Protective effect of hydrogen sulphide against 6-OHDA-induced cell injury in SH-SY5Y cells involves PKC/PI3K/Akt pathway

Abstract

Background and purpose: Hydrogen sulphide (H(2)S) is a novel neuromodulator. The present study aimed to investigate the protective effect of H(2)S against cell injury induced by 6-hydroxydopamine (6-OHDA), a selective dopaminergic neurotoxin often used to establish a model of Parkinson's disease for studying the underlying mechanisms of this condition.

Experimental approach: Cell viability in SH-SY5Y cells was measured using MTT assay. Western blot analysis and pharmacological manipulation were employed to study the signalling mechanisms.

Key results: Treatment of SH-SY5Y cells with 6-OHDA (50-200 microM) for 12 h decreased cell viability. Exogenous application of NaHS (an H(2)S donor, 100-1000 microM) or overexpression of cystathionine beta-synthase (a predominant enzyme to produce endogenous H(2)S in SH-SY5Y cells) protected cells against 6-OHDA-induced cell apoptosis and death. Furthermore, NaHS reversed 6-OHDA-induced loss of tyrosine hydroxylase. Western blot analysis showed that NaHS reversed the down-regulation of PKCalpha, epsilon and Akt and the up-regulation of PKCdelta in 6-OHDA-treated cells. Blockade of PKCalpha with Gö6976 (2 microM), PKCepsilon with EAVSLKPT (200 microM) or PI3K with LY294002 (20 microM) reduced the protective effects of H(2)S. However, inhibition of PKCdelta with rottlerin (5 microM) failed to affect 6-OHDA-induced cell injury. These data suggest that the protective effects of NaHS mainly resulted from activation of PKCalpha, epsilon and PI3K/Akt pathway. In addition, NaHS-induced Akt phosphorylation was significantly attenuated by Gö6976 and EAVSLKPT, suggesting that the activation of Akt by NaHS is PKCalpha, epsilon-dependent.

Conclusions and implications: H(2)S protects SH-SY5Y cells against 6-OHDA-induced cell injury by activating the PKCalpha, epsilon/PI3K/Akt pathway.

Figure 1

MTT assay showing the effect of NaHS and/or 6-OHDA on SH-SY5Y cell viability. (A) Effect of 6-OHDA on cell viability of SH-SY5Y cells. Cells were treated with 6-OHDA at different concentrations for 12 h. (B) Effect of NaHS on cell viability in SH-SY5Y cells treated with 6-OHDA. Cells were pretreated with various concentrations (10–1000 µM) of NaHS for 10 min before 6-OHDA (50 µM) was added for another 12 h. (C) Pretreatment of NaHS for 10 min and 1 h showed a similar protective effect against 6-OHDA-induced cell injury. Data are presented as mean ± SEM, n = 5, ***P < 0.001 versus control; #P < 0.05, ##P < 0.01, ###P < 0.001 versus 6-OHDA-treated cells. 6-OHDA, 6-hydroxydopamine.

Figure 2

Effect of NaHS on TH expression in SH-SY5Y cells treated with 6-OHDA. Cells were pretreated with NaHS for 10 min before addition of 6-OHDA (50 µM) for another 12 h. Proteins were extracted and subjected to Western blot analysis using the anti-TH and β-tubulin (as a loading control) antibodies. Data are presented as mean ± SEM, n = 5, ***P < 0.001 versus control; ##P < 0.01 versus 6-OHDA-treated cells. 6-OHDA, 6-hydroxydopamine; TH, tyrosine hydroxylase.

Figure 3

Role of PKC isoforms in the neuroprotective effects of NaHS in SH-SY5Y cells treated with 6-OHDA. (A–B) a: PKCδ, b: PKCα, c: PKCε. (A) Time course for the effect of 6-OHDA on the translocation of PKC isoforms. (B) Effect of NaHS (100 µM) on the translocation of PKC isoforms caused by 6-OHDA (50 µM, 2 h). Right panel shows the effect of various PKC isoform inhibitors on the translocation of PKC isoforms. The ratios of membrane/cytosol fraction are normalized to that of control group. β-Actin was used as a loading control. Data are presented as mean ± SEM, n = 5–8, *P < 0.05, **P < 0.01, ***P < 0.001 versus control; ###P < 0.001 versus 6-OHDA-treated cells. 6-OHDA, 6-hydroxydopamine; PKC, protein kinase C.

