Neuroprotective Properties of Rutin Hydrate against Scopolamine-Induced Deficits in BDNF/TrkB/ERK/CREB/Bcl2 Pathways

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1.-Please include the following bibliographical citation in the introduction:

Çelik, H., Kandemir, F.M., Caglayan, C. et al. Neuroprotective effect of rutin against colistin-induced oxidative stress, inflammation and apoptosis in rat brain associated with the CREB/BDNF expressions. Mol Biol Rep 47, 2023–2034 (2020). https://doi.org/10.1007/s11033-020-05302-z

2.-For subsequent work, although the results are clear, I suggest not overexposing the membrane to luminol.

Author Response

Comments 1: Please include the following bibliographical citation in the introduction:

Çelik, H., Kandemir, F.M., Caglayan, C. et al. Neuroprotective effect of rutin against colistin-induced oxidative stress, inflammation and apoptosis in rat brain associated with the CREB/BDNF expressions. Mol Biol Rep 47, 2023–2034 (2020). https://doi.org/10.1007/s11033-020-05302-z

Response 1: Thank you for your valuable feedback on our study. Based on your suggestion, the reference you recommended was considered valuable for supporting our research and has been added to the introduction, Line 70-71.

Comments 2: For subsequent work, although the results are clear, I suggest not overexposing the membrane to luminol.

Response 2: As you suggested, we will adjust the exposure time in future experiments to achieve the best results.

Reviewer 2 Report

Comments and Suggestions for Authors

Your study showed RH increased memory and fEPSTP in scopolamine-induced mouse. Also, RH increased the levels of BDNF/TrkB/ERK/CREB singaling.  I have a few questions.

1. in Figure 2, fEPSP activity increased by RH treatment in normal condition. Also in normal mouse (not scopolamie injection), RH administraion is increased fEPSP activity. Does RH have inducer effect to activate fEPSP? If RH treatment has high fEPSP acivity,  Isn't there a possibility that this could cause hyperactivity behavior or ADHD due to it's high fEPSP?

2. It would be good to clarify whether RH regulates BDNF/TrkB signals as an acetylcholine agonist or whether RH direct regulates signals by confirming the change in acetylcholine. It seems likely that RH is similar to acetylcholine or may act as an inducer.

Author Response

Comments 1: in Figure 2, fEPSP activity increased by RH treatment in normal condition. Also in normal mouse (not scopolamie injection), RH administraion is increased fEPSP activity. Does RH have inducer effect to activate fEPSP? If RH treatment has high fEPSP acivity,  Isn't there a possibility that this could cause hyperactivity behavior or ADHD due to it's high fEPSP?

Response 1: Our results indicate that RH treatment at various dosages (1, 10, and 100 μM) significantly increases fEPSP activity in the CA1 region, both under normal conditions and with scopolamine, which is known to impair long-term potentiation. Enhanced fEPSP activity suggests that RH may facilitate synaptic plasticity, which is crucial for learning and memory processes. fEPSP measurements in the hippocampus primarily reflect synaptic strength and plasticity, which are related to cognitive functions such as memory formation and retrieval. The CA1 region, in particular, is essential for spatial memory and contextual learning. Hyperactivity and ADHD are complex neurodevelopmental disorders influenced by a variety of brain regions, neurotransmitter systems like dopamine and norepinephrine, and genetic factors. They are not solely attributable to synaptic activity in the hippocampus. Although RH increases fEPSP activity in the hippocampus, this does not necessarily translate to global neuronal hyperexcitability. The hippocampus is just one part of the brain, and increased synaptic activity here doesn't directly imply heightened excitability in other regions linked with hyperactivity or ADHD.

Comments 2: It would be good to clarify whether RH regulates BDNF/TrkB signals as an acetylcholine agonist or whether RH direct regulates signals by confirming the change in acetylcholine. It seems likely that RH is similar to acetylcholine or may act as an inducer.

Response 2: we were impressed by your insightful comment. Measuring ACh/AChE levels would have clearly determined whether RH acts directly on acetylcholine or as an agonist. However, we can verify this through several references even without conducting the measurements ourselves. According to the reference below, rutin reduces acetylcholinesterase activity and inhibits it in certain brain regions, while increasing choline acetyltransferase, thereby elevating acetylcholine levels. Based on this, it seems likely that rutin regulates BDNF/TrkB signaling as an acetylcholine agonist. We have added this information to the discussion section.

The references are as follows:

1. Anesti, M., Stavropoulou, N., Atsopardi, K., Lamari, F. N., Panagopoulos, N. T., & Margarity, M. (2020). Effect of rutin on anxiety-like behavior and activity of acetylcholinesterase isoforms in specific brain regions of pentylenetetrazol-treated mice. Epilepsy & behavior : E&B, 102, 106632. https://doi.org/10.1016/j.yebeh.2019.106632
2. Xianchu, L., Huan, P., Kang, L., Beiwang, D., & Ming, L. (2022). Protective effect of rutin against diabetes-associated cognitive decline in rats. Pakistan journal of pharmaceutical sciences, 35(3), 769–775.

