Protective effect of docosahexaenoic acid on lipotoxicity-mediated cell death in Schwann cells: Implication of PI3K/AKT and mTORC2 pathways

Abstract

Background and aim: Docosahexaenoic acid (DHA) exhibits neuroprotective properties and has been shown to preserve nerve cells following trauma and ischemic injury. Recently, we showed that DHA pretreatment improved locomotion and reduced neuropathic pain after acute spinal cord injury in adult rats. These improvements were associated with an increase in the levels of AKT in spinal cord injury neurons. In this study, we investigate the implication of PI3K/AKT and mTOR pathway in DHA-mediated protection of primary cultured Schwann cells (pSC) undergoing palmitic acid-induced lipotoxicity (PA-LTx).

Methods: Primary cultured Schwann cells were treated with PA (PA:BSA, 2:1) in the presence or absence of DHA (1-200 µM) for 24-48 hr. Cell viability was determined by crystal violet staining and nuclear morphology was examined using Hoechst staining.

Results: We found that pSC cultures exposed to palmitic acid (PA) overload showed chromatin condensation, a decrease in cell viability and an inhibition of AKT phosphorylation in a time-dependent manner. Next, pSC exposed to PA overload were treated with DHA. The data show that co-treatment with DHA inhibited the loss of cell viability and apoptosis caused by PA. Moreover, treatment with DHA inhibited chromatin condensation, significantly stimulated p-AKT phosphorylation under PA-LTx condition, and DHA alone increased AKT phosphorylation. Additionally, when these pSC cultures were treated with PI3K inhibitors LY294002 and, BKM120 and mTOR inhibitors Torin 1 (mTORC1/mTORC2), but not rapamycin (mTORC1), the protective effects of DHA were not observed.

Conclusion: These findings suggest PI3K/AKT and mTORC2 kinase pathways are involved in the protective function (s) of DHA in PA-induced Schwann cell death.

Keywords: AKT phosphorylation; PA-induced lipotoxicity; docosahexaenoic acid; primary cultured Schwann cells.

Figure 1

PA‐LTx induced apoptosis and a decrease in AKT phosphorylation in primary Schwann cells (pSC). pSC were treated with BSA alone (control), PA:BSA (2:1) for 24–48 hr. (a) Cell viability was assessed by crystal violet assay. (b) The effect of PA:BSA (2:1) on AKT phosphorylation was examined by Western blot. The pSC cell lysates from control cultures (CTL) exposed to BSA alone and cultures exposed to PA:BSA (2:1) at 1, 3, 6, 12, and 24 hr (hrs.) were prepared and subjected to specific antibodies against AKTp‐Ser473, AKTp‐Thr308 and to total AKT. The blots were then analyzed using the Li‐Cor Odyssey system. A representative Western blot is shown above each bar graph. The data represent mean ± SE of at least four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 shown above the lines when compared between two linked groups

Figure 2

The Effect of PI3K/AKT inhibitors on primary Schwann cells (pSC) viability. pSC cultures were treated with PI3K inhibitors LY290042 (a), and BKM120 (b) for 48 hr followed by crystal violet assay to measure cell viability. The effect of LY290042 (c) and BKM120 (d) on AKT phosphorylation was examined by Western blot. The pSC cells lysates were prepared and subjected to specific antibodies against AKTp‐Ser473, AKTp‐Thr308, and total AKT protein. The blots were then analyzed using the Li‐Cor Odyssey system. A representative Western blot is shown above each bar graph. The data represent mean ± SE of at least four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. CTL

Figure 3

The Effect of co‐treatment and post‐treatment of DHA on primary cultured Schwann cells (pSC) viability under PA‐LTx. pSC cultures were treated with BSA alone (CTL), with PA:BSA (2:1) alone, with DHA (50–200 µM) alone (a) or co‐treated with PA:BSA (2:1) and DHA (50–200 µM) (b) for 48 hr and then cell viability was measured by crystal violet assay to measure cell viability. Alternately, the cells were post‐treated with DHA (50 µM) at 1, 3, 6, 12, or 24 hr after an initial PA exposure, and cell viability was measured at 48 hr (c). The data represent mean ± SEM of at least four independent experiments. **p < 0.01 and ***p < 0.001 are shown above the bars when compared to control groups. ^### p < 0.001 are shown above the lines when compared between two linked groups

Figure 4

DHA eliminates apoptotic cell death and restores AKT phosphorylation in primary cultured Schwann cells (pSC) under PA‐LTx. pSC were treated with BSA alone (CTL), with PA:BSA (2:1) alone, with DHA (50 µM) alone or co‐treated with PA:BSA (2:1) and DHA (50 µM) for 48 hr. Nuclear morphology was determined by Hoechst staining. Nuclear condensations are indicated with white arrows (a). The pSC culture lysates were prepared and subjected to Western blot analysis using specific antibodies against AKTp‐Ser473, AKTp‐Thr308, and total AKT protein. The blots were then analyzed using the Li‐Cor Odyssey system. A representative Western blot is shown by each bar graph (b). The data represent mean ± SEM of at least four independent experiments. *p < 0.05 are shown above the bars when compared to control groups. ^# p < 0.05 and ^## p < 0.01 are shown above lines when compared between two linked groups

Figure 5

The increase in AKT phosphorylation in primary cultured Schwann cells (pSC) by DHA is abolished by PI3K inhibitors. pSC were treated with DHA (50 µM) for (1–24 hr) (a). pSC were treated with DHA in the presence or absence of LY294002(40 µM), BKM120 (2 µM) and for 6 hrs (b). pSC cell lysates were prepared and subjected to Western blot analysis using specific antibodies against AKTp‐Ser473, AKTp‐Thr308, and total AKT. The blots were then analyzed using the Li‐Cor Odyssey system. The data represent mean ± SEM of at least three independent experiments. A representative Western blot is shown above each bar graph. *p < 0.05, when compared to the control groups. ^# p < 0.05 and ^### p < 0.01 are shown above lines when compared between two linked groups

Figure 6

The protective effect of DHA on primary cultured Schwann cells (pSC) under PA‐LTx is abolished by PI3K/AKT inhibitors. pSC cultures were treated with DHA (50 µM) in the presence or absence of PI3K inhibitors LY290042 (40 µM) or BKM120 (2 µM) (a). The pSC cultures were treated with PA:BSA (2:1), PA + DHA in a presence or absence of PI3K inhibitors LY290042 (40 µM), or BKM120 (2 µM) (b). Cell viability was measured at 48 hr by crystal violet assay. Cells morphology was visualized at 48 hr by Hoffman modulation contrast microscopy. Membrane blebs and cell shrinkage are indicated with white arrows (c). The data represent mean ± SEM of at least five independent experiments **p < 0.05, ***p < 0.001 when compared between two linked groups

Figure 7

The protective effect of DHA on primary cultured Schwann cells (pSC) under PA‐LTx is partially inhibited by mTOR inhibitors. pSC cultures were treated with DHA (50 µM) in a presence or absence of mTOR inhibitors rapamycin (50 and 100 nM) or Torin 1 (50 and 100 nM) (a, b). The pSC cultures were treated with PA:BSA (2:1), PA + DHA in the presence or absence of mTOR inhibitors rapamycin (50 and 100 nM), or Torin 1 (50 and 100 nM) (c, d). Cell viability was measured at 48 hr by crystal violet assay. The data represent mean ± SEM of at least five independent experiments ^## p < 0.01, ^### p < 0.001 when compared between two linked groups and * p < 0.05, *** p < 0.001 when compared with the control