Hydrogen Sulfide Inhibits Formaldehyde-Induced Cellular Senescence In HT22 Cells Via Upregulation Of Leptin Signaling Pathway

[Background and Objective] Profound studies have shown that formaldehyde(FA) has toxic effects on central nervous system. Hydrogen sulfide(H2S), recognized as a neuroprotectant, can inhibit FA-induced cell apoptosis, cytotoxicity, and endoplasmic reticulum stress. This indicates that H2 S can inhibit FA-caused neurotoxicity, but the underlying mechanisms need further study. Senescent neurons play an important role in the process of neurodegenerative diseases, so we hypothesis that cellular senescence exerts significant value on FA-induced neurotoxicity. Leptin has neuroprotective effect on central nervous system. Our previous study has shown that up-regulated leptin signaling pathway mediates H2S-induced neuroprotective effects against arecoline-caused apoptosis and endoplasmic reticulum stress in PC12 cells. Thus, in the present study, we will explore whether cellular senescence involves in FA-induced neurotoxicity, whether H2 S can inhibit FA-caused cellular senescence, and whether leptin signaling pathway mediates this effect of H2 S.[Methods] Cell viability was assayed by Cell Counting kit-8(CCK-8). SA-?-gal(Senescence Associated acidic-?-Galactosidas) staining was used to determine senescent cells. Cell growth curves were measured by Trypan blue stain assays to determine the growth situation of cells. The expressions of leptin, lepRb, and senescence-associated proteins p16INK4 a and p21CIP1 were detected by Western blot. The content of leptin in the cell culture supernatant was determined by ELISA assay.[Results] 1. After treatment with FA(100, 200 ?M) for 48 h, the viability of HT22 cells was significantly decreased. This result indicates the cytotoxicity effect of FA on HT22 cells. 2. Treatment of HT22 cells with FA(50, 100, 200 ?M, 48 h) caused significantly increased staining for senescence-associated ?-galactosidase(SA-?-gal), increased expressions of senescence mark proteins(p16INK4a and p21CIP1), and suppressed cell growth. These results indicate that FA can accelerate cellular senescence in HT22 cells. 3. Pretreatment with NaHS(a donor of H2 S, 400 ?M, 30 min) obviously inhibited FA-induced decrease in cell viability of HT22 cells. This result proves the inhibitory effect of H2 S on FA-caused cytotoxicity in HT22 cells. 4. Pretreatment with NaHS(400 ?M, 30 min) significantly reversed the FA(100 ?M, 48 h)-induced senescent features in HT22 cells including increased staining for SA-?-gal, increased expressions of p16INK4 a and p21CIP1, and suppressed cell growth. These indicate that H2 S can prevent FA-induced cellular senescence in HT22 cells. 5. FA(50, 100, 200 ?M, 48 h) down-regulated the expressions of leptin and lepRb in HT22 cells as well as the content of culture supernatant's leptin in a dose dependent manner. This indicates the possible relationship between FA-caused neurotoxicity and down-regulated leptin signaling. 6. Treatment of HT22 cells with NaHS(100, 200, 400 ?M) for 48 h up-regulated the expressions of leptin and lepRb as well as the culture supernatant's leptin content in a does-dependent manner. Furthermore, pretreatment with NaHS significantly reversed FA-induced down-regulation of leptin and lepRb, and decrease in culture supernatant's leptin content. Thus, it is possible that the inhibitory effect of H2 S on FA-caused cellular senescence is related to the up-regulated leptin signaling. 7. Pretreatment with leptin tA(50 nM, an inhibitor of leptin signaling pathway) for 30 min reversed the inhibitory effect of NaHS on FA-caused decrease of cell viability and increase of cellular senescence. These indicate that leptin signaling pathway mediates the protective effect of H2 S against FA-caused cellular senescence in HT22 cells.[Conclusion] 1. FA accelerates cellular senescence in HT22 cells; 2. H2 S inhibits FA-induced cellular senescence in HT22 cells. 3. Leptin signaling pathway mediates the inhibitory effect of H2 S on FA-caused cellular senescence in HT22 cells.