6'- O-Galloylpaeoniflorin Attenuates Cerebral Ischemia Reperfusion-Induced Neuroinflammation and Oxidative Stress via PI3K/Akt/Nrf2 Activation

Abstract

6'-O-galloylpaeoniflorin (GPF), a galloylated derivative of paeoniflorin isolated from peony root, has been proven to possess antioxidant potential. In this present study, we revealed that GPF treatment exerted significant neuroprotection of PC12 cells following OGD, as evidenced by a reduction of oxidative stress, inflammatory response, cellular injury, and apoptosis in vitro. Furthermore, treatment with GPF increased the levels of phosphorylated Akt (p-Akt) and nuclear factor-erythroid 2-related factor 2 (Nrf2), as well as promoted Nrf2 translocation in PC12 cells, which could be inhibited by Ly294002, an inhibitor of phosphoinositide 3-kinase (PI3K). In addition, Nrf2 knockdown or Ly294002 treatment significantly attenuated the antioxidant, anti-inflammatory, and antiapoptotic activities of GPF in vitro. In vivo studies indicated that GPF treatment significantly reduced infarct volume and improved neurological deficits in rats subjected to CIRI, as well as decreased oxidative stress, inflammation, and apoptosis, which could be inhibited by administration of Ly294002. In conclusion, these results revealed that GPF possesses neuroprotective effects against oxidative stress, inflammation, and apoptosis after ischemia-reperfusion insult via activation of the PI3K/Akt/Nrf2 pathway.

Figure 1

The antioxidative and anti-inflammatory activities of GPF in the OGD model. To determine the antioxidant and anti-inflammatory effects of GPF, PC12 cells were pretreated with different doses of GPF (0, 10, 50, or 100 μM, resp.) for 1 h before subjected to OGD. PC12 cells without GPF and OGD treatment were set as a control. Typical representative DCFH-DA staining (ROS level detection) is shown in (a), revealing that the ROS level (green fluorescence intensity) was remarkably higher in PC12 cells subjected to OGD (2nd panel) than that in the control group (1st panel). After treatment with 10, 50, or 100 μM GPF (3rd, 4th, and 5th panels, resp.), the fluorescence intensity was lower in PC12 cells than that in cells without GFP treatment (2nd panel), in a dose-dependent manner. The MMP (b) and SOD activity (right panel of (c)) decreased in PC12 cells subjected to OGD (2nd bar) compared to that in the control cells, whereas 10, 50, and 100 μM GPF (3rd, 4th, and 5th panels, resp.) treatment significantly upregulated the OGD-decreased MMP and SOD activity in a dose-dependent manner in PC12 cells. GPF downregulated the OGD-induced upregulation of the MDA level in a dose-dependent manner (left panel of (c)). GPF significantly decreased the OGD-induced upregulation of IL-1β (left panels of (d) and (e)) and TNF-α (right panels of (d) and (e)) by ELISA (d) and qPCR (e) in PC12 cells. The data of each group were from three independent assays. ^∗P < 0.05 versus 0 mg/kg GFP with OGD; ^∗∗P < 0.01 versus 0 mg/kg GFP with OGD.

Figure 2

GPF protected the hypoxic-ischemic injury in PC12 cells. To determine the neuroprotective effects of GPF, PC12 cells were pretreated with different doses of GPF (0, 10, 50, or 100 μM, resp.) for 1 h before being subjected to OGD. PC12 cells without GPF and OGD treatment were set as a control. The cellular injury induced by OGD was analyzed by a cellular viability assay (CCK-8 assay) (a) and a LDH activity assay (b). GPF treatment at the indicated doses relieved OGD-induced cellular injury. The amount of apoptotic PC12 cells was determined by TUNEL staining. TUNEL-positive and total (DAPI-positive) number of the cells in each group were counted manually by two independent observers in 6 random microscopic fields (400x), and the percentage of TUNEL-positive cells were subsequently calculated (c). GPF decreased the ratio of TUNEL-positive cells (purple fluorescence, labeled with white arrows) in a dose-dependent manner. Nuclei were stained by DAPI (blue fluorescence). GPF downregulated the OGD-induced levels of caspase-3 in a dose-dependent manner in PC12 cells. The data of each group were from three independent assays. ^∗P < 0.05 versus 0 mg/kg GFP with OGD; ^∗∗P < 0.01 versus 0 mg/kg GFP with OGD.

Figure 3

GPF upregulates and activates Nrf2. PC12 cells were treated with different doses of GPF (0, 10, 50, or 100 μM, resp.) for 6 h or treated with PI3K/Akt inhibitor Ly294002 (50 μM) for 1 h followed by treatment with GPF (100 μM) for 6 h. The results of Western blot analysis showed that Nrf2 and p-Akt expression levels were upregulated by 10–100 μM GPF, which were inhibited by pretreatment with the PI3K/Akt inhibitor Ly294002. GPF treatment has no effect on Akt expression in PC12 cells (a). Fluorescence staining showed that 10–100 μM GPF treatment induced Nrf2 (red fluorescence) translocation into the nuclei (blue). Pretreatment with Ly294002 significantly attenuated the GPF-induced nuclear translocation of Nrf2 (b). The data of each group were from three independent assays. ^∗P < 0.05 versus the control group; ^∗∗P < 0.01 versus the control group; ^#P < 0.01 versus the 100 μM GPF group.

