## 3 Results  
Enhanced Reduction in Heps2 Cell Viability by Combined Treatment with Doxorubicin and Muscari comosum Extract (MET70)  
Chemical characterization of the bulb of _Musari comosum_ revealed a diverse range of phytochemicals, including phenolic acids, fatty acids, flavonoids, triterpenes, phytosterols, and homoisoflavones [20,23]. Our previous study demonstrated that the 70% methanol extract (MET70) had the highest total phenolic content (58.72 \(\pm\) 1.11 mg gallic acid equivalents/g extract) and one of the highest flavonoid contents (20.37 \(\pm\) 1.24 mg quercetin equivalents/g extract) among the tested extracts. Additionally, MET70 exhibited strong antioxidant activity in vitro and reduced HepG2 cell viability by approximately 30% at a concentration of 100 \(\upmu\)g/mL after 72 h of exposure [25]. To assess whether the bulb extract could enhance the effect of doxorubicin on cell viability, HepG2 cells were treated with increasing doses of doxorubicin, both alone and in combination with MET70 (100 \(\upmu\)g/mL). The MTT assay (Figure 1) revealed that doxorubicin reduced cell viability in a dose-dependent manner after 72 h, with an IC50 of 0.98 \(\upmu\)M (95% confidence interval, 0.645-1.461 \(\upmu\)M). Notably, the combination of doxorubicin and MET70 significantly reduced cell viability, with an IC50 of 0.01675 \(\upmu\)M (95% confidence interval,0.0106-0.02514 \(\upmu\)M). Treatment with the MET70 extract alone resulted in only a slight reduction in cell viability, whereas the untreated control group maintained consistently high viability values, supporting the robustness of the assay. These results indicate that, while MET70 exhibits limited cytotoxicity by itself, it significantly enhances the susceptibility of HepG2 cells to doxorubicin. Based on these results, subsequent experiments were conducted using a sublethal dose of 0.5 \(\upmu\)M doxorubicin in combination with 100 \(\upmu\)g/mL MET70. Impact of Combined Doxorubicin and Muscari comosum Extract (MET70) Treatment on Intracellular ROS Levels and Redox/Inflammatory Gene Expression  
To evaluate the effect of MET70 treatment in combination with doxorubicin on intracellular reactive oxygen species (ROS) accumulation, we measured the oxidation of a non-fluorescent probe (2\({}^{\prime}\),7\({}^{\prime}\)-dichlorydrofluorescein diacetate, DCFH-DA) to its fluorescent form (2\({}^{\prime}\),7\({}^{\prime}\)-dichlor fluorescein, DCF). As shown in Figure 2, after 72 h, the combination of the drug and extract resulted in a significant increase in intracellular ROS levels, enhancing the effect of doxorubicin. Furthermore, treatment with MET70 alone did not alter the level of cellular ROS. We analyzed the expression of genes involved in the regulation of redox balance. The expression level of Nrf2, a key transcription factor that regulates the production of ROS-scavenging enzymes, increased after treatment with doxorubicin alone but did not change significantly in combination with MET70. MET70 itself increased the expression level of Nrf2 (Figure 3a). The combined treatment had no effect on the catalase and NAD(P)H Quinone Dehydrogenase 1 (NQO1) expression; on the contrary, it decreased both superoxide dismutase 2 (SOD2) and Glutathione peroxidase 1 (GPX-1) expression levels (Figure 3b,c). The expression level of mitochondrial uncoupling protein 2 (UCP2), a mitochondrial protein involved in the resolution of oxidative stress, decreased following the combined treatment (Figure 3b,d). Ultimately, the reduced expression of SOD2 and, in particular, of both GPX-1 and UCP2 could be responsible for the greater oxidative stress and therefore the lower viability of cells treated with doxorubicin combined with MET70. Figure 1: Dose–response curves of doxorubicin, MET70 and their combination on HepG2 cells. HepG2 cells were cultured for 72 h in the presence of increasing concentrations of doxorubicin, either alone or in combination with the _Muscari comosum_ extract (MET70). MET70 alone and untreated control groups were also included.