Protective Effects of One 2,4-Dihydro-3H-Pyrazol-3-one Derivative against Posterior Capsular Opacification by Regulation of TGF-β2/SMADs and Non-SMAD Signaling, Collagen I, and Fibronectin Proteins
Round 1
Reviewer 1 Report
Please, rewrite entirely tha paper in order to get a minimum standard of language quality. Ask for the help of a English native or an expert. Automatic translator are not of any help.
Author Response
Response to Reviewer 1 Comments
Point 1:
English language and style Extensive editing of English language and style required
Response 1: Please provide your response for Point 1. (in red)
The article of English language and style Extensive editing of English language and style has been edited by AJE (American Journal Expert) as the following attached file.
Point 2:
Please, rewrite entirely this paper in order to get a minimum standard of language quality. Ask for the help of a English native or an expert. Automatic translator are not of any help.
Response 2: Please provide your response for Point 2. (in red)
The article has been rewritten entirely this paper in order to get a minimum standard of language quality and has been asked for the help of a English native or an expert AJE (American Journal Expert) s the following attached file.
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Liu and co-workers findings reveal the benefits of TSE by regulation of TGF-β/SMAD signaling and non-SMAD signaling related gene proteins to display anti-fibrosis activity in cells for the possibility of prevention of PCO after cataract surgery. However, a large number of related works have been reported (e.g., Bioactive materials, 2022, 9: 343-357; Investigative Ophthalmology & Visual Science, 2021, 62(10): 24-24.), the innovation of this paper is very limited. Therefore, I do not recommend this paper for publication in Curr. Issues Mol. Biol..
In addition, there are issues that the authors need to address in order to convince the audience of the proposed claims.
Author Response
Response to Reviewer 2 Comments
Point 1:
English language and style are fine/minor spell check required
Response 1: Please provide your response for Point 1. (in red)
The article of English language and style are fully edited by AJE (American Journal Expert) as the following attached file.
Point 2:
Liu and co-workers findings reveal the benefits of TSE by regulation of TGF-β/SMAD signaling and non-SMAD signaling related gene proteins to display anti-fibrosis activity in cells for the possibility of prevention of PCO after cataract surgery. However, a large number of related works have been reported (e.g., Bioactive materials, 2022, 9: 343-357; Investigative Ophthalmology & Visual Science, 2021, 62(10): 24-24.), the innovation of this paper is very limited. Therefore, I do not recommend this paper for publication in Curr. Issues Mol. Biol.
Response 2:
Our TSE compound is synthesized by means of medicinal chemistry according SAR (structure-activity-relationship) similar to the clinic pirfenidone (PFD) drug used for anti-fibrosis in idiopathic pulmonary fibrosis (IPF) but expensive) and nevertheless TSE displays better activity than PFD on anti-PCO.
Explanation:
Each paper had its own innovation and individual views. The Reviewer pointed the first article belongs to Pharmaceutics. The Reviewer pointed the second article belongs to clinic Cohort study. Our TSE compound is synthesized by means of medicinal chemistry according SAR (structure-activity-relationship) similar to the pirfenidone (PFD) compound (a new drug used for anti-fibrosis in lung but it is expensive) and nevertheless TSE displays better efficacy than PFD on anti-PCO.
The first article ”Zhang et al., 2022. Drug-eluting intraocular lens (IOL) with sustained bromfenac release for conquering posterior capsular opacification. Bioactive materials. 9: 343-357” demonstrated that a non-steroidal anti-inflammatory drug (NSAID), bromfenac, displaying inhibition of cell migration, overexpression of EMT markers including fibronectin (FN), MMP2, α-SMA and Snail, and cellular signal-regulated kinase (ERK)/ glycogen synthase kinase -3β (GSK-3β) signaling induced by TGF-β2. And they developed a drug-eluting IOL using poly (PLGA) with sustained bromfenac release ability via ERK/GSK-3β /Snail signaling for the prevention of PCO development. This technology needs operation (IOL) which coupled with bromfenac’s releasing. There exist different molecular mechanisms between our TSE and bromfenac. Our innovation is that TSE displays anti-PCO activity by its targeted genes expressions, which are complete, abundant, and detailed in addition to bromfenac‘s ERK signaling. Their authors also pointed out that “other signaling pathways and many targeted genes “should be further studied” in the final paragraph of Discussion. Therefore, our article is suitable for published in this Journal “Current issues in molecular biology”;
The second article “Patnaik et al., 2021. The protective effect of metformin use on early Nd:YAG laser capsulotomy. Investigative Ophthalmology & Visual Science. 62(10): 24-24” The clinic anti-diabetic drug, metformin, coupled with operation (Nd:YAG laser capsulotomy) their authors concluded that future studies are required to more fully understand how metformin may interfere with the development of PCO including molecular mechanisms (merely ERK signaling done and cited previous evidences showing metformin’s activation of AMPK to display anti-fibrosis in kidney and lung) and in nondiabetic population following with lens extraction. Our innovation is that TSE displays anti-PCO activity by its targeted genes expressions are complete and detailed in addition to metformin‘s ERK signaling pathway. Therefore, our article is suitable for published in this Journal “Current issues in molecular biology”;
Point 3:
In addition, there are issues that the authors need to address in order to convince the audience of the proposed claims.
