Gas Chromatography–Mass Spectrometry-Based Cerebrospinal Fluid Metabolomics to Reveal the Protection of Coptisine against Transient Focal Cerebral Ischemia–Reperfusion Injury via Anti-Inflammation and Antioxidant

Round 1

Reviewer 1 Report

I wonder the authors used Coptisine or Coptisine chloride. From Fig1 A, I think it should be Coptisine chloride or other form of salt. Please double-check.

In line 78 and elsewhere in the text, the authors misspelled 2,3,5-triphenytetrazolium chloride, it should be 2,3,5-triphenyltetrazolium chloride.

I am not sure I fully understand the meanings of lines 47 and 283. Please make them clearly for readers.

What are the biomarkers the authors found in this study?

The authors need to carefully check their grammars and spellings.

A lot of nouns should be used plurals instead the authors used singulars.

Author Response

We feel great thanks for your professional review work on our manuscript. As you are concerned, there are several problems that need to be addressed. According to your nice suggestion, we have made extensive corrections to our manuscript, the details corrections are listed below.

1. I wonder the authors used Coptisine or Coptisine chloride. From Fig1 A, I think it should be Coptisine chloride or other form of salt. Please double-check.

Response: Thanks for your indication. This mistake has been corrected in the section of material and methods. “Cop chloride(purity>98%) was purchased by Yirui Biotechnology Co.Ltd.(Chengdu, China)”

2. In line 78 and elsewhere in the text, the authors misspelled 2,3,5-triphenytetrazolium chloride, it should be 2,3,5-triphenyltetrazolium chloride.

Response: Thanks for your comments. This mistake has been corrected.

3. I am not sure I fully understand the meanings of lines 47 and 283. Please make them clearly for readers.

Response: Thank you for your advice. (1) The sentence in line 47 has been corrected as follows: “In recent, a liquid chromatography tandem mass spectrometry based-metabolomic approach was applied to investigate the protective mechanism of Bai-Mi-decoction on is-chemic stroke rats, and found 30 differential metabolites were restored by Bai-Mi-decoction intervention, e.g., phenylalanine, valine and LysoPI(18:0/0:0).” (2) The sentence in line 283 has been corrected as follows: “ Zhai and their colleagues also illustrated that the antioxidative effect of Cop was linked with upregulation in the expression of nuclear factor-erythroid-2 related factor 2 and NADPH quinone oxidoreductase 1. ”. We hope these revisions would make readers understand.

4. What are the biomarkers the authors found in this study?

Response: Thanks for your advice. The information of CSF biomarkers between control, cerebral IR injury and Cop treated add in the manuscript and supplementary materials (Tab S3, S4). Finally, five CSF metabolites were screen as target of Cop against cerebral IR(Tab S5 and Fig 5A).

