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Aging, as an inevitable natural process in living organisms, has long been a focal point of scientific research. As organisms age, cells gradually enter a senescent state, and the accumulation of these senescent cells can negatively impact the normal functioning of tissues and organs, leading to various age-related diseases. FOX04 – DRI is beneficial for clearing senescent cells.
Overview of FOX04 - DRI
FOX04 - DRI is a synthetic peptide, with the full name fork head box O transcription factor 4 - D - Retro - Inverso. Its discovery stems from in-depth research into the regulatory mechanisms of senescent cells. During cellular senescence, multiple molecular mechanisms are involved, and the FOX04 transcription factor plays a key role in this process. FOX04 exhibits specific expression patterns and functional changes in senescent cells. By elucidating its mechanism of action, scientists designed and synthesized FOX04-DRI to specifically target senescent cells, aiming to address age-related issues.
Figure 1 Schematic illustration of the elimination of cigarette smoke-induced senescent lung fibroblasts using FOXO4 siRNA-loaded DNA nanoparticles.
Mechanism of FOX04-DRI in clearing senescent cells
Interference with FOX04-p53 interaction: In senescent cells, FOX04 interacts with the p53 protein. The p53 protein plays a central role in cellular stress responses, cell cycle regulation, and apoptosis. Under normal conditions, p53 promotes apoptosis in damaged or senescent cells by activating the expression of a series of apoptosis-related genes, thereby maintaining the health of the cell population. In senescent cells, FOX04 binds to p53, inhibiting its transport to the cell nucleus, preventing p53 from performing its pro-apoptotic function, resulting in the survival and accumulation of senescent cells. FOX04-DRI can specifically interfere with this interaction between FOX04 and p53, blocking their binding, allowing p53 to re-enter the cell nucleus, activate the apoptosis signaling pathway, and induce apoptosis in senescent cells, thereby achieving the clearance of senescent cells.
Regulation of intracellular signaling pathways: In addition to interfering with the FOX04-p53 interaction, FOX04-DRI may also influence the fate of senescent cells by regulating other intracellular signaling pathways. The PI3K/AKT signaling pathway plays a key regulatory role in various aspects of cell growth, proliferation, survival, and metabolism. Studies have shown that the activity of the PI3K/AKT signaling pathway is often altered in senescent cells, affecting their normal functions. FOX04-DRI may regulate the activity of the PI3K/AKT signaling pathway, altering the balance of signal transduction within senescent cells, thereby creating an unfavorable intracellular environment for their survival and inducing apoptosis. Regulation of this signaling pathway may also influence cellular metabolic states, further driving senescent cells toward apoptosis.
Influence on the Senescence-Associated Secretory Phenotype (SASP): Senescent cells secrete a series of cytokines, chemokines, and proteases, forming the senescence-associated secretory phenotype (SASP). SASP not only negatively affects surrounding normal cells, disrupting their normal functions, but also triggers chronic inflammatory responses, further accelerating the aging process of tissues and organs. FOX04-DRI may mitigate the adverse effects of senescent cells on the surrounding microenvironment by inhibiting the expression and secretion of SASP-related factors in senescent cells. In the study, it was found that after treating senescent cells with FOX04-DRI, the expression levels of SASP-related factors such as IL-1α, IL-1β, tumor necrosis factor α (TNFα), and MMP2 in senescent cells were significantly reduced. This indicates that FOX04-DRI can effectively regulate the SASP of senescent cells, improve the cellular microenvironment, and indirectly promote the clearance of senescent cells and the recovery of the tissue microenvironment.
The Role of FOX04-DRI in Different Tissues and Diseases
Role in the Reproductive System: Male late-onset hypogonadism is an age-related disease whose core mechanism is dysfunction of aged testicular interstitial cells. FOX04 is specifically expressed in human testicular interstitial cells, and in the elderly, the nuclear translocation of FOX04 is associated with reduced testosterone synthesis. Using hydrogen peroxide-induced aged TM3 testicular interstitial cells as an in vitro model, it was observed that FOX04 can maintain the viability of aged testicular interstitial cells and inhibit their apoptosis. FOX04-DRI selectively induces p53 nuclear export and apoptosis of aged testicular interstitial cells by disrupting the FOX04-p53 interaction. In a natural aging mouse model, administration of FOX04-DRI improved the testicular microenvironment and alleviated age-related testosterone secretion deficiency. Therefore, FOX04-DRI may be beneficial for the treatment of male late-onset hypogonadism.
Figure 2 Expression of FOXO4 in human testes detected by immunofluorescent staining.
Role in the respiratory system
Pulmonary fibrosis: Pulmonary fibrosis (PF) is a progressive interstitial lung disease. Its incidence and prevalence increase with age, and cellular senescence is considered one of the key driving factors in its pathogenesis. In a bleomycin (BLM)-induced PF mouse model, FOX04-DRI demonstrated effects similar to those of the approved drug pirfenidone. It reduces the number of senescent cells, downregulates the expression of the senescence-associated secretory phenotype (SASP), and alleviates BLM-induced morphological changes and collagen deposition in lung tissue. Additionally, FOX04-DRI increases the proportion of type 2 alveolar epithelial cells (AEC2) and fibroblasts while reducing the number of myofibroblasts. In vitro experiments showed that compared to mouse and human lung fibroblast cell lines, FOX04-DRI was more inclined to kill transforming growth factor-β (TGF-β)-induced myofibroblasts, and by inhibiting myofibroblasts, it led to the downregulation of extracellular matrix (ECM) receptor interaction pathways in BLM-induced PF.
