Hypoxia and TNF-α reduced BDNF production in rat hippocampal neurons and astrocytes.
Rat hippocampal neurons and astrocytes were cultured and exposed to hypoxia (5% O2) or TNF-α (40ng/mL) treatment for different times (0, 1, 2, 3, 6, 12h), and the production of BDNF was measured. As shown in Figure 1, we reported that in hippocampal neurons and astrocytes, hypoxia reduced BDNF production in a time-dependent manner, with the significant effects appearing at 3h in hippocampal neurons (Figure 1a, p<0.01 vs control) and at 6h in astrocytes (Figure 1b, p<0.01 vs control). In addition, we found that TNF-α also reduced BDNF production in a time-dependent manner, and the significant effects appeared at 3h in both hippocampal neurons and astrocytes (Figure 1c and 1d, p<0.01 vs control). Thereafter, these treatment conditions were applied in the following experiments to study the potential mechanisms.
Propofol reversed hypoxia- and TNF-α-modulated BDNF reduction in rat hippocampal neurons
To observe the effects of propofol on hypoxia- and TNF-α- modulated BDNF reduction in hippocampal neurons and astrocytes, we pretreated cells with different concentrations of propofol (1, 5, 10, 25, 50, 100μM) for 1h, followed by hypoxia or TNF-α treatment. As shown in Figure 2, in hippocampal neurons, propofol (25, 50 and 100μM) induced BDNF production, which was inhibited by hypoxia (5% O2, 3h) treatment (Figure 2a, p<0.01vs control, p<0.05 vs hypoxia). Propofol (25, 50 and 100μM) also induced BDNF production, which was inhibited by TNF-α (40ng/mL, 3h) treatment (Figure 2b, p<0.01 vs control, p<0.05 vs TNF-α). In contrast, we found that even 100μM propofol had no or minor effect on BDNF production in astrocytes in response to hypoxia or TNF-α (Figure 2c and 2d). Also, please note that 0.1% DMSO, the solvent for propofol, had no effect on BDNF production in hippocampal neurons or astrocytes (Figure 2). Therefore, we ruled out the role of DMSO. More importantly, we inferred that 25μM propofol might be the minimally effective concentration that reversed hypoxia- and TNF-α- inhibited BDNF production in hippocampal neurons, and accordingly we focused on the mechanism responsible for the beneficial effect of 25μM propofol.
The beneficial effect of popofol on BDNF production was mediated through regulating the phosphorylation of ERK and CREB
We revealed that in rat hippocampal neurons, hypoxia (5% O2, 3h) and TNF-α (40ng/mL, 3h) increased the phosphorylaion of ERK ,which was attenuated by 25μM propofol, 10μM PD98059 (a selective ERK inhibitor) or 10μM KO-947 (a potent and specific ERK inhibitor) (Figure 3a). We also detected that hypoxia (5% O2, 3h) and TNF-α (40ng/mL, 3h) increased the phosphorylation of CREB at Ser142 (p-CREBSer142) while reduced the phosphorylation of CREB at Ser133 (p-CREBSer133), which were both reversed by 25μM propofol, 10μM PD98059 or 10μM KO-947 (Figure 3b and 3c). Consistently, we demonstrated that 10μM PD98059 and 10μM KO-947 could attenuate the inhibitory effect of hypoxia and TNF-α on BDNF production, which is similar to the effect of propofol (Figure 3d). In addition, we reported that the beneficial effect of propofol on hypoxia- and TNF-α-inhibited BDNF production was abolished by the presence of 10μM ERK activator (Ceramide C6) (Figure 3d).
Hypoxia and TNF-α had no effect on TrkB expression, truncation or phosphorylation in rat hippocampal neurons and astrocytes.
Rat hippocampal neurons and astrocytes were cultured and exposed to hypoxia (5% O2) or TNF-α (40ng/mL) treatment for different times (0, 1, 2, 3, 6, 12h), and the expression, truncation, as well as phosphorylation of TrkB were measured. As shown in Figure 4, we reported that hypoxia had no effect on the expression, truncation or phosphorylation of TrkB in rat hippocampal neurons (Figure 4a) and in astrocytes (Figure 4b). Also, TNF-α had no effect on the expression, truncation or phosphorylation of TrkB in rat hippocampal neurons (Figure 4c) and astrocytes (Figure 4d).
Propofol induced TrkB phosphorylation in rat hippocampal neurons
We treated rat hippocampal neurons and astrocytes with different concentrations of propofol (1, 5, 10, 25, 50, 100μM) for 1h, followed by hypoxia (5% O2, 3h) or TNF-α (40ng/mL, 3h) treatment, and examined the expression, truncation and phosphorylation of TrkB. Interestingly, we noticed that in rat hippocampal neurons propofol had no effect on TrkB expression or truncation, while propofol (50 and 100μM) induced TrkB phosphorylation no matter cells were exposed to hypoxia, TNF-α or not (Figure 5a, p<0.05 vs control). However propofol had no effect on TrkB expression, truncation or phosphorylation in astrocytes (Figure 5b). Thereafter, we intended to investigate the mechanism responsible for 50μM propofol-induced TrkB phosphorylation in hippocampal neurons.
Propofol-induced TrkB phosphorylation was carried out via modulating p35 expression and Cdk5activation
We found that in hippocampal neurons, hypoxia (5% O2, 3h) and TNF-α (40ng/mL, 3h) did not affect p35 expression, while, 50μM propofol, rather than 0.1%DMSO, induced the expression of p35 regardless of the exposure to hypoxia or TNF-α (Figure 6a). Consistently, although hypoxia and TNF-α had no effect on the activation of Cdk5 (Figure 6b), it was activated by 50μM propofol but not 0.1%DMSO. In addition, hypoxia, TNF-α, propofol and DMSO had no effect on the expression of Cdk5 and p39 (Figure 6c). Then, we applied siRNA technology to confirm the involvement of p35 and Cdk5 in propofol-mediated TrkB phosphorylation. As shown in Figure 6d, we demonstrated that the siRNA targeting p35, p39 and Cdk5 could effectively diminish the expression of p35, p39 and Cdk5, respectively. More importantly, we revealed that blockade of p35 and Cdk5 alleviated propofol-induced TrkB phosphorylation, while blockade of p39 had no such effect (Figure 6e).