The Effects of Psilocybin on Lipopolysaccharide-Induced Inflammation in THP-1 Human Macrophages
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The paper cannot be accepted without disclosing the source of psilocybin, how it was obtained and the related chemical characterization. Was it synthetic? Which kind of synthesis was used? How was it purified? What was the purity?
If on the other hand was from natural origine, the extraction method must be reported as well as the chemical characterization to ensure purity especially in respect of other tryptamines that are likely to be copurified and can play a role in the reported anti-inflamatory responce.
Author Response
Answer: The information regarding the source and purity of psilocybin has been included in the paper. It should be mentioned that psilocybin used in this study was synthetic. The following certificate of analysis verifies its high-performance liquid chromatography (HPLC) grade and purity level of 99.12%. However, the specific details or steps involved in the synthesis process are not disclosed or provided. The certificate is attached in the letter.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
Since methods and results look appropriate and promising, I believe the manuscript has a good perspective, but it is in the draft form currently. There are some problems, which must be solved before it is considered for publication.
1.The figures in your paper are a bit blurry. Please consider replacing them with clearer ones.
2.Relevant research background needs to be added to the introduction. You should also present an introduction to the pathways involved in the experiment.
3.You need to explain your simulation results in detail and why you got such results.
In figure2, after treating the cells,10 μM and 15 μM psilocybin reduced the levels and 5μM psilocybin increased the level. Please explain the reasons for this result.
4.The conclusions need more content, and authors are advised to highlight important findings.
5.Please explain further the design of the experimental method.
The 96-well plate has a cell culture capacity of 106 per well, far exceeding the number of cells cultured in a conventional 96-well plate. Please explain the experimental methodology further.
Comments on the Quality of English Language
There are no obvious word misuse or grammatical errors in the essay. Please kindly proofread the article based on its content.
Author Response
1.The figures in your paper are a bit blurry. Please consider replacing them with clearer ones.
Answer: Some figures have been replaced with clearer ones.
2.Relevant research background needs to be added to the introduction. You should also present an introduction to the pathways involved in the experiment.
Answer: Some additions have been made to the introduction in this regard. It is important to highlight that the exploration of the pharmaceutical properties of psilocybin is an emerging scientific field, resulting in a scarcity of papers specifically addressing the anti-inflammatory impact of psilocybin.
3.You need to explain your simulation results in detail and why you got such results.
In figure2, after treating the cells,10 μM and 15 μM psilocybin reduced the levels and 5μM psilocybin increased the level. Please explain the reasons for this result.
Answer: The following explanation has been added to the discussion section of the paper.
“In general, the impact of psilocybin on the production of proinflammatory cytokines in LPS/LA-induced macrophages can be complex, as evidenced by our experimental results. The findings suggest that the effect of psilocybin on the production of proinflammatory cytokines is dose dependent. For instance, 10 and 15 mM concentrations of psilocybin decreased the levels of COX-2 and IL-1b proteins, while 5 mM increased the level of these proteins. Additionally, 10 mM psilocybin reduced the level of IL-6, but 15 mM increased the level of this protein. Moreover, while 10 mM psilocybin did not impact the level of Pro-TNFa, 5 and 15 mM concentrations increased its level. The dose-dependent effects of psilocybin on the production of proinflammatory cytokines could be attributed to its complex interactions with the immune system. Additional research is required for a comprehensive understanding of the underlying mechanisms involved.”
4.The conclusions need more content, and authors are advised to highlight important findings.
Answer: We implemented adjustments accordingly. In this regard, Figure 9 was relocated to the dissection section, accompanied by an additional explanation highlighting the crucial findings of the study.
5.Please explain further the design of the experimental method.
Answer: An explanation regarding the design of experimental method has been added to the materials and methods. It is as follows:
“In this study, we examined the effects of three different doses of psilocybin on both LPS-stimulated and unstimulated THP-1 macrophages. The objective was to understand the impact of psilocybin on general inflammatory responses and on LPS-mediated inflammatory responses in THP-1 macrophages. To ensure that psilocybin's activity does not interfere with LPS induction, we pre-treated the cells with psilocybin before the induction with LPS. It is important to highlight that we conducted optimization studies to determine the optimal dose of LPS and the duration of LPS and ATP incubation. Additionally, since ATP is mainly involved in the assembly of the NLRP3 inflammasome, we studied the response of NLRP3, Pro-IL-1β, and IL-1β to psilocybin in LPS+ATP (LA)-induced THP-1 macrophages whereas for other proteins and gene expression studies, we used LPS-induced THP-1 macrophages.”
The 96-well plate has a cell culture capacity of 106 per well, far exceeding the number of cells cultured in a conventional 96-well plate. Please explain the experimental methodology further.
Answer: Thank you for your comment. It has been revised to 1*10^4/ml in the paper. Additionally, an explanation has been incorporated into the Materials and Methods section as follows:
“It is worth mentioning that we seeded a higher number of THP-1 monocyte cells in each well. This decision is based on the fact that THP-1 monocytes, upon differentiation into macrophages, acquire macrophage-like properties, including the loss of their ability to undergo cell division”
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
In the M&M section the info regarding who synthesized the psilocybin are still missing.
These info needs to be included, for culture plates you include even the catalog number and for something so important nothing is disclosed.
Moreover the certificate of analysis is three and a half years old.
At least you should include the storage conditions you used.
Author Response
The information about psilocybin was in the Methods. We wrote:
"....psilocybin (Product Number: 520-52-50, Applied Pharmaceutical Innovation, Edmonton, AB, Canada), .... Note that the psilocybin employed in this study was synthesized, purified by HPLC and its purity, as per HPLC analysis, was found to be 99.12%."
We added that after purchase, psilocybin was stored at 20C in a dark place. Please note that the original research was performed nearly two years ago.
Reviewer 2 Report
Comments and Suggestions for Authors
The study can be accepted at this version.
Author Response
Thanks.
