The protective effect of lithium on the morphine induced apoptosis in PC12 cell line

Opioids are powerful analgesics with widespread usage. Morphine induces apoptosis in many of cells such as neurons and immune cells. In this study the protective effect of lithium against apoptosis induced by morphine on PC12 cell line was investigated. We used MTT assay, annexin V-FITC test and real time RT-PCR to measure viability, apoptosis and gene expression, respectively. Different concentrations (0.2, 0.4, 0.8, 1.6 mmol/L) of morphine were used to induce apoptosis. Also, we incubated cells with 1.2 mmol/L lithium chloride for 72 hours and then added 0.8 and 1.6 mmol/L of morphine. Our results indicate that morphine (0.8 and 1.6 mmol/L) induces cell death after both 24 h and 12h exposure when using MTT test and annexin V test, respectively. The data obtained by real-time RT-PCR show that after 6 hours exposure to 0.8 (not 1.6) mmol/L of morphine, mRNA expression of Bax (a proapoptotic protein) and Bcl-2 (an antiapoptotic protein) increased and decreased, respectively. In the other part of experiment, pretreatment of PC12 cells by lithium chloride (1.2 mmol/L) for 72 hours decreased the mRNA expression of Bax which was elevated by morphine (0.8 and 1.6 mmol/L). Interestingly, mRNA expression of Bcl-2 which was elevated by morphine (0.8 mmol/L) during 6 hours was increased by pretreatment of lithium 1.2 mmol/L. On the other hand, incubation of cells with 1.2 mmol/L lithium chloride for 72 hours did not alter the mRNA expression of Bax or Bcl-2 and had no toxic effect on the survival of PC12 cells. Pretreatment of lithium protects PC12 cells from morphine induced apoptosis. This effect can be related to Bax and Bcl-2 genes expression.