Table S1 Retention time and similarity of analytes in SD rats’ CSF by GC-MS

Compound	tR（min）	Similarity (%)	Compound	tR（min）	Similarity (%)
Cyclobutane	2.027	80	Pentanedioic acid	17.315	88
3-Buten-2-ol	2.415	81	Glutamine	17.904	92
alpha-Pinene	3.185	96	Phenylalanine	17.959	95
Ethane	3.225	97	Ribonic acid	18.205	80
2-Thenaldehyde	3.38	81	Dodecanoic acid	18.345	83
Benzene	3.435	86	Lyxose	18.37	86
borate	3.605	93	Ribose	18.4	92
ethane-1,2-diol	3.67	92	Asparagine	18.521	93
Dimethylamine	4.05	93	Arabinose	18.579	89
Pentane	4.655	95	Lysine	18.715	81
Propanoic acid	4.775	93	Sorbitol	18.805	89
Acetic acid	5.315	93	Arabitol	19.035	90
n-Butylamine	5.705	92	Phosphoric acid	19.482	87
Alanine	5.885	92	Galactopyranose	19.94	96
1,4-Butanediol	5.92	84	1,2,3-PA	20.03	86
Butanoic acid	6.415	94	D-Ribo-Hexitol	20.075	85
1,2-Butanediol	6.73	83	Gluconic acid	20.11	83
Ethanedioic acid	6.78	82	Erythrotetrofuranose	20.325	84
Valine	8.628	88	Fructose	20.47	93
m-ethynylaniline	8.7	84	Galactose	20.595	93
Urea	9.415	93	Glucose	20.69	89
Glycerol	10.185	93	Dulcitol	21.01	91
2(3H)-Furanone	10.25	92	Ribitol	21.011	84
Isoleucine	10.676	93	Tyrosine	21.085	87
Threonine	10.695	90	Sucrose	21.18	85
Glycine	10.98	94	Cholesterol	21.266	86
Butanedioic acid	11.295	94	Pantothenic acid	21.45	82
Chlorphentermine	11.373	80	Mannose	21.639	83
2,3-DBA	11.992	91	Palmitelaidic acid	21.711	83
Serine	12.48	92	Hexadecanoic acid	21.825	91
Cadaverine	13.753	84	Inositol	22.026	85
AA	15.322	83	Uric acid	22.079	84
Malic acid	15.762	90	Stearic acid	22.795	84
Threitol	16.13	90	Oleic acid	22.93	89
Proline	16.33	96	2-monopalmitin	23.35	84
Aspartic acid	16.376	90	2-Monostearin	23.745	86
Pentanoic acid	16.545	82	13-Docosenamide	24.195	89
Creatinine	16.87	91	1,2-BA	24.813	86
2,3,4-TBA	17.03	95

Abbreviation：1,2,3-PA:1,2,3-Propanetricarboxylic acid;2,3,4-TBA:2,3,4-Trihydroxybutyric acid；1,2-BA: 1,2-Benzenedicarboxylic acid; 2,3-DBA:2,3-Dihydroxybutanoic acid;

 

Table S2 Reproducibility of analytes in Quality Control from CSF by GC-MS 

 

Metabolites	tR(min)			Content（ng/mL)
	Mean	SD	RSD(%)	Mean	SD	RSD(%)
Alanine	5.889	0.002	0.037	0.158	0.009	5.873
Serine	12.489	0.005	0.040	0.237	0.007	2.853
Cadaverine	13.754	0.005	0.037	0.058	0.002	4.139
Glucose	20.692	0.003	0.013	10.506	0.475	4.523
Sucrose	21.182	0.003	0.014	0.195	0.007	3.578
Stearic acid	22.794	0.004	0.016	0.063	0.002	2.584

Abbreviation:RSD: relative standard deviation.SD:standard deviation.

 

 

 

Tab S3 Screen in CSF marker between control and cerebral IR

Compound	Log2(FC)	Log10(p)	Compound	Log2(FC)	Log10(p)
Lysine	-5.432	2.703	Arabitol	-1.890	5.254
Propanoic acid	-5.066	9.415	2(3H)-Furanone	-1.737	1.302
13-Docosenamide	-3.794	5.836	Dodecanoic acid	-1.338	2.119
Sucrose	-3.222	5.474	n-Butylamine	-1.288	1.543
Gluconic acid	-3.100	2.913	Acetic acid	-0.821	4.083
2,3,4-TBA	-3.036	7.270	1,2,3-PA	-0.811	2.231
2-monopalmitin	-2.887	7.536	Hexadecanoic acid	-0.712	2.109
D-Fructose	-2.881	6.850	ethane-1,2-diol	0.831	2.794
Dulcitol	-2.782	8.923	Pantothenic acid	0.942	1.452
Threitol	-2.717	6.489	Ethanedioic acid	1.161	1.905
Lyxose	-2.614	11.446	Glucose	1.650	7.664
2-Monostearin	-2.573	1.932	2-Thenaldehyde	2.490	3.221
Ribonic acid	-2.561	11.687	Alanine	2.689	3.122
Glycerol	-2.540	9.927	borate	2.698	10.277
Galactopyranose	-2.540	1.811	Tyrosine	2.744	8.730
Ethane	-2.514	13.378	Ribose	2.914	8.069
Cadaverine	-2.490	1.939	Erythrotetrofuranose	3.032	7.199
Dimethylamine	-2.421	13.737	Butanoic acid	3.193	0.656
3-Buten-2-ol	-2.390	15.900	Creatinine	3.555	6.126
Sorbitol	-2.323	3.251	Serine	3.577	6.291
Oleic acid	-2.309	1.387	Pentane	3.772	6.677
D-Ribo-Hexitol	-2.050	1.723	1,4-Butanediol	4.049	6.168
Glycine	-2.007	3.464	Galactose	4.068	1.691
1,2-Butanediol	-1.957	1.993

 

 

Tab S4 The candidate markers of CSF between cerebral IR and Cop treated.

