Review for 10.1186/s12876-025-03863-6 (2025)

Title: Comparative Analysis of #-Estradiol and Testosterone on Lipid Droplet Accumulation,
and Regulatory Protein Expression in Palmitate/Oleate-Induced Fatty HepG2 Cells
Version: 0 Date: 10 Feb 2025
Reviewer:  Anwar Ashraf Abouelnasr
Reviewer's report:
Dear Authors, please address the following points below
-The introduction could be further enhanced by expanding on how #-estradiol and testosterone
are hypothesized to interact with the various key proteins regulating LD metabolism (PLIN2,
ATGL, CGI-58, etc.). A brief discussion of potential molecular pathways or signaling
mechanisms through which these hormones may influence lipid accumulation and metabolism
in hepatocytes would add depth to the rationale for this study.
-While the introduction mentions conflicting reports on the impact of testosterone on fatty liver
disease, it would be helpful to outline some of these discrepancies or findings in more detail.
This would provide a clearer context for the reader about the uncertainty in the field and the
need for this comparative analysis.
-While the methodology clearly states the use of the MTT assay to determine IC50 values for
palmitate and oleate, it would be helpful to provide actual numerical values for the IC50 doses
of each fatty acid. This would give readers a clearer understanding of the concentration ranges
used in the experiments.
-A bit more detail on the specific experimental groups and the rationale behind the selection of
the doses of #-estradiol and testosterone would be useful. For example, the rationale for selecting
cell viability levels (V40, V50, and V60) for hormone treatment could be expanded. Is this based
on prior research or an exploratory factor to find the optimal treatment conditions?
-In the gene expression analysis, the manuscript mentions specific forward and reverse primers
used for RT-qPCR. Including a reference to Table 1 for primer sequences would improve clarity.
Additionally, it would be beneficial to provide more details on why these particular genes were
chosen. While PLIN2, ATGL, CIDE, and CGI-58 are critical regulators of lipid metabolism, a
brief explanation of why they were selected (e.g., based on their known interactions with sex
hormones or involvement in NAFLD pathogenesis) could be added to justify their inclusion.
-While the discussion provides a solid theoretical framework, there is room to clarify some of
the proposed mechanisms further. For example, while the role of PPAR# is discussed about
lipid droplet formation and fatty acid beta-oxidation, it would be helpful to provide more direct
evidence or hypotheses linking PPAR# activation to the observed effects of #-estradiol and
testosterone on PLIN2 and CIDEB expression. More mechanistic details could enhance the
reader’s understanding of how these hormones interact with lipid metabolic pathways at a
molecular level.
-The role of hormonal dose dependence, particularly regarding testosterone, is mentioned but not
fully explored. Given the finding that testosterone’s effect on genes like CGI-58 appears dose-
independent in steatotic cells, a deeper analysis of the molecular pathways involved would be
beneficial. Are there alternative signaling mechanisms or receptor interactions that might explain
this behavior? This aspect could be better unpacked, potentially by addressing the possibility of
non-classical receptor interactions or the involvement of other signaling molecules.
-The manuscript discusses the combined treatment of #-estradiol and testosterone but doesn't
fully explore the potential synergistic or antagonistic effects of these hormones. While you
suggest that #-estradiol may override testosterone’s effects, additional experimental data
or references could be included to support this hypothesis. For example, are there known
mechanisms by which these hormones may influence each other’s receptor binding or
downstream signaling pathways?