Bcl‐2 is a negative regulator of interleukin‐1β secretion in murine macrophages in pharmacological‐induced apoptosis

BACKGROUND AND PURPOSE Cucurbitacin R, a natural anti‐inflammatory product, has been shown to exhibit activity against both adjuvant‐induced arthritis and delayed‐type hypersensitivity reactions induced by various agents. Previous studies have demonstrated that the effects of cucurbitacin R stem from its inhibition of both cytokine production and lymphocyte proliferation. EXPERIMENTAL APPROACHES Effects of cucurbitacin R were investigated on lipopolysaccharide‐stimulated RAW 264.7 cells. Cell cycle evolution was analysed by flow cytometry, detection of apoptosis by DNA ladder, Bcl‐2, p21, p53, Bax, cleaved caspase‐1 (p10), caspase‐9, and caspase‐3, cleaved caspase (p17) and interleukin‐1β detection was followed by Western blot analysis and mRNA expression with quantitative real time reverse transcription‐polymerase chain reaction (qRT‐PCR). KEY RESULTS Cucurbitacin R was found to induce apoptosis in lipopolysaccharide‐stimulated RAW 264.7 macrophages through the inhibition of Bcl‐2 expression, which regulates pro‐inflammatory caspase‐1 activation and interleukin‐1β release. Also, cucurbitacin R arrested the cell cycle in the G2/M phase and increased the subG0 population in lipopolysaccharide‐stimulated RAW 264.7 macrophages. Moreover, it increased the expression of proteins p53 and p21, down‐regulated the expression of Bcl‐2, activated the activity of caspase‐1 and augmented the production of interleukin‐1β. Finally, the transfection of RAW 264.7 macrophages with a Bcl‐2 expression plasmid produced the inhibition of apoptosis and caspase‐1 activation/interleukin‐1β release induced by cucurbitacin R in RAW 264.7 cells. CONCLUSIONS AND IMPLICATIONS Taken together, these results point to a new apoptotic process in which interleukin‐1β release is directly regulated by Bcl‐2 status; this contributes to the evidence that apoptotic processes do not induce inflammation.