CoCl 2 increased EAAC1 protein expression in SH-SY5Y cells
We used CoCl2 to mimic hypoxia in SH-SY5Y cells. First, we examined whether CoCl2 altered the protein levels of EAAC1 in SH-SY5Y cells. We found that there was a dose-dependent increase in EAAC1 expression after 24 hrs of CoCl2 (50–500 µM) treatment (Fig. 1a). Quantification of the data demonstrated that CoCl2 significantly increased EAAC1 expression (CON, 1.04 ± 0.14; 50 µM CoCl2, 1.13 ± 0.29; 100 µM CoCl2, 1.71 ± 0.12; 150 µM CoCl2, 1.88 ± 0.18; 200 µM CoCl2, 2.58 ± 0.56; 300 µM CoCl2, 3.58 ± 0.56; 500 µM CoCl2, 5.87 ± 0.34; n = 8; ***P < 0.001; Fig. 1b). CoCl2 treatment significantly increased EAAC1 protein expression at each subsequent time point (0, 1, 3, 12, 24, 36, and 48 hrs). EAAC1 protein expression was significantly increased after exposure to 100 µM CoCl2 for > 24 hrs compared with that of the controls (n = 6; *P < 0.05, ***P < 0.001; Fig. 1c and d).
NRG1 alleviated CoCl 2 -induced overexpression of EAAC1 in SH-SY5Y cells
To determine whether NRG1 affected the CoCl2-induced increase in EAAC1 expression, we pretreated cells with NRG1 (5 nM or 10 nM) for 15 minutes before CoCl2 administration. Treatment with 100 µM CoCl2 for 36 hrs significantly upregulated EAAC1 expression (CON, 0.99 ± 0.21; CoCl2, 5.61 ± 0.87, n = 8; ***P < 0.001; Fig. 2a and b). Treatment with 5 nM or 10 nM NRG attenuated the increase in EAAC1 expression induced by 100 µM CoCl2 (CoCl2, 5.61 ± 0.87; CoCl2 + 5 nM NRG1, 4.05 ± 0.09; CoCl2 + 10 nM NRG1, 3.39 ± 0.43; n = 8, #P < 0.05, ##P < 0.01; Fig. 2a and b). As shown in Fig. 2a, c and d, treatment with 100 µM CoCl2 for 36 hrs significantly upregulated HIF-1α (CON, 1.01 ± 0.19; 100 µM CoCl2, 4.56 ± 0.41, n = 6; ***P < 0.001) and p53 (CON, 0.92 ± 0.27; 100 µM CoCl2, 3.62 ± 0.38, n = 6; ***P < 0.001) expression. Pretreatment with NRG1 for 36 hrs attenuated this increase in HIF-1α accumulation induced by 100 µM CoCl2 (CoCl2, 4.56 ± 0.41; CoCl2 + 5 nM NRG1, 2.55 ± 0.35; CoCl2 + 10 nM NRG1, 1.47 ± 0.28, n = 6; ##P < 0.01, ###P < 0.001; Fig. 2c). Moreover, pretreatment with 5 nM or 10 nM NRG1 for 36 hrs attenuated the increase in p53 stabilization induced by 100 µM CoCl2 (CoCl2, 3.62 ± 0.38; CoCl2 + 5 nM NRG1, 3.10 ± 0.46; CoCl2 + 10 nM NRG1, 1.85 ± 0.15, n = 6; #P < 0.05; Fig. 2d). These results are consistent with those of our previous studies demonstrating the effects of NRG1 on HIF-1α or p53 [23].
NRG1 inhibited CoCl 2 -induced increases in EAAC1 and Tau immunoreactivity
We examined the immunoreactivity of EAAC1 in SH-SY5Y cells using immunofluorescence staining. To measure the effects of NRG1 on SH-SY5Y cells, cells were pretreated for 15 minutes with 10 nM NRG1 and then treated with 100 µM CoCl2 (Fig. 3a). Treatment with 100 µM CoCl2 for 24 hrs significantly upregulated EAAC1 expression in comparison to that of the control group (CON, 1.02 ± 0.10; 100 µM CoCl2, 4.45 ± 0.64, n = 8; **P < 0.01; Fig. 3b). We also confirmed that the pretreatment of SH-SY5Y cells with 10 nM NRG1 for 24 hrs significantly attenuated EAAC1 overexpression (CoCl2, 4.45 ± 0.64; CoCl2 + 10 nM NRG1, 1.83 ± 0.37, n = 8, #P < 0.05; Fig. 3b) compared with that of the control group. Interestingly, treatment with 100 µM CoCl2 for 24 hrs markedly increased the accumulation of Tau in comparison with that of the control group (CON, 1.00 ± 0.20; CoCl2, 2.58 ± 0.27, n = 8; **P < 0.01; Fig. 3c). Furthermore, 10 nM NRG1 attenuated the CoCl2-induced increase in Tau expression (CoCl2, 2.58 ± 0.27; CoCl2 + NRG1, 1.45 ± 0.15, n = 8; #P < 0.05; Fig. 3c).
