Année : 2025

In the Nature publication Signal Transduction and Targeted Therapy, researchers have described how glibenclamide, a drug used to treat type 2 diabetes, partially reverses epigenetic alterations and fights cellular senescence in mice.

A system tightly tied together

This paper begins with a discussion of the relationship between epigenetic alterations and cellular senescence. The histone H3K4me3 upregulates the senescence-related genes Cdkn1a, which is responsible for the biomarker p21 [1], and Cdkn2a, which is responsible for the biomarker p16 [2]. H3K27me3, on the other hand, downregulates these genes. Another histone, H3K9me3, suppresses repetitive genetic elements that cause an inflammatory response related to senescence [3].

While research has been done on directly targeting these histones [4], doing this with small molecules is difficult because they are structurally similar [3]. These researchers point to evidence suggesting that it may be more effective to target metabolism instead, as fundamental aspects of metabolism are linked to histone methylation [5].

These researchers had previously found that chlorpropamide provides a rejuvenation effect in C. elegans worms through a mitochondrial pathway [6]. They began this work to more definitively determine how and why this was happening, looking for a therapeutic target.

A metabolic target

This work began with a study on lung fibroblasts. Using a chemical probe based on chlorpropamide, the researchers looked at protein functions in order to determine what was being affected by this compound. They found MDH2 to be a potential target, as it had similar mitochondrial effects as chlorpropamide.

Further work in lung fibroblasts found that MDH2 was directly related to cellular senescence, whether it was induced by doxycycline or by excessive replication. The researchers created two cell lines, one with suppressed MDH2 and the other with overexpressed MDH2, to determine whether this relationship was causative in nature. They found that suppressing MDH2 reduced key senescence biomarkers, including SA-β-gal and p16, while overexpressing it increased them. The difference was not large, but it was statistically significant.

The researchers then tested how MDH2 interacts with five different sulfonylureas, a class of drugs that includes chlorpropamide. Of these drugs, the researchers found that glibenclamide has the strongest interaction with MDH2, far stronger than that of chlorpropamide.

In doxycycline-induced senescent lung fibroblasts, glibenclamide was found to reduce senescence biomarkers, including SA-β-gal, p16, and interleukins. Its overall effects in this area seemed to be roughly on par with those of metformin, another diabetes drug; it was not as good at reducing the inflammatory cytokine IL-6, but it reduced IL-1β in these cells, which metformin did not do. These beneficial effects were confirmed to be dependent on MDH2, as glibenclamide had no effects in cells with silenced MDH2.

Increases in histones and ROS

While the effects on lifespan and frailty were positive, glibenclamide increased, rather than decreased, mitochondrial reactive oxygen species (ROS) in lung fibroblasts. The researchers found that this was due to the inhibition of the TCA metabolic cycle, which forced the mitochondria to use oxygen-burning glycolysis instead [7]. The researchers hold that these effects on the TCA cycle are core to its beneficial effects, as they relate to the methionine cycle, which affects methylation.

On the first day of treatment in lung fibroblasts, glibenclamide immediately upregulated the senescence suppressor H3K27me3. Interestingly, when given for five days, glibenclamide upregulated both H3K4me3 and H3K27me3 in these cells while having no effect on H3K9me3.

Benefits for mice

The researchers also experimented on Black 6 mice. One group of 12-month-old mice was given glibenclamide, another was given NMN, and a third served as a control group. At 26 and 27 months of age, the glibenclamide group had significantly less frailty than either of the other groups. Mice given NMN appeared to live slightly longer on average, but the effect was not statistically significant; mice given glibenclamide, on the other hand, lived significantly longer.

Another experiment found that, while its physical benefits were not apparent at this age, glibenclamide reduces liver fibrosis and senescence in 20.5-month-old mice. In these animals, the drug was found to have significant effects on H3K27me3 but not H3K4me3.

