Anti-Inflammatory Effects of Abeliophyllum distichum Nakai (Cultivar Okhwang 1) Callus through Inhibition of PI3K/Akt, NF-κB, and MAPK Signaling Pathways in Lipopolysaccharide-Induced Macrophages

Round 1

Reviewer 1 Report

The presented message lacks the depth of discussion. Some of the sentences are trivial.

However, the topic is interesting. In my opinion major revision is needed and the authors should improve the manuscript.

The manuscript need a revision of English form and language:

Line 17 “were investigated THROUGH immunoblotting.” (immunoblotting was employed to find out…?)

Line 49 “Recent studies have OBSERVED that…”

Line 64, 328: “Reportedly,”

Lines 303-4: “To mediate and initiate the NF-κB signal transduction pathway, the activation of 303 MAPK (ERK1/2, JNK1/2, and p38) and PI3K-Akt signaling pathway is crucial.”

Line 336” “In the HPLC analysis, acteoside and isoacteoside were analyzed …..”

Some sentences are not clear. The authors must clarify their idea:

Lines 20-21: “Further, alleviating translocation to nuclear of NF‐κB p65 by the LPS‐triggered and degradation of inhibitor of NF-кB (IкB) by suppressing phosphorylation.”

Lines 25-26: “…CAO can serve as a potential anti-inflammatory agent alleviating TO CAUSING inflammation factors in cell signaling pathways.”

Line 320: “…. were stained with DAPI”

Line 386: “data from individual cells…”

Some sentences lack novelty and the depth of discussion:

Lines 24-25: “…signaling pathways, probably via GLYCOSIDE BIOACTIVITY.” – too general explanation

Lines 30-31: “Inflammation serves as a response to harmful stimuli causing by injured cells, pathogens, endogenous signals….,”

Line 46: “After phosphorylation by LPS stimulation,…” – not precise enough

Lines 52-53: “INVESTIGATING PI3K/Akt and MAPK signaling pathways WILL PROVIDE AN EFFECTIVE THERAPEUTIC APPROACH for alleviating inflammatory (inflammation?)” (will contribute to…?)

Lines 323-4: “According to Bremer [36], several “Oleaceae family” plants, such as Jasminum, 323 Abeliophyllum, and Forsythia genera, contain glycosides”

Some other points were listed below:

Introduction

Lines 67-8: “Both cultured plant cells and whole plants cells METABOLIZE compounds in a qualitatively similar manner [30]. “ (synthesize?)

Materials and Methods

2.8. Nitric Oxide (NO) Production Assay and 2.9. Western blotting analysis

Lines 139, 145: Did you use CAO and LPS simultaneously for 2 hours or the RAW 264.7 cells were pretreated with one of the compounds1q?

2.10. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) - since RT-PCR was used to analyze mRNA level, the description of the method is incomplete

Results

Figure 3, 5 captions: please, indicate the method used to measure gene expression

Line 143: please, give name and company of the microplate reader manufacturer

Line 226 “…which was then analysed to be 39.93% and 5.67% (w/w),” – the result is not clear (possibly split it into two separate sentences)

Line 228: “…and probably the active constituents present in CAO” – these are speculations, please, refer rather to the quantity of the compounds

Line 237: MTS assay was not described in Materials and Methods section

Line 240: “These results indicate that CAO did not significantly affect cell proliferation.” It should be rather moved to discussion section

Line 243: Figure 3. „Cells were pre-treated with CAO” why pre-treated? The cells were just incubated with CAO

Line 248: Do you mean: “pro-inflammatory cytokines”?

Figure 6: 50 mg/ml? – please, show the units

Discussion

Line 334: “IDENTIFY THE MECHANISM of the expression on anti-inflammatory factors using” was such mechanistic study the aim? or rather the assessment whether CAO may interfere with some pro-inflammatory factors?

Lines 358-9: “We identified the mechanism of action by which CAO reduces the production of inflammatory mediators” the Authors presented some activities of the CAO, not the complete effect it exerts on whole pathways (e.g AKT). Please, be careful using “the mechanism”

The concluding remarks highlighting the main findings of the study might be helpful for the readers

Author Response

Thanks for the advice.

It has been carefully reviewed and answered faithfully.

Revisions are highlighted in red.

Line 17 “were investigated THROUGH immunoblotting.” (immunoblotting was employed to find out…?)

• Immunoblotting was employed to find out the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide (iNOS), activation of MAPK molecules, NF-κB, and Akt. Cytokines, COX-2, and iNOS gene expression were assessed using polymerase chain reaction techniques.

Line 49 “Recent studies have OBSERVED that…”

• According to recent studies, phosphatidylinositol 3-kinase/Akt (PI3K-Akt) signaling pathways are related to the expression of pro-inflammatory mediators through IκB degradation and phosphorylation of NF-κB in LPS-induced macrophages.

Line 64, 328: “Reportedly,”

• In particular, acteoside possesses several pharmacological properties.

