OBJECTIVE Estrogen replacement therapy can decrease the risk of developing Alzheimer's disease. Phytoestrogens have been proposed as potential alternatives to estrogen replacement therapy. The purpose of this study was to evaluate the in vitro protective effects of coumestrol on mice astrocytes. METHODS Different concentrations of coumestrol were tested for their protective efficacy against two toxic insults, lipopolysaccharide (LPS) and amyloid-beta peptide, on astrocytes. The mitochondrial activity of astrocytes was determined, and the protective efficacy and pathway were examined by their specific gene expression and protein change. RESULTS The results showed that coumestrol induced a modest but significant increase in viability of astrocytes, while the viability of astrocytes was reduced following exposure to LPS and amyloid-beta peptide. The addition of coumestrol could reverse the toxic effect induced by LPS and amyloid-beta peptide. Both the LPS and amyloid-beta peptide enhanced interleukin 1, interleukin 6, and tumor necrosis factor-alpha synthesis and these effects were inhibited by 10(-9)M coumestrol. This effect was more obvious on the LPS-induced inflammation. The estrogen receptor expression was upregulated by coumestrol, while the effect was more obvious on estrogen receptor-beta (ER-beta). These effects can be inhibited by extracellular signal-regulated kinase and c-Jun N-terminal kinase inhibitors but not p38 inhibitor. CONCLUSIONS The current data support a possible role for astrocytes in the mediation of neuroprotection by coumestrol. An indirect extracellular signal-regulated kinase/c-Jun N-terminal kinase signaling pathway to downregulate the expression of interleukin 1, interleukin 6, and the tumor necrosis factor-alpha cytotoxic effect may act in concert with the proposed direct ER-beta biosynethsis pathway to achieve a widespread, global protection of ER-beta positive neurons.