MeHg has been a kind of dangerous environmental contaminant, since the sensational outbreak of Minamata Disease in Japan in 1956. The researchers have never stopped studying the toxicity of MeHg. At present, a large number of population data and experimental studies have confirmed that MeHg can cause serious and irreversible nervous system damage (Harada, 1995; Wu et al., 2020). There are also in vitro studies showing that MeHg has a certain toxic effect on Neuro-2a, F3, C17.2, neural stem cells and other nerve cells, and can induce cell death through a variety of ways (Chang et al., 2013; Sato et al., 2020), but the mechanisms are still not very clear. The Caspase-dependent pathway is a classic apoptosis signaling pathway. In recent years, studies have found that PARP related signaling molecules also play a key role in the process of cell apoptosis (Wang, et al., 2016). In this study, SH-SY5Y cells were used as the target cells. In this study, we explored the role and mechanism of PARP/AIF signaling molecules in the process of apoptosis that induced by MeHg in SH-SY5Y cells, and revealed the influence of ROS on the activation of downstream apoptosis signaling molecules.
Cell viability and morphology are important indicators of cell state. This study confirmed the toxic effect of MeHg on SH-SY5Y cells. With the increase of the time and dose of MeHg, the cell viability decreased significantly. Among them, the survival rate of the highest dose group treated for 24 h was even lower than 50%, while the low-dose groups could promote cell growth in a short time, which might be due to the bactericidal effect of MeHg. Chang et al. detected that the 48 h survival rate of human NSCs cells treated with MeHg decreased slightly (Chang, et al., 2013). Chung et al. observed that the 24 h survival rate of Neuro-2a cells treated with MeHg decreased significantly (Chung et al., 2019). In this study, we found that the 24 h survival rate of SH-SY5Y cells treated with MeHg reached 30%. Nerve cells were more sensitive to toxic substances than other cells. It was confirmed that SH-SY5Y cells decreased with MeHg in a time and dose-dependent manner. At the same time, we further confirmed the strong toxicity of MeHg in cell morphology. Under the light microscope, with the increase of MeHg concentration, the cell morphology changed significantly, and finally fell off or even died.
The role of ROS in DNA damage response is multifaceted and pleomorphic (Srinivas, et al., 2019). In previous studies, it was confirmed that there was also ROS accumulation during the apoptosis of SH-SY5Y cells induced by silica nanoparticles (Yang et al., 2017). It can be seen that ROS plays a major role in cell survival and apoptosis. Therefore, it is predicted that reducing ROS production by using inhibitors may affect the outcome of cell apoptosis. In many cases, the production of ROS is accompanied by the increase of Ca2+. ROS and Ca2+ can accelerate the process of apoptosis and participate in a variety of apoptosis pathways. The increase of ROS was detected in SH-SY5Y cells treated with MeHg only for 3 h, and even after 30 min of DCFH-DA staining, the green fluorescence increased in MeHg group, but hardly detected in the control group. The concentration of Ca2+ also increased after 24 h. In addition to the accumulation of ROS, in previous studies, it was believed that the toxicity of cells would lead to ER stress and unfolded protein reaction (Hou et al., 2021). Through the detection of mitochondrial damage related proteins VDAC1 and SOD2, it was found that there was a dose-dependent relationship between the expression of these proteins and the concentration of MeHg. This study confirmed that MeHg can induce oxidative stress in cells, increase intracellular ROS and Ca2+ level, cause mitochondrial damage and lead to apoptosis.
