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

doi:10.12122/j.issn.1673-4254.2025.08.19

Abstract

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

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.

Figures/Tables 6

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

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.

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.

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.

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.

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.

References 25

[1]	Zhang Y, Wang XM, Li XK, et al. Sirtuin 2 deficiency aggravates ageing-induced vascular remodelling in humans and mice[J]. Eur Heart J, 2023, 44(29): 2746-59. doi：10.1093/eurheartj/ehad381
[2]	Campisi J, d’Adda di Fagagna F. Cellular senescence: when bad things happen to good cells[J]. Nat Rev Mol Cell Biol, 2007, 8(9): 729-40. doi：10.1038/nrm2233
[3]	Sun DY, Wu WB, Wu JJ, et al. Pro-ferroptotic signaling promotes arterial aging via vascular smooth muscle cell senescence[J]. Nat Commun, 2024, 15(1): 1429. doi：10.1038/s41467-024-54088-2
[4]	Lin YG, Lin CX, Cao Y, et al. Caenorhabditis elegans as an in vivo model for the identification of natural antioxidants with anti-aging actions[J]. Biomed Pharmacother, 2023, 167: 115594. doi：10.1016/j.biopha.2023.115594
[5]	Nihira NT, Kudo R, Ohta T. Inflammation and tumor immune escape in response to DNA damage[J]. Semin Cancer Biol, 2025, 110: 36-45. doi：10.1016/j.semcancer.2025.02.005
[6]	Jiménez-Loygorri JI, Villarejo-Zori B, Viedma-Poyatos Á, et al. Mitophagy curtails cytosolic mtDNA-dependent activation of cGAS/STING inflammation during aging[J]. Nat Commun, 2024, 15(1): 830. doi：10.1038/s41467-024-46010-7
[7]	徐东川, 杨宗统, 苏敏. 何首乌的现代药理作用及机制研究进展[J]. 西安文理学院学报: 自然科学版, 2022, 25(2): 79-83.
[8]	颜子杰, 张琳, 韩鑫瑶, 等. 益智仁-防风通过PI3K/Akt/mTOR信号通路调控NLRP3炎性小体改善糖尿病肾病小鼠炎症反应[J]. 中国中药杂志, 2025, 50(10): 2798-809. doi：10.19540/j.cnki.cjcmm.20250214.502
[9]	Zhu JH, Jia YM, Wang C, et al. Lonicera japonica polysaccharides improve longevity and fitness of Caenorhabditis elegans by activating DAF-16[J]. Int J Biol Macromol, 2023, 229: 81-91. doi：10.1016/j.ijbiomac.2022.12.289
[10]	Ru Q, Li YS, Chen L, et al. Iron homeostasis and ferroptosis in human diseases: mechanisms and therapeutic prospects[J]. Signal Transduct Target Ther, 2024, 9(1): 271. doi：10.1038/s41392-024-01969-z
[11]	张凤奎. 缺铁性贫血的现代认识与治疗进展[J]. 临床血液学杂志, 2023, 36(11): 763-7. doi：10.13201/j.issn.1004-2806.2023.11.001
[12]	Koppula P, Zhuang L, Gan BY. Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy[J]. Protein Cell, 2021, 12(8): 599-620. doi：10.1007/s13238-020-00789-5
[13]	Zhao TT, Guo XJ, Sun Y. Iron accumulation and lipid peroxidation in the aging retina: implication of ferroptosis in age-related macular degeneration[J]. Aging Dis, 2021, 12(2): 529-51. doi：10.14336/ad.2020.0912
[14]	Fu HJ, Zhou XY, Qin DL, et al. Inhibition of ferroptosis delays aging and extends healthspan across multiple species[J]. Adv Sci, 2025, n/a(n/a): 2416559. doi：10.1002/advs.202416559
[15]	Lakowski B, Hekimi S. The genetics of caloric restriction in Caenorhabditis elegans [J]. Proc Natl Acad Sci USA, 1998, 95(22): 13091-6. doi：10.1073/pnas.95.22.13091
[16]	Juozaityte V, Pladevall-Morera D, Podolska A, et al. The ETS-5 transcription factor regulates activity states in Caenorhabditis elegans by controlling satiety[J]. Proc Natl Acad Sci USA, 2017, 114(9): E1651-8. doi：10.1073/pnas.1610673114
[17]	Watts JL, Ristow M. Lipid and carbohydrate metabolism in Caenorhabditis elegans [J]. Genetics, 2017, 207(2): 413-46.
[18]	Liang DG, Minikes AM, Jiang XJ. Ferroptosis at the intersection of lipid metabolism and cellular signaling[J]. Mol Cell, 2022, 82(12): 2215-27. doi：10.1016/j.molcel.2022.03.022
[19]	杨帆, 肖曼, 陈柏岑, 等. 益智仁提取物延缓秀丽隐杆线虫衰老的研究[J]. 中华中医药学刊, 2022, 40(2): 43-6, 265. doi：10.13193/j.issn.1673-7717.2022.02.010
[20]	Babu S, Jayaraman S. An update on β-sitosterol: a potential herbal nutraceutical for diabetic management[J]. Biomed Pharmacother, 2020, 131: 110702. doi：10.1016/j.biopha.2020.110702
[21]	Sajad M, Ali R, Kumar R, et al. β-Sitosterol ameliorates the cognitive deficits and neuropathological hallmarks in an Alzheimer’s disease model[J]. Arab J Chem, 2025, 18(1): 106072. doi：10.1016/j.arabjc.2024.106072
[22]	Maurer P, T’Sas F, Coutte L, et al. FEV acts as a transcriptional repressor through its DNA-binding ETS domain and alanine-rich domain[J]. Oncogene, 2003, 22(21): 3319-29. doi：10.1038/sj.onc.1206572
[23]	Liang YX, Liang YK, Zou ZH, et al. Tumor suppressor role and clinical significance of the FEV gene in prostate cancer[J]. Dis Markers, 2022, 2022: 8724035. doi：10.1155/2022/8724035
[24]	Zhang X, Yang LS, Liu JN, et al. FEV-mediated WNT2 transcription is involved in the progression of colorectal cancer via the Wnt signaling[J]. Cytotechnology, 2024, 76(6): 683-96. doi：10.1007/s10616-024-00643-0
[25]	Shiota M, Izumi H, Miyamoto N, et al. Ets regulates peroxiredoxin1 and 5 expressions through their interaction with the high-mobility group protein B1[J]. Cancer Sci, 2008, 99(10): 1950-9. doi：10.1111/j.1349-7006.2008.00912.x