Response 3:
In the last paragraph of Introduction: Pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone; PFD) is demonstrated to be an anti-inflammatory drug with anti-fibrosis activity, and it has used in clinical agent for the management of idiopathic pulmonary fibrosis (IPF) [18], and nevertheless it is expensive. Therefore, we try to find one substitute for PFD. ……Thus, we evaluate whether TSE treatment could be effective in anti-fibrosis and display better efficacy than PFD and without toxic effect.
In the first paragraph of Discussion: Therefore, we thought if there are therapeutic interventions with no toxic side effect and low cost by blocking TGFβ-2 and SMAD pathway to led to antifibrotic effects, and finally contribute to prevent PCO. The present study showed that TSE is non-toxic by LDH assay.
Please see the attachment:
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The paper is now readable, but further English editing is needed.
The scientific results are on the overall acceptable.
Author Response
Response to Reviewer 1 Comments
Point 1:
The paper is now readable, but further English editing is needed.
The scientific results are on the overall acceptable.
Response 1: Please provide your response for Point 2. (in red)
My edited file F51VC2Y77 has been completed by American Journal Experts (AJE) and Lea Moore (American Journal Experts) replied that
Oct 11, 2022, 08:28 EDT
Dear Professor Chun-Ching Shih,
Thank you for contacting us regarding the journal's comments. We use this feedback to ensure that our editing is the high quality we promise.
Point 2:
Are the results clearly presented? Must be improved
Response 2: Please provide your response for Point 1. (in red)
And the Results have been Revised to－>
3.1. Effects of Five Compounds on the Expressions of TGF-β2, SMAD3, or SMAD4 in SRA01/04 Cells
There was no significant difference in the protein expression levels of TGF-β2, SMAD3, and SMAD4 between the DMSO group and the CON group. There is a significant decrease in the expression levels of TGF-β2 in the TSE-1-, TSE-2 (TSE)-, TSE-3-, TSE4-, and TSE-5-treated groups compared to the CON group (p < 0.001, p < 0.001, p < 0.01, p < 0.001, p < 0.01, respectively) (Figure 2A,B). There is a significant decrease in the expression levels of SMAD3 in the PFD-, TSE-1-, TSE-2 (TSE)-, TSE-3-, TSE4-, and TSE-5-treated groups compared to the CON group (p < 0.001, p < 0.001, p < 0.001, p < 0.001, p < 0.001, p < 0.001, respectively) (Figure 2A,C). There is a significant decrease in the expression levels of SMAD4 in the PFD-, TSE-1-, TSE-2 (TSE)-, TSE-3-, and TSE-5-treated groups compared to the CON group (p < 0.001, p < 0.05, p < 0.001, p < 0.001, p < 0.01, respectively) (Figure 2A,D). We found that TSE-2 (TSE) displayed a significant decrease in TGF-β2 expression compared with that in the CON group.
3.2. Cell Viability of Five Compounds
In the trypan blue exclusion test, after 24, 48, 72, or 96 hours of treatment with 5 compounds of DHPO, the percentages of living cells were 108.6 ± 2.7%, 105.3 ± 1.4%, 103.6 ± 2.1%, and 103.2 ± 2.4% for the control and the 10 μg/ml TSE groups, respectively (Figure 3A). No significant difference was found between the groups (p > 0.05).
3.3. LDH Assay
Following treatment with 5 compounds 24 hours later, the cell-mediated lysis percentages were 4.47 ± 0.9% for the control and the 10 μg/ml TSE groups and 7.36 ± 1.1% for the control and the 10 μg/ml TSE groups 48 hours later. TSE did not display a significant cytotoxicity action (Figure 3B).
3.4. Effects of TSE on the mRNA Levels of Targeted Genes in Cells
The mRNA levels of TGF-β2, SMAD3, and SMAD4 were markedly enhanced in SRA01/04 cells compared to the CON group (p < 0.001, p < 0.001, p < 0.001, respectively). A reduction in the mRNA levels of TGF-β2 was observed in the 5, 10, and 20 mg/ml TSE-treated groups compared to the vehicle-treated TGF-β2 group (p < 0.05, p < 0.001, p < 0.001, respectively) (Figure 4A,B). A reduction in the mRNA levels of SMAD3 was observed in the 5, 10, and 20 mg/ml TSE-treated groups compared to the vehicle-treated TGF-β2 group (p < 0.05, p < 0.001, p < 0.001, respectively) (Figure 4A,B). A reduction in the mRNA level of SMAD4 was observed in the 5, 10, and 20 mg/ml TSE-treated groups compared to the vehicle-treated TGF-β2 group (p < 0.01, p < 0.001, p < 0.001, respectively) (Figure 4A,B).
