Curcumin exerts protective effects against hypoxia‑reoxygenation injury via the enhancement of apurinic/apyrimidinic endonuclease 1 in SH‑SY5Y cells: Involvement of the PI3K/AKT pathway

Abstract

Curcumin, a polyphenolic compound extracted from the plant Curcuma longa, has been reported to exert neuroprotective effects against cerebral ischemia reperfusion (I/R) injury. However, the mechanisms underlying these effects remain to be fully elucidated. Emerging evidence indicated that apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional enzyme, participates in neuronal survival against I/R injury. Therefore, the aim of the present study was to investigate whether curcumin alleviates oxygen‑glucose deprivation/reperfusion (OGD/R)‑induced SH‑SY5Y cell injury, which serves as an in vitro model of cerebral I/R injury, by regulating APE1. The results revealed that curcumin increased cell viability, decreased LDH activity, reduced apoptosis and caspase‑3 activity, downregulated the pro‑apoptotic protein Bax expression and upregulated the anti‑apoptotic protein Bcl‑2 expression in SH‑SY5Y cells subjected to OGD/R. Simultaneously, curcumin eliminated the OGD/R‑induced decreases in APE1 protein and mRNA expression, as well as 8‑hydroxy‑2'‑deoxyguanosine (8‑OHdG) level and AP sites in SH‑SY5Y cells. However, APE1 knockdown by siRNA transfection markedly abrogated the protective effects of curcumin against OGD/R‑induced cytotoxicity, apoptosis and oxidative stress, as illustrated by the decreases in reactive oxygen species production and NADPH oxidase 2 expression, and the increase in superoxide dismutase activity and glutathione levels in SH‑SY5Y cells. Furthermore, curcumin mitigated the OGD/R‑induced activation of phosphatidylinositol 3‑kinase/protein kinase B (PI3K/AKT) signaling pathway. Treatment with LY294002, an inhibitor of PI3K/AKT pathway activity, attenuated the protective effects of curcumin on cytotoxicity and apoptosis, and reversed the curcumin‑induced upregulation of APE1 protein expression in SH‑SY5Y cells subjected to OGD/R. Taken together, these results demonstrated that curcumin protects SH‑SY5Y cells against OGD/R injury by inhibiting apoptosis and oxidative stress, and via enhancing the APE1 level and activity, promoting PI3K/AKT pathway activation.

Figure 1

Effects of curcumin on cytotoxicity and the apoptosis of OGD/R-injured SH-SY5Y cells. (A) SH-SY5Y cells were treated with various concentrations of curcumin (1, 5, 10 and 15 µl) for 24 h, and the cell viability was measured by CCK-8 assay. Data were normalized to the control as 100%. (B) Cell viability examined by CCK-8 assay and (C) LDH activity in the culture supernatant were tested in SH-SY5Y cells with curcumin (1, 5, 10 and 15 µl) for 24 h immediately after 1 h of OGD exposure. LDH activity data were normalized to the control, which was set as 1. Subsequently, SH-SY5Y cells were treated with curcumin (10 µl) for 24 h immediately after 1 h of exposure to OGD and then assessed for apoptosis. (D) Apoptosis was measured by Annexin V-FITC/PI double staining, followed by flow cytometry. (E) Caspase-3 activity was measured with an assay kit. (F) Protein expression was detected by western blot analysis. (G) Bax and (H) Bcl-2 protein levels were quantitatively analyzed. Data are expressed as the mean ± standard deviation of at least three independent experiments. ^**P<0.01 and ^***P<0.001, vs. control; ^#P<0.05 and ^##P<0.01, vs. OGD/R exposure alone. OGD/R, oxygen-glucose deprivation/reperfusion; CCK-8, Cell Counting Kit-8; LDH, lactate dehydrogenase; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2.

Figure 2

Effects of curcumin on the APE1 level and oxidative DNA damage in OGD/R-injured SH-SY5Y cells. SH-SY5Y cells were treated with curcumin (10 µl) for 24 h immediately after 1 h of exposure to OGD. (A) APE1 protein expression was measured by western blot analysis, and data were normalized to the control, which was set as 100%. (B) APE1 mRNA expression was detected by reverse transcription-quantitative polymerase chain reaction. The levels of the oxidative DNA damage markers, (C) 8-OHdG and (D) AP sites, were measured with a ELISA kit from Oxis Health Products and biotin-labeled aldehyde reactive probe with a colorimetric assay, respectively. Data are expressed as the mean ± standard deviation of at least three independent experiments. ^**P<0.01 vs. control; ^#P<0.05 and ^##P<0.01, vs. OGD/R exposure alone. OGD/R, oxygen-glucose deprivation/reperfusion; APE1, apurinic/apyrimidinic endonuclease 1; 8-OHdG, 8-hydroxy-2′-deoxyguanosine.

