Background: Cigarette smoke is a well-known reproductive toxicant that causes structural and functional damage to the testis through oxidative stress and apoptotic pathways. This study aimed to evaluate whether apple seed extract (ASE) can mitigate testicular damage induced by tobacco smoke extract (TSE) in a mouse model. Methods: Adult male mice were orally administered TSE to induce testicular injury. ASE was delivered intraperitoneally, either as a post-treatment or concurrently with TSE. Histological evaluation (H&E staining), immunohistochemistry (IGF-1 and VEGF), and immunofluorescence (TNF-α, Caspase-3, and BCL-2) were conducted to assess tissue morphology, growth factor expression, and apoptotic signaling. Results: TSE exposure led to degeneration of the seminiferous tubules, suppressed IGF-1 and VEGF expression, and increased pro-apoptotic markers (TNF-α, Caspase-3), along with reduced BCL-2 expression. ASE treatment restored seminiferous tubule architecture, enhanced the expression of growth and anti-apoptotic factors, and attenuated apoptotic signals. These restorative effects were particularly significant in the post-treatment groups. Conclusions: ASE demonstrated protective and reparative effects against TSE-induced testicular toxicity by modulating oxidative and apoptotic pathways. These findings highlight ASE as a promising natural therapeutic candidate for ameliorating smokerelated male reproductive dysfunction.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
    Animal ethics and housing
    Preparation of tobacco smoke extract (TSE) and appleseed extract (ASE)
    TSE and ASE injections in mice
    Testicular tissue processing and H&E staining
    Alcian blue staining of testicular sections
    In situ zymography
    Immunohistochemistry for IGF-1 and VEGF
    Immunofluorescence double-staining
    Quantitative analysis of IHC staining
RESULTS
    Histological analysis of testicular tissue after TSE andASE treatment
    Analysis of growth factors and tissue enzymaticactivity in testicular tissue
    Effects of ASE on TSE-induced inflammatory andapoptotic markers in testes
DISCUSSION
CONCLUSION
REFERENCES

• Sang-Hwan Kim(General Graduate School of Animal Life Convergence Science, Hankyong National University, Ansung 17579, Korea, Institute of Applied Humanimal Science, Hankyong National University, Ansung 17579, Korea) Corresponding author