Reviewer 3 Report

Comments and Suggestions for Authors

                                                                                                       Date: 08-Sept-2024

Journal: Neurology International (ISSN 2035-8377)

Manuscript ID: neurolint-3185680

Type: Article

Title: Neuroprotective properties of Rutin hydrate against scopolamine-induced learning and memory deficits in rats

Authors: Inturu Sreelatha, Ga-Young Choi, In-Seo Lee, Omkaram Inturu, Hyun-Sook Lee, Yea-Na Park, Cheol-Won Lee, Sungho Maeng, Ji-Ho Park *

Brief Summary of the article

The study suggests that rutin hydrate (RH) could be a promising therapeutic agent for Alzheimer's disease (AD). RH improved learning and memory, enhanced synaptic plasticity, and modulated neurotrophic factors in rats. Further research is needed to fully understand its mechanisms of action and evaluate its efficacy and safety in human clinical trials. The overall study is well planned and implemented which reflects throughout the manuscript. However, I have a few general suggestions.

11. The article title appears overly broad. I recommend the authors incorporate specific pathway markers like BDNF/TrkB/ERK/CREB/Bcl2 in the title.

  2. Studies conducted in the past report the neuroprotective effects of rutin hydrate (RH) against neurodegeneration, neuroinflammation, and AD. So, the authors should report how the current study differs from the previous studies and the future scope of the current research. (DOI: https://doi.org/10.1016/j.neuroscience.2012.02.046 ; https://doi.org/10.3390/ijms24054863 ; https://doi.org/10.1016/j.yebeh.2019.106632 ; https://link.springer.com/article/10.1007/s11064-016-1863-7 ; 10.1186/s12974-021-02182-3 )

33. What is the rationality of selecting a single dose of RH for efficacy study? The study should have been more complete if RH would have been selected at three or at least two different doses.

  4. Please describe which region of the hippocampus was affected due to scopolamine and how RH increased the neurogenesis or alleviates neuroinflammation in that region of the hippocampus.

55. Please explain in the manuscript the rationality of using Adult male Sprague-Dawley (SD) rats in the study and why were female rats neglected.

66. Throughout the manuscript the authors have written as Fig. 1A, 1B, however in some sentences of the result section such as Page 9, Line-389, 390 etc, shows as Figure 4C, 4D. Please maintain a uniform consistency throughout the manuscript.  

 7. Page-10, Figure- 4, L-399, L-453, L-160, L-350, L-373, L- 423, Rutin hydrate is written as routine hydrate. Please correct the same throughout the manuscript.

88. Immunoblot images in the supplementary should be marked with ladder, and represented according to journals requirements. Please refer to the article to article to implement the changes. (https://doi.org/10.1038/s41401-020-00539-7 )

Comments on the Quality of English Language

The manuscript requires minor grammatical and English corrections. 

1.     Throughout the manuscript the authors have written as Fig. 1A, 1B, however in some sentences of the result section such as Page 9, Line-389, 390 etc, shows as Figure 4C, 4D. Please maintain a uniform consistency throughout the manuscript.   

2.     Page-10, Figure- 4, L-399, L-453, L-160, L-350, L-373, L- 423, Rutin hydrate is written as routine hydrate. Please correct the same throughout the manuscript.

Author Response

Comments 1: The article title appears overly broad. I recommend the authors incorporate specific pathway markers like BDNF/TrkB/ERK/CREB/Bcl2 in the title.

Response 1: We modified the title by adding pathway markers.

“Neuroprotective Effects of Rutin Hydrate Against Scopolamine-Induced Deficits in BDNF/TrkB/ERK/CREB/Bcl2 Pathways”

Comments 2: Studies conducted in the past report the neuroprotective effects of rutin hydrate (RH) against neurodegeneration, neuroinflammation, and AD. So, the authors should report how the current study differs from the previous studies and the future scope of the current research. (DOI: https://doi.org/10.1016/j.neuroscience.2012.02.046 ; https://doi.org/10.3390/ijms24054863 ; https://doi.org/10.1016/j.yebeh.2019.106632 ; https://link.springer.com/article/10.1007/s11064-016-1863-7 ; 10.1186/s12974-021-02182-3 )

Response 2: This study is different from previous research by focusing on the use of electrophysiological methods to induce long-term potentiation (LTP) and specifically assess changes in fEPSP activity. While past studies have reported the neuroprotective effects of RH against neurodegeneration, neuroinflammation, and Alzheimer's disease, our research emphasizes synaptic plasticity and its relationship with LTP. By concentrating on fEPSP activity, we aim to provide new insights into how RH influences synaptic plasticity, which is a key aspect of cognitive function that has not been the primary focus of the studies cited. This approach allows us to explore the mechanistic details of how RH affects synaptic plasticity and could contribute to a deeper understanding of its potential therapeutic benefits.

