Many neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's, and amyotrophic lateral sclerosis, are linked to oxidative stress-induced neuronal cell death (Alexi et al., 2000; Wu, Re, Nagai, Ischiropoulos, & Przedborski, 2006; Zhang, Dawson, & Dawson, 2000). Toxins and chemicals contribute to oxidative stress (Xicoy et al., 2017).In the present study, we demonstrated a protective effect of TA against H2O2-treated SH-SY5Y cells. TA reduced H2O2-induced cell death at low concentrations while TA itself at high concentrations (above 50 mM) caused cell death. Reduction of Bax and Caspase-3 and increase of Bcl-2 and p-AKT were detected as the underlying mechanism of TA protective effect. Further, high concentrations of TA concentrations caused the death of the neuroblastoma cells by increasing Bax/Bcl-2 ratio and Caspase-3. Several studies demonstrated that H2O2-induced apoptotic cell death depends on the concentration and duration of H2O2 exposure (Chen, Liu, Yin, Luo, & Huang, 2009). In the present study, we confirmed that organized cells treated with 0 to 800µM of H2O2 showed dose-dependent cytotoxicity.
Pre-treatment with 6.25 and 25 mM of TA significantly protected SH-SY5Y viability. These findings were further confirmed by occurring alterations in the percentage of cells at late apoptosis and necrosis in TA pretreated cells. The anti-apoptotic Bcl-2 family members (Bcl-2 and Bcl-x) and the pro-apoptotic Bcl-2 family members (Bax, Bid, and Bak) are key players in the mitochondrial-related apoptosis pathway(Banjara, J, Hinds, & Kvansakul, 2020; Fei & Ethell, 2008). Bcl-2 which is positioned in the mitochondrial outer membrane is an anti-apoptotic protein and prevents the release of cytochrome c (Borner, 2003). Bax, on the other hand, is a pro-apoptotic element that is transferred from cytosol and to the outer mitochondrial membrane following Bcl-2 reduction. This may result in the loss of mitochondrial membrane potential, an increase in mitochondrial membrane permeability, and the release of cytochrome c into the cytosol which triggers the programmed cell death mechanism (Chinnaiyan et al., 1996). the Bcl-2 family stabilizes pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) products to regulate the apoptotic process (Youle & Strasser, 2008). Thus, Bax/Bcl-2 ratio is a useful predictor of apoptotic cell death (Cregan et al., 1999; Youle & Strasser, 2008). In this study, we observed Bcl-2 was down-regulated and Bax mRNA expression up-regulated in H2O2-received SH-SY5Y cells. Pre-treatment with TA partially ameliorated the expression of Bcl-2 and Bax transcripts. Treatment with a high concentration of TA also caused changes in Bcl-2 and Bax mRNA expression in a way that the Bax/Bcl-2 ratio was increased. It has been shown that TA has protective effects at low concentrations (100 µM) on rotenone-induced cell stress in the SH-SY5Y cell line (Kim et al., 2009). Our study showed a protective effect of TA against H2O2-induced cell stress at low concentrations (6.25-50 mM). However, TA itself at high concentrations (above 50 mM) caused cell apoptosis.
Caspase-3 is the main executive caspase-dependent apoptotic pathways. During apoptotic processes, cytochrome c is released from mitochondria to the cytosol. The released cytochrome c activates Caspase-9, which in turn activates Caspase-3 and induce cell death program (McIlwain, Berger, & Mak, 2013). The increase in Caspase-3 is directly related to the increase in Bax/Bcl-2 ratio (Hussar, 2022). Our findings show that H2O2- and the high dose of TA-induced apoptosis were accompanied by an increase in Caspase-3 expression and Bax/Bcl-2 ratio. However, pre-treatment with a low concentration of TA inhibited Caspase-3. These findings are in line with other studies demonstrating the neuroprotective potential of TA via modulating the Bax/Bcl-2 ratio and Caspase-3 expression (Kim et al., 2009).
Numerous studies have demonstrated that the PI3K/AKT signaling pathway regulates cell survival (Vauzour, Vafeiadou, Rice-Evans, Williams, & Spencer, 2007). PI3K/AKT activation has also anti-apoptotic effects. It has been shown that tetramethyl pyrazine and GDNF protect dopaminergic neurons from 6-OHDA-induced neurotoxicity through activation of the PI3K/AKT pathway (Chen et al., 2017; Shorvon, 2011). Our observations showed that treatment with H2O2 significantly dephosphorylated Akt, but pretreatment of cells with low concentrations of TA could augment Akt phosphorylation and improve SH-SY5Y cell survival. In the meantime, high concentrations of TA reduced Akt phosphorylation and declined cell survival.
Epilepsy is divided into several categories, one of which is provoked epilepsy, which plays a role in this type of epilepsy in addition to genetic factors, as well as pharmaceutical agents. Various factors, including seizures caused by drugs, are the cause of provoked epilepsy (Shorvon, 2011). Hisato TAKAGI et al showed that tranexamic acid, which is used in heart surgery, increases the risk of seizures (Takagi, Ando, Umemoto, & All-Literature Investigation of Cardiovascular Evidence, 2017). Our observations in vitro show that tranexamic acid at high concentrations causes the death of neuroblastoma cells. In brief, the results of the present study indicated TA has protective effects at low concentrations and neurodegenerative effects at high concentrations on the neuroblastoma SH-SY5Y cell line.