Lycium barbarum polysaccharide regulates mitochondrial dynamics to improve H2O2-induced apoptosis of SH-SY5Y cells

doi:10.12307/2025.049

Chinese Journal of Tissue Engineering Research ›› 2025, Vol. 29 ›› Issue (13): 2736-2743.doi: 10.12307/2025.049

Lycium barbarum polysaccharide regulates mitochondrial dynamics to improve H2O2-induced apoptosis of SH-SY5Y cells

Wang Jiwei1, 2, Li Yanbing1, 2, Guo Minfang2, Meng Tao2, Yu Jingwen2, Liu Xiaoqin2, Mu Bingtao2, Jia Siwei1, 2, Ma Cungen1, 2, Yu Jiezhong1, 2, 3

1Research Center of Neurobiology/Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine, Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China; 2Institute of Brain Science, Shanxi Datong University, Datong 037009, Shanxi Province, China; 3Datong Fifth People’s Hospital, Datong 037009, Shanxi Province, China

Received:2023-12-05 Accepted:2024-03-14 Online:2025-05-08 Published:2024-09-11
Contact: Yu Jiezhong, Professor, Doctoral supervisor, Research Center of Neurobiology/Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine, Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China; Institute of Brain Science, Shanxi Datong University, Datong 037009, Shanxi Province, China; Datong Fifth People’s Hospital, Datong 037009, Shanxi Province, China; Co-corresponding author: Ma Cungen, Doctoral supervisor, Research Center of Neurobiology/Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine, Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China; Institute of Brain Science, Shanxi Datong University, Datong 037009, Shanxi Province, China
About author:Wang Jiwei, Master candidate, Research Center of Neurobiology/Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine, Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China; Institute of Brain Science, Shanxi Datong University, Datong 037009, Shanxi Province, China Li Yanbing, Master candidate, Research Center of Neurobiology/Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine, Shanxi University of Chinese Medicine, Jinzhong 030619, Shanxi Province, China; Institute of Brain Science, Shanxi Datong University, Datong 037009, Shanxi Province, China Wang Jiwei and Li Yanbing contributed equally to this article.
Supported by:
“Four Batches” Science and Technology Innovation Project of Shanxi Province in 2022, No. 2022XM33 (to YJZ); Shanxi Province Natural Science Research General Project No. 202303021211244 (to YJZ); Shanxi Provincial Administration of Traditional Chinese Medicine Scientific Research Project No.2023ZYYB2042 (to YJZ); Shanxi Provincial Key Laboratory of Traditional Chinese Medicine Construction project No. zyyyjs2024027 (to YJZ); Shanxi Basic Research Plan Project, No. 20210302123476, 20210302123478 (to GMF); Medical Science and Technology Leader Team of Shanxi Health Commission, No. 2020TD05 (to MCG); College Students Innovation Entrepreneurship Training Program of Shanxi Datong University, No. XDC2022174 (to GMF); Basic Research Project of Shanxi Datong University, No. 2022K17 (to YJW); Youth Scientific Research Project of Shanxi Basic Research Program, No. 20210302124276 (to LXQ)