Comments 3: What is the rationality of selecting a single dose of RH for efficacy study? The study should have been more complete if RH would have been selected at three or at least two different doses.

Response 3: We chose RH-treated groups given a dose of 100 mg/kg/day by referring to several studies. Even in studies using various concentrations (50, 100, 200 mg/kg), the effect at RH 100 mg/kg was the most outstanding compared to other concentrations. Since our study aimed to determine whether RH could improve SCOP-induced impairment rather than comparing different concentrations, we focused on a single dose. Following your suggestion, we agree that investigating the effects of different RH concentrations in a follow-up study would be valuable.  

The references are as follows:

1. Ismail, Topal., Aslı, Özbek, Bilgin., Ferda, Keskin, Cimen., Nezahat, Kurt., Zeynep, Suleyman., Yasin, Bilgin., Adalet, Ozcicek., Durdu, Altuner. (2018). The effect of rutin on cisplatin-induced oxidative cardiac damage in rats.. Anatolian Journal of Cardiology, 20(3):136-142. doi: 10.14744/ANATOLJCARDIOL.2018.32708
2. C, Perez, Guerrero., M., J., Martin., E., Marhuenda. (1994). Prevention by rutin of gastric lesions induced by ethanol in rats: role of endogenous prostaglandins.. General Pharmacology-the Vascular System, 25(3):575-580. doi: 10.1016/0306-3623(94)90217-8

Comments 4: Please describe which region of the hippocampus was affected due to scopolamine and how RH increased the neurogenesis or alleviates neuroinflammation in that region of the hippocampus.

Response 4: This study focused on the effects of scopolamin and RH in the CA1 region of the hippocampus, specifically examining the impact on LTP, a key mechanism for synaptic plasticity and memory formation. Scopolamine is an anticholinergic drug that blocks acetylcholine receptors, leading to cognitive impairments by disrupting cholinergic signaling. The CA1 region of the hippocampus is highly sensitive to acetylcholine and plays a crucial role in memory and spatial learning. In this study, scopolamine reduced the fEPSP in the CA1 region, thereby impairing synaptic plasticity and LTP induction, which is a cellular mechanism critical for learning and memory. This suggests that scopolamine causes deficits in synaptic transmission and cognitive functions by interfering with hippocampal activity, particularly in the CA1 region.

The results of this study suggest that RH may enhance neurogenesis and reduce neuroinflammation in the CA1 region of the hippocampus by modulating the BDNF/TrkB/ERK/CREB/Bcl2 signaling pathway. Given the CA1 region's key role in synaptic plasticity, memory formation, and learning, RH appeared to upregulate BDNF and its receptor TrkB, which are important for promoting neurogenesis and supporting neuronal survival. This activation of the BDNF/TrkB pathway initiates downstream cascades involving ERK and CREB, both critical for boosting synaptic plasticity and memory-related processes. In addition, RH seems to offer neuroprotection by increasing the expression of Bcl-2, an anti-apoptotic protein that helps prevent neuronal cell death. Through these mechanisms, RH not only encourages the survival and differentiation of new neurons but also dampens neuroinflammation, as neurogenesis and synaptic activation are closely linked to inflammatory processes in the hippocampus. 

Comments 5: Please explain in the manuscript the rationality of using Adult male Sprague-Dawley (SD) rats in the study and why were female rats neglected.

Response 5: The choice to use adult male Sprague-Dawley rats in this study was made to reduce variability associated with hormonal fluctuations, which could affect the interpretation of hippocampal function and neuroplasticity. Female rats experience hormonal cycles that could introduce additional variables, potentially complicating the analysis of Rutin Hydrate (RH) treatment effects on fEPSP activity and long-term potentiation (LTP). By using male rats, we aimed to ensure greater consistency and control, allowing for clearer insights into the specific impacts of RH on synaptic plasticity.

We added this description to the Animals part of Materials and Methods section, line 141-143.

Comments 6: Throughout the manuscript the authors have written as Fig. 1A, 1B, however in some sentences of the result section such as Page 9, Line-389, 390 etc, shows as Figure 4C, 4D. Please maintain a uniform consistency throughout the manuscript.  

Response 6: We modified Figure 4C, 4D to Fig. 4C,4D. And the manuscript has been revised consistently overall.

Comments 7:. Page-10, Figure- 4, L-399, L-453, L-160, L-350, L-373, L- 423, Rutin hydrate is written as routine hydrate. Please correct the same throughout the manuscript.

Response 7: We modified it to Rutin hydrate in the manuscript.

Comments 8: Immunoblot images in the supplementary should be marked with ladder, and represented according to journals requirements. Please refer to the article to article to implement the changes. (https://doi.org/10.1038/s41401-020-00539-7 )

Response 8: We inserted the images with molecular weight marker in the supplementary material and made revisions based on the editor's feedback.