Compound	Log2(FC)	-Log10(p)
Ribose Stearic acid Glycerol 2,3,4-TBA Glycine Oleic acid	-2.502 -2.432 2.742 0.587 2.852 4.170	4.533 1.830 3.095 1.435 1.834 4.674

 

 

 

 

Tab S5 Identification in CSF markers between control, cerebral IR and Cop treated groups

Compounds	Control vs Cerebral IR		Cerebral IR vs Cop treated
	Log2(FC)	-Log10(p)	Log2(FC)	-Log10(p)
Ribose Glycerol 2,3,4-TBA Glycine Oleic acid	2.914 -2.540 -3.036 -2.007 -2.309	8.069 9.927 7.270 3.464 1.387	-2.502 2.742 0.587 2.852 4.170	4.533 3.095 1.435 1.834 4.674

 

5. The authors need to carefully check their grammars and spellings. A lot of nouns should be used plurals instead the authors used singulars

Response: Thanks for your indication. These mistakes have been corrected.

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

 

The paper entitled “GC-MS based cerebrospinal fluid metabolomics to reveal the protection of Coptisine against transient focal cerebral ischemia-reperfusion injury via ant-inflammation and antioxidant” is a well-designed study that demonstrated the protective effect of Coptisine in the focal ischemia cerebral model in rat. The authors demonstrated that Coptisine reduces the infarct area and partially prevents neurobehavioral dysfunction. The treatment also decreases some inflammatory mediators and attenuates oxidative stress. Additionally, it was demonstrated that 5 metabolites are modulated by the Coptisine treatment. These were identified by the GC-MS metabolomics analysis. Results are clear and consistent. The discussion is excellent, the authors found many related papers that corroborated and support their findings in the metabolomics analysis.

I didn’t have access to supplementary tables.

I don't have important observations, just two comments:

 ·         Definition of CSF is missing in the introduction (line 72), it only appears in the abstract

·         In the description of results obtained in Figure 4, the authors should indicate the panel to facilitate comprehension.

·         Conclusion should be changed before methods

      

Although in general, the authors use Standard English, there are some words that are not used properly. I suggest reviewing carefully the text. For example:

Line 82: “the cerebral ischemia size was apparently increased in cerebral…” The common form to refer to the damaged area is “infarct area” instead of “ischemia size”. The authors found statistical differences between control and ischemic groups, so, it is not necessary to say “apparently”.

Line 85: “…to investigate whether the protective effect...”. The use of "whether" is incomplete in the sentence.

 

Lines 142 and 144, have similar errors.

Author Response

We feel great thanks for your professional review work on our manuscript. As you are concerned, there are several problems that need to be addressed. According to your nice suggestion, we have made extensive corrections to our manuscript, the details correction are listed below.

 

1. I didn’t have access to supplementary tables.

Response: Thanks for your indication. Due to our negligence, the supplementary tables were forgot to attach. Now we have attached them in our manuscript. The information of CSF biomarkers between control, cerebral IR injury and Cop treated add in the manuscript and supplementary materials (Tab S3 and S4). Finally, five CSF metabolites may be target of Cop against cerebral IR (Tab S5 and Fig 5A).