Abstract

Abstract: BACKGROUND: A large number of studies have shown that neurodegenerative diseases are closely related to oxidative stress injury and the imbalance of mitochondrial dynamics. Lycium barbarum polysaccharides have a neuroprotective effect. However, it is not clear whether lycium barbarum polysaccharides can ameliorate apoptosis induced by oxidative stress injury by regulating abnormal mitochondrial dynamics.
OBJECTIVE: To study the effect of lycium barbarum polysaccharides on apoptosis induced by H2O2 in SH-SY5Y human neuroblastoma cells.
METHODS: SH-SY5Y cells were cultured in three groups. The control group was cultured for 24 hours. The hydrogen peroxide group was treated with H2O2 for 24 hours, and the lycium barbarum polysaccharide group was treated with lycium barbarum polysaccharide for 2 hours and then treated with H2O2 for 24 hours. After treatment, the levels of malondialdehyde, glutathione, and superoxide dismutase in the precipitation of the cells were detected by kit. Mitochondrial membrane potential was detected by JC-1 kit. Cell viability was detected by MTT assay. Apoptosis was detected by TUNEL. The expression levels of mitochondrial dynamics-related proteins (phosphorylated promoter protein 1, mitochondrial fission protein 1, mitochondrial fusion protein 1, mitochondrial fusion protein 2, and optic atrophy protein 1) and apoptotic proteins (Bax, Bcl-2, and Caspase-3) were detected by immunofluorescence staining and western blot assay.
RESULTS AND CONCLUSION: (1) Compared with the control group, the levels of malondialdehyde were increased (P < 0.05), and the levels of superoxide dismutase and glutathione were decreased (P < 0.05) in the H2O2 group. Compared with the H2O2 group, the malondialdehyde level was decreased (P < 0.05), and the superoxide dismutase and glutathione levels were increased (P < 0.05) in the lycium barbarum polysaccharide group. (2) The mitochondrial membrane potential in the H2O2 group was lower than that in the control group (P < 0.05), and that of lycium barbarum polysaccharide group was higher than that of the H2O2 group (P < 0.05). (3) Compared with the control group, the apoptosis rate and the expression of Bax and Caspase-3 protein were increased (P < 0.05), while the cell viability and the expression of Bcl-2 protein were decreased (P < 0.05) in the H2O2 group. Compared with the H2O2 group, the apoptosis rate and the expression of Bax and Caspase-3 protein were decreased (P < 0.05), while the cell viability and the expression of Bcl-2 protein were increased (P < 0.05) in the lycium barbarum polysaccharide group. (4) Compared with the control group, the protein expression levels of phosphorylated promoter protein 1 and mitochondrial fission protein 1 were increased (P < 0.05), and the protein expression levels of mitochondrial fusion protein 1, mitochondrial fusion protein 2, and optic atrophy protein 1 were decreased (P < 0.05) in the H2O2 group. Compared with the H2O2 group, the protein expression levels of phosphorylated promoter protein 1 and mitochondrial fission protein 1 were decreased (P < 0.05), and the protein expression levels of mitochondrial fusion protein 1, mitochondrial fusion protein 2, and optic atrophy protein 1 were increased (P < 0.05) in the lycium barbarum polysaccharide group. (5) These results indicate that lycium barbarum polysaccharide can improve SH-SY5Y cell apoptosis caused by oxidative stress damage by regulating mitochondrial dynamics.

Key words: 枸杞多糖, 氧化应激, 线粒体动力学, 神经退行性疾病, 细胞凋亡

CLC Number:

Wang Jiwei, Li Yanbing, Guo Minfang, Meng Tao, Yu Jingwen, Liu Xiaoqin, Mu Bingtao, Jia Siwei, Ma Cungen, Yu Jiezhong. Lycium barbarum polysaccharide regulates mitochondrial dynamics to improve H2O2-induced apoptosis of SH-SY5Y cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(13): 2736-2743.

