Activating SRC/MAPK signaling via 5-HT1A receptor contributes to the effect of vilazodone on improving thrombocytopenia

Reviewer #1 (Public Review):

Summary:
This is well-performed research with solid results and thorough controls. The authors did a good job of finding the relationship between the 5-HT1A receptor and megakaryocytopoiesis, which demonstrated the potential of vilazodone in the management of thrombocytopenia. The paper emphasizes the regulatory mechanism of 5-HT1A receptor signaling on hematopoietic lineages, which could further advance the field of thrombocytopenia for therapeutic purposes.

Strengths:
This is comprehensive and detailed research using multiple methods and model systems to determine the pharmacological effects and molecular mechanisms of vilazodone. The authors conducted in vitro experiments using HEL and Meg-01 cells and in vivo experiments using Zebrafish and Kunming-irradiated mice. The experiments and bioinformatics analysis have been performed with a high degree of technical proficiency. The authors demonstrated how vilazodone binds to 5-HTR1A and regulates the SRC/MAPK pathway, which is inhibited by particular 5-HTR1A inhibitors. The authors determined this to be the mechanistic underpinning for the effects of vilazodone in promoting megakaryocyte differentiation and thrombopoiesis.

Weaknesses:
1. Which database are the drug test sets and training sets for the creation of drug screening models obtained from? What criteria are used to grade the results?

2. What is the base of each group in Figure 3b for the survival screening of zebrafish? The positivity rate of GFP-labeled platelets is too low, as indicated by the quantity of eGFP+ cells. What gating technique was used in Figure 3e?

3. In Figure 4C, the MPV values of each group of mice did not show significant downregulation or upregulation. The possible reasons for this should be explained.

4. The PPI diagram and the KEGG diagram in Figure 6 both provide a possible mechanism pathway for the anti-thrombocytopenia effect of vilazodone. How can the authors analyze the differences in their results?

5. 5-HTR1A protein expression is measured only in the Meg-01 cells assay. Similar quantitation through western blot is not shown in other cell models.

Reviewer #2 (Public Review):

Summary:
The authors tried to understand the mechanism of how a drug candidate, VLZ, works on a receptor, 5-HTR1A, by activating the SRC/MAPK pathway to promote the formation of platelets.

Strengths:
The authors used both computational and experimental methods. This definitely saves time and funds to find a useful drug candidate and its therapeutic marker in the subfield of platelets reduction in cancer patients. The authors achieved the aim of explaining the mechanism of VLZ in improving thrombocytopenia by using two cell lines and two animal models.

Weaknesses:
Only two cell lines, HEL and Meg-01 cells, were evaluated in this study. However, using more cell lines is really depending on the workflow and the grant situations of the current research team.