3.5. Effects of TSE on the Expression Levels of TGF-β2, SMADs, Fibronectin, and Collagen I
The protein expression levels of TGF-β2, SMAD3, SMAD4, fibronectin, and collagen I were significantly increased in TGF-β2-induced cells compared to the CON group (p < 0.001, p < 0.001, p < 0.001, p < 0.001, p < 0.001, respectively) (Figure 4C,D,E,F). Decreases in the expression levels of TGF-β2 were observed in the 5, 10, and 20 mg/ml TSE-treated groups compared with the vehicle-treated TGF-β2 group (p < 0.05, p < 0.001, p < 0.001, respectively) (Figure 4C,D). Decreases in the expression levels of SMAD3 were observed in the 5, 10, and 20 mg/ml TSE-treated groups compared with the vehicle-treated TGF-β2 group (p < 0.05, p < 0.001, p < 0.001, respectively) (Figure 4C,D). Decreases in the expression levels of SMAD4 were observed in the 5, 10, and 20 mg/ml TSE-treated groups compared with the vehicle-treated TGF-β2 group (p < 0.05, p < 0.001, p < 0.001, respectively) (Figure 4C,D). Decreases in the expression levels of fibronectin were observed in the 10 and 20 mg/ml TSE-treated groups compared with the vehicle-treated TGF-β2 group (p < 0.001, p < 0.001, respectively) (Figure 4E,F). Decreases in the expression levels of collagen I were observed in the 10 and 20 mg/ml TSE-treated groups compared with the vehicle-treated TGF-β2 group (p < 0.001, p < 0.001, respectively) (Figure 4E,F).
3.6. TSE Inhibits the Nuclear Translocation of SMADs
By immunofluorescence assay, the expression levels of SMAD2/3, and SMAD4 protein in SRA01/04 cells were scanned and examined. SMAD2/3, and SMAD4 may be expressed within the cells and nearly in the cytoplasm. As shown in Figure 5 and Figure 6, the nuclear SMAD2/3 or SMAD4 staining in the control group (in the absence of TGF-β2) in SRA01/04 cells was very weak. These results were evaluated under confocal microscopy. Following treatment with 5, 10, or 20 mg/ml TSE, nuclear SMAD2/3 and nuclear SMAD4 expression was decreased in the SRA01/04 cell line compared with the control under microscopy at 200× magnification, and the suppressive activity of 20 mg/ml TSE was the most effective (Figure 5 and Figure 6). Thus, these findings demonstrated that TSE displayed a suppressive effect on SMAD2/3 or SMAD4 translocation to the nuclear in SRA01/04 cells.
3.7. Effects of TSE on the Protein Expression of p- JUN/ JUN, p-ERK1/2/ ERK1/2, p-p38/ p38, p-Akt (Ser473)/ t-Akt, and p-Akt (Thr308)/ t-Akt in SRA01/04 cells
The expression levels of p- JUN/ JUN, p-ERK1/2/ ERK1/2, p-p38/ p38, p-Akt (Ser473)/ t-Akt, and p-Akt (Thr308)/ t-Akt were significantly increased in TGF-β2-induced cells compared with the CON cells (p < 0.05, p < 0.05, p < 0.05, p < 0.05, p < 0.01, respectively) (Figure 7A,B,C,D). A decrease in p- JUN/ JUN and p-ERK1/2/ ERK1/2 expression was found in the 5, 10 and 20 mg/ml TSE-treated groups compared to the vehicle-treated TGF-β2 group (Figure 7A,B). A decrease in p-p38/ p38 expression was found in the 10 and 20 mg/ml TSE-treated groups compared to the vehicle-treated TGF-β2 group (p < 0.001, p < 0.001, respectively) (Figure 7A,B). A decrease in p-PI3K/ PI3K and p-Akt (Thr308)/ t-Akt expression was found in the 5, 10, and 20 mg/ml TSE-treated groups compared to the vehicle-treated TGF-β2 group (Figure 7C,D). A decrease in p-mTOR/ mTOR and p-Akt (Ser473)/ t-Akt expression was found in the 10 and 20 mg/ml TSE-treated groups compared to the vehicle-treated TGF-β2 group (Figure 7C,D).
Author Response File: Author Response.pdf
Reviewer 2 Report
The authors have made careful revisions, so I recommend this paper for publication.
Author Response
Response to Reviewer 2 Comments
Point 1: The authors have made careful revisions, so I recommend this paper for publication.
Response 1: Please provide your response for Point 2. (in red)
Dear Reviewer, thank you very much.
Author Response File: Author Response.pdf