While its anti-aging effects have not been tested in human beings, glibenclamide is a drug that is already being prescribed in the clinic. If these beneficial effects can be confirmed in human beings, this drug might be more widely prescribed to slow cellular senescence, particularly in the liver. The researchers also suggest that derivatives of this drug could be developed to more precisely target MDH2 to further slow cellular senescence.

Literature

[1] Yan, K., Ji, Q., Zhao, D., Li, M., Sun, X., Wang, Z., … & Liu, G. H. (2023). SGF29 nuclear condensates reinforce cellular aging. Cell Discovery, 9(1), 110.

[2] Kotake, Y., Zeng, Y., & Xiong, Y. (2009). DDB1-CUL4 and MLL1 mediate oncogene-induced p16 INK4a activation. Cancer research, 69(5), 1809-1814.

[3] Zhang, B., Long, Q., Wu, S., Xu, Q., Song, S., Han, L., … & Sun, Y. (2021). KDM4 orchestrates epigenomic remodeling of senescent cells and potentiates the senescence-associated secretory phenotype. Nature aging, 1(5), 454-472.

[4] Hsu, C. L., Lo, Y. C., & Kao, C. F. (2021). H3K4 methylation in aging and metabolism. Epigenomes, 5(2), 14.

[5] Salminen, A., Kauppinen, A., Hiltunen, M., & Kaarniranta, K. (2014). Krebs cycle intermediates regulate DNA and histone methylation: epigenetic impact on the aging process. Ageing research reviews, 16, 45-65.

[6] Mao, Z., Liu, W., Huang, Y., Sun, T., Bao, K., Feng, J., … & Li, J. (2022). Anti-aging effects of chlorpropamide depend on mitochondrial complex-II and the production of mitochondrial reactive oxygen species. Acta Pharmaceutica Sinica B, 12(2), 665-677.

[7] Li, X., Yang, Y., Zhang, B., Lin, X., Fu, X., An, Y., … & Yu, T. (2022). Lactate metabolism in human health and disease. Signal transduction and targeted therapy, 7(1), 305.

In Molecular Therapy, a team of researchers has described how increasing the expression of a form of Klotho, a protein that has been frequently found to have rejuvenative effects, leads to longer lifespans in male mice.

Klotho has various forms

In their introduction, the researchers distinguish between the various forms of Klotho. The full mRNA sequence that generates Klotho creates two homologous effective sections (KL1 and KL2) and a membrane that is meant for transportation between cells: this full version is m-KL [1]. Once enzymes take away this membrane, however, it becomes p-KL, with each section being p-KL1 and p-KL2.

However, this full version interferes with the function of FGF23, a protein that manages the metabolism of minerals [2]. Artificially upregulating this protein, therefore, is not safe [3].

Another form of Klotho, secreted Klotho (s-KL), does not have this problem. s-KL has been found to have multiple anti-aging effects according to a review of 65 studies [4]. Of course, without the transmembrane protein, it is not fit for intercellular transportation. Therefore, the researchers used an adeno-associated virus (AAV) to deliver a gene therapy that upregulates Klotho in the mice in this experiment.

Improvements in lifespan

The researchers used a total of 96 mice of the Black 6 strain: a group that received the AAV at 6 months, a group that received the AAV at 12 months, and a control group that received an ineffective AAV at 6 months. Both male and female mice were included in this experiment.

The treatment had far different effects in males and females. In female mice, the treatment had similar effects at 6 months and 12 months, but the increase in s-KL was accompanied by serious health problems during the course of the experiment, including skin ulcers and bleeding from the anus. In male mice, the AAV upregulated s-KL much more than in female mice, and it was much more effective at 12 months than at 6 months. Despite having far more s-KL, the males did not experience any of the health problems that the females did; instead, they received significant improvements to their lifespan.

Improvements to tissues and performance

Interestingly, at the age of 24 months, females showed improvements on the rotarod balance test that males did not. Both sexes given the s-KL AAV at 12 months were able to hold onto a horizontal bar longer than their control groups. In a three-trial grip strength test, the AAV-treated males performed far better, while the females performed better during only the first trial. The males also had significant reductions in fibrosis.

