## 3 Results
### MLD Synergistically Attenates Hepatocyte Apoptosis
**\(p<0.01\) between the vehicle and CCl\({}_{4}\) groups; **\(p<0.01\), **\(p<0.05\)** versus CCl\({}_{4}\)-injected mice; **\(p<0.01\), + \(p<0.05\)** between Sily- and MLD-treated mice; **\(p<0.01\), + \(p<0.05\)** between LB- and MLD-treated mice; **\(p<0.01\), + \(p<0.05\)** between DL- and MLD-treated mice. CV, central vein. increased the number of nitrotyrosine-positive cells in the liver (\(p<0.01\); Figure 4a, left and Figure 4b, upper). Although administration of silymarin or DL attenuated the CCl4-mediated increases in number of nitrotyrosine-positive cells, these differences were not statistically significant. However, all three doses of MLD and LB significantly decreased the number of nitrotyrosine-positive cells, relative to CCl4 treatment (\(p<0.01\), CCl4 versus MLD; \(p<0.05\), CCl4 versus LB), and the magnitude of the reduction in number of nitrotyrosine-positive cells by MLD was greater than that by silymarin or either of the single herbal extracts alone (\(p<0.01\); Figure 4b, upper). Immunohistochemistry-idimensional staining of 4-hydroxynonenal and quantification of malondialdehydedehyde were conducted to assess the effects of MLD on hepatic lipid peroxidation. CCl4-mediated increases in lipid peroxidation were significantly inhibited by the three different doses of MLD (\(p<0.01\); Figure 4a, right; Figure 4b, lower; and Figure 4c). The inhibition of 4-hydroxynonenal-positive cells and malondialdehydedehyde by 100 or 200 mg/kg MLD was more potent than that seen in response to silymarin or either of the herbal extracts alone (\(p<0.01\); Figure 4b, lower and Figure 4c). Although the magnitude of the reduction in number of 4-hydroxynonenal-positive cells induced by 50 mg/kg MLD was greater than that induced by DL (\(p<0.01\)), the number of 4-hydroxynonenal-positive cells observed in the 50 mg/kg MLD group did not differ from that in either the silymarin or LB group (Figure 4b, lower). In addition, 50 mg/kg MLD synergistically decreased malondialdehydedehyde levels in comparison to silymarin or either of the herbal extracts alone (\(p<0.05\), LB versus 50 mg/kg MLD; \(p<0.01\), other comparisons; Figure 4c). Figure 4: MLD alleviates CCl4-mediated oxidative stress. Hepatic tissues were immunostained using anti-nitrotyrosine or anti-4-hydroxynonenal antibodies (**a**), and positive cells were counted (**b**). Scale bars, 200 \(\upmu\)m. (**c**) Lipid peroxidation. Malondialdehydedehyde concentration was determined using liver homogenates. All values are expressed as mean \(\pm\) SD of ten mice. **+**\(p<0.01\) between the vehicle and CCl4 groups; **+**\(p<0.01\), **+**\(p<0.05\) versus CCl4-injected mice; **+**\(p<0.01\) between Sily- and MLD-treated mice; **+**\(p<0.01\), **+**\(p<0.05\) between LB- and MLD-treated mice; **+**\(p<0.01\) between DL- and MLD-treated mice. CV, central vein. Next, we explored the effects of MLD on antioxidant activity in CCl\({}_{4}\)-treated mice. Compared to the vehicle, CCl\({}_{4}\) injection significantly reduced the mRNA levels of nuclear factor erythroid 2-related factor 2 (Nrrf2) (\(p<0.01\); Figure 5a), an important transcription factor for antioxidant gene expression [21]. Administration of MLD, silymarin, LB, and DL significantly reversed the reduction in levels of Nrf2 mRNA (\(p<0.05\), CCl\({}_{4}\) versus DL; \(p<0.01\), other comparisons). This restoration of Nrf2 mRNA by three different doses of MLD was more potent than that by silymarin or either the herbal extracts alone (\(p<0.01\); Figure 5a). Consistent with the reduction in Nrf2 mRNA levels, CCl\({}_{4}\) also significantly depleted glutathione (an endogenous antioxidant) levels and inhibited SOD and catalase activities (\(p<0.01\); Figure 5b-d).