D-半乳糖

是一种由六个碳和一个醛组成的单糖。

In Vitro

Cell（BMSCs，different concentrations of D-gal，incubated at 37℃for another 48 h）

Cell viability analysis was performed using the Cell Counting Kit-8 reagent. According to the instructions, cells were seeded in 96-well plates at a density of 5×10³ cells/well and incubated for 24 h. Then, the cells were treated with or without different concentrations of D-gal (Solarbio, Beijing, China) or NAD+ (Solarbio, Beijing, China) or knockdown Sirt1 by si-RNA and then incubated at 37℃for another 48 h. After that, the culture media were removed, then mixing CCK-8 and the culture media in a volume ratio of 1: 10 which was then added to each well and incubated at 37℃for 2h. The absorbance was measured at a wavelength of 450 nm using a microplate reader in order to assess cell viability.

来源文献：Wang J, Liu L, Ding Z, Luo Q, Ju Y, Song G. Exogenous NAD+ Postpones the D-Gal-Induced Senescence of Bone Marrow-Derived Mesenchymal Stem Cells via Sirt1 Signaling. Antioxidants (Basel). 2021 Feb 7;10(2):254. doi: 10.3390/antiox10020254. PMID: 33562281; PMCID: PMC7915830.

In Vitro 

Cell（Small Interfering RNA Transfection，BMSCs，10 g/L of D-gal for 48 h）

For the small interfering RNA (si-RNA) transfection experiments, BMSCs were seeded in 12-well plates at a density of 2×10⁴ cells/well. After inducing cell senescence by use of 10 g/L of D-gal for 48 h, according to the si-RNA instruction manual , diluted si-RNA, si-RNA negative control, and lipofectamine 3000 with a FBS-free medium at a ratio of 2.5 µL/100 µLwere added, respectively.

来源文献：Wang J, Liu L, Ding Z, Luo Q, Ju Y, Song G. Exogenous NAD+ Postpones the D-Gal-Induced Senescence of Bone Marrow-Derived Mesenchymal Stem Cells via Sirt1 Signaling. Antioxidants (Basel). 2021 Feb 7;10(2):254. doi: 10.3390/antiox10020254. PMID: 33562281; PMCID: PMC7915830.

In Vitro 

Cell（MHSC cells，200 mmol/L D-gal for 24 h）

MHSC cells were cultured in high-sugar medium supplemented with 10 % FBS in DMEM at 37℃ in a 5 % CO₂ incubator. The cells were divided into the NC, Dgal and experimental groups (Fabp1-siRNA, Acaa1b-siRNA, Hmgcs1-siRNA, Rac2-siRNA, and Map3k5-siRNA), and the sequences are shown in Supplementary Material Table S4. The cells were transfected with Lipofectamine 2000 reagent, and the Dgal group was induced with 200 mmol/L D-gal for 24 h. The experimental group

was transfected with siRNA and cultured for 48 h. Some of the cells were used to verify the effect of transfection after total RNA extraction, and the rest were photographed under a fluorescence microscope to detect the ROS levels.

来源文献：Zhang J, Zheng W, Zhou Z, An Y, Zheng H, Zhang Y, Wei Y, Zhang Q, Zheng J, Wang F. Aqueous Polygalae Radix extract (PRE) prolongs the lifespan of C. elegans and alleviates D-galactose-induced oxidative stress in the mouse liver and brain by modulating PPARγ/MAPK. J Ethnopharmacol. 2025 May 28;348:119878. doi: 10.1016/j.jep.2025.119878. Epub 2025 Apr 24. PMID: 40287113.

Cell（Sertoli cells）

Before the experiment was initiated, the cells were pretreated with a serum-free DMEM/F12 medium for 12 h to prepare them for the subsequent procedures. Various final concentrations of D-gal (5, 10, 20, 40, or 80 mg/mL) were added to the cells to simulate aging effects,along with 5, 10, 20, or 40 μM curcumin. The cells were then exposed to these substances for 48 h. Furthermore, to further investigate the cellular responses, the cells were preincubated with 50 µM chloroquine , and 10 µM compound C , for 1 h before the main treatments were applied.

文献来源：Xue C, Yan Z, Cheng W, Zhang D, Zhang R, Duan H, Zhang L, Ma X, Hu J, Kang J, Ma X. Curcumin ameliorates aging-induced blood-testis barrier disruption by regulating AMPK/mTOR mediated autophagy. PLoS One. 2025 Apr 24;20(4):e0321752. doi: 10.1371/journal.pone.0321752. PMID: 40273194; PMCID: PMC12021166.

In Vivo
Mouse（Male ICR mice,6 weeks of age, weighing 18–22 g,intraperitoneally injected,1 g/kg Dgal for 8 weeks）

After 1 week of adaptation, they were randomly divided into six groups: the normal

control group (NC), D-galactose group (Dgal), positive control group (VC) and low-, medium- and high-dose PRE groups (PRE-L, PRE-M, and PRE-H, respectively). Except for those in the NC group, all of the mice were intraperitoneally injected with 1 g/kg Dgal for 8 weeks and gavaged with normal saline or PRE (50, 100, or 200  mg/kg) at different times daily.

来源文献：Zhang J, Zheng W, Zhou Z, An Y, Zheng H, Zhang Y, Wei Y, Zhang Q, Zheng J, Wang F. Aqueous Polygalae Radix extract (PRE) prolongs the lifespan of C. elegans and alleviates D-galactose-induced oxidative stress in the mouse liver and brain by modulating PPARγ/MAPK. J Ethnopharmacol. 2025 May 28;348:119878. doi: 10.1016/j.jep.2025.119878. Epub 2025 Apr 24. PMID: 40287113.

Mice（male Kunming mice； 200 mg/kg/day；60 days ；subcutaneously injected）

The mice were randomly assigned to four groups: control, curcumin + D-gal treatment, rapamycin + D-gal treatment, and D-gal (purity ≥ 99.0%, Solarbio,

Beijing, China) treatment, with each group containing 10 individuals. The D-gal group was subcutaneously injected with 200 mg/kg/day D-gal for 60 days Concurrently, the control group administered an equal volume of saline solutions. The curcumin+D-gal group administered 200 mg/kg/day curcumin at 12 h after the daily D-gal injections. The RAPA+D-gal group administered 2 mg/kg RAPA at 12 h after the daily D-gal injections.

文献来源：Xue C, Yan Z, Cheng W, Zhang D, Zhang R, Duan H, Zhang L, Ma X, Hu J, Kang J, Ma X. Curcumin ameliorates aging-induced blood-testis barrier disruption by regulating AMPK/mTOR mediated autophagy. PLoS One. 2025 Apr 24;20(4):e0321752. doi: 10.1371/journal.pone.0321752. PMID: 40273194; PMCID: PMC12021166.

Zhang J,et al. J Ethnopharmacol. 2025 May 28;348:119878. doi: 10.1016/j.jep.2025.119878.

Zhang J,et al. J Ethnopharmacol. 2025 May 28;348:119878. doi: 10.1016/j.jep.2025.119878.