Regeneration capability was tested by transplanting muscles from old mice into younger mice. The muscle fibers in the AAV-treated animals became much larger than those from the control group. Muscles derived from the animals treated at 6 months old grew a wider variety of fiber sizes than those derived from animals treated at 12 months. Proliferation markers, and markers relating to a muscle-related fate of stem cells, were increased in the muscles derived from the mice that had received the s-KL AAV.

The researchers also tested bone tissue, seeing significant improvements to bone structure in females treated at 6 months and non-significant improvements in males treated at 12 months. Curiously, while FGF23 was upregulated along with many other bone-related factors in male mice, it was downregulated in female mice. This may be beneficial for females, as age-related increases in FGF23 have been linked to osteoporosis [5].

While no behavior testing was done in this study, the researchers did examine the mice’s brains. They found that, in the treated animals, there were more functional neurons and a thicker cellular layer, and markers of cellular proliferation were increased in the hippocampus. An examination of differently expressed genes revealed that the treated animals had fewer age-related changes than the control group.

The researchers note that this is the first time an AAV for s-KL has demonstrated lifespan increases in wild-type mice; previous experiments used transgenic mice. They believe that further experiments should test mice with different genetic backgrounds, because the side effects they saw in this experiment may or may not be limited to the AAV’s effects on Black 6 mice. Further work may elucidate exactly why klotho treatment has such different effects on males and females.

Literature

[1] Chen, C. D., Tung, T. Y., Liang, J., Zeldich, E., Tucker Zhou, T. B., Turk, B. E., & Abraham, C. R. (2014). Identification of cleavage sites leading to the shed form of the anti-aging protein klotho. Biochemistry, 53(34), 5579-5587.

[2] Kurosu, H., Ogawa, Y., Miyoshi, M., Yamamoto, M., Nandi, A., Rosenblatt, K. P., … & Kuro-o, M. (2006). Regulation of fibroblast growth factor-23 signaling by klotho. Journal of Biological Chemistry, 281(10), 6120-6123.

[3] Roig-Soriano, J., Sánchez-de-Diego, C., Esandi-Jauregui, J., Verdés, S., Abraham, C. R., Bosch, A., … & Chillón, M. (2023). Differential toxicity profile of secreted and processed α-Klotho expression over mineral metabolism and bone microstructure. Scientific reports, 13(1), 4211.

[4] Abraham, C. R., & Li, A. (2022). Aging-suppressor Klotho: Prospects in diagnostics and therapeutics. Ageing Research Reviews, 82, 101766.

[5] Sirikul, W., Siri-Angkul, N., Chattipakorn, N., & Chattipakorn, S. C. (2022). Fibroblast growth factor 23 and osteoporosis: evidence from bench to bedside. International Journal of Molecular Sciences, 23(5), 2500.

The authors of a recent study describe Ginkgolide B, a compound with senotherapeutic potential that improved muscle health, metabolism, frailty, inflammation, and senescence metrics and increased lifespan in female mice [1].

From East Asia to the clinic

Ginkgolide B is a compound that can be extracted from Ginkgo biloba, an East Asian tree known as the maidenhair tree. Previous research indicates that Ginkgolide B may offer many health benefits, such as improvements in osteoporosis and muscle regeneration in aged mice [2-7].

Therefore, these researchers hold that Ginkgolide B’s good safety, tolerability, and pharmacokinetic profile in humans [8, 9] and promising beneficial effects seen in model organisms make it a good candidate for healthspan and lifespan studies.

Extending lifespan

The researchers tested Ginkgolide B’s impact on female mouse lifespan. They started Ginkgolide B administration at 20 months (equivalent to 70- to 80-year-old humans). Ginkgolide B significantly extended the median lifespan by 8.5% and “extended the mean maximal lifespans of the 10% and 20% longest-lived mice by approximately 55” days.

