益智仁提取物谷甾醇通过抑制铁死亡中的ETS-5基因表达延长秀丽隐杆线虫的寿命

doi:10.12122/j.issn.1673-4254.2025.08.19

南方医科大学学报 ›› 2025, Vol. 45 ›› Issue (8): 1751-1757.doi: 10.12122/j.issn.1673-4254.2025.08.19

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益智仁提取物谷甾醇通过抑制铁死亡中的ETS-5基因表达延长秀丽隐杆线虫的寿命

李军仪²(), 陈思源⁴, 谢力遥², 王劲³, 程奥², 张绍伟², 林继瑜³, 方志涵³, 潘一锐³, 崔翀鹤⁵, 陈庚鑫⁵, 张超³, 李栎¹()

^1.海南医科大学基础医学与生命科学学院热带转化医学教育部重点实验室//病原生物学教研室，海口 571199；南方医科大学2. 第一临床医学院
^5.第二临床医学院，广东广州 510000
^3.南方医科大学基础医学院生物化学与分子生物学系//广东省单细胞技术与应用重点实验室，广东广州 510000
^4.广东药科大学健康学院，广东广州 510006

收稿日期:2024-11-21 出版日期:2025-08-20 发布日期:2025-09-05
通讯作者: 李栎 E-mail:1449321559@qq.com;liliml@163.com
作者简介:李军仪，在读本博连读八年制，E-mail: 1449321559@qq.com
基金资助:
海南省重点研究发展基金(ZDYF2021SHFZ223);国家自然科学基金(82473569)

β-sitosterol, an important component in the fruits of Alpinia oxyphylla Miq., prolongs lifespan of Caenorhabditis elegans by suppressing the ferroptosis pathway

Junyi LI²(), Siyuan CHEN⁴, Liyao XIE², Jin WANG³, Ao CHENG², Shaowei ZHANG², Jiyu LIN³, Zhihan FANG³, Yirui PAN³, Chonghe CUI⁵, Gengxin CHEN⁵, Chao ZHANG³, Li LI¹()

^1.Department of Pathogenic Biology & Key Laboratory of Tropical Translational Medicine of Ministry of Education, School of Basic Medicine and Life Sciences, Hainan Medical University. Haikou 571199, China
^2.First School of Clinical Medicine
^5.Second School of Clinical Medicine, Southern Medical University, Guangzhou 510000, China
^3.Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Southern Medical University; Guangdong Provincial Key Laboratory of Single Cell Technology and Application, Guangzhou 510000, China
^4.School of Health, Guangdong Pharmaceutical University, Guangzhou 510006, China

Received:2024-11-21 Online:2025-08-20 Published:2025-09-05
Contact: Li LI E-mail:1449321559@qq.com;liliml@163.com
Supported by:
National Natural Science Foundation of China(82473569)

摘要/Abstract

摘要：

目的阐明益智仁（AOF）提取物的抗衰老潜力，重点关注其对秀丽隐杆线虫（C. elegans）寿命的调控作用及ETS-5基因在铁死亡中的功能。方法用10 µg/mL的谷甾醇（BS）处理秀丽隐杆线虫，记录线虫的生存时间并计算平均寿命，同时检测体长、运动能力和生殖功能等生理指标。利用RNAi技术敲低ETS-5基因的表达，统计敲低后线虫的生存时间和平均寿命，并利用油红O染色、MDA水平和GSH/GSSG比值评估线虫体内的脂肪积累和脂质氧化还原稳态。通过qPCR检测铁死亡相关基因（FTN-1、GPX-1、AAT-9）的表达水平，通过酶活性分析检测谷甾醇处理和ETS-5 RNAi对AAT-9酶活性的影响，并在HUVEC细胞模型中验证BS对FEV核定位的影响。结果益智仁提取物BS延长线虫寿命（P<0.05）并促进脂质蓄积并降低脂质过氧化水平（P<0.05），对谷甾醇响应的关键基因ETS-5敲低也有相同的效果（P<0.05）；ETS-5 RNAi导致铁死亡抑制因子GPX-1、AAT-9及FTN-1表达上调（P<0.05），GSH/GSSG上调表达（P<0.05）。结论 BS通过影响ETS-5转录因子的表达进而影响铁死亡关键基因AAT-9酶的活性进而影响线虫的铁死亡，进而对线虫的衰老产生影响。

关键词: β-谷甾醇, 衰老, 铁死亡, ETS-5

Abstract:

Objective To elucidate the anti-aging effect of β-sitosterol (BS), an important component in the fruits of Alpinia oxyphylla Miq., in C. elegans and its regulatory effect on ETS-5 gene to modulate ferroptosis. Methods C. elegans treated with 10 µg/mL BS were monitored for survival time and changes in body length, motility, and reproductive function. The effect of ETS-5 gene knockdown on survival time of C. elegans was observed, and the changes in fat accumulation and lipid redox homeostasis in the transfected C. elegans were assessed using Oil Red O staining and by detecting MDA levels and the GSH/GSSG ratio. The mRNA expression levels of ferroptosis-related genes (FTN-1, GPX-1 and AAT-9) were detected using qPCR. The effects of BS treatment and ETS-5 knockdown on AAT-9 enzyme activity in C. elegans were examined. The effect of BS on nuclear localization of FEV (the human homolog of ETS-5) was validated in cultured human umbilical venous endothelial cells (HUVECs). Results Both BS treatment and ETS-5 knockdown significantly prolonged the lifespan, promoted lipid accumulation and reduced lipid peroxidation in C. elegans. ETS-5 knockdown resulted in upregulated expressions of the ferroptosis repressors GPX-1, AAT-9 and FTN-1 and increased the GSH/GSSG ratio in C. elegans. Conclusion BS inhibits ferroptosis in C. elegans by suppressing the expression of ETS-5 transcription factor and hence the activity of AAT-9 enzyme, a key gene for ferroptosis, which in turn prolongs the lifespan of C. elegans.