Lines 303-4: “To mediate and initiate the NF-κB signal transduction pathway, the activation of 303 MAPK (ERK1/2, JNK1/2, and p38) and PI3K-Akt signaling pathway is crucial.”

• The activation of MAPK (ERK1/2, JNK1/2, and p38) and PI3K-Akt signaling pathway is crucial, to initiating the NF-κB signal transduction pathway.

Line 336” “In the HPLC analysis, acteoside and isoacteoside were analyzed …..”

• As a result of HPLC analysis, two components are predominant in CAO, which are analyzed to acteoside and isoacteoside compared to purchased standard products.

Lines 20-21: “Further, alleviating translocation to nuclear of NF‐κB p65 by the LPS‐triggered and degradation of inhibitor of NF-кB (IкB) by suppressing phosphorylation.”

• Further, translocation to nuclear of NF‐κB p65 and degradation of inhibitor of NF-кB (IкB) alleviated by suppressing phosphorylation.

Lines 25-26: “…CAO can serve as a potential anti-inflammatory agent alleviating TO CAUSING inflammation factors in cell signaling pathways.”

• Thus, CAO can serve as a potential anti-inflammatory agent alleviating inflammation factors in cell signaling pathways.

Line 320: “…. were stained with DAPI”

• The sentence has been deleted.

Line 386: “data from individual cells…”

• Furthermore, immunofluorescence data from cells confirmed that the phosphorylation of Akt was inhibited by CAO treatment.

Lines 24-25: “…signaling pathways, probably via GLYCOSIDE BIOACTIVITY.” – too general explanation

• These results indicate that the anti-inflammatory effect of CAO is mediated via the inhibition of MAPK, PI3K/Akt, and NF-κB signaling pathways, probably via glycosides, phenolics, flavonoids bioactivity derived from plants.

Lines 30-31: “Inflammation serves as a response to harmful stimuli causing by injured cells, pathogens, endogenous signals….,”

• Inflammation serves as a response to harmful stimuli. These stimuli are caused by injured cells, pathogens, endogenous signals, which are generally resulted in cellular healing to restore normal function.

Line 46: “After phosphorylation by LPS stimulation,…” – not precise enough

• The common NF-κB signaling pathway is activated within stimulation to pro-inflammatory mediators such as molecular stimulus, pathogens, and cytokines. Activation of NF-κB could occur through depends on ubiquitination of IκB proteins. Phosphorylation of IκB proteins followed by degradation and ubiquitination releases the NF-κB p50:p65 dimers. The released p65 is then translocated to the nucleus, where it binds to target genes, brings about the transcription of related mediators.

Lines 52-53: “INVESTIGATING PI3K/Akt and MAPK signaling pathways WILL PROVIDE AN EFFECTIVE THERAPEUTIC APPROACH for alleviating inflammatory (inflammation?)” (will contribute to…?)

• Hence, investigating PI3K/Akt and MAPK signaling pathways will contribute to providing an effective therapeutic approach for alleviating inflammation.

Lines 323-4: “According to Bremer [36], several “Oleaceae family” plants, such as Jasminum, 323 Abeliophyllum, and Forsythia genera, contain glycosides”

• According to a study on chemotaxonomy of the Oleaceae, the Oleaceae family synthesizes and distributes iridoid glucosides. Abeliophyllum, one of the Oleaceae family, synthesizes caffeoyl phenylethanoid glycosides (cornoside and acteoside).

Introduction

Lines 67-8: “Both cultured plant cells and whole plants cells METABOLIZE compounds in a qualitatively similar manner [30]. “ (synthesize?)

• Both cultured plant cells and whole plant cells synthesize compounds in a qualitatively similar manner.

Materials and Methods

• 2.8, 2.9, 2.10, and 2.11 are revised briefly for reducing similarity.

Results

Figure 3, 5 captions: please, indicate the method used to measure gene expression

• The changed sentences are highlighted in red.

Line 143: please, give name and company of the microplate reader manufacturer

• It added.

Line 226 “…which was then analysed to be 39.93% and 5.67% (w/w),” – the result is not clear (possibly split it into two separate sentences)

• It separates the sentence into two sentences.

Line 228: “…and probably the active constituents present in CAO” – these are speculations, please, refer rather to the quantity of the compounds

• The changed sentences are highlighted in red.

Line 237: MTS assay was not described in Materials and Methods section

• It described in 2.7.

Line 240: “These results indicate that CAO did not significantly affect cell proliferation.” It should be rather moved to discussion section

• The sentence was moved to Discussion.

Line 243: Figure 3. „Cells were pre-treated with CAO” why pre-treated? The cells were just incubated with CAO.

• The changed sentences are highlighted in red.

Line 248: Do you mean: “pro-inflammatory cytokines”?

• The changed words are highlighted in red.

Figure 6: 50 mg/ml? – please, show the units

• The unit was explained to the legend.