Chronic obstructive pulmonary disease (COPD): The pathogenesis and progression of COPD are closely associated with cellular senescence. In cigarette smoke-induced senescent pulmonary fibroblasts, FOX04 expression is elevated. To knockdown FOX04 in senescent fibroblasts, self-assembling DNA nanotubes (siFOXO4-NT) loaded with single-stranded FOX04 siRNA were designed and synthesized. Studies have shown that siFOXO4-NT can enter human lung fibroblasts (HFL-1 cells) in a concentration- and time-dependent manner, thereby reducing FOX04 levels in vitro. siFOXO4-NT selectively eliminates aged HFL-1 cells by reducing the elevated BCLXL expression and BCL2/BAX ratio in cigarette smoke extract (CSE)-induced aged HFL-1 cells.
Role in cartilage tissue: Autologous chondrocyte implantation (ACI) is a method for treating joint cartilage damage and preventing post-traumatic osteoarthritis. During the in vitro expansion of chondrocytes, a necessary step in ACI, senescent cells are generated, which negatively impact the quality and quantity of newly formed cartilage. Researchers hypothesized that FOX04-DRI treatment could remove senescent cells from expanded chondrocytes, thereby enhancing their potential to generate high-quality cartilage. To simulate the in vitro expansion process in ACI, chondrocytes isolated from healthy donors were expanded to population doubling level (PDL) 9, representing chondrocytes ready for implantation, while cells at PDL3 served as a minimal expansion control. Results showed that FOX04-DRI treatment removed over half of the cells at PDL9 but had no significant effect on the number of PDL3 chondrocytes. The senescence level of PDL9 chondrocytes treated with FOX04-DRI was significantly reduced. Based on standard micro-cluster culture results, FOX04-DRI pretreatment did not enhance the chondrogenic potential of PDL9 chondrocytes, but chondroid tissue derived from FOX04-DRI-pretreated PDL9 cells exhibited lower expression of aging-associated secretory factors. FOX04-DRI effectively removed senescent cells from PDL9 chondrocytes.
Role in radiation-induced pulmonary fibrosis (RIPF): RIPF is a common and severe complication following thoracic tumor radiotherapy. C57BL/6 mice were randomly divided into irradiation and irradiation + FOX04-DRI groups, with the right hemithorax of each group receiving 17 Gy X-ray irradiation. FOX04-DRI group and irradiation + FOX04-DRI group mice were administered FOX04-DRI via intraperitoneal injection at weeks 16 and 20 post-irradiation. Results showed that FOX04-DRI reduced collagen deposition in lung tissue of RIPF mice, decreased expression of col1α1 and α-SMA, and reduced the number of β-galactosidase (β-gal) positive cells (a marker of senescent cells) in lung tissue. FOX04-DRI was able to inhibit the expression of P21 and P16^(Ink4a) genes and proteins in lung tissue of RIPF mice, as well as the expression of SASP genes IL-1α, IL-1β, tumor necrosis factor α (TNFα), and MMP2. FOX04-DRI also reduces reactive oxygen species (ROS) levels in lung tissue of RIPF mice and promotes the activation of p-AKT and p-PI3K proteins. This suggests that FOX04-DRI reduces oxidative stress and inhibits cellular senescence by activating the PI3K/AKT signaling pathway, thereby reversing RIPF.
Figure 3 FOXO4-DRI alleviates myofibroblast differentiation in BLM-induced PF mice.
Conclusion
In summary, FOX04-DRI, as a revolutionary drug capable of specifically eliminating senescent cells, plays an important role in the treatment of age-related diseases and aging intervention.
Sources
[1] Han Y, Wu Y, He B, et al. DNA nanoparticles targeting FOXO4 selectively eliminate cigarette smoke-induced senescent lung fibroblasts[J]. Nanoscale Advances, 2023,5(21):5965-5973.DOI:10.1039/d3na00547j.
[2] Han X, Yuan T, Zhang J, et al. FOXO4 peptide targets myofibroblast ameliorates bleomycin-induced pulmonary fibrosis in mice through ECM-receptor interaction pathway[J]. Journal of Cellular and Molecular Medicine, 2022,26(11):3269-3280.DOI:10.1111/jcmm.17333.
[3] Huang Y, He Y, Makarcyzk M J, et al. Senolytic Peptide FOXO4-DRI Selectively Removes Senescent Cells From in vitro Expanded Human Chondrocytes[J]. Frontiers in Bioengineering and Biotechnology, 2021,9:677576.DOI:10.3389/fbioe.2021.677576.
[4] Zhang C, Xie Y, Chen H, et al. FOXO4-DRI alleviates age-related testosterone secretion insufficiency by targeting senescent Leydig cells in aged mice[J]. Aging (Albany Ny), 2020,12(2):1272-1284.DOI:10.18632/aging.102682.
[5] Baar M P. DNA Damage Induced Cell Fates during Aging, 2019[C]. https://api.semanticscholar.org/CorpusID:196682180
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