NRG1 rescued CoCl 2 -induced ROS generation and the reduction in antioxidant enzymes in SH-SY5Y cells
We tested the protective effect of NRG1 against CoCl2-induced ROS generation. We found that treatment with 100 µM CoCl2 for 24 hrs significantly increased ROS levels (CON, 1.13 ± 0.20; CoCl2, 4.46 ± 0.44, n = 6; ***P < 0.001; Fig. 4a and b) compared with the levels in the control group. However, pretreatment with 5 nM or 10 nM NRG1 significantly attenuated CoCl2-induced ROS generation (CoCl2, 4.46 ± 0.44; CoCl2 + 5 nM NRG1, 2.70 ± 0.37; CoCl2 + 10 nM NRG1, 1.67 ± 0.16, n = 6, #P < 0.05, ##P < 0.01; Fig. 4a and b). To determine whether NRG1 affects the antioxidant defense system, we analyzed the activity of the antioxidant enzymes GPx and SOD. Treatment with 100 µM CoCl2 significantly reduced the activity of GPx (CON, 32.37 ± 1.63; CoCl2, 19.31 ± 1.77, n = 6; **P < 0.01; Fig. 4c) compared with that of the control group. Pretreatment with 5 nM or 10 nM NRG1 for 36 hrs attenuated the CoCl2-induced reduction in Gpx activity (CoCl2, 19.31 ± 1.77; CoCl2 + 5 nM NRG1, 34.38 ± 1.94; CoCl2 + 10 nM NRG1, 30.46 ± 1.99, n = 6, ###P < 0.001; Fig. 4c). Moreover, after the cells were exposed to 100 µM CoCl2 in the presence or absence of NRG1 for 36 hrs, SOD activity was measured. We also demonstrated that after the cells were exposed to CoCl2 for 36 hrs, there were distinct decreases in SOD activity (CON, 121.78 ± 2.88; CoCl2, 98.91 ± 5.02, n = 8; ***P < 0.001; Fig. 4d). Moreover, pretreatment of cells with 5 nM or 10 nM NRG1 attenuated the CoCl2-induced decrease in SOD activity (CoCl2, 98.91 ± 5.02; CoCl2 + 5 nM NRG1, 116.74 ± 2.51; CoCl2 + 10 nM NRG1, 114.189 ± 3.76, n = 8, #P < 0.05, ##P < 0.01; Fig. 4d).
NRG1 rescued CoCl 2 -induced apoptosis and cell death
We examined whether NRG1 affects CoCl2-induced apoptosis in SH-SY5Y cells. To detect apoptotic nuclei in SH-SY5Y cells, we used TUNEL staining. Treatment with 100 µM CoCl2 significantly increased the proportion of apoptotic nuclei (CON, 2.00 ± 0.58; CoCl2, 21.33 ± 2.03, n = 6; ***P < 0.01; Fig. 5a and b) compared with that of the control group. Pretreatment with 10 nM NRG1 for 24 hrs reduced the number of CoCl2-induced TUNEL-positive cells (CoCl2, 21.33 ± 2.03; CoCl2 + 10 nM NRG1, 6.33 ± 2.03, n = 6, ##P < 0.01, Fig. 5a and b).
Next, we examined CoCl2-induced cytotoxicity in SH-SY5Y cells. The cells were incubated with 10 nM NRG1 and then exposed to 100 µM CoCl2 for 36 hrs (CON, 10.48 ± 2.10; CoCl2, 31.97 ± 3.21; CoCl2 + 10 nM NRG1, 16.18 ± 2.05, n = 6, #P < 0.05, Fig. 5c).
Effects of NRG1 on CoCl 2 -induced changes in apoptotic or antiapoptotic proteins
We next investigated whether caspase-3 cleavage is increased by CoCl2. SH-SY5Y cells were treated with 100 µM CoCl2 for 24 hrs before fixation and immunofluorescence detection of cleaved caspase-3. We found that CoCl2 increased the cleavage of caspase-3, and quantitative analysis showed that the number of cleaved caspase-3-positive cells was increased (CON, 1.00 ± 0.51; CoCl2, 3.40 ± 0.34, n = 6; **P < 0.01; Fig. 6a and b). Furthermore, pretreatment with 10 nM NRG1 for 24 hrs rescued the CoCl2-induced increase in the number of cleaved caspase-3-positive cells (CoCl2, 3.40 ± 0.34; CoCl2 + 10 nM NRG1, 1.93 ± 0.35, n = 6, #P < 0.05, Fig. 6a and b). To determine whether NRG1 regulates CoCl2-induced caspase-3 cleavage, we performed western blotting. We observed that the level of cleaved caspase-3 (17 and 19 kD) was significantly increased after CoCl2 treatment (CON, 1.28 ± 0.16; CoCl2, 2.56 ± 0.29, n = 6; **P < 0.01; Fig. 6c and d). NRG1 attenuated the CoCl2-induced increase in cleaved caspase-3 (CoCl2, 2.56 ± 0.29; CoCl2 + 5 nM NRG1, 1.52 ± 0.17; CoCl2 + 10 nM NRG1, 1.13 ± 0.10, n = 6; #P < 0.05, ##P < 0.01; P < 0.05; Fig. 6c and d). Furthermore, the expression of Bcl-XL (an antiapoptotic protein) was decreased in CoCl2-induced cells (CON, 1.02 ± 0.14; CoCl2, 0.4 ± 0.08, n = 6; *P < 0.05; Fig. 6c and e). NRG1 protected against the CoCl2-induced reduction in Bcl-xL protein expression (CoCl2, 0.4 ± 0.08; CoCl2 + 5 nM NRG1, 0.66 ± 0.05; CoCl2 + 10 nM NRG1, 1.01 ± 0.11, n = 6; #P < 0.05, Fig. 6c and e). These results suggest that NRG1 may have a protective role under hypoxic conditions by regulating apoptosis.