Key words: β-sitosterol, senescence, ferroptosis, ETS-5

李军仪, 陈思源, 谢力遥, 王劲, 程奥, 张绍伟, 林继瑜, 方志涵, 潘一锐, 崔翀鹤, 陈庚鑫, 张超, 李栎. 益智仁提取物谷甾醇通过抑制铁死亡中的ETS-5基因表达延长秀丽隐杆线虫的寿命[J]. 南方医科大学学报, 2025, 45(8): 1751-1757.

Junyi LI, Siyuan CHEN, Liyao XIE, Jin WANG, Ao CHENG, Shaowei ZHANG, Jiyu LIN, Zhihan FANG, Yirui PAN, Chonghe CUI, Gengxin CHEN, Chao ZHANG, Li LI. β-sitosterol, an important component in the fruits of Alpinia oxyphylla Miq., prolongs lifespan of Caenorhabditis elegans by suppressing the ferroptosis pathway[J]. Journal of Southern Medical University, 2025, 45(8): 1751-1757.

图/表 6

表1 mRNA引物序列

Tab.1 Primer sequences for qPCR in this study

Primer	Sequence
ETS-5 forward	GGGGAGAGCGGAAGAGTAAG
ETS-5 reverse	GATGACAGATCTCCGTTGGG
GPX-1-top	GTCACTTTCGGATTACAAAGGAAA
GPX-1-bot	GGGAAGGCAAGAACTTCGAGA
ACS-17-top	GTCGGAGAGAGTCAAGGCTG
ACS-17-bot	ACGTCGGACACATTCTTCCC
FTN-1-top	CGAGTGGGGAACTGTCCTTG
FTN-1-bot	TCATTGATCGAATGTACCTGCTCT
AAT-9-top	ACAAATGGCATGGCACTTGT
AAT-9-bot	CGACCCACATGAACGAGAAC
DAF-16-top	CGCCGCTACCATCTGACATCAC
DAF-16-bot	TCCGCCAAAAGAAGCCGAACG

图1 BS处理对秀丽隐杆线虫的衰老表型的影响

Fig. 1 Effects of BS treatments on senescence phenotype of C. elegans. A: There was no significant difference in survival between BS-treated group and the control group at 24 h. B: Survival curves showing significantly prolonged C. elegans survival in BS group. C: Comparison of mean lifespan between BS and control groups. D-F: BS treatment does not significantly affect the total reproductive population (D), body length (E) or head bobbing frequency (F) of C. elegans. *P<0.05 vs Control group.

图2 敲低ETS-5对秀丽隐杆线虫的衰老表型的影响

Fig.2 Effect of ETS-5 interference on senescence phenotype of C. elegans. A: qPCR showing that BS treatment significantly lowers the expression of ETS-5 mRNA. B, C: ETS-5 interference increases survival time of C. elegans. D: BS and si-ETS-5 both significantly inhibit senescence marker DAF-16 mRNA expression in C. elegans. **P<0.01, ***P<0.001 vs Control group and si-Control group.

图3 不同处理后线虫体内脂肪含量以及脂质过氧化水平

Fig.3 Effect of BS treatment and ETS-5 interfernece on body fat content and lipid peroxidation in C. elegans. A-C: Body fat content in C. elegans treated with BS and si-ETS-5. B, C: Quantitative analysis showing trhat both BS treatment si-ETS-5 decreases body fat content in C. elegans.D: Lipid peroxidation level is significantly decreased both in BS-treated C. elegans and those treated with si-ETS-5. **P<0.01, ***P<0.001 vs Control group and si-Control group.

图4 秀丽隐杆线虫寿命的延长与铁死亡相关蛋白有关

Fig.4 The extension of C. elegans lifespan is related to ferroptosis-associated proteins. A-F: Quantitative PCR results for mRNA expressions of AAT-9, FTN-1, and GPX-1 in C. elegans treated with BS and si-ETS-5. G: GSH/GSSG ratios in C. elegans treated with BS and si-ETS-5. *P<0.05, **P<0.01,***P<0.001 vs Control group and si-Control group.

图5 BS通过影响ETS-5的表达影响下游通路

Fig.5 BS inhibits ETS-5 expression to affect the downstream pathways. A: Assessment of AAT-9 enzyme activity in C. elegans treated with BS and si-ETS-5. B: FEV expression in BS-treated HUVECs. ** P<0.01, ***P<0.001 vs Control group and si-Control group.

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益智仁提取物谷甾醇通过抑制铁死亡中的ETS-5基因表达延长秀丽隐杆线虫的寿命

β-sitosterol, an important component in the fruits of Alpinia oxyphylla Miq., prolongs lifespan of Caenorhabditis elegans by suppressing the ferroptosis pathway

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