In order to further explore the molecular mechanism of apoptosis, we detected the apoptosis pathway related proteins. Caspase dependent apoptosis signaling pathway is one of the classic apoptosis pathways (Shi, 2002). Caspase-9, Apaf-1 and Cyt C together form an apoptotic complex to induce apoptosis. This study found that after MeHg treated cells for 24 h, the related proteins of the pathway were activated, and the formation of apoptotic complex further activated the downstream apoptosis signal molecule Caspase-3 to form cleavage fragment, which finally led to apoptosis. These results suggest that MeHg induces apoptosis of SH-SY5Y cells through classical caspase dependent apoptosis pathway. In recent years, studies have found that there is another key signal in the process of apoptosis, namely PARP. PARP is a DNA damage repair enzyme. It is activated by recognizing DNA fragments with structural damage and is generally considered as a receptor of apoptosis. PARP and the apoptosis inducing factor AIF released from mitochondrial endomembrane can promote apoptosis (Jang et al., 2017; Regdon et al., 2019). This pathway often occurs in the brain and nervous tissues and other tissues with high metabolism. Yu et al. found that PARP was activated in mouse embryonic fibroblasts treated with DNA alkylating agent MNNG, and then AIF transferred from mitochondria to nucleus, resulting in cell apoptosis (Yu, et al., 2002). AIF was considered to be a completely independent and caspase independent active factor with apoptotic effect at the initial stage of discovery. However, many studies have shown that AIF is a double-edged sword, which can not only participate in the oxidative phosphorylation and respiratory chain circulation of normal cells, but also can release from mitochondria and induce nucleus lysis leading to apoptosis (Murari et al., 2020; Nan et al., 2020). Through the detection of PARP and AIF proteins, we found that PARP/AIF pathway was also activated in the process of SH-SY5Y cell apoptosis induced by MeHg. These results confirmed that MeHg could induce apoptosis at least through caspase and PARP/AIF pathways. At the same time, the proportion of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-xL increased, indicating the outcome of MeHg inducing apoptosis.
This study is the first time to explore the role of ROS in apoptosis and the relationship between caspase and PARP/AIF apoptosis pathway through three intervention methods. Firstly, by inhibiting the generation of ROS, the role of ROS in activating downstream apoptotic pathways was discussed, NAC has been widely used in clinic because it can effectively inhibit oxidative stress (Halasi et al., 2013; Luczak and Zhitkovich, 2013; Rushworth and Megson, 2014). Fluorescence analysis results showed that NAC could effectively inhibit the generation of ROS. The apoptosis rate of the MeHg and NAC co-incubation group was significantly lower than that of the MeHg group. On this basis, apoptosis related proteins were detected, and it was found that the expressions of these proteins decreased to varying degrees in the NAC and MeHg co-incubation group compared with the MeHg alone group. In general, the reduction of ROS has a certain regulatory effect on apoptosis, but it seems that it cannot completely inhibit the occurrence of apoptosis. It indicates that the downstream apoptotic signal molecules are still functioning at the same time or one of them. So which signal plays the main role, we choose to add inhibitors to explore. Z-VAD-FMK is a pan-Caspase inhibitor that can inhibit the function of Caspase 1–10 proteins (Martinet et al., 2006; Moretti et al., 2009). The reason is that fluoromethyl ketone derived peptides can effectively mimic the caspase cleavage site, so that Z-VAD-FMK can bind to the caspase catalytic site and inhibit the induction of apoptosis. Studies in vivo have shown that the administration of Z-VAD-FMK was not toxic and could inhibit apoptosis in animal models (Xu et al., 2019). Our experiments also confirmed that Z-VAD-FMK could inhibit the increase in apoptosis rate of SH-SY5Y cells caused by MeHg, effectively reduced the expression of Caspase-3, 9 and Cyt C, and blocked the caspase pathway. However, Z-VAD-FMK had little effect on PARP activation and AIF expression. That is to say, MeHg can still cause SH-SY5Y cell apoptosis after using pan-Caspase inhibitors, indicating that the activation of PARP/AIF signal is independent of caspase dependent apoptosis pathway. On the contrary, whether the inhibition of the PARP/AIF pathway will affect the activation of caspase apoptotic signal needs further study. In this study, the use of olaparib effectively reduced the increase of the apoptosis rate induced by MeHg, the expression of PARP and its cleavage fragments and AIF protein were inhibited. Interestingly, we also detected caspase related proteins and found that olaparib meanwhile inhibited the expressions of Caspase-3 activation fragments, Caspase-9 and Cyt C proteins. It is speculated that the activation of PARP/AIF signal plays an important role in ROS mediated apoptosis of SH-SY5Y cells induced by MeHg, which is independent of caspase dependent apoptosis pathway, but affects caspase dependent apoptosis pathway (Fig. 6). It shows that the PARP/AIF apoptotic signal plays a major role in the apoptosis process of SH-SY5Y caused by MeHg.