The finding from the cell viability assayThe finding from the cell viability assay

The main finding of this study is that RMT and n-3 PUFAs synergistically reverses oxidative and neuro-inflammatory pathophysiology and protects the neuronal cells from further oxidative stress damage. To our knowledge, this is the first report to demonstrate the underlying synergistic mechanism of RMT and n-3 PUFAs EPA and DHA counter acting by preventing and rescuing the neurons from the pathogenic effect of oxidative stress.

 

Consistent with the previous studies 51,52, cell viability of SH-SY5Y cells was likely not affected with lower concentrations of H2O2; on the other hand, high-level H2O2 initiated cell death. In addition, our finding from the cell viability assay infers significant synergism existing between RMT and EPA by stimulating viability and providing an additional insight on their preventive and therapeutic effects. Several studies ascertain the specific mode of action of EPA promoting neuronal survival, which further supports our finding 53-55. Interestingly, the present data also shows that DHA in combination with FLX were able to improve the viability in the rescue group through inhibition of apoptosis. DHA treatment is found to be differentially affecting the viability with different cell lines 56-58. Previous studies have shown protective actions of DHA 59-61 and FLX 62,63 against neuronal injury by various mechanisms supporting our results.

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Generally, the fate of the neuronal cells, directly and indirectly, is modulated by the accumulation of ROS during oxidative stress. In our study, a significant decrease in ROS was observed with the cells pretreated with RMT and EPA demonstrates their ROS inhibitory effect. This initial blockage of ROS generation might offer significant protection of neurons by RMT and EPA indicates their anti-inflammatory and anti-oxidative effects. Interestingly, DHA showed ROS inhibiting effect with the rescue group which may elucidate its neuroprotective nature. However, we still do not know precisely the therapeutic dynamics of RMT and n-3 PUFAs integrated into ROS inhibition involved in cell survival. On the other hand, an increased ROS level was seen with H2O2 pretreatment followed by FLX and with combination of n-3 PUFAs, needs further experiments to investigate this paradoxical effect.

 

Exogenous H2O2 and generated ROS have been reported to be a potent stimulus for NF-?B activation in the cytoplasm and translocation into the nucleus 64. Our results of immunocytochemistry showed, RMT, FLX and n-3 PUFAs induced and translocated NF??B from the cytoplasm into the nucleus in response to H2O2 stimulation. The activation of NF-?b seems to be associated with mechanisms of antidepressant effects of several traditional antidepressants (e.g., specific serotonin reuptake inhibitors and tricyclic antidepressants) which supports our study 65-67.  Our findings show that n-3 PUFAs triggers the activation of NF-?B in response to exogenous oxidative stimuli is similar to our previous study 35, adding up a strong evidence of their anti-inflammatory status. Interestingly, our study also shows that EPA and DHA along with RMT activated PPAR? and suppressed NF-?B, this crosstalk seem to be apparent reason in promoting the cell survival from oxidative stress and followed inflammatory cascade. As n-3 PUFAs being a PPAR? ligand, they antagonize the pro-inflammatory capability of NF-?B and promote cell survival 68.

 

Our results on antioxidant enzymes demonstrates a cause in imbalance of antioxidant defense system caused by exogenous H2O2 paved way to a reduction of the activity of CAT and increased SOD1 and SOD2 activity as supported by previous reports 69,70. Furthermore, RMT and n-3 PUFAs enhanced the antioxidant enzyme activities of CAT and SOD1 and SOD2, exhibits their neuroprotective roles against the oxidative damage caused by H2O2 and ROS production. This finding suggest that RMT and n-3 PUFAs ameliorates H2O2 induced neuronal damage via its antioxidant defense mechanism and might be used to treat oxidative stress mediated neuronal disorders. The results of this study also show that the already activated NF-?B would have led to the induction of SOD2 protein levels preventing apoptosis and neuronal damage 71,72. Additionally, PPAR? is also said to accelerate the removal of excessive superoxide by increasing SOD transcription 50. Therefore, prevention of neuronal cell death by RMT and n-3 PUFAs might be partly mediated via a decrease of intracellular ROS production, activation of NF-?B and PPAR? and recovery of anti-oxidant enzymes.

 

Tyrosine hydroxylase (TH) a marker of SH-SY5Y cell differentiation is also known to generate ROS and have been proposed to contribute to the neurodegeneration of dopaminergic neurons 73. In our current study, an increase in TH expressions was observed after pretreatment with RMT and n-3 PUFAs concordant with previous studies with the SH-SY5Y and other cell lines 74,75. Similarly, a significant increase in c-Fos belonging to AP-1 family of transcription factors was significantly increased with RMT and n-3 PUFAs prevented and rescued the neuronal cells from apoptosis following oxidative stress. This current finding is further supported by previous studies with RMT 76, FLX 77,78, n-3 PUFAs 79,80. Altogether, these results with TH and c-Fos proteins help us to explain the cellular protective properties of RMT and n-3 PUFAs against oxidative stress induced neuronal degeneration.

 

Our results undoubtedly reveal that RMT showed preventive mechanism; whereas, FLX and DHA possess rescue mechanism, but EPA showed both rescue and preventive effect by significantly increasing the cell viability of the neuronal cells and marked changes with the scrutinized biomarkers. Our results is supported by a recent evidence showing EPA to be more effective than DHA, in reversing damage caused by oxidative stress 81. Further, to back up our results, clinical studies related to n-3 PUFAs, provide evidences that DHA monotherapy show conflicting antidepressant effects 82,83. Our present work also, show that RMT combined with EPA has a positive effect on protecting the cells from oxidative damage, than combined with DHA. Interestingly, recent meta-analyses suggested that EPA has better antidepressant effects than DHA in combination with antidepressant medications which supports our finding 84,85.  Within the framework of our results we observe that EPA and DHA have differential interaction with RMT. However, RMT and n-3 PUFAs EPA and DHA has yet not gained the status of an antidepressant, further scrutinization is awaited even if several studies have proven their antidepressant like properties.

 

Collectively, our study elucidates how RMT and EPA synergistically are able to reverse oxidative and neuro-inflammatory pathophysiology and protect the neuronal cells from oxidative damage. This study provides a strong pre-clinical evidence of RMT/ EPA combination to be translated to the clinical setting is urgently warranted.