Inhibition of Phagocytosis by Silibinin in Mouse Macrophages

Round 1

Reviewer 1 Report

The authors concentrated their work on the inhibition of phagocytosis by silibinin using mouse macrophages as a model. 

The introduction is sufficient to understand the context of the study.

In the materials and methods section, it says "human phosphor-MAPK array", would it not be "phospho"?

Why use mouse macrophages? There are human cell lines, such as THP-1 cells, which can be differentiated into macrophages, or human PBMCs can be used. The choice of this model should be justified. 

for figure 5, it would be good to indicate how many times the manipulations were carried out and whether what is shown corresponds to one manipulation or the synthesis of several. The experiments were carried out with one hour of silibinin and 20 min of LPS. Why were these conditions chosen? Figure 1 shows that the cells change morphology later on. Why were protein activation kinetics not used? or have I missed something?

Author Response

Reviewer 1:

The authors concentrated their work on the inhibition of phagocytosis by silibinin using mouse macrophages as a model.

The introduction is sufficient to understand the context of the study.

Q: In the materials and methods section, it says "human phosphor-MAPK array", would it not be "phospho"?

A: We corrected the typographical error.

Q: Why use mouse macrophages? There are human cell lines, such as THP-1 cells, which can be differentiated into macrophages, or human PBMCs can be used. The choice of this model should be justified.

A: We used the mouse macrophage cell line, RAW264.7 cells, because the cells respond well by LPS. And we set up the system to investigate. We can further investigate with in vivo study with mice. For example ip injection of LPS and silibinin and check the peritoneal macrophages. We agree to the reviewer that human cells study will help to understand the effects of silibinin and make the results solid. We tried to use THP-1 cells, we could not differentiate the THP-1 monocyte to macrophages by PMA. So, we could not further study with THP-1 cells. The other cells like PBMC or BMDM will be another choice for further studies.

Q: for figure 5, it would be good to indicate how many times the manipulations were carried out and whether what is shown corresponds to one manipulation or the synthesis of several. The experiments were carried out with one hour of silibinin and 20 min of LPS. Why were these conditions chosen? Figure 1 shows that the cells change morphology later on. Why were protein activation kinetics not used? or have I missed something?

A: We previously tested the two MAPKs such as ERK1/2 and p38 and found out 20 min was peak of phospho-signal during 2 hrs kinetics. And the Human phospho-MAPK array kit data sheet showed the HeLa cell with UV 30 min and HepG2 with IL-1b 30 min. So we choose 20 min time point to check phosphorylation signal of the MAPKs. In Fig. 2 we showed that some cells starts to change the morphology. But massive changes were happen in 24 hrs. So the mechanism of the earlier MAPKs activation and morphological activation later on will be our further study subject.

Reviewer 2 Report

In their manuscript, Sun et al. determined the role of silibinin on LPS-dependent mouse macrophage activation. To achieve this, the authors used the murine macrophage cell line RAW 264.7. Using silibinin and LPS co-treatment, RAW264.7 cells showed a reduced LPS-activation. However, silibinin and LPS-treatment did not lead to cell death. Moreover, silibinin blocked macrophage phagocytosis of pHrodo green E. coli particles. Finally, the authors determined the effect of silibinin on LPS-induced activation of the MAP kinase pathway.

The manuscript might be interesting for the scientific community, but some concerns should be carefully addressed:

Figure 4: A quantification of the shown changes in morphology and phagocytosis activates should be provided with and without silbinin co-treatment.

Figure 5: How can the murine proteins be stained with an antibody panel developed towards human proteins? Please clarify!

Figure 6: It is mandatory to include silibinin in this Figure.

It would be nice to perform some of the experiments in primary macrophages, such as bone marrow derived or blood monocyte derived macrophages.

Author Response

Reviewer 2:

In their manuscript, Sun et al. determined the role of silibinin on LPS-dependent mouse macrophage activation. To achieve this, the authors used the murine macrophage cell line RAW 264.7. Using silibinin and LPS co-treatment, RAW264.7 cells showed a reduced LPS-activation. However, silibinin and LPS-treatment did not lead to cell death. Moreover, silibinin blocked macrophage phagocytosis of pHrodo green E. coli particles. Finally, the authors determined the effect of silibinin on LPS-induced activation of the MAP kinase pathway.

The manuscript might be interesting for the scientific community, but some concerns should be carefully addressed:

Q: Figure 4: A quantification of the shown changes in morphology and phagocytosis activates should be provided with and without silbinin co-treatment.

A: Although quantification of the morphological change and phagocytosis were suggested in Fig. 3 and 4, respectively, quantitative analysis will be important for the investigation of the M1 and M2 polarization. These analysis should be performed with the M1 and M2 marker analysis. This will be our future study. We added the following sentences.

“While silibinin effectively inhibited the macrophage activation, the morphological changes including M1 and M2-ploarization were also found in silibinin co-treated group (data not shown). To comprehensively investigate the effects of silibinin on macrophage differentiation, it would be beneficial to quantify both M1 and M2-polarized cells and assess markers associated with these polarization states.”

Q: Figure 5: How can the murine proteins be stained with an antibody panel developed towards human proteins? Please clarify!

A: Due to the similarity of the peptide sequences of human and mouse, some anti-human proteins detect mouse proteins too. The company raised antibodies against human phospho-kinases and checked cross reactivity. Some antibodies were confirmed that they cross react to mouse proteins, like AKT1, pan AKT, JNK2, JNKpan, p38a, p38r, p70S-6K, and ERK1. Some antibodies were not confirmed the cross-reaction including AKT2. We added following sentences to the results. “While some antibodies indicated cross-reactivity with mouse phospho-MAPK, and LPS stimulation notably induced phosphorylation, it's important to acknowledge the potential for nonspecific antibody detection. Therefore, we primarily analyzed the MAPK array results in terms of their broader impact on MAPK pathways rather than focusing solely on specific kinases.”

Q: Figure 6: It is mandatory to include silibinin in this Figure.

A: Thank you for the suggestion. We added the silibinin in the Fig. 6.

Q: It would be nice to perform some of the experiments in primary macrophages, such as bone marrow derived or blood monocyte derived macrophages.

A: We agree with the reviewer. We discussed the requirements of the other cells including THP1, PBMC, and BMDM.

Round 2

Reviewer 2 Report

All of my concerns have been adequately addressed.