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. 2004 Aug 13:4:11.
doi: 10.1186/1472-6882-4-11.

Rubia cordifolia, Fagonia cretica linn and Tinospora cordifolia exert neuroprotection by modulating the antioxidant system in rat hippocampal slices subjected to oxygen glucose deprivation

Avinash K Rawal  1 , Manohar G Muddeshwar, Saibal K Biswas
Affiliations

Rubia cordifolia, Fagonia cretica linn and Tinospora cordifolia exert neuroprotection by modulating the antioxidant system in rat hippocampal slices subjected to oxygen glucose deprivation

Avinash K Rawal et al. BMC Complement Altern Med. .

Abstract

Background: The major damaging factor during and after the ischemic/hypoxic insult is the generation of free radicals, which leads to apoptosis, necrosis and ultimately cell death. Rubia cordifolia (RC), Fagonia cretica linn (FC) and Tinospora cordifolia (TC) have been reported to contain a wide variety of antioxidants and have been in use in the eastern system of medicine for various disorders. However, their mechanism of action was largely unknown. We therefore selected these herbs for the present study to test their neuroprotective ability and the associated mechanism in rat hippocampal slices subjected to oxygen-glucose deprivation (OGD).

Methods: Hippocampal Slices were subjected to OGD (oxygen glucose deprivation) and divided into 3 groups: control, OGD and OGD + drug treated. Cytosolic Cu-Zn superoxide dismutase (Cu-Zn SOD), reduced glutathione (GSH), glutathione peroxidase (GPx), nitric oxide (NO) was measured as nitrite (NO2) in the supernatant and protein assays were performed in the respective groups at various time intervals. EPR was used to establish the antioxidant effect of RC, FC and TC with respect to superoxide anion (O2.-), hydroxyl radicals (. OH), nitric oxide (NO) radical and peroxynitrite anion (ONOO) generated from pyrogallol, menadione, DETA-NO and Sin-1 respectively. RT-PCR was performed for the three groups for GCLC, iNOS, Cu-Zn SOD and GAPDH gene expression.

Results: All the three herbs were effective in elevating the GSH levels, expression of the gamma-glutamylcysteine ligase and Cu-Zn SOD genes. The herbs also exhibited strong free radical scavenging properties against reactive oxygen and nitrogen species as studied by electron paramagnetic resonance spectroscopy. In addition all the three herbs significantly diminished the expression of iNOS gene after 48 hours which plays a major role in neuronal injury during hypoxia/ischemia.

Conclusions: RC, FC and TC therefore attenuate oxidative stress mediated cell injury during OGD and exert the above effects at both the cytosolic as well as at gene expression level and may be an effective therapeutic tool against ischemic brain damage.

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Figures

Figure 1
Figure 1
Effect of RC, FC and TC on the cytosolic GSH levels during OGD in rat hippocampal slices. * p <0.001 compared to stabilized, and + p < 0.001 compared to OGD. Results expressed are a mean of 6 independent experiments ± SEM.
Figure 2
Figure 2
a: Representative GCLC mRNA expression in relation to GAPDH in control, OGD and OGD + drug treated rat hippocampal slices. Lane 1 – Control, Lane 2 – OGD 24 hr, Lane 3 – OGD 48 hr, Lane 4 – OGD + FC, Lane 5 – OGD+TC, Lane 6 – OGD+RC. b: Percentage densitometric expression of GCL mRNA expression in relation to GAPDH in control, OGD and OGD + drug treated rat hippocampal slices. * p < 0.01 and ** p < 0.001 versus control, + p < 0.01 versus * & ** and ++ p < 0.001 versus * & **. Results expressed are a mean of 6 independent experiments ± SEM.
Figure 3
Figure 3
Non-linear curve fit analysis of the effect of RC, FC and TC on scavenging free radicals such as A) O2-, B) OH., C) NO and D) ONOO- generated by pyrogallol, menadione, DETA-NO and SIN-1 respectively. The reference curve fit is depicted in the respective groups. Results expressed are a mean of 3 independent experiments ± SEM.
Figure 4
Figure 4
Effect of RC, FC and TC on the glutathione peroxidase levels in rat hippocampal slices. + p < 0.01 versus stabilized and ++ p < 0.001 versus OGD. Results expressed are a mean of 4 independent experiments ± SEM.
Figure 5
Figure 5
Effect of RC, FC and TC on Cu-Zn SOD gene expression and cytosolic free levels during OGD in rat hippocampal slices. RT-PCR pictograph of Cu-Zn SOD is shown aligned to the corresponding groups. * p < 0.001 versus stabilized, + p < 0.001 versus OGD. Results expressed are a mean of 6 independent experiments ± SEM.
Figure 6
Figure 6
Effect of RC, FC and TC and amounts of ascorbic acid and GSH equivalent to that present in the herbs (RC*, FC* and TC* respectively) on NO2 generation in hippocampal slices. + p <0.001 versus control (100%) and ** p < 0.001 versus OGD. Results expressed are a mean of 3 independent experiments ± SEM.
Figure 7
Figure 7
Effect of RC, FC and TC on the iNOS gene expression in rat hippocampal slices subjected to OGD and OGD+RC/FC/TC. Lane 1 – Control, Lane 2 – OGD 24 hr, Lane 3 – OGD 48 hr, Lane 4 – OGD + FC, Lane 5 – OGD+TC, Lane 6 – OGD+RC. *p < 0.05 vs control, **p < 0.001 vs control, +p < 0.001 vs **. Results expressed are a mean of 6 independent experiments ± SEM.
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