In this study, we utilized PC12 cells to explore the protective properties of COA-Cl against oxidative damage induced by H2O2 and 6-OHDA. Both H2O2 and 6-OHDA are cytotoxic agents. H2O2 is used to cause oxidative damage in cellular models and 6-OHDA is used as an in vitro model of DA neuron degeneration. COA-Cl is known to possess antioxidant and neuroprotective effects in both in vitro and in vivo ischemia models (Feng et al., 2016). In addition, COA-Cl has been shown to induce striatal dopamine release both in vivo and in vitro (Jamal et al., 2019). Considering these effects, we wanted to test whether COA-Cl could exert neuroprotective effects on dopaminergic neurons in cellular model of PD. Therefore, we chose the PC12 cell line to study the effects of COA-Cl on H2O2-induced neurotoxicity. Our results demonstrate that H2O2 triggered cell injury, accompanied by an increase in intracellular ROS generation and apoptosis via increasing the Bcl-2/Bax ratio and caspase-3 activity, consistent with previous studies (Matsura et al., 1999; Dumont et al., 1999; Redza-Dutordoir and Averill-Bates, 2017). However, COA-Cl attenuated these effects, indicating that COA-Cl can inhibit H2O2-induced neurotoxicity, most likely by preventing oxidative stress and apoptosis. In accordance with the above findings, 6-OHDA dose-dependently decreased cell viability and the ratio of Bcl-2/Bax protein, and increased LDH release. However, these changes were also attenuated by treatment with COA-Cl. These data provide the first evidence that COA-Cl protects PC12 cells against H2O2- and 6-OHDA-induced oxidative injury and cell demise. Thus, our findings raise the possibility of the therapeutic application of COA-Cl for treating PD.
Several lines of evidence have confirmed that H2O2 and 6-OHDA produce oxidative stress, which is closely associated with neuronal damage in various neurodegenerative diseases, including PD (Milton, 2004; Simola et al., 2007; Yang et al., 2016; Lee et al., 2021). COA-Cl exhibits neuroprotective and anti-oxidative effects, which may prove beneficial in intracerebral hemorrhage and ischemic stroke (Okabe et al., 2013; Feng et al., 2016). PC12 cells are widely used as neuronal cell models in vitro and exhibit some features of mature dopaminergic neurons (Greene and Tischler, 1976; Duan et al., 2015; Heusinkveld et al., 2017). To explore whether COA-Cl might protect dopaminergic neurons against oxidative stress-induced damage, we investigated the effects of COA-Cl in H2O2- and 6-OHDA-treated PC12 cells. It was found that treatment with COA-Cl produced significant neuroprotection against injury in PC12 cells.
PC12 cells were treated with H2O2 and 6-OHDA at concentrations ranging from 50–500 µM for 24 h. Our data revealed that H2O2 and 6-OHDA produced a loss of cell viability in a concentration-dependent manner, with significant cytotoxicity being observed at a concentration of 200 µM (Figs. 1B,C). The 200 µM of H2O2 and 6-OHDA resulted in decrease in cell viability of 42.3 to 43.1% compared to untreated cells (viability of 100%), which are in agreement with other studies (Cho et al, 2008; Oraki Kohshour et al., 2013; Zou et al., 2016; Ramazani et al., 2019). Therefore, we used 200 µM H2O2 and 6-OHDA (56.9–57.7% inhibition) to examine the protective effects of COA-Cl in PC12 cells. Our data showed that COA-Cl (100 µM) pretreatment significantly attenuated the decreased cell viability induced by H2O2 and 6-OHDA (Figs. 1D,E). We also assayed the LDH released to further investigate the protective effects of COA-Cl against cell injury induced by H2O2 and 6-OHDA in PC12 cells. The results revealed that exposure to H2O2 and 6-OHDA increased LDH release. Of note, treatment with COA-Cl attenuated the LDH release induced by H2O2 and 6-OHDA (Figs. 2A,B). We also found that COA-Cl (10–200 µM) was non-toxic to PC12 cells (Fig. 1A), indicating that COA-Cl itself did not cause any cell injury, consistent with our previous study (Jamal et al., 2019). H2O2- and 6-OHDA-induced alteration of cell viability and LDH release was almost similar, which is in line with earlier evidence demonstrate that H2O2 and 6-OHDA produced similar morphological changes in various sites of the brains of cats and rats (Poirier, 1975). Together, these results show that COA-Cl protects PC12 cells against injury caused by H2O2 and 6-OHDA and led us to hypothesize that COA-Cl could protect against injury by inhibiting ROS generation.
ROS production causes oxidative stress and mitochondrial dysfunction, leading to cell apoptosis and contributing to many neurodegenerative diseases, including PD (Cui et al., 2004). H2O2 generates hydroxyl radicals, which are highly toxic and can cause serious damage to cells, leading to apoptosis or necrosis (McKeague et al., 2003; Minjie et al., 2005). Hence, the removal of excess ROS and the inhibition of its production may have a protective effect on cell death caused by oxidative stress. Thus, in this study, H2O2 was used to stimulate PC12 cells to explore whether COA-Cl could prevent H2O2-stimulated ROS generation and the resulting oxidative stress. Our data confirmed that H2O2 markedly increases intracellular ROS production, which initiates DNA and mitochondrial damage, resulting cell apoptosis (Rhee, 1999; Ding et al., 2016). However, treatment with COA-Cl suppressed the production of intracellular ROS in H2O2-treated cells (Fig. 3), suggesting that COA-Cl acts as an anti-apoptotic agent by suppressing ROS under H2O2-induced neurotoxic conditions. Collectively, our data strongly suggest that COA-Cl promotes PC12 cells survival by decreasing the amount of ROS, which, in turn, preserves mitochondrial activity.
