Hydrogen Sulfide Alleviastes Arecoline Via Upregulating Leptin Signaling Pathway In PC12 Cells

[Backgroud and Objective]Recent studies have shown that arecoline, a major alkaloid of the areca nut, induces neurotoxicity by enhancing oxidative stress and apoptosis. Hydrogen sulfide(H2S) has been recognized not only as a neuromodulator, but also as a neuroprotectant against oxidative stress and apoptosis in the central nervous system(CNS). Leptin is a hormone mainly secreted by adipocytes, exerting its biological activity through its long leptin receptor(lep Rb) in CNS. Emerging evidences demonstrate that leptin has neuroprotective effect. Therfore, we want to investigate whether H2 S inhibits arecoline-induced neurotoxicity in PC12 cells and whether the underling protective mechanism of H2 S is related to regulating leptin signaling pathway in present study. [Methods]Cell viability was assessed by Cell Counting kit-8(CCK-8). The morphological of apoptotic cell was observed by Hoechst 33258 staining. The rate of cell apoptosis was measured by flow cytometry(FCM) after Annexin V-FITC /PI staining. The activity of Caspase-3 in cells and the level of leptin in the culture supernatant were determined by Elisa assay. The level of H2 S in the culture supernatant was measured by Unisense H2 S sensors. The expressions of two key enzymes for generation of endogenous H2S(Cystathionine-bata-synthase(CBS) and 3-mercaptopyruvate sulphurtransferase(3-MST) proteins, leptin, Lep Rb, apoptosis-related proteins(Bax and Bcl-2), and the marker proteins of endoplasmic reticulum stress(Glucose-regulated protein 78(GRP78), CCAAT/enhancer binding protein(C/EBP) homologous protein(CHOP) and Cleaved caspase-12) were detected by Western Blot. [Results]After treatment with arecoline(0.5, 1, and 2 m M) for 24 h, the viability of PC12 cells was obviously attenuated, the activity of Caspase-3 and the expression of pro-apoptotic protein Bax in PC12 cells were upregulated, while the expression of anti-apoptotic protein Bcl-2 was suppressed. In addition, arecoline caused excessive endoplasmic reticulum(ER) stress in PC12 cells, as indicated by the upregulations of GRP78, CHOP, and Cleaved caspase-12. Notably, arecoline decreased the levels of CBS and 3-MST in PC12 cells and the content of H2 S in culture supernatant. However, Na HS(400 and 800 μM), a H2 S donor, significantly rescued arecoline-induced cytotoxicity, apoptosis and ER stress in PC12 cells. Intriguingly, Na HS markedly attenuated arecoline-induced downregulation of leptin and lep Rb proteins in PC12 cells, as well as the level of leptin in culture supernatant. Inhibition of leptin pathway by leptin t A(50 n M) reversed the protection of H2 S against arecoline-induced cytotoxicity, apoptosis and ER stress in PC12 cells. [Conclusion]H2S attenuates arecoline-induced neurotoxicity by upregulating leptin signaling pathway.