Additionally, the researchers observed a reduced incidence of tumors; however, even the Ginkgolide B-treated mice with tumors still had longer lives, suggesting that Ginkgolide B extends lifespan not only by reducing tumors but through its beneficial impact on multiple organs and molecular processes.

Strengthening muscle

Apart from increasing lifespan, an increase in healthspan was also observed.

First, the researchers tested the impact of Ginkgolide B on muscle mass and strength. Ginkgolide B treatment improved female mice’s muscle strength, exercise capacity, and balance. It also reversed aging-related muscle wasting symptoms, such as a decreased skeletal muscle-to-body ratio, alterations to protein content in muscle, and muscle atrophy markers in aged skeletal muscle. Ginkgolide B also led to enlargement in the thigh’s main (femoral) artery and capillary density, which allowed for increased accessibility of oxygen and nutrients in muscles. However, it didn’t improve fatigue resistance or muscle recovery rate.

On the molecular level, Ginkgolide B treatment reversed several age-related changes associated with declining physical performance and muscle contraction; for example, it reduced aging-related increases in intramuscular lipid infiltration and collagen deposition.

Since the researchers focused on female mice, they also investigated sex hormones’ role in age-dependent muscle functioning. They surgically removed the mice’s ovaries, resulting in estrogen-deficient mice. Loss of estrogen led to muscle deterioration and decreased physical performance. Ginkgolide B treatment restored those functions in a dose-dependent manner, with high doses of Ginkgolide B almost completely restoring measured muscle functions.

Improved aging markers

Apart from declining muscular health, aging results in changes to metabolism, increased frailty, the chronic, low-grade inflammation known as inflammaging, and declining organ health. Ginkgolide B treatment helped alleviate those symptoms, such as by reducing the frailty index by 64.8% and benefiting the heart, kidney, spleen, and liver.

After two months of Ginkgolide B treatment, the body composition of aged mice resembled that of young mice. Similarly, it reversed disruption in biochemical measurements, such as serum triglyceride and total cholesterol levels, in aged mice and improved glucose tolerance and disruptions in glucose metabolism-related genes in skeletal muscle and liver.

Treatment with Ginkgolide B also positively impacted the inflammatory profile of aged mice, making it similar to that of young mice. The researchers also observed changes in the profiles of immune cells in aged mice, such as decreases in pro-inflammatory M1 macrophages and increases in anti-inflammatory M2 macrophages.

Inflammaging, among other aging-related processes, is linked to senescent cells. The researchers observed that Ginkgolide B treatment positively impacted the expression of several senescence-associated markers, such as the senescence-associated secretory phenotype (SASP) along with DNA damage, cell cycle, cell size, and cell proliferation in different organs and in cell culture models of induced senescence.

Molecular pathways

The authors of this study also examined aging-induced molecular changes by conducting multiple analyses of gene expression in the mouse leg muscle using either bulk expression data or expression data from single nuclei.

The results indicated that both aging and Ginkgolide B impacted gene expression. Ginkgolide B treatment had slight but measurable impacts in this area, partially reversing some of the changes that are brought about by normal aging in mice.

An analysis that focused on the hallmarks of aging showed that multiple genes related to these hallmarks are disrupted during normal aging. Ginkgolide B “partially restored intercellular communication, cellular senescence, nutrient sensing deregulation and mitochondrial dysfunction.”

Further gene expression analysis was performed separately for different subtypes of nuclei. The authors observed that one of the subtypes, called Runx1+ type 2B myonuclei, which appear in muscle cells, had the most significant alterations to gene expression. They refer to this subtype of cells as having a “host of age-related and GB-rescued signatures at the single-nucleus level.”

These myonuclei were enriched with apoptosis and ROS markers during aging, which were reversed by Ginkgolide B treatment. The authors hypothesize that the enrichment of apoptosis markers suggests that age-related apoptosis in Runx1+ type 2B myonuclei contributes to muscle degeneration.