Antioxidant enzymes, including SOD, play a major role in ROS scavenging and preventing the damage caused by oxidation. Overexpression of SOD protects against oxidative stress-induced cell death and injury (Kiningham et al., 1999). As previously shown, H2O2 likely provokes astrocyte death, in part, through a reduction in the activity of the antioxidant enzymes SOD and catalase (Sokolova et al., 2001; Lopez et al., 2007). Thus, we hypothesized that COA-Cl-induced neuroprotection is mediated through a modulation of antioxidant enzymes. Indeed, it was found that H2O2 decreased the activity of SOD (Fig. 6), and that this effect was reversed by COA-Cl, suggesting that the neuroprotective effect of COA-Cl is also related to its antioxidant effects. A previous study showed that COA-Cl could effectively reduce the oxidative stress marker 8-OHdG in a rat intracerebral hemorrhage model (Lu et al., 2016). Consistent with this previous finding, the present data strongly suggest that COA-Cl can protect against PD-related neuronal injury by enhancing the antioxidant status via a lowering of the level of ROS and inhibiting the production of oxidative enzymes. It has been shown that expression of SOD in cultured glial cells depends of protein kinase C activation (Huang et al., 2001). Hence, we suggest that COA-Cl is able to stimulate SOD activity in PC12 cells possibly through activation of protein kinase C; however, further investigation is needed to determine how COA-Cl activates SOD.
We also examined the effects of COA-Cl on PC12 cell apoptosis induced by H2O2 and 6-OHDA. Previous work has found that H2O2 and 6-OHDA induce apoptosis in PC12 cells (Jiang et al., 2003; Hanrott et al., 2006; Hadipour et al., 2020). Apoptosis occurs by a series of molecular events, including an up-regulation of Bcl-2, a down-regulation of Bax, decreased cytoplasmic release of cytochrome C, and reduced cleavage of caspases 9 and 3. For example, Bcl-2 inhibits apoptosis (Tsujimoto, 1998), whereas Bax promotes apoptosis (Lindenboim et al., 2000. Therefore, the ratio of Bcl-2 to Bax plays a pivotal role in cell survival and death. We hypothesized that COA-Cl could effectively attenuate the apoptosis caused by H2O2 and 6-OHDA via an increase in the Bcl-2/Bax ratio and an inactivation of caspase-3. Thus, we investigated if COA-Cl could attenuate apoptosis in H2O2-and 6-OHDA-treated PC12 cells. As shown in Fig. 4, both H2O2 and 6-OHDA decreased the Bcl-2/Bax expression ratio in PC12 cells, indicating that H2O2 and 6-OHDA are able to stimulate mitochondria-dependent apoptosis. However, COA-Cl markedly increased the Bcl-2/Bax ratio (Fig. 4B,D), suggesting that the anti-apoptotic effect of COA-Cl may be associated with its modulation of the expression of the Bcl-2 family. In addition, we evaluated apoptotic cells using TUNEL assays. It was found that COA-Cl significantly decreased the percentage of TUNEL-positive cells (Fig. 5A,B), suggesting that COA-Cl consistently reduces apoptosis in PC12 cells. Our results suggest that the H2O2- and 6-OHDA-induced decrease in the Bcl-2/Bax ratio alters mitochondrial membrane permeability, which, in turn, activates the caspase-3 cascade, leading to apoptosis. Several studies have reported that H2O2 induces apoptosis through activation of caspase-3 in vitro (Juknat et al., 2005; Shin et al., 2009; Matsura et al., 1999; Wu et al., 2011). Thus, we investigated the effects of COA-Cl on H2O2-induced caspase-3 activation in PC12 cells. Consistent with previous findings, the present results showed that H2O2 induces caspase-3 cleavage in PC12 cells (Fig. 5C). However, COA-Cl attenuated caspase-3 activity, indicating that the protective effect of COA-Cl can be accounted for by an inhibition of caspase-3 activity, likely resulting from oxidative stress suppression. These findings indicate that COA-Cl suppression of the Bcl-2 family in PC12 cells is mediated, at least in part, through the caspase 3-dependent apoptosis pathway. COA-Cl-induced attenuation of the apoptotic and necrotic processes induced by H2O2 is mostly in line with the findings from the model of 6-OHDA-induced cell damage. In combination, these results provide the first evidence that COA-Cl can protect against H2O2- and 6-OHDA-induced cell death, possibly through the induction of antioxidant enzymes and a regulation of oxidative stress, mitochondrial dysfunction, and apoptotic events.
In conclusion, the novel finding of the current study is that COA-Cl exerts neuroprotective effects against H2O2- and 6-OHDA-induced neuronal injury in PC12 cells. The protective effects of COA-Cl may be mediated by preventing oxidative stress and neuronal apoptosis. On the basis of these findings, COA-Cl could be a plausible therapeutic agent for the treatment of PD. However, further research is needed to verify and explore potential mechanisms underlying these findings.