Figure 4

The protective effect of NaHS on cell viability in the presence and absence of various PKC isoform inhibitors. (A) Blockade of PKCα with Gö6976 (2 µM, 30 min pretreatment) or PKCε with EAVSLKPT (200 µM, 30 min pretreatment) attenuated the protective effect of NaHS (100 µM, 12 h) on 6-OHDA (50 µM, 12 h)-induced cell injury. (B) Blockade of PKCδ with rottlerin did not reverse cell injury caused by 6-OHDA. Data are presented as mean ± SEM, n = 5, ***P < 0.001 versus control; #P < 0.05, ###P < 0.001 versus 6-OHDA-treated cells; ⁺P < 0.05, ⁺⁺P < 0.01 versus NaHS + 6-OHDA-treated cells. 6-OHDA, 6-hydroxydopamine; PKC, protein kinase C.

Figure 5

Involvement of PI3K/Akt pathway in the neuroprotective effects of NaHS. (A–B) Time-courses for the effect of 6-OHDA (50 µM, A) and NaHS (100 µM, B) on Akt activity. (C) NaHS reversed the inhibitory effect of 6-OHDA (6 h and 12 h) on Akt phosphorylation. The histograms represent the ratio of phosphorylated protein over total Akt. Data are presented as mean ± SEM, n = 5, *P < 0.05, ***P < 0.001 versus control; ###P < 0.001 versus 6-OHDA-treated cells. 6-OHDA, 6-hydroxydopamine.

Figure 6

Blockade of PI3K with LY294002 (20 µM, 30 min pretreatment) abolished the protective effect of NaHS (100 µM, 12 h) on cell injury induced by 6-OHDA (50 µM, 12 h). Data are presented as mean ± SEM, n = 5, ***P < 0.001 versus control; ##P < 0.01 versus 6-OHDA-treated cells; ⁺⁺⁺P < 0.001 versus NaHS + 6-OHDA-treated cells. 6-OHDA, 6-hydroxydopamine.

Figure 7

Effect of NaHS on Akt activation was dependent on PKC activity. Blockade of PKCα with Gö6976 (2 µM, A) or PKCε with EAVSLKPT (200 µM, B) attenuated NaHS-up-regulated Akt phosphorylation in SH-SY5Y cells treated with 6-OHDA for 6 h. Whole cell lysates were prepared for Western blot analysis of total Akt and phosphorylated Akt level. The histograms represent the ratio of phosphorylated protein to total Akt. Results shown are the mean ± SEM, n = 5, **P < 0.01 versus control; ###P < 0.001 versus 6-OHDA-treated cells; ⁺⁺P < 0.01, ⁺⁺⁺P < 0.001 versus NaHS + 6-OHDA-treated cells. 6-OHDA, 6-hydroxydopamine; PKC, protein kinase C.

Figure 8

Effect of endogenous H₂S on 6-OHDA-induced cell apoptosis in SH-SY5Y cells. (A) Transfection of CBS cDNA into SH-SY5Y cells increased the protein expression of CBS. β-Tubulin was used as a loading control. (B) Effect of CBS overexpression and exogenous application of NaHS on endogenous H₂S level. Mean ± SEM, n = 8, *P < 0.05, ***P < 0.001 versus GFP; ###P < 0.001 versus CBS. (C) CBS overexpression alleviated 6-OHDA-induced apoptosis. Green: early apoptosis indicated by FITC fluorescence; red: late phase apoptosis stained by propidium iodide; blue: nuclei stained by Hoechst 33342. Photos were taken at ×20 magnification. Scale bar: 200 nm. 6-OHDA, 6-hydroxydopamine; CBS, cystathionine β-synthase; GFP, green fluorescent protein.