| [Backgroud and Objective] Arecoline, the most abundant alkaloid of the areca nut, can induce toxicity to neurons. Hydrogen sulfide（H2S） has recently been identified as an endogenous gas with neuroprotective effects. Our laboratory has found that H2 S protects PC12 cells against arecoline-induced neurotoxicity. Howerver, the underlying mechanism of this protection induced by H2 S is still unclear. Increasing evidence has demonstrated that autophagic flux is involved in neuroprotection. Therefore, the present work is aim to explore whether autophagic flux mediates the protection of H2 S against arecoline-caused neurotoxicity. [Methods]LDH release was measured by Lactate dehydrogenase（LDH） assay kit. The rate of cell apoptosis was evaluated by flow cytometry（FCM） after Annexin V-FITC/PI staining. The morphological of apoptotic cell was observed under a fluorescence microscope after Hoechst 33258 staining. The mitochondrial membrane potential（MMP） in cells was observed under a fluorescence microscope after JC-1 staining. The structure of autophagy was observed under transmission electron microscopy（TEM）. The levels of two key indicator of autophagic flux such as microtubule-associated protein 1A/1B-light chain 3（LC3） including LC3 I as well as LC3 II, and sequestosome 1（p62/SQSTM1） protein, and the expressions of the molecular markers of endoplasmic reticulum（ER） stress including Glucose-regulated protein 78（GRP78） and Cleaved cysteinyl aspartate specific proteinase-12（Cleaved caspase-12） were measured by Western Blot. [Results]Treatment of PC12 cells with arecoline（0.5, 1, and 2 m M） for 24 h, the number of autophagic vacuoles, the ratio of LC3II/LC3 I, and the level of p62 protein were markedly elevated, suggesting that arecoline can block autophagy flux in PC12 cells. Pretreatment with 10 μM of chloroquine（CQ）, the inhibitor of autophagy flux, for 30 min did not aggravate the number of autophagic vacuoles, the ratio of LC3II/LC3 I, and the level of p62 protein caused by arecoline（1 m M, 24 h） in PC12 cells. 200 and 400 μM of sodium hydrosulfide（Na HS）, the donor of H2 S, increased autophagic flux, as evidenced by diminished number of autophagic vacuoles, and downregulated LC3II/LC3 I as well as p62 expression in PC12 cells. We also found that preincubation with Na HS（200 or 400 μM, for 30 min） ameliorated arecoline-induced accumulation of autophagic vacuoles and increases in the rate of LC3II/LC3 I as well as the expression of p62 in PC12 cells, indicating that H2 S can improve arecoline-disrupted autophagy flux. Furthermore, we found that blocking autophagic flux by pretreatment with CQ（10 μM, for 30 min） abolished the inhibitory effects of H2 S on arecoline-elicited accumulation of autophagic vacuoles and upregulations of LC3II/LC3 I as well as p62 protein, indicating that inhibiting autophagic flux can reserve the improvement of H2 S on arecoline-induced disruptions in autophagic flux. Blocking autophagic flux by pretreatment with CQ（10 μM, for 30 min） antagonized the inhibitory role of Na HS（400 μM, 30 min） on arecoline-induced LDH release in PC12 cells, which indicates that inhibiting autophagic flux can reserve H2S-atteunated toxicity of arecoline in PC12 cells. Blockade of autophagic flux by pretreatment with CQ（10 μM, for 30 min） ameliorated Na HS-reserved increases in the rate of cell apoptosis and the occurrence of apoptotic morphology, and dissipation of MMP caused by arecoline in PC12 cells, which reveals that suppressing autophagic flux can abolish the protection of H2 S on arecoline-triggered apotosis. We also found that inhibiting autophagic flux by pretreatment with CQ（10 μM, for 30 min） attenuated Na HS-reserved upregulations of GRP78 and Cleaved caspase-12 induced by arecoline, suggseting that disruption of autophagic flux can antagonize the inhibitory action of H2 S on arecoline-induced ER stress. [Conclusion] H2 S improves arecoline-caused disruption in autophagic flux, which contributes to the protection of H2 S against the neurotoxicity of arecoline. |
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