Terminalia chebula extract protects OGD-R induced PC12 cell death and inhibits lps induced microglia activation

Abstract

Terminalia chebula, native to Southeast Asia, is a popular medicinal plant in Ayurveda. It has been previously reported to have strong antioxidant and anti-inflammatory efficacy. In this study, we aimed to investigate if fruit extract from T. chebula might protect neuronal cells against ischemia and related diseases by reduction of oxidative damage and inflammation in rat pheochromocytoma cells (PC12) using in vitro oxygen-glucose deprivation followed by reoxygenation (OGD-R) ischemia and hydrogen peroxide (H2O2) induced cell death. Cell survival was evaluated by a 2-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Free radical scavenging, lipid peroxidation and nitric oxide inhibition were measured by diphenyl-1-picrylhydrazyl (DPPH), thiobarbituric acid (TBA) and Griess reagent, respectively. We found that T. chebula extract: (1) increases the survival of cells subjected to OGD-R by 68%, and H2O2 by 91.4%; (2) scavenges the DPPH free radical by 96% and decreases malondialdehyde (MDA) levels from 237.0 ± 15.2% to 93.7 ± 2.2%; (3) reduces NO production and death rate of microglia cells stimulated by lipopolysaccharide (LPS). These results suggest that T. chebula extract has the potential as a natural herbal medicine, to protect the cells from ischemic damage and the possible mechanism might be the inhibition of oxidative and inflammatory processes.

Figure 1

3D HPLC spectrum of T. chebula extract showing gallic acid and ellagic acid.

Figure 2

Effect of T. chebula extract on PC12 cells. (A) Cytotoxic effect of T. chebula extract on PC12 cells. Different concentrations of T. chebula extract were treated on PC12 cells for 28 h in normal condition. (B) Effect of T. chebula extract against OGD-R induced cell death on PC12 cells. PC12 cells were exposed to OGD for 4 h followed by re-oxygenation for 24 h. Different concentrations of T. chebula extract were treated 30 min before and during 4 h of OGD. PC12 cells viability was measured by MTT assay. Control group served as 100%, and data obtained in other groups were calculated as percentage of control accordingly. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 3

Effect of T. chebula extract on H₂O₂-induced cell death and lipid peroxidation assay. PC12 cells were exposed to 200 μM H₂O₂ for 24 h. Different concentrations of T. chebula extract were treated 2 h before and during H₂O₂ exposure. Control group served as 100%, and data obtained in other groups were calculated as percentage of control accordingly. (A) PC12 cells viability was measured by MTT assay. (B) Lipid peroxidation assay (MDA level) was measured by thiobarbutic assay. ** p < 0.01, and *** p < 0.001.

Figure 4

Effect of T. chebula extract on LPS-induced cell death and NO inhibition assay. BV2 cells were exposed to LPS (1 μg/mL) for 24 h. Different concentrations of T. chebula extract were treated 2 h before and during LPS exposure. (A) Cell viability was measured by MTT assay. Control group served as 100%, and data obtained in other groups were calculated as percentage of control accordingly. (B) Cell-conditioned supernatants were collected and the production of NO in the supernatants was measured using Griess reagent. NO production was calculated by using standard nitrite. * p < 0.05, ** p < 0.01, and *** p < 0.001.