TPNA10168, an Nrf-2 activator, stimulates glutathione production by inducing system Xc-, and g-glutamate cysteine ligase subunits in cultured mouse astrocytes.

Abstract Nuclear factor-erythroid 2-related factor 2 (Nrf-2) is a transcription factor involved in cellular defense mechanisms against oxidative stress and mediates the expression of various antioxidant-related molecules. We previously showed that TPNA10168, a novel Nrf-2 activator, has neuroprotective effects. To elucidate the mechanisms underlying the cytoprotective action of TPNA10168, its effects on the expression of glutathione biosynthesis-related molecules in cultured mouse astrocytes were examined. Cultured astrocytes were prepared from the cerebra of 1-day-old C57BL/6N mice. mRNA and protein levels were measured by quantitative PCR and immunoblotting, respectively. Treatment with TPNA10168 (30 µM) increased the astrocytic Nrf-2 protein level. Nuclear translocation of Nrf-2 and its binding to the Nrf-2 recognition DNA fragment were stimulated by TPNA10168. The mRNA and protein levels of system Xc−, a cystine/glutamate exchange transporter, in cultured astrocytes were increased by TPNA10168. TPNA10168 also increased the expression of γ-glutamate cysteine ligase subunits (γ-GCLm and γ-GCLc). Accompanied by the increased expression of system Xc−, γ-GCLm, and γ-GCLc, the production of astrocytic glutathione was increased by TPNA10168. The effects of TPNA10168 on the expression of system Xc−, γ-GCLm, and γ-GCLc were reduced by the Nrf-2 inhibitor ML385 (10 µM). Rotenone (2.5 nM) induced cell death in cultured astrocytes; however, this effect was alleviated by TPNA10168. The results suggest that Nrf-2-mediated astrocytic glutathione biosynthesis is one of the mechanisms underlying the cytoprotective action of TPNA10168.