Counteractive Effect Of Interleukin-6 On NMDA-Induced Neuronal Apoptosis And Intracellular Signal-Transduction Mechanisms Mediating The Effect

Objectives: The previous studies in our laboratory have shown that interleukin-6 (IL-6) can protect neurons against glutamate- and N-methyl-D-aspartate(NMDA)-induced excitatory neurotoxicity. Although neuronal apoptosis has been known to be close related to pathogenesis of brain diseases, effect of IL-6 on neuronal apoptosis and mechanisms involved in the effect are primarily unclear. Thus, in this study, we established a neuronal apoptotic model induced by NMDA, and investigated the neuroprotection of IL-6 from NMDA-induced apoptosis. On the other hand, the mechanisms involved in the IL-6 neuroprotection were explored from the profile of IL-6 intracellular signal-transduction pathways. Methods: The cerebellar granule neurons from postnatal 8-day infant rats were chronically exposed to IL-6 (40 or 120 ng/ml), JAK inhibitor AG490 (5 or 10μmol/L) and PI3K inhibitor LY294002 (5 or 10μmol/L) for 8 days, respectively. NMDA (100μmol/L) was then added to the cultures and stimulated the neurons for 30 min to induce the neuronal apoptosis. Real-time PCR and Western blot were employed to detect the expression of gene and protein related to neuronal apoptosis, including Bcl-2, Bax and caspase-3. Meanwhile, the expression of phospho-STAT3 and phospho-Akt were measured by Western blot assay after the neurons were treated with IL-6, AG490 and LY294002. Results: NMDA stimulation of the cultured cerebellar granule neurons without IL-6 pretreatment led to a notable reduction of Bcl-2 mRNA and protein expressions, as well as a remarkable enhancement of Bax and caspase-3 mRNA and protein expressions. IL-6 itself had a significant and dose-dependent enhancement of the expressions of Bcl-2 mRNA and protein, as well as reduction of the expressions of Bax and caspase-3 mRNAs and proteins. The NMDA stimulation of the neurons that had been pretreated with IL-6 caused a remarkable increase in Bcl-2 mRNA and protein expressions, as well as a marked decrease in Bax and caspase-3 mRNA and protein expressions in comparison with those of neurons lacking IL-6 pretreatment. The effects of IL-6 were concentration-dependent, and the high concentration of IL-6 (120 ng/ml) caused the NMDA-induced changes in the gene and protein expressions to return to the intact control levels. The NMDA stimulation of the neurons that had been chronically pretreated with AG490 and IL-6 presented a dramatic decrease in Bcl-2 mRNA and protein expressions, as well as a striking increase in Bax and caspase-3 mRNA and protein expressions relative to those of the neurons pretreated with IL-6 alone. Although the actions of AG490 were dose-dependent, the higher dose of AG490 (10μmol/L) only partially blocked the effects of IL-6 on the neuronal expressions of the genes and proteins related to the apoptosis. Simultaneously, AG490 blocked the effect of IL-6 up-regulation on the phospho-STAT3 expression. The NMDA stimulation of the neurons that had been pretreated with PI3K inhibitor LY294002 and IL-6 resulted in a significant lower expression of Bcl-2 and a higher expression of bax and caspase-3 than those of the neurons pretreated with IL-6 alone. LY294002 of 10μmol/L had a stronger effect on IL-6 neuroprotection than 5μmol/L LY294002, but it still did not completely block the IL-6 neuroprotection. Moreover, LY294002 blocked the action of IL-6 up-regulation on the phospho-Akt expression. Conclusions: NMDA down-regulated Bcl-2 expression and up-regulated Bax and caspase-3 expressions in the neurons, showing that NMDA can induce neuronal apoptosis. IL-6 antagonized the NMDA-induced expression down-regulation of Bcl-2 and expression up-regulation of Bax and caspase-3 in the neurons, revealed that IL-6 can protect neurons against neuronal apoptosis. After the activities of intracellular signal-transduction molecules JAK and PI3K in the neurons were inhibited by AG490 and LY294002 respectively, the phosphorylation of their downstream proteins STAT3 and Akt were depressed and the neuroprotection of IL-6 against apoptosis were blocked. The findings suggest that both the intracellular signal-transduction pathways for IL-6, JAK-STAT3 and PI3K-Akt, participate in mediating the neuroprotection of IL-6 against neuronal apoptosis.