Discussion

Line 334: “IDENTIFY THE MECHANISM of the expression on anti-inflammatory factors using” was such mechanistic study the aim? or rather the assessment whether CAO may interfere with some pro-inflammatory factors?

• In the present study, we tried to identify the assessment of the expression on pro-inflammatory factors by the CAO treatment.

Lines 358-9: “We identified the mechanism of action by which CAO reduces the production of inflammatory mediators” the Authors presented some activities of the CAO, not the complete effect it exerts on whole pathways (e.g AKT). Please, be careful using “the mechanism”

• The changed words are highlighted in red.

Reviewer 2 Report

The authors of this manuscript present an interesting research review on Anti-inflammatory effects of Abeliophyllum distichum Nakai (cultivar Okhwang 1) callus through inhibition of PI3K/Akt, NF-κB, and MAPK signaling pathways in lipopolysaccha- ride-induced macrophages. The text needs very few revisions. The comparison and the analysis of the anti-inflammatory effects of Abeliophyllum distichum Nakai (cultivar Okhwang 1) can add further research interest.

Abstract

COMMENT:

Introduction

Line 55       Nakai, 

Materials and Methods

Line 99     and

Line 146     radioimmunoprecipitation assay

Results

Error bars in figures are not clear due to its balck color.

Discussion

Conclusions

I wonder if a conclusion section is need.

Author Contributions

References

COMMENT: Please check the reference list according to the author’s instruction.

Reference 7 is not justified

Please check the capital letter of the Journal title. for i.e in references 10 and 11

Line 481    methods

latin names for example Sargassum hemiphyllum in line 438,    A. distichum.   443    Abeliophyllum distichum     446, 457, 461, 490, etc

Please check the style and of the reference list

Author Response

Thanks for the advice.It has been carefully reviewed and answered faithfully.

Line 55       Nakai, 

• Fixed italics.

Line 99     and

• Fixed.

Line 146     radioimmunoprecipitation assay

• Revised the words

Error bars in figures are not clear due to its black color.

• All graphs are marked with "*" through statistical test. The error expressed by the error bar is insignificant, and important details are explained in Direct Results and Discussion.

 Conclusions

• added conclusion section.

 References

• checked the references diligently and carefully.

Round 2

Reviewer 1 Report

In my opinion minor revision is needed. English form and language must be improved. Some sentences are not clear enough. Conclusions must be more extensive and specific to data obtained. In my opinion, after changes the manuscript is suitable for the publication.

Kind regards,

Minor considerations were listed below:

Line 20: “CAO inhibited producing NO…” “CAO inhibited the production (or generation) of NO…”

Line 26:please, remove “Thus”

Line 27: please, change “Thus, CAO can serve as a potential anti-inflammatory agent alleviating inflammation factors in cell signaling pathways.” to “CAO can serve as a potential anti-inflammatory agent, which alleviates inflammation factors and act through specific cell signaling pathways ”

Line 32: “which are generally resulted in” “which resulted in”

Line 50: please, change the sentence “Activation of NF-κB could occur through depends on ubiquitination of IκB proteins [13,15].”  

“Activation of NF-κB occurs through (OR depends on) ubiquitination of IκB proteins [13,15].”

”

Line 51: “Phosphorylation of IκB proteins followed by degradation and ubiquitination releases the 51 NF-κB p50:p65 dimers [16,17].” – do you mean: “Phosphorylation of IκB protein is followed by its degradation and ubiquitination, which results in releasing NF-κB p50:p65 dimers [16,17].” ?

Line 294 “CAO 50, 50 μg/mL of callus of A. distichum var. Okhwang 1.” Please, remove “50”

Line 308: please, change “In the present study, we tried to identify the assessment of the expression on pro-inflammatory factors by the CAO treatment.” to  “In the present study, we tried to assess whether the treatment of RAW 264.7 macrophages with CAO may affect the expression on selected pro-inflammatory factors.”

Line 361: “immunofluorescence data from cells….” – please, be more specific about the method used

Conclusions are weak – please, refer to the main findings of the study

Line 369: please, refer to the experimental model “Treatment of CAO can reduce the levels of cytokines, iNOS, and COX-2 in murine RAW 264.7 macrophages.”

Line 370: “Also, these reductions are related to its alleviation of MAPK, NF-kB, and PI3K-Akt signaling pathways” - the Authors presented some activities of the CAO, NOT the complete effect it exerts on whole pathways. Maybe such sentence will be more adequate: “Moreover, CAO affected particular proteins regulating MAPK, NF-kB, and PI3K-Akt signaling pathways””

Line 371: “Furthermore, the regulation of NF-kB signaling may be associated with translocation to the nucleus in the step of expressions.” – this sentence is not clear enough

Author Response

Thanks for the revision.

What you mentioned has been faithfully applied.

In addition, the "Conclusion" section has been added to strengthen the basis for the conclusion.

Thank you.