Table S1 Retention time and similarity of analytes in SD rats’ CSF by GC-MS

Compound	tR（min）	Similarity (%)	Compound	tR（min）	Similarity (%)
Cyclobutane	2.027	80	Pentanedioic acid	17.315	88
3-Buten-2-ol	2.415	81	Glutamine	17.904	92
alpha-Pinene	3.185	96	Phenylalanine	17.959	95
Ethane	3.225	97	Ribonic acid	18.205	80
2-Thenaldehyde	3.38	81	Dodecanoic acid	18.345	83
Benzene	3.435	86	Lyxose	18.37	86
borate	3.605	93	Ribose	18.4	92
ethane-1,2-diol	3.67	92	Asparagine	18.521	93
Dimethylamine	4.05	93	Arabinose	18.579	89
Pentane	4.655	95	Lysine	18.715	81
Propanoic acid	4.775	93	Sorbitol	18.805	89
Acetic acid	5.315	93	Arabitol	19.035	90
n-Butylamine	5.705	92	Phosphoric acid	19.482	87
Alanine	5.885	92	Galactopyranose	19.94	96
1,4-Butanediol	5.92	84	1,2,3-PA	20.03	86
Butanoic acid	6.415	94	D-Ribo-Hexitol	20.075	85
1,2-Butanediol	6.73	83	Gluconic acid	20.11	83
Ethanedioic acid	6.78	82	Erythrotetrofuranose	20.325	84
Valine	8.628	88	Fructose	20.47	93
m-ethynylaniline	8.7	84	Galactose	20.595	93
Urea	9.415	93	Glucose	20.69	89
Glycerol	10.185	93	Dulcitol	21.01	91
2(3H)-Furanone	10.25	92	Ribitol	21.011	84
Isoleucine	10.676	93	Tyrosine	21.085	87
Threonine	10.695	90	Sucrose	21.18	85
Glycine	10.98	94	Cholesterol	21.266	86
Butanedioic acid	11.295	94	Pantothenic acid	21.45	82
Chlorphentermine	11.373	80	Mannose	21.639	83
2,3-DBA	11.992	91	Palmitelaidic acid	21.711	83
Serine	12.48	92	Hexadecanoic acid	21.825	91
Cadaverine	13.753	84	Inositol	22.026	85
AA	15.322	83	Uric acid	22.079	84
Malic acid	15.762	90	Stearic acid	22.795	84
Threitol	16.13	90	Oleic acid	22.93	89
Proline	16.33	96	2-monopalmitin	23.35	84
Aspartic acid	16.376	90	2-Monostearin	23.745	86
Pentanoic acid	16.545	82	13-Docosenamide	24.195	89
Creatinine	16.87	91	1,2-BA	24.813	86
2,3,4-TBA	17.03	95

Abbreviation：1,2,3-PA:1,2,3-Propanetricarboxylic acid;2,3,4-TBA:2,3,4-Trihydroxybutyric acid；1,2-BA: 1,2-Benzenedicarboxylic acid; 2,3-DBA:2,3-Dihydroxybutanoic acid;

 

Table S2 Reproducibility of analytes in Quality Control from CSF by GC-MS 

 

Metabolites	tR(min)			Content（ng/mL)
	Mean	SD	RSD(%)	Mean	SD	RSD(%)
Alanine	5.889	0.002	0.037	0.158	0.009	5.873
Serine	12.489	0.005	0.040	0.237	0.007	2.853
Cadaverine	13.754	0.005	0.037	0.058	0.002	4.139
Glucose	20.692	0.003	0.013	10.506	0.475	4.523
Sucrose	21.182	0.003	0.014	0.195	0.007	3.578
Stearic acid	22.794	0.004	0.016	0.063	0.002	2.584

Abbreviation:RSD: relative standard deviation.SD:standard deviation.

 

 

 