Figure 4

Nrf2 siRNA and Ly294002 attenuated the antioxidative and cell protective activities of GPF. PC12 cells pretreated with Ly294002 (50 μM) for 1 h or transfected with Nrf2 siRNA (50 nM) or scramble RNA was administrated with GPF (100 μM) for 1 h and then subjected to OGD as mentioned above. PC12 cells only treated with GPF and OGD were set as the control group. The ROS level (green fluorescence, in (a)) and MDA level (right panel of (c)) were remarkably increased after Nrf2 siRNA or Ly294002 treatment, compared with the control cells. The MMP (b) and SOD activity (right panel of (c)) were downregulated by Nrf2 siRNA or Ly294002 administration. The mRNA expression (detected by qRT-PCR; upper panel of (d)) in the PC12 cells as well as the protein levels (detected by ELISA; lower panel of (d)) of TNF-α and IL-1β in the cell culture medium was remarkably upregulated by Nrf2 siRNA or Ly294002 treatment, compared with the control group. The results of the CCK-8 assay and LDH activity detection revealed that Nrf2 siRNA or Ly294002 weakened the protective effects of GPF against OGD (e). Finally, Nrf2 siRNA or Ly294002 administration upregulated the ratio of TUNEL-positive cells and the generation of caspase-3 (f). The data of each group were from three independent assays. ^∗∗P < 0.01 versus the control group.

Figure 5

GPF mitigates the cerebral infarction volume and neurological impairment through the PI3K/Akt pathway in a rat CIRI model in vivo. Before being subjected to CIRI, rats were pretreated with different dose of GPF (2.5, 5, or 10 mg/kg/day) for 14 days. In some cases, Ly294002 (20 mM, 5 μL) was injected into the lateral ventricle daily for 7 days before CIRI model establishment. The infarct volume was remarkably decreased in the GPF treatment groups, compared with rats without GPF treatment. Yet, the infarct volume further increased after treatment with Ly294002 (a) and (b). The neurological score was reduced in the GPF treatment groups, compared with the CIRI rats without GPF treatment, indicating that GPF could protect nerve function and improve brain function in a dose-dependent manner. Nevertheless, the nerve-protecting and nerve-improving activities of GPF were diminished after treatment with Ly294002 (c). Each group contains at least six rats. ^∗P < 0.05 versus 0 mg/kg GFP in the CIRI models; ^∗∗P < 0.01 versus 0 mg/kg GFP in the CIRI models; ^#P < 0.01 versus 10 mg/kg GFP in the CIRI models.

Figure 6

GPF decreases oxidative stress through the PI3K/Akt pathway in a rat CIRI model. The SOD, GSH, and GSH-Px activities as well as the MDA level were analyzed in the indicated groups in the brain tissue of MCAO rats (a). (b) Nrf2 nuclear translocation was determined by a Western blot assay in the isolated nuclear fraction of the brain tissue of MCAO rats in each group (upper left panels of (b)). Densitometric analysis of the Nrf2 Western blot images is presented in the upper right panels of (b). The HO-1 expression and the corresponding densitometry of the indicated groups were analyzed as well (lower panels of (b)). Each group contains at least six rats. ^∗P < 0.05 versus 0 mg/kg GPF in the CIRI models; ^∗∗P < 0.01 versus 0 mg/kg GPF in the CIRI models; ^&P < 0.01 versus 10 mg/kg in the CIRI models; ^#P < 0.01 versus 10 mg/kg in the CIRI models.

Figure 7

GPF diminished the inflammatory reaction through the PI3K/Akt pathway in a rat CIRI model in vivo. Iba1, phosphorylated p38 (p-p38), and phosphorylated JNK (p-JNK) were immunohistochemically stained in the rat brain slides of the indicated groups, respectively (a). The number of Iba1, p-p38, and p-JNK positively stained cells was counted (b). The gene expression levels of IL-1β and TNF-α in the indicated groups were detected by qPCR (c). Each group contains at least six rats. ^∗P < 0.05 versus 0 mg/kg GPF in the CIRI models; ^∗∗P < 0.01 versus 0 mg/kg GPF in the CIRI models; ^#P < 0.01 versus 10 mg/kg GPF in the CIRI models.

Figure 8

GPF relieved cell apoptosis in a rat CIRI model in vivo. Rat brain slices of the indicated groups were stained with a TUNEL assay kit (a). The TUNEL-positive cells (yellow-brown, elliptical, or round) of each corresponding group were counted (b). The activity of caspase-3 was detected in the rat brain samples of the indicated groups (c). Each group contains at least six rats. ^∗P < 0.05 versus 0 mg/kg GPF in the CIRI models; ^∗∗P < 0.01 versus 0 mg/kg GPF in the CIRI models; ^#P < 0.01 versus 10 mg/kg GPF in the CIRI models.