# Attenuating Oxidative Damage with Macelignan in Glutamate-Induced HT22 Hippocampal Cells
## 4 Discussion  
Living cells continuously generate and accumulate free radicals and ROS during aerobic metabolism, resulting in oxidative stress [41]. In this abnormal state, oxidative stress damages cellular constituents, leading to cell death, which is one of the major causes of neurodegenerative disorders [41; 42]. In this work, the neuroprotective properties of macelignan against glutamate-induced oxidative damage, along with its effect on apoptosis via modulation of the MAPK signaling pathway in HT22 cells, were investigated. Figure 5: Effect of macelignan on glutamate-stimulated activation of MAPK pathway. (**a**) Expression of MAPK pathway-related proteins and GAPDH; (**b**) levels of p-ERK/ERK, p-JNK/JNK, and p-p38/p38. GAPDH was detected as loading control. Value represents the mean \(\pm\) S.D. (\(n=3\)). # \(p<0.05\) compared to the untreated cells, \({}^{*}p<0.05\) compared to glutamate-treated cells (ANOVA, Tukey’s test). Mac, macelignan; Glu, glutamate. _M. fragrans_ has long been used as an essential ingredient for food and cosmetics, as well as in traditional medicines [43]. Macelignan is the significant bioactive component of _M. fragrans_, and recent studies have shown both in vitro and in vivo that it may have neuroprotective properties. [PERSON] et al. [44] revealed the neuroprotective property in macelignan from interferon-\(\gamma\) and lipopolysaccharide-inducible dopaminergic neuron degeneration in midbrain slice cultures. According to [PERSON] et al. [30], macelignan has antioxidant and anti-inflammatory properties. They also showed that macelignan protected HT22 cells from ROS and glutamate-induced neurotoxicity. [PERSON] et al. [45] revealed that the administration of macelignan to rats significantly reduced hippocampal microglial activation and attenuated spatial memory impairment. Macelignan suppressed Tau hyperphosphorylation and amyloid \(\beta\)-protein production in N2a/SweAPP cells (a 3\(\times\) Transgene mouse primary nerve cell line) [33]. In addition, macelignan exhibited dopaminergic neuroprotection via the activation of peroxisome-proliferator-activated receptors and the induction of arginase-1 in midbrain slice cultures [44]. Based on these findings, macelignans are assumed to exert protective effects against neurodegenerative disorders. One of the most popular techniques for assessing the antioxidative effect of food and pharmaceutical substances is the capacity to scavenge DPPH free radicals, which has the benefits of a quick reaction time and an easy process [46]. According to the findings of a prior study [30], with an IC\({}_{50}\) value of 36.98 uM, macelignan demonstrated concentration-dependent scavenging activity and reduced DPPH radicals. This suggests that macelignan has an antioxidant effect on DPPH radical scavenging activity. As HT22 cells do not express NMDA receptors, they are resistant to glutamate-dependent excitotoxicity [47]. Evidence showed that differentiated HT22 cells are sensitive to excitotoxicity, representing a better model for hippocampal neurons [17]. Nonetheless, several studies have shown that glutamate can induce oxidative cell death in undifferentiated HT22 cells, making them a commonly used cell model of oxidative stress [18; 40; 48]. Therefore, undifferentiated HT22 cells were used in the present study. Several studies have revealed that high concentrations of glutamate induce neurotoxicity by increasing ROS levels and activating caspase pathways [16; 18]. A rapid increase in ROS levels is the initial event of oxidative glutamate toxicity in HT22 cells [49]. Excess ROS generation brought on by glutamate exposure causes oxidative damage, which in turn triggers cell death through apoptosis [50]. The production of ROS is crucial for oxidative stress and is more likely to attack neuronal cells that possess a vulnerable antioxidant defense system [49; 51]. In this study, fluorescence was enhanced in HT22 cell exposure to glutamate, which demonstrated the accumulation of ROS.