The authors conducted further database searches and experiments to find a molecular pathway linking Ginkgolide B treatment and Runx1, a transcription factor that controls the expression of multiple genes. They identified miR-27b-3p, a microRNA whose levels are decreased in aged muscles and restored by Ginkgolide B treatment. Restoration of miR-27b-3p levels leads to reduced expression of Runx1.

Senotherapeutic potential

The researchers concluded that Ginkgolide B has a strong senotherapeutic potential, even when started late in life, and can help address aging-related conditions that current senotherapeutics fail to address, such as sarcopenia. However, the obtained results should be investigated in different mouse strains and eventually in humans to confirm their therapeutic value.

Literature

[1] Lee, C. W., Wang, B. Y., Wong, S. H., Chen, Y. F., Cao, Q., Hsiao, A. W., Fung, S. H., Chen, Y. F., Wu, H. H., Cheng, P. Y., Chou, Z. H., Lee, W. Y., Tsui, S. K. W., & Lee, O. K. (2025). Ginkgolide B increases healthspan and lifespan of female mice. Nature aging, 5(2), 237–258.

[2] Wu, T., Fang, X., Xu, J., Jiang, Y., Cao, F., & Zhao, L. (2020). Synergistic Effects of Ginkgolide B and Protocatechuic Acid on the Treatment of Parkinson’s Disease. Molecules (Basel, Switzerland), 25(17), 3976.

[3] Zhao, Y., Xiong, S., Liu, P., Liu, W., Wang, Q., Liu, Y., Tan, H., Chen, X., Shi, X., Wang, Q., & Chen, T. (2020). Polymeric Nanoparticles-Based Brain Delivery with Improved Therapeutic Efficacy of Ginkgolide B in Parkinson’s Disease. International journal of nanomedicine, 15, 10453–10467.

[4] Yao Y. (2020). Ginsenosides reduce body weight and ameliorate hepatic steatosis in high fat diet‑induced obese mice via endoplasmic reticulum stress and p‑STAT3/STAT3 signaling. Molecular medicine reports, 21(3), 1059–1070.

[5] Zhu, B., Xue, F., Zhang, C., & Li, G. (2019). Ginkgolide B promotes osteoblast differentiation via activation of canonical Wnt signalling and alleviates osteoporosis through a bone anabolic way. Journal of cellular and molecular medicine, 23(8), 5782–5793.

[6] Lee, C. W., Lin, H. C., Wang, B. Y., Wang, A. Y., Shin, R. L., Cheung, S. Y. L., & Lee, O. K. (2021). Ginkgolide B monotherapy reverses osteoporosis by regulating oxidative stress-mediated bone homeostasis. Free radical biology & medicine, 168, 234–246.

[7] Wang, B. Y., Chen, Y. F., Hsiao, A. W., Chen, W. J., Lee, C. W., & Lee, O. K. (2023). Ginkgolide B facilitates muscle regeneration via rejuvenating osteocalcin-mediated bone-to-muscle modulation in aged mice. Journal of cachexia, sarcopenia and muscle, 14(3), 1349–1364.

[8] Shen, C., Jin, X., Wu, M., Huang, X., Li, J., Huang, H., Li, F., Liu, J., Rong, G., & Song, S. (2020). A sensitive LC-MS/MS method to determine ginkgolide B in human plasma and urine: application in a pharmacokinetics and excretion study of healthy Chinese subjects. Xenobiotica; the fate of foreign compounds in biological systems, 50(3), 323–331.

[9] Shao, F., Zhang, H., Xie, L., Chen, J., Zhou, S., Zhang, J., Lv, J., Hao, W., Ma, Y., Liu, Y., Ou, N., & Xiao, W. (2017). Pharmacokinetics of ginkgolides A, B and K after single and multiple intravenous infusions and their interactions with midazolam in healthy Chinese male subjects. European journal of clinical pharmacology, 73(5), 537–546.