Study of the Effect of Neutral Polysaccharides from Rehmannia glutinosa on Lifespan of Caenorhabditis elegans

Reviewer Number

Original comments of the reviewer

Reply by the author(s)

1

Line 152, Page 7: what do you mean by MDA?

Changed it to CAT.

2

Line 202, Page 9: what does Figure 14 refer to?

Please check the number of Figure 12 and 13 .

Changed it to Figure 13,

I have checked the number of Figure 12 and 13 to ensure correct.

3

Line 197, Page 9; Line 105, Page 4: C. elegans should be written in italic.

Correction has been made.

4

Line 192, Page 9:  what do you mean by oleanolic acid?

Changed it to NPRG.

5

Figure 11: there is no P-value or error bar for each group especially for daf-2 group. Do you mean none of the comparison is statistically significant?

Sorry, in the beginning, I plotted with average, but after your reminding, I realized that it was not rigorous, so I plotted with the original data and adopted the analysis of variance (ANOVA) by SAS 8.2.

6

Figure 2: have you done replicate experiments for each time-point?

Yes, I have done replicate experiments for three times for each time-point, and plotted with average.

7

Result 2.10 on Page 9, what is the relationship between daf-16 mutant TJ356, CF1038 and DR26? The authors need to discuss more about this in discussion part.

I am so sorry, in result 2.10 there shouldn't have been TJ356.I have changed it to CF1038. In addition, We have explained to daf-16 mutant TJ356 in 4.13.

8

Result 2.9 on Page 8: have you done the experiment to test whether NPGR can inhibit daf-2 expression under oxidative stress?

We didn't do it. Our aim was to investigate the mechanism by which NPRG prolongs the lifespan of nematodes under normal conditions. But testing whether NPGR can inhibit daf-2 expression under oxidative stress can further enrich the research results. We will study it in the following experiment.

9

In the Method part 4.3 on Page 11: the authors need to provide detailed preparation process of NPRRP and NPRR.

We added the following: RR and RRP will be cut into 0.5 cm2 small pieces, and 10 times more acetone was added to purge fat‑soluble components. We added 10 times more water to pigment and decoct it on a radiant cooker for 3 times, each time for 1 h. Then, we merged the decoctions and concentrated the filtrate by reduced pressure distillation at 70°C to an appropriate amount. Next, we added 95% ethanol until the content of it in filtrate as 80%, placing it for 12 h under 4°C. Then, we filtered and froze it to dry. We dissolved it in distilled water and deproteinized by Sevage (chloroform: n‑butanol = 4:1) and repeated more than 10 times until no protein absorption was detected by UV spectrum analysis. Finally, the PRG were obtained by freeze‑drying.

10

The structure of the two NPRGs were not fully characterized as the information of molecular weight, sugar chain structure and linkage form are missing. The authors can refer to the literature below.

Thanks very much for your valuable comment. Molecular weight, sugar chain structure and linkage form of NPRG are important. However, the paper mainly focuses on pharmacological studies, supplemented by chemical structure studies. We will study it in the following experiment.

11

Result 2.3 on Page 3: The authors need to show out the LC chromatograms of NPRR and NPRP for monosaccharide composition analysis.

I added the figure 2 and figure 3 to show out the LC chromatograms of NPRR and NPRP for monosaccharide composition analysis in result 2.3.

12

There are too many figures included in this manuscript. I suggest to combine Figure 2-5, Figure 6-8 and Figure 9-13 according to the content described in each.

I combined Figure 6-7, Figure 8-10, Figure 11-12 and Figure 14-15 according to the content described in each.

Reviewer Number

Original comments of the reviewer

Reply by the author(s)

1

Authors suggested “anti-aging” effects of NPRG in C. elegans. However, they just showed beneficial effects of NPRG on oxidative stress and survival. Survival prolongation is not equal to anti-aging. If authors would like to suggest “anti-aging effect”, they have to examine other aging or senescence-associated markers such as p21 and senescence-associated β-galactosidase. Or, they need to remove the term “anti-aging effect”.

We adopted your valuable opinions and suggestions.

And some modifications have been made in the title, abstract, keywords discussion.

