Without a Hesperetin-only control group, how can you definitively rule out that the observed restoration of SIRT1/Nrf2 pathway components is not due to Hesperetin independently elevating these pathways in a normal physiological state? Could the effects seen in the ADHD/HSP groups be a combination of blocking MSGL’s effects and an inherent, baseline-upgrading action of Hesperetin?
Consequently, wouldn’t it be more accurate to conclude that Hesperetin “prevented the MSGL-induced downregulation” of the SIRT1/Nrf2 pathway, rather than claiming it caused “upregulation”? The current language implies an enhancing effect that the experimental design cannot verify.
Beyond the valid point raised in the comment, a more fundamental error in the methodology and results reporting significantly compromises the study’s reliability and internal consistency. The study conflates and misrepresents the Keap1 protein’s functional role throughout the manuscript.
1. In the Abstract and Introduction, the authors state they are examining the “SIRT1/Nrf2/Keap1/OH-1 signaling” pathway, implying Keap1 is a positive, activated component like SIRT1 and Nrf2. For example, the abstract concludes that HSP “upregulated SIRT1/Nrf2/Keap1/OH-1 proteins.” This is biologically incorrect.
2. Keap1 (Kelch-like ECH-associated protein 1) is well-established in molecular biology as the negative regulator of Nrf2. Under normal conditions, Keap1 binds to Nrf2 in the cytoplasm and targets it for proteasomal degradation. Under oxidative stress, this interaction is disrupted, allowing Nrf2 to translocate to the nucleus and activate antioxidant genes like HO-1. Therefore, a therapeutic agent’s success would be associated with the inactivation or downregulation of Keap1’s repressive function, not its “upregulation.”
3. The results themselves inadvertently reveal this error.
+ Table 1 (Protein Levels): It reports that Keap1 protein levels are not measured. This is a major omission, as it is a central protein in the hypothesized pathway.
+ Figure 7 (Gene Expression): This figure shows the mRNA expression of Keap1. The data indicates that the ADHD model group has higher Keap1 mRNA expression compared to the control, and Hesperetin treatment lowers it back towards the control level.
+ This gene expression data aligns perfectly with the established mechanism: the toxic insult (MSGL) may increase Keap1, suppressing Nrf2, and the protective treatment (Hesperetin) counteracts this by reducing Keap1, thereby freeing Nrf2.