Ginsenosides Rb1 and Rg1 Protect Primary Cultured Astrocytes against Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury via Improving Mitochondrial Function

Round 1

Reviewer 1 Report

Manuscript ID: ijms-645283

Ginsenoside Rb1 and Rg1 Protect Primary Cultured Astrocytes Against Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury via Improving Mitochondrial Function

Which is the mechanism by which OGD/R damages the astrocytes? Does apoptosis play a role?

Which was the effect of Rg1and of  Rb1 on the mt membrane potential

Why did the increase of ROS caise a decrease I catalase activity? One would expect that catalas activity increase with increasing oxidative stress..

The English needs to be revised

11/19

Author Response

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Reviewer 2 Report

The authors studied how the ginsenosides R1 and Rg1 protect astrocytes after coupled oxygen-glucose deprivation/reoxygenation-induces Injury. This effect is mainly mediated by protecting mitochondrial function and reducing oxidative stress.

The manuscript is well written and the results are interesting. However, some aspects expressed below, have been taking into account to recommend major revision.

First, in the experiment of cell viability, the number of cells that are rescued after the OGD/R is quite small. I am afraid that the authors are using a CCK8, which is dependent on dependent on NAD(P)H-dependent oxidoreductase enzymes. It is important to keep in mind that the OGD/R and ginsenosides R1 and Rg1-treatment alter metabolic activity in the astrocytes and thus tetrazolium dye reduction without affecting cell viability. I recommend using viability assays independent of cell metabolism.

The authors have measured ROS production by using DCFH-DA. However, using a probe like MitoSOX would be very informative. With MitoSOX, we could better understand if there is an upregulation of the ROS-production in the mitochondria, which in turn, will be depleted by the catalase, which was the enzyme selected for the authors.

Determination of the REDOX state of the cells by measuring GSH and GSSG would be very interesting.

The authors have found a depletion of ATP levels after OGD/R that can be reversed by incubation with ginsenosides. They propose that this effect is due to the rescue of the OXPHOS activity; however, they did not study the anaerobic glycolysis, which after the OGD/R could be further modulated. Actually, astrocytes are well known for the lactate shuttle to feed neurons even in hypoxic conditions.

The author showed that the activity of the complex V, which is responsible for the ATP production, is reduced from 15 U/mg prot to 5 U/mg prot after OGD/R treatment, and then rescued until 10 U/mg prot with the ginsenosides treatment. However ATP, levels are no so deeply modified, how can the author explain these differences?

Finally, using a co-culture with neurons could be very informative to determine how the modulation in the REDOX and OXPHOS state of the astrocytes could modulate neuron viability.

No information about the kit used for determining the complexes activity was found on the website of the manufacturer. Please include a brief sum-up of the protocols.

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

I thank the reply of the authors.

Author Response

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