2

Authors demonstrated that NPRG increased total superoxide dismutase (SOD) activity. However, they did not show that the increased SOD activity was mainly ascribed for upregulation of SOD3 but not SOD2. Authors have to examine the expression of not only SOD3 but also SOD1 and SOD2, and clearly explain why they focused on SOD3 expression in this study.

Coleen t. Murphy et al. analyzed the expression of downstream genes of C. elegans daf-16 using DNA microarray analysis and RNAi technology, and proposed the role of daf-2/daf-16 in regulating the aging and longevity of C. elegans, and proved that sod-3 gene was the downstream factor of daf-16 gene [1].YOKO HONDA's study showed that the expression of sod-3 gene was significantly up-regulated in daf-2 mutant nematode, but the expression of sod-1 and sod-2 gene was not significantly changed. Therefore, it could be inferred that the sod-3 gene was regulated by daf-2/daf-16 gene, and was related to the life span of C. elegans[2].

1.                Murphy, C. T.; McCarroll, S. A.; Bargmann, C. I.; Fraser, A.; Kamath, R. S.; Ahringer, J.; Li, H.; Kenyon, C., Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. 424, (6946), 277-283.

2.                Honda Y , Honda S . The daf-2 gene network for longevity regulates oxidative stress resistance and Mn-superoxide dismutase gene expression in Caenorhabditis elegans.[J]. The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1999, 13(11):1385-1393.

Because part of our research results showed that NPRG acted through the IIS signaling pathway, we only focused on examine SOD-3 expression. This is similar to the experimental method in the following paper.

3.                Jia, W.; Guangzhi, Z.; Xiaobing, H.; Zhe, W.; Ninghua, T., 1,4-Naphthoquinone Triggers Nematode Lethality by Inducing Oxidative Stress and Activating Insulin/IGF Signaling Pathway in Caenorhabditis elegans. Molecules 22, (5), 798-.

4.                Lee, E. B.; Xing, M. M.; Kim, D. K., Lifespan-extending and stress resistance properties of brazilin from Caesalpinia sappan in Caenorhabditis elegans. Archives of Pharmacal Research.

5.                Feng, S.; Cheng, H.; Xu, Z.; Yuan, M.; Huang, Y.; Liao, J.; Yang, R.; Zhou, L.; Ding, C., Panax notoginseng polysaccharide increases stress resistance and extends lifespan in Caenorhabditis elegans. Journal of Functional Foods 45, 15-23.

3

Quantification of GFP fluorescence seems to be arbitrary. Nuclear localization of DAF-16 should be confirmed by other methods such as western blot after the separation of nuclear fraction from cytoplasmic fraction. In addition, as nuclear localization of DAF-16 is regulated by phosphorylation process, western blot for phospho-DAF-16 relative to total DAF-16 should be helpful.

When DAF-16::GFP enters the nucleus, it produces a bright spot of light under a fluorescence microscope. We measured the amount of fluorescence points in each group at the same time to indicate the expression of DAF-16. We think this is a convenient operation to reduce the fluorescence error of software measurement.

Thanks very much for your valuable suggestions. TJ356 strain carrying a daf-16::GFP was used to test the translocation of DAF-16 in the nucleus. The main purpose of this paper is to prove that NPRG functions on daf-16. It has been well demonstrated in the daf-16 mutant lifetime experiment. It would be perfect with content determination of phospho-daf-16, which will be supplemented in future studies.

4

In Figure 11, why were standard deviations not shown? How many samples were examined?

I’m sorry, in the beginning, I plotted with average, but after your reminding, I realized that it was not rigorous, so I plotted with the original data and adopted the analysis of variance (ANOVA) by SAS 8.2.

There were three samples for every group.

5

In statistical analysis, t-test is inappropriate for comparing the differences among multiple groups. ANOVA with post hoc tests should be used. Authors should retry their statistical analyses in this study

In this paper, t-test was widely used to analyze the difference between two groups of data, such as the blank group with NPRR group or the blank group with NPRRP. But for RT‑PCR assay, t-test is inappropriate for comparing the differences among multiple groups, I used ANOVA for analyzing those data.

And in statistical analysis, I added that and differences between groups were determined by using analysis of variance.