Hydrogen Sulfide Prevents Homocysteine-induced Endoplasmic Reticulum Stress In PC12Cells By Upregulation SIRT1

[Backgroud and Objective]Homocysteine (Hcy) produces neurotoxicity and is an independent risk factor forAlzheimer’s disease. Our previous studies have shown that Hydrogen sulfide (H2S) hasthe protective effect on Hcy-induced neurotoxicity, but the mechanism remains to beclarified. Excessive endoplasmic reticulum (ER) stress leads to the apoptosis of neuronalcells. Silent informationation regulator1(SIRT1) plays an important role inneuroprotection. Therefore we assume that H2S antagonize Hcy-induced neurotoxicity byinhibition of ER stress via regulating the expression of SIRT1.To verify the hypothesis, we will use PC12cell treated with Hcy as the cell model of Hcyneurotoxicity to investigate the protection of H2S against Hcy-induced ER stress in PC12cells and the mediate role of SIRT1in the protective effects of H2S on Hcy-induced ERstress and neurotoxicity in PC12cells.[Methods]Cell Counting Kit-8(CCK8) assay was used to detect the viability of PC12cells. Flowcytometry (FCM) was used to measure the apoptosis of PC12cells; Western blot wasused to detect the expression of ER stress related protein (Glucose-regulated protein78,GRP78; Cleaved caspase-12, Cleaved caspase12) and the expression of SIRT1.[Results]1. Hcy promotes ER stress and down-regulates the expression of SIRT1in PC12cells.After PC12cells were treated with Hcy (1.25,2.5,5mM) for24h, the expressions ofGRP78and Cleaved caspase12were increased in PC12cells in a concentration-dependentmanner, indicating that Hcy induces neurotoxicity via inducing ER stress. After PC12cells were treated with Hcy (1.25,2.5,5mM) for24h, the expression ofSIRT1was decreased in PC12cells concentration-dependently, indicating that Hcy-induced ER stress is related to downregulation of SIRT1in PC12cells.2. H2S antagonizes ER stress induced by Hcy in PC12cellsAfter PC12cells were cotreated with NaHS (100,200or400μM) and Hcy (5mM) for24h, the upregulatory effects of Hcy (5mM,24h) on GRP78and Cleaved caspase12expressions in PC12cells were reduced. These dates indicated that H2S can antagonizeER stress induced by Hcy.3. SIRT1mediates the protection of H2S against Hcy-induced ER stress andneurotoxicity in PC12cells.3.1H2S promotes the high expression of SIRT1in PC12cellsAfter PC12cells were treated with NaHS (100,200and400μM) for24h, the expressionof SIRT1is upregulated in PC12cells, which indicated that H2S can promote the highexpression of SIRT1.3.2H2S reduces the inhibition of SIRT1expression induced by Hcy in PC12cellsAfter PC12cells were treated with NaHS (400μM) and Hcy (5mM) for24h, theinhibition of SIRT1expression induced by Hcy was reversed.3.3Sirtinol, the inhibitor of SIRT1, blocks the protective effect of H2S against ERstress induced by Hcy in PC12cellsAfter pretreatment of PC12cells with Sirtinol (20nM) for2h and then coexposed to Hcy(5mM) and NaHS (400μM) for24h, the upregulated expressions of GRP78and Cleavedcaspase12by Hcy were significantly blocked by pretreatment with Sirtinol, indicatingthat H2S antagonizes Hcy-induced ER stress via upregulating the expression of SIRT1inPC12cells.3.4Sirtinol, the inhibitor of SIRT1, blocks the protective effect of H2S againstcytotoxcity and apoptosis induced by Hcy in PC12cellsAfter pretreatment of PC12cells with Sirtinol (20nM) for2h and then coexposed to Hcy (5mM) and NaHS (400μM) for24h, Hcy-exerted cytotoxicity and apoptosis weresignificantly prevented by pretreatment with Sirtinol, indicating that SIRT1mediates theprotection of H2S against Hcy-induced neurotoxicity in PC12cells.[Conclusions]1. Hcy promotes ER stress in PC12cells by down-regulation of SIRT1.2. H2S prevents Hcy-induced neurotoxicity and ER stress in PC12cells via upregulatingthe expression of SIRT1.