Cytoprotective propensity of Bacopa monniera against hydrogen peroxide induced oxidative damage in neuronal and lung epithelial cells

Abstract

Hydrogen peroxide (H2O2), a major reactive oxygen species (ROS) produced during oxidative stress, is toxic to the cells. Hence, H2O2 has been extensively used to study the effects of antioxidant and cytoprotective role of phytochemicals. In the present investigation H2O2 was used to induce oxidative stress via ROS production within PC12 and L132 cells. Cytoprotective propensity of Bacopa monniera extract (BME) was confirmed by cell viability assays, ROS estimation, lipid peroxidation, mitochondria membrane potential assay, comet assay followed by gene expression studies of antioxidant enzymes in PC12 and L132 cells treated with H2O2 for 24 h with or without BME pre-treatment. Our results elucidate that BME possesses radical scavenging activity by scavenging 2,2-diphenyl-1-picrylhydrazyl, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), superoxide radical, and nitric oxide radicals. The IC50 value of BME against these radicals was found to be 226.19, 15.17, 30.07, and 34.55 µg/ml, respectively). The IC50 of BME against ROS, lipid peroxidation and protein carbonylation was found to be 1296.53, 753.22, and 589.04 µg/ml in brain and 1137.08, 1079.65, and 11101.25 µg/ml in lung tissues, respectively. Further cytoprotective potency of the BME ameliorated the mitochondrial and plasma membrane damage induced by H2O2 as evidenced by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase leakage assays in both PC12 and L132 cells. H2O2 induced cellular, nuclear and mitochondrial membrane damage was restored by BME pre-treatment. H2O2 induced depleted antioxidant status was also replenished by BME pre-treatment. This was confirmed by spectrophotometric analysis, semi-quantitative RT-PCR and western blot studies. These results justify the traditional usage of BME based on its promising antioxidant and cytoprotective property.

Keywords: Antioxidant enzymes; Bacopa monniera; Cytotoxicity; Hydrogen peroxide; L132 cells; Oxidative stress; PC12 cells; Reactive oxygen species.

Fig. 1

a Protective role of BME on 20 mM AAPH induced BSA oxidative fragmentation. Lane 1: BSA; lane 2: BSA + AAPH; lane 3: BSA + AAPH + 5 μg/ml BME; lane 4: BSA + AAPH + 10 μg/ml BME; lane 5: BSA + AAPH + 20 μg/ml BME; lane 6: BSA + AAPH + 40 μg/ml extract; lane 7, BSA + AAPH + 10 μg/ml Gallic acid. b The band intensity is quantified by NIH Image J analysis software. The data are presented as mean ± SEM of three independent experiments. *p < 0.05 versus the respective control group and ^# p < 0.05 versus the respective AAPH treatment

Fig. 2

a Dose-dependent effects of H₂O₂ (0–600 µM) on PC12 and L132 cell viability. b Dose dependent protective effect of BME on H₂O₂ (300 µM for PC12 and 500 µM for L132 cells) induced cytotoxicity. The data are presented as mean ± SEM of three independent experiments

Fig. 3

a Dose-dependent effects of H₂O₂ (0–600 µM) on LDH leakage. b Protective effect of BME (100 µg/ml) on H₂O₂ (300 µM for PC12 and 500 µM for L132 cells) induced LDH leakage. The data are presented as mean ± SEM of three independent experiments. *p < 0.05 versus the respective control group and ^# p < 0.05 versus the respective H₂O₂ treated group

Fig. 4

a Estimation of ROS production induced by H₂O₂ in PC12 and L132 cells. The data are represented as mean ± SEM of three independent experiments. *p < 0.05 versus the respective control group and ^# p < 0.05 versus the respective H₂O₂ treated group. b ROS production in PC12 and L132 cells was monitored by fluorescence microscopy. (from left to right) a Control cells without any treatment, b 100 μg/ml BME, c H₂O₂ (300 µM for PC12 and 500 µM for L132) for 24 h d cells pre-treatment with 100 μg/ml BME for 1 h and then exposed to H₂O₂ (300 µM for PC12 and 500 µM for L132) for 24 h

Fig. 5

a Protective effect of BME (100 µg/ml) against H₂O₂ (300 µM for PC12 and 500 µM for L132 cells) induced lipid peroxidation in PC12 and L132 cells. b Effect of BME (100 µg/ml) on H₂O₂ (300 µM for PC12 and 500 µM for L132 cells) induced decrease of mitochondrial membrane potential determined by spectrofluorimetric method. The data are presented as mean ± SEM of three independent experiments. *p < 0.05 versus the respective control group and ^# p < 0.05 versus the respective H₂O₂ treated group

Fig. 6

a Effect of BME on DNA damage induced by H₂O₂ in PC12 and L132 cells. a Control cells without any treatment, b 100 μg/ml BME, c H₂O₂ (300 µM for PC12 and 500 µM for L132) for 24 h d cells pre-treatment with 100 μg/ml BME for 1 h and then exposed to H₂O₂ (300 µM for PC12 and 500 µM for L132) for 24 h and (b). The tail length of the comet was measured in each cell using image pro^® plus software and represented as percent tail movement. The data are presented as mean ± SEM of three independent experiments. *p < 0.05 versus the respective control group and ^# p < 0.05 versus the respective H₂O₂ treated group

Fig. 7

a Protective effect of BME (100 μg/ml) against H₂O₂ treatment on expression of oxidative stress marker SOD, CAT, GPx and GR was analyzed by semi-quantitative PCR analysis. b The band intensity is quantified by NIH Image J software. The data are represented as mean ± SEM of three independent experiments. *p < 0.05 versus the respective control group and ^# p < 0.05 versus the respective H₂O₂ treated group

Fig. 8

a Protective effect of BME (100 μg/ml) against H₂O₂ treatment on expression of oxidative stress marker proteins SOD, CAT, GPx and GR analyzed by immunobloting. b The band intensity was calculated by NIH Image J software. The data are represented as mean ± SEM of three independent experiments. *p < 0.05 versus the respective control group and ^# p < 0.05 versus the respective H₂O₂ treated group