Cystone - An ayurvedic polyherbal formulation inhibits adherence of uropathogenic E. coli and modulates H2O2-induced toxicity in NRK-52E cells

Abstract

Gentamicin is a widely used antibiotic for the treatment of adverse urinary tract infections (UTI), which in turn causes nephrotoxicity to uroepithelial cells and hence an alternative safe herbal remedy is much desired to compensate these toxic effects. The bacterial adhesion to the uroepithelial cells is the primary step in UTI and it induces various immunogenic reactions leading to the generation of reactive oxygen species (ROS), which are detrimental to the cells survival. Inhibition of bacterial adherence to urinary tract epithelial cells has been assumed to account for the beneficial action ascribed to cystone (an ayurvedic polyherbal formulation) in the prevention of UTI. In this study, we have examined the effect of cystone on the adherence of pathogenic [2-(14)C]-acetate labeled Escherichia coli (MTCC-729) to rat proximal renal tubular cells (NRK-52E cells). Further, the antioxidant property of cystone was studied using hydrogen peroxide (400 μM) as a pro-oxidant in NRK-52E cells. The results showed that cystone inhibited the adherence of E. coli to NRK-52E cells significantly. Additionally cystone effectively combats the toxicity induced by H2O2 in NRK-52E cells. The cytoprotective effect of cystone is brought about by inhibiting lipid peroxidation by 36% in cells treated with cystone compared to H2O2-treated cells without cystone. The antioxidant enzymes catalase, glutathione were increased by 53% and 68% respectively and superoxide dismutase activity was increased 3-fold. The glutathione content was significantly increased by 2.4-fold in NRK-52E cells treated with cystone compared to H2O2 control group. These results suggest that cystone effectively inhibits bacterial adherence to NRK-52E cells and attenuates H2O2-induced toxicity in NRK-52E cells by inhibiting lipid peroxidation and increasing the antioxidant defense mechanism.

Keywords: antioxidant enzymes; bacterial adhesion; hydrogen peroxide; lipid peroxidation; urinary tract infection.

Figure 1

Dose-dependent effect of (A) Cystone, (B) Gentamicin and (C) H₂O₂ on NRK-52E cell viability. NRK-52E cells were treated for 24 h with or without test substances at different concentrations and the cell viability was determined by MTT. Notes: Values are Mean ± SEM of 18 samples taken from three independent experiments. *Significant difference at P < 0.01 compared to control.

Figure 2

Effect of gentamicin and cystone on the adherence of E. coli to NRK-52E cells. (A) Time course of incorporation of [2-¹⁴C]-acetate to E.coli. (B) Inhibition of bacterial adhesion to NRK-52E cells by gentamicin and cystone. (C) Effect of sub-inhibitory concentration of gentamicin and (D) cystone. The labeled E. coli cells (1 × 10⁵ E. coli cells/mL) were incubated with 1 × 10⁵ NRK-52E cells for 30 min with or with gentamicin/cystone and the results are given as described in materials and methods. Notes: Values are mean ± SEM of 18 samples taken from three independent experiments. *Significant difference at P < 0.01 compared to control.

Figure 3

The cytoprotective and antioxidant effect of cystone in NRK-52E cells. (A) The NRK-52E cells were treated with or without 1% cystone along with 400 μM H₂O₂ and Gentamicin (125 μg/mL) and the cell viability was determined by MTT assay. In another set of experiments (B) Lipid peroxidation and (C) reduced glutathione (GSH) content were measured in NRK-52E cells. Notes: Values are mean ± SEM of 18 samples taken from three independent experiments. *Significant difference at P < 0.01 compared to control.