.After cell adhesion and passaging,the RAW264.7 cells were divided into control group and tentacle extract(TE)group.The cells in the control group received no additional treatment,while the cells in the TE group were treated with nemopilema nomurai TE at concentrations(0–100μg/ml).Cell viability was assessed in both groups using the CCK-8 assay.Cell apoptosis at the half-maximal inhibitory concentration(IC_(50))was measured by flow cytometry in the control and TE groups.Intracellular reactive oxygen species(ROS)levels at IC_(50) were observed using the 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescent probe.Malondialdehyde(MDA)levels,superoxide dismutase(SOD)and catalase(CAT)activities at IC_(50) were detected by the thiobarbituric acid method,xanthine oxidase method,and ultraviolet absorption method,respectively.The mRNA expression levels of C-X-C motif chemokine 10(Cxcl10),interleukin-6(IL-6),and tumor necrosis factor(TNF-α)at IC_(50) were detected by RT-qPCR.The protein expression levels of p38 and phosphorylated p38(p-p38)at IC_(50) were analyzed by Western blotting.Results:The TE group exhibited significantly lower cell viability compared with the control group,with a concentration-dependent decrease.The IC_(50) of the TE group cells was 21.9μg/ml,and the apoptosis rate[(22.58±0.15)%]was significantly higher than that of the control group[(4.62±0.05)%],with a statistically significant difference(P<0.001).At IC_(50),the TE group showed significantly higher levels of ROS and MDA,elevated CAT activity,and reduced SOD activity compared with the control group,with statistically significant differences(P<0.01 or P<0.001).Compared with the control group,the TE group cells at IC_(50) showed significant increases in the relative mRNA expression levels of Cxcl10[(265.7±94.5)-fold],IL-6[(342.9±94.5)-fold],and TNF-α[(26.2±5.5)-fold],with statistically significant differences(all P<0.001).Compared with the control group,the TE group cells at IC_(50) exhibited significantly elevated p-p38 protein expression level,with a statistically significant difference(P<0.01).Conclusion:Nemopilema nomurai toxin induces RAW264.7 cell apoptosis by mediating p38 phosphorylation,which triggers oxidative stress and upregulates pro-inflammatory factor levels.
作者
耿晓宇
王增发
王明科
华晓雨
于菲菲
万君怡
王艺
曹珂芸
肖良
杨积顺
Geng Xiaoyu;Wang Zengfa;Wang Mingke;Hua Xiaoyu;Yu Fejfei;Wan Junyi;Wang Yi;Cao Keyun;Xiao Liang;Yang Jishun(Naval Medical Center,Naval Medical University,Shanghai 200052,China;Faculty of Naval Medicine,Naval Medical University,Shanghai 200433,China;College of Traditional Chinese Medicinal Materials,Jilin Agricultural University,Changchun 130118,China)
出处
《中华航海医学与高气压医学杂志》
2025年第7期685-691,共7页
Chinese Journal of Nautical Medicine and Hyperbaric Medicine
基金
海军特色医学中心卓越人才项目(21TPZYL01)
XX军事研究项目(CB2023A03)。
关键词
沙海蜇毒素
炎症
氧化应激
P38
细胞凋亡
Nemopilema nomurai toxin
Inflammation
Oxidative stress
p38
Apoptosis
