# Attenuating Oxidative Damage with Macelignan in Glutamate-Induced HT22 Hippocampal Cells
## 4 Discussion
However, this was reversed in a concentration-dependent manner following macelignan treatment. Consequently, macelignan may exhibit antioxidant activity by inhibiting glutamate-induced ROS accumulation. Neuronal apoptosis is linked to oxidative damage and a lack of balance between ROS and antioxidant defenses [52]. Generally, apoptotic cell death can be observed morphologically to distinguish it from healthy cells or other types of cell death [53]. In the present study, chromatin condensation was observed using Hoechst 33342 staining. As mentioned previously, the nuclei are round in healthy cells and fragment and stain more intensely in apoptotic cells [35]. We found that glutamate toxicity resulted in apoptotic cell death by showing the characteristics of apoptosis; however, these morphological changes were significantly improved by pretreatment with macelignan. Furthermore, apoptotic conditions were detected using a Tali assay, which is more accurate in distinguishing viable cells from dead cells [54]. In the current investigation, glutamate led to a notable rise in both apoptotic cells (stained with annexin V-Alexa Fluor 448 dye) and dead cells (stained with PI); however, this rise in the number of abnormal cells was effectively improved by treatment with macelignan. These results suggest that macelignan attenuates glutamate-induced apoptosis. Changes in oxidative stress are linked to neuronal death [55]. Stress-induced upregulation and phosphorylation in MAPK have been implicated in neuronal apoptosis [16; 56]. JNK, p38, and ERK are the major subgroups of MAPK; JNK and p38 regulate apoptosis and autophagy, whereas ERK supports cell survival or promotes cell death [57; 58; 59]. Treatment with JNK or p38 inhibitors attenuated neuronal cell death induced by glutamate, as previously reported [40]. According to earlier research, ERK activation during glutamate-induced oxidative toxicity is pivotal for neuronal cell death, especially in HT22 cells [60; 61]. Consistently, in this investigation, glutamate significantly induced the phosphorylation of p38, JNK, and ERK; however, pretreatment with macelignan effectively down-regulated the expression of these proteins. These findings showed that macelignan protects HT22 cells from glutamate-induced apoptosis via suppressing MAPK activation. However, further investigations should be focused on various pathways linked to oxidative stress, including mitochondrial dysfunction, Nrf2/HO-1, and antioxidant enzymes, using different cell lines, which can widen the understanding of macelignan on oxidative damage. In conclusion, macelignan, which was identified and isolated from _M. fragrans_, exhibited a protective activity against glutamate-induced oxidative damage in HT22 cells by scavenging DPPH radicals, inhibiting ROS generation, preventing apoptotic cell death, and regulating the MAPK signaling pathway. According to these findings, macelignan may be a potential medicinal substance to treat neurodegenerative disorders associated with oxidative stress. Our data provide novel insights into the possible therapeutic use of macelignan in neurodegenerative disorders and point out the significance of the MAPK pathway in mediating its neuroprotective effects. Nevertheless, further studies in animal models or clinical trials are needed based on the results in the present study. For example, we found that macelignan at 5 and 10 \(\upmu\)M had a good inhibitory effect on oxidative damage and apoptosis in vitro. Referring to these concentrations, a proper dose for oral administration can be converted in an oxidative stress-induced mouse model, then the molecular mechanisms underlying antioxidant action as well as the bioavailability could be investigated. Furthermore, clinical trials on macelignan can help to investigate whether it has actual side effects in human participants and widen its application.