Icariin Inhibits AGE-Induced Injury in PC12 Cells by Directly Targeting Apoptosis Regulator Bax

Abstract

Diabetic encephalopathy (DE) is a serious complication caused by long-term cognitive impairment in diabetic patients. At present, there is no effective treatment for DE. Icariin (ICA) is a bioactive ingredient isolated from Epimedium. Previous research indicated that ICA was neuroprotective against Aβ-induced PC12 cell insult; however, the effect of ICA on an advanced glycosylation end product- (AGE-) induced neural injury model has not been studied. In this study, we investigated the neuroprotective effects of ICA on AGE-induced injury in PC12 cells. Our findings revealed that ICA could effectively protect PC12 cells from AGE-induced cell apoptosis by suppressing oxidative stress. Moreover, we observed that ICA could significantly protect against mitochondrial depolarization following AGE stimulation and inactivate the mitochondria-dependent caspase-9/3 apoptosis pathway. Most notably, we identified the direct target protein of ICA as apoptosis regulator Bax by a pulldown assay. We found that ICA could specifically target Bax protein and inhibit Bax dimer formation and migration to mitochondria. Furthermore, a siRNA knockdown experiment revealed that ICA could inhibit PC12 cell apoptosis and oxidative stress through targeting Bax. Taken together, our findings demonstrated that ICA could attenuate AGE-induced oxidative stress and mitochondrial apoptosis by specifically targeting Bax and further regulating the biological function of Bax on mitochondria.

Figure 1

The chemical structure of Icariin (ICA).

Figure 2

ICA protected PC12 cells against AGE-induced apoptosis. (a) PC12 cells were exposed to AGEs and treated with ICA (5, 10, and 20 μM) for 48 h. Cell viability was assessed by MTT and expressed relative to control. (b) PC12 cells were treated as in (a), and LDH release was detected with a commercial kit. (c) Apoptotic nuclei were identified using Hoechst 33258 staining. Scale bar = 50 μm. (d) Apoptotic cells were identified by double staining with AO and EB. The cells which took up both dyes were classified as apoptotic (indicated by arrows in the merged images). Scale bar = 50 μm. All data were presented as mean ± S.D. from at least three independent experiments. ^###P < 0.001 relative to the control group; ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 relative to the model group.

Figure 3

ICA inhibited oxidative stress in AGE-induced PC12 cells. (a) PC12 cells were exposed to AGEs and treated with ICA (5, 10, and 20 μM) for 48 h. Detection of intracellular and mitochondrial ROS using DCFH-DA and MitoSOX. Scale bar = 50 μm. (b and c) PC12 cells were treated as in (a), and the intracellular level of SOD and MDA were detected. Data were presented as mean ± S.D. from independent experiments performed in triplicate. ^###P < 0.001 relative to the control group; ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 relative to the model group.

Figure 4

ICA protected PC12 cells against AGE-induced apoptosis via the caspase-9/3 pathway. (a) PC12 cells were subjected to AGEs with or without ICA extract for 48 h. Mitochondrial depolarization was investigated by the JC-1 staining assay. The cells with depolarized mitochondria were identified by green fluorescence. Scale bar = 50 μm. (b) The cells were treated as in (a). The protein levels of caspase-3, PRAR, cleaved caspase-9, cleaved caspase-3, and cleaved PARP were examined by western blot. Data were presented as mean ± S.D. from independent experiments performed in triplicate. ^###P < 0.001 relative to the control group; ^∗P < 0.05 and ^∗∗∗P < 0.001 relative to the model group.

Figure 5

Bax is a direct target protein for ICA. (a) The PC12 lysates were incubated with ICA beads or control beads, and then the potential proteins bound to the beads were resolved by SDS-PAGE, followed by silver staining. The asterisk-marked bands indicated nonspecific bead-bound vimentin and actin. (b) SPR analysis of ICA binding to Bax. The kinetic parameters of k_a, k_d, and K_D were derived by fitting to a 1 : 1 Langmuir binding model. (c) ICA beads were incubated with PC12 lysates or recombinant Bax protein in the absence or presence of ICA for competitive binding, and the proteins bound to the beads were analyzed by western blot or silver staining. (d) ICA promoted Bax resistant to pronase (DARTS). PC12 lysates were incubated with pronase in the absence or presence of ICA, and Bax was detected by western blot. (e) PC12 lysates were incubated with ICA (20 μM) or vehicle followed by the cellular thermal shift assay (CETSA). Data were presented as mean ± S.D. from independent experiments performed in triplicate.

Figure 6

ICA regulated the biological activity of Bax. (a) ICA inhibited Bax transfer to mitochondria. (b) Bax dimer was detected by chemical cross-linking technology. (c and d) The reverse effects of Bax gene silencing on ICA-mediated antioxidative stress and neuroprotection were detected using MitoSOX and MTT assay. Scale bar = 50 μm. Data were presented as mean ± S.D. from independent experiments performed in triplicate. ^###P < 0.001 relative to the control group; ^∗P < 0.05 relative to the model group. N.S., not significant.