Figure 3

Effects of APE1 knockdown on cytotoxicity and apoptosis in the presence or absence of curcumin in SH-SY5Y cells exposed to OGD/R. SH-SY5Y cells were transfected with APE1t or APE1s, followed by treatment with curcumin (10 µl) for 24 h immediately after 1 h of OGD exposure. (A) Western blot and (B) quantified protein expression of APE1. ^**P<0.01 vs. control. (C) Cell viability was measured by Cell Counting Kit-8 assay. (D) LDH activity was detected with a cytotoxicity detection kit. (E) Caspase-3 activity was measured using an assay kit. (F) The protein expression levels of Bax and Bcl-2 were determined by western blot analysis. Quantitative analysis of the (G) Bax and (H) Bcl-2 protein bands. Data are expressed as the mean ± standard deviation of at least three independent experiments. ^*P<0.05 and ^**P<0.01, vs. APE1s + control; ^#P<0.05 and ^##P<0.01, vs. APE1s + OGD/R exposure alone; ^&P<0.05 and &&P<0.01, vs. APE1s + OGD/R + curcumin treatment. OGD/R, oxygen-glucose deprivation/reperfusion; APE1, apurinic/apyrimidinic endonuclease 1; APE1t, siRNA targeting APE1; APE1s, scrambled siRNA; LDH, lactate dehydrogenase; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; NS, no significant difference.

Figure 4

Effects of APE1 knockdown on oxidative stress in the presence or absence of curcumin in SH-SY5Y cells exposed to OGD/R. SH-SY5Y cells were transfected with APE1t or APE1s followed by treatment with curcumin (10 µl) for 24 h immediately after 1 h of OGD exposure. (A) Intracellular ROS generation determined by the DCFH-DA method (magnification, ×200). (B) The fluorescence intensity was analyzed by flow cytometry. (C) NOX2 expression was determined by western blot analysis. (D) SOD activity and (E) GSH levels were measured using commercial assay kits. Data are expressed as the mean ± standard deviation of at least three independent experiments. ^*P<0.05 and ^**P<0.01, vs. APE1s + control; ^#P<0.05 and ^##P<0.01 vs. APE1s + OGD/R exposure alone; ^&P<0.05 and ^&&P<0.01, vs. APE1s + OGD/R + curcumin treatment. OGD/R, oxygen-glucose deprivation/reperfusion; APE1, apurinic/apyrimi-dinic endonuclease 1; APE1t, siRNA targeting APE1; APE1s, scrambled siRNA; NOX2, NADPH oxidase 2; SOD, superoxide dismutase; GSH, glutathione.

Figure 5

Effects of curcumin on the PI3K/AKT pathway in OGD/R-injured SH-SY5Y cells. SH-SY5Y cells were treated with curcumin (10 µl) for 24 h immediately after OGD exposure for 1 h. (A) Protein expression levels were measured by western blot analysis. Quantitative analysis of the (B) p-PI3K/PI3K and (C) p-AKT/AKT ratio is shown. Data are expressed as the mean ± standard deviation of at least three independent experiments. ^**P<0.01 vs. control; ^#P<0.05 and ^##P<0.01, vs. OGD/R exposure alone. OGD/R, oxygen-glucose deprivation/reperfusion; PI3k, phosphatidylinositol 3-kinase; AKT, protein kinase B.

Figure 6

Effects of LY294002 on cytotoxicity, apoptosis and APE1 expression in OGD/R-injured SH-SY5Y cells. Cells were pre-treated with a PI3K/Akt inhibitor (LY294002; 10 µM) for 2 h following incubation with curcumin (10 µl) for 24 h immediately after OGD exposure for 1 h. (A) Cell viability was measured by Cell Counting Kit-8 assay. (B) LDH activity in the culture supernatant was detected using a Cytotoxicity Detection kit. (C) Caspase-3 activity was determined by a caspase-3 activity assay kit. (D) Bax, (E) Bcl-2 and (F) APE1 protein expression levels were detected by western blot analysis. Data are expressed as the mean ± standard deviation of at least three independent experiments. ^**P<0.01 vs. control; ^##P<0.01 vs. OGD/R exposure alone; ^&P<0.05 and ^&&P<0.01, vs. curcumin + OGD/R co-treatment. OGD/R, oxygen-glucose deprivation/reperfusion; APE1, apurinic/apyrimidinic endonuclease 1; PI3k, phosphatidylinositol 3-kinase; AKT, protein kinase B; LDH, lactate dehydrogenase; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2.