Figures/Tables 2

2.1 枸杞多糖浓度和过氧化氢浓度筛选结果 MTT法检测结果显示，与对照组比较，0.5，1 g/L枸杞多糖处理后细胞活力提高(P < 0.05，P < 0.01)，1 g/L以上枸杞多糖处理后的细胞活力则降低(P < 0.001，P < 0.000 1)，见图1。因此，后续实验选择1 g/L枸杞多糖处理细胞。 MTT法检测结果显示，与对照组比较，200 μmol/L过氧化氢处理后细胞活力已降低到75%左右(P < 0.01)，200 μmol/L以上过氧化氢处理后细胞活力已降到70%以下(P < 0.01)，见图2，因此，后续实验选择250 μmol/L过氧化氢处理细胞。 2.2 枸杞多糖保护过氧化氢诱导的SH-SY5Y细胞活力 MTT检测结果显示，与对照组比较，过氧化氢组细胞活力降低(P < 0.000 1)；与过氧化氢组比较，枸杞多糖组细胞活力升高(P < 0.01)，见图3。 2.3 枸杞多糖对过氧化氢诱导SH-SY5Y细胞氧化应激指标的影响与对照组比较，过氧化氢组谷胱甘肽和超氧化物歧化酶水平减少(P < 0.001，P < 0.000 1)，丙二醛水平增加(P < 0.01)；与过氧化氢组比较，枸杞多糖组谷胱甘肽、超氧化物歧化酶水平增加(P < 0.001，P < 0.000 1)，丙二醛水平减少(P < 0.05)，见图4。 2.4 枸杞多糖保护过氧化氢诱导SH-SY5Y细胞线粒体膜电位水平观察免疫荧光染色结果显示，过氧化氢组绿色荧光最强而红色最弱，说明在细胞凋亡初期线粒体膜电位呈现减小的趋势；与过氧化氢组比较，枸杞多糖组红色荧光强度升高而绿色荧光强度却降低；定量分析结果显示，对照组线粒体膜电位大于过氧化氢组(P < 0.000 1)，枸杞多糖组线粒体膜电位大于过氧化氢组(P < 0.001)，见图5，说明枸杞多糖治疗后对SH-SY5Y细胞线粒体膜电位有保护作用。 2.5 枸杞多糖对过氧化氢诱导SH-SY5Y细胞凋亡的影响 TUNEL染色结果显示，与对照组比较，过氧化氢组细胞凋亡数量增加(P < 0.000 1)；与过氧化氢组比较，枸杞多糖组细胞凋亡数量减少(P < 0.000 1)，见图6，表明枸杞多糖能够有效地抵御由过氧化氢引发的细胞凋亡现象。 2.6 枸杞多糖对过氧化氢诱导SH-SY5Y细胞凋亡相关蛋白表达的影响 Western Blot检测结果显示，与对照组比较，过氧化氢组蛋白Bax和Caspase-3表达增加(P < 0.01，P < 0.000 1)，蛋白Bcl-2表达减少(P < 0.000 1)；与过氧化氢组相比，枸杞多糖组蛋白Bax和Caspase-3表达减少(P < 0.05，P < 0.001)，蛋白Bcl-2 表达增加(P < 0.01)，见图7。免疫荧光染色结果显示，与对照组比较，过氧化氢组Caspase-3 蛋白表达升高(P < 0.01)；与过氧化氢组比较，枸杞多糖组Caspase-3蛋白表达降低 (P < 0.05)，见图8，说明枸杞多糖能够改善过氧化氢诱导细胞凋亡蛋白的表达。 2.7 枸杞多糖对过氧化氢诱导SH-SY5Y细胞线粒体动力学相关蛋白表达的影响免疫荧光染色结果显示，与对照组比较，过氧化氢组分裂蛋白p-Drp1表达增加(P < 0.001)，融合蛋白Mfn1和OPA1表达减少(P < 0.000 1，P < 0.01)；与过氧化氢组比较，枸杞多糖组分裂蛋白p-Drp1表达减少 (P < 0.001)，Mfn1和OPA1表达增加(P < 0.001，P < 0.05)，见图9。 Western Blot检测结果显示，与对照组比较，过氧化氢组蛋白分裂蛋白p-Drp1和Fis1(P < 0.01，P < 0.001)，融合蛋白Mfn1、Mfn2和OPA1表达减少(P < 0.01，P < 0.001，P < 0.000 1)；与过氧化氢组比较，枸杞多糖组p-Drp1和Fis1表达减少(P < 0.01)，Mfn1、Mfn2和OPA1(P < 0.001，P < 0.01，P < 0.01)，见图10。通过免疫荧光染色和 Western Blot检测结果得出，枸杞多糖可以影响过氧化氢诱导细胞线粒体分裂和融合蛋白的表达。"

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Lycium barbarum polysaccharide regulates mitochondrial dynamics to improve H2O2-induced apoptosis of SH-SY5Y cells

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