## 4 Discussion
In addition, CCl\({}_{4}\) directly produces ROS and RNS via the uncoupling of oxidative phosphorylation in the mitochondrial membrane and induction of inducible nitric oxide synthase [30,37]. In parallel with our previous report [16], we showed that hepatic lipid peroxidation (e.g., 4-hydroxynonenal and malondialdehydedehyde) and nitrotyrosine levels were increased in the CCl\({}_{4}\)-treated mice, suggesting that CCl\({}_{4}\) induces hepatic oxidative stress, which was synergistically attenuated by MLD administration. Nrf2 interacted with Keap1 is continuously degraded by the ubiquitin-proteasome system. Oxidative stress allows Nrf2 to dissociate from Keap1 and translocate into thenucleus, where Nrf induces the transcription of diverse antioxidant genes, such as glutamate cysteine ligase (an enzyme involved in the glutathione biogenesis), SOD, catalase, and NAD(P)H: quinone oxidoreductase 1, which act as a defense against oxidative stress-mediated cell injury [21]. Because genetic ablation of Nrf2 potentiates the severity of liver injury in experimental animals [38; 39; 40], Nrf2 is regarded as a key transcription factor coping with oxidative stress and inflammation in the liver. Consistent with previous reports [41; 42], the results presented here showed that CCl\({}_{4}\) downregulated hepatic mRNA levels of Nrf2, resulting in the depletion of hepatic glutathione, and reduced SOD and catalase activities, which may facilitate liver injury via the disruption of the antioxidant response. Moreover, our preliminary results using HepG2 cells (hepatocyte-surrogate cells) indicate that MLD significantly transactivated antioxidant response element-harboring reporter genes and induced the mRNA level of NAD(P)H: quinone oxidoreductase 1 in a concentration-dependent manner [23]. Together, these results indicate that MLD synergistically restored impaired Nrf2 mRNA expression as well as antioxidant defense responses, suggesting that MLD-mediated Nrf2 activation protects against CCl\({}_{4}\)-mediated liver injury by alleviating oxidative stress. Present results revealed that MLD significantly decreased the CCl\({}_{4}\)-mediated infiltration of inflammatory cells and the production of proinflammatory cytokines. Among the proinflammatory cytokines produced, TNF-\(\alpha\) is unique in terms of its rapid induction and ability to induce cell death after CCl\({}_{4}\) injection [30; 43]. The targeted disruption of TNF-\(\alpha\) or TNF receptor I has been shown to attenuate hepatic injury and decrease ALT activity induced by CCl\({}_{4}\)[44], implying that TNF-\(\alpha\) is the most important cytokine for the progression of CCl\({}_{4}\)-mediated acute liver injury. In addition, TNF-\(\alpha\) accelerates the infiltration of inflammatory cells into the damaged lesions by upregulating the expression of adhesion molecules [44]. The subsequent binding of TNF-\(\alpha\) to receptors expressed by these inflammatory cells and damaged hepatocytes activates diverse signaling cascades including that of nuclear factor-\(\kappa\)B (NF-\(\kappa\)B) which further stimulate the production of proinflammatory cytokines (e.g., IL-1\(\beta\) and IL-6) [30; 45]. Moreover, the CCl\({}_{4}\)-mediated release of damage-associated molecular patterns from injured hepatocytes activates pyroptosis [25] and may promote the maturation and secretion of IL-1\(\beta\). In parallel with a previous report [46], our preliminary results also showed that MLD synergistically blocked the increases in mRNA levels of Rel A, a major subunit of NF-\(\kappa\)B, induced by CCl\({}_{4}\) (data not shown). Furthermore, it has been reported that the induction of Nrf2-dependent antioxidant genes regulates the production of proinflammatory cytokines by inhibiting NF-\(\kappa\)B [47]. More interestingly, Nrf2 inhibits the transcription of proinflammatory cytokines via direct binding to the promoter [48].