Apple Seed Extract Post-Treatment Alters Selected IGF-Related and Extracellular Matrix-Associated Markers Following Tobacco Leaf Extract-Induced Histological Liver Injury

Tobacco leaf extract (TLE) exposure can induce liver injury-associated responses involving cell death, inflammatory signaling, and extracellular matrix (ECM)-related changes. This study examined whether apple seed extract (ASE) post-treatment changes apoptosis-, inflammation-, ECM-, and insulin-like growth factor (IGF)-related markers after TLE exposure. Primary mouse hepatocytes were exposed to TLE, ASE alone, or TLE followed by ASE, and mouse liver tissues were examined after TLE exposure with or without ASE post-treatment. TLE reduced hepatocyte viability in a concentration-dependent manner, with an IC50 of 4.4 mg/mL. Annexin V/propidium iodide analysis showed that early apoptosis increased from 2.20% in untreated cells to 5.50% after 2 mg/mL TLE and 85.65% after 4 mg/mL TLE. ASE alone at 40 µg/mL increased the early apoptotic fraction to 53.65%, indicating that this concentration was not biologically neutral under basal culture conditions. After TLE exposure followed by ASE post-treatment, the live-cell fraction remained high in T2HA and T4HA, whereas T5HA retained a high early apoptotic fraction. In mice, TLE exposure was accompanied by visible liver appearance changes and histological alterations. ASE post-treatment changed Alcian blue staining, gelatinase activity, TIMP-associated signals, and IGF-related signals. These findings indicate treatment-dependent changes in selected injury-associated markers rather than a consistently protective effect of ASE. The study does not assign these effects to a specific ASE constituent because compound-level chemical standardization was not performed.