Tab S3 Screen in CSF marker between control and cerebral IR

Compound	Log2(FC)	Log10(p)	Compound	Log2(FC)	Log10(p)
Lysine	-5.432	2.703	Arabitol	-1.890	5.254
Propanoic acid	-5.066	9.415	2(3H)-Furanone	-1.737	1.302
13-Docosenamide	-3.794	5.836	Dodecanoic acid	-1.338	2.119
Sucrose	-3.222	5.474	n-Butylamine	-1.288	1.543
Gluconic acid	-3.100	2.913	Acetic acid	-0.821	4.083
2,3,4-TBA	-3.036	7.270	1,2,3-PA	-0.811	2.231
2-monopalmitin	-2.887	7.536	Hexadecanoic acid	-0.712	2.109
D-Fructose	-2.881	6.850	ethane-1,2-diol	0.831	2.794
Dulcitol	-2.782	8.923	Pantothenic acid	0.942	1.452
Threitol	-2.717	6.489	Ethanedioic acid	1.161	1.905
Lyxose	-2.614	11.446	Glucose	1.650	7.664
2-Monostearin	-2.573	1.932	2-Thenaldehyde	2.490	3.221
Ribonic acid	-2.561	11.687	Alanine	2.689	3.122
Glycerol	-2.540	9.927	borate	2.698	10.277
Galactopyranose	-2.540	1.811	Tyrosine	2.744	8.730
Ethane	-2.514	13.378	Ribose	2.914	8.069
Cadaverine	-2.490	1.939	Erythrotetrofuranose	3.032	7.199
Dimethylamine	-2.421	13.737	Butanoic acid	3.193	0.656
3-Buten-2-ol	-2.390	15.900	Creatinine	3.555	6.126
Sorbitol	-2.323	3.251	Serine	3.577	6.291
Oleic acid	-2.309	1.387	Pentane	3.772	6.677
D-Ribo-Hexitol	-2.050	1.723	1,4-Butanediol	4.049	6.168
Glycine	-2.007	3.464	Galactose	4.068	1.691
1,2-Butanediol	-1.957	1.993

 

 

Tab S4 The candidate markers of CSF between cerebral IR and Cop treated.

Compound	Log2(FC)	-Log10(p)
Ribose Stearic acid Glycerol 2,3,4-TBA Glycine Oleic acid	-2.502 -2.432 2.742 0.587 2.852 4.170	4.533 1.830 3.095 1.435 1.834 4.674

 

 

 

 

Tab S5 Identification in CSF markers between control, cerebral IR and Cop treated groups

Compounds	Control vs Cerebral IR		Cerebral IR vs Cop treated
	Log2(FC)	-Log10(p)	Log2(FC)	-Log10(p)
Ribose Glycerol 2,3,4-TBA Glycine Oleic acid	2.914 -2.540 -3.036 -2.007 -2.309	8.069 9.927 7.270 3.464 1.387	-2.502 2.742 0.587 2.852 4.170	4.533 3.095 1.435 1.834 4.674

 

2. I don't have important observations, just two comments:

• Definition of CSF is missing in the introduction (line 72), it only appears in the abstract
• In the description of results obtained in Figure 4, the authors should indicate the panel to facilitate comprehension.

Response: Thanks for your suggestion. (1) The definition of CSF has been written in the introduction section and highlighted in the manuscript (“CSF is a biological fluid that circulates in the brain, and it is in direct contact with the extracellular space of the brain, and its changes reflect different functional states of the brain”). (2) The description of results obtained in Fig 4 add in the section of result as follow: “There were 47 differential metabolites were discovered between IR and control groups in CSF, including 16 increased (ribose, ethanedioic acid and butanoic acid etc) and 31 decreased(glycine, oleic acid, gluconic acid and cadaverine, etc), otherwise there were 6 differential metabolites in CSF (4 increased(oleic acid, glycerol, glycine and 2,3,4-trihydroxybutyric acid) and 2 decreased(ribose and stearic acid)) after Cop-treated.” and a marker list between control, cerebral IR and Cop treated was attached in Fig S3.

3. Conclusion should be changed before methods

Response: Thanks for your advice. However, the format order of 《Molecules》 is introduction-results-discussion-materials and methods-conclusion.

4. Although in general, the authors use Standard English, there are some words that are not used properly. I suggest reviewing carefully the text. For example:

Line 82: “the cerebral ischemia size was apparently increased in cerebral…” The common form to refer to the damaged area is “infarct area” instead of “ischemia size”. The authors found statistical differences between control and ischemic groups, so, it is not necessary to say “apparently”.

Line 85: “…to investigate whether the protective effect...”. The use of "whether" is incomplete in the sentence.

Lines 142 and 144, have similar errors.

Response: Thanks for your comments. These mistakes have been corrected, for example: we used “infract size” instead of “ischemia size”. 

Author Response File: Author Response.docx