The hypothesis is that the therapeutic window created by dasatinib+quercetin (D+Q)–induced chromatin remodeling dictates the optimal dosing interval for senescent cell clearance. Evidence shows D+Q induces a transient “rejuvenation” of chromatin—increased nuclear regularity and reduced heterogeneity—that reverts within 24 h in young cells after drug removal【2】. This remodeling coincides with downregulation of SASP factors and precedes the actual removal of senescent cells, suggesting that the drug’s primary action may be to reset senescent cells to a reversible state before they are cleared by subsequent immune or apoptotic mechanisms. If the chromatin state must be maintained to prevent re‑entry into a senescent phenotype, then dosing should occur at intervals shorter than the 24‑h reversion period to keep cells in a labile, clearance‑prone condition. Current preclinical regimens use weekly D+Q dosing in mice, whereas human trials employ monthly cycles, creating a mismatch that may leave a gap where senescent cells recover and re‑establish a stable SASP.

We propose that aligning D+Q administration with the chromatin remodeling half‑life—specifically, delivering a low‑dose pulse every 12 h for three consecutive days each week—will sustain the chromatin‑open state, thereby increasing the proportion of senescent cells susceptible to clearance without raising cumulative dasatinib exposure. This approach tests two predictions: (1) sustained chromatin remodeling will correlate with greater reduction in senescence markers (p16^INK4a^, SA‑β‑gal, SASP cytokines) compared to standard weekly dosing; (2) total dasatinib exposure measured by AUC will be lower or equivalent in the frequent‑low‑dose schedule because each pulse uses a reduced dose (e.g., 2.5 mg/kg dasatinib + 25 mg/kg quercetin) that still achieves target kinase inhibition due to reduced drug clearance between doses.

To test this, we will use the SM/J mouse model of age‑related intervertebral disc degeneration. Groups will receive: (A) standard weekly D+Q (5 mg/kg dasatinib + 50 mg/kg quercetin for 3 days); (B) pulsed low‑dose D+Q (2.5 mg/kg dasatinib + 25 mg/kg quercetin every 12 h for 3 days each week); (C) vehicle control. Treatment will start at 6 weeks of age and continue for 12 weeks. Primary outcomes: disc histology (safranin‑O staining), quantitative PCR for senescence‑associated genes, and serum SASP IL‑6 and MMP13 levels. Secondary outcomes: chromatin state assessed by ATAC‑seq on isolated nucleus pulposus cells to measure nuclear regularity and heterogeneity at 0 h, 6 h, 12 h, and 24 h after each dose; dasatinib plasma pharmacokinetics to compute AUC.

If group B shows significantly lower senescence marker expression and SASP secretion than group A while exhibiting comparable or reduced dasatinib AUC, the hypothesis is supported. Conversely, if chromatin remodeling metrics do not differ between schedules or if frequent low dosing fails to improve clearance despite maintained chromatin openness, the hypothesis is falsified. This experiment directly tests whether the transient epigenetic window is a rate‑limiting senolytic step and whether dosing precision can improve efficacy while mitigating long‑term dasatinib safety concerns.