Protective Effect Of Hydrogen Sulfide Against Formaldehyde-induced Neurotoxicity To PC12 Cells And The Underlying Mechanisms

【Backgroud and Objective】Formaldehyde is a kind of volatile toxic substances. Exposure to formaldehyde in vivo can hazard central nervous system. In addition, an increase in endougenous formaldehyde level by upregulation of semicarbazide-sensitive amine oxidase (SSAO) contributes to the pathology of Alzheimer's disease (AD). Therefore, it is of utmost importance to develop novle therapeutic approaches to halt the neurotoxicity of formaldehyde.Hydrogen sulfide (H₂S) broadly participates in a variety of physiological pathological processes and is considered to be the third gasotransmitter following the nitric oxide and carbon monoxide. In recent years, more and more evidences support that endogenous H₂S has an important role in modulating the function of central nervous system, especially in antioxidant and neuroprotective action.Increasing evidence has shown that oxidative damage is one of the most critical effects of formaldehyde exposure. Whether dose H₂S prevent the neurotoxicity of formaldehyde? Therefore, in the current study, we used PC12 cells exposed to formaldehyde as the cell model of formaldehyde neurotoxicity to investigate the effects of H₂S on formaldehyde-induced cytotoxicity to PC12 cells and explore the underlying mechanisms.【Methods】The viability of PC12 cells was observed by the trypan blue dye exclusion assay. Elisa assay was used to detecting the release of lactate dehydrogenase (LDH). The apoptotic PC12 cells was observed under a fluorescence microscope after hochest 33258 staining and detected by flow cytometry (FCM) after propidium iodide (PI) staining. The level of MMP was assessed under a fluorescence microscope after JC-1 staining and measured by FCM after rhodamine 123 (Rh123) staining. The level of intracellsular ROS in PC12 cells was observed under a fluorescence microscope and detected by FCM after 2, 7-dichloro-fluorescein diacetate (DCFH-DA) staining. The release of cytochrome c (Cyt-c) and the expressions of Bcl-2 and paraoxonase-1 (PON-1) in the PC12 cells were detected by Western Blot. The activity of PON-1 in the PC12 cells was detected by spectrophotometric method.【Results】1. Formaldehyde-triggered neurotoxicity to PC12 cells and the underlying mechanisms.Treatment of PC12 with formaldehyde (60, 120, and 240μmol/L) for 24 h inhibited the viability of PC12 cells and induced the release of LDH in a concerntration-depondent manner. Exposure of PC12 cells to 120μmol/L of formaldehyde for 24 significantly induced morphological defect and apoptosis. These data indicated that formaldehyde can cause neurotoxicity to PC12 cells.After PC12 cells were dealed with 120μmol/L of formaldehyde for 9 h, the level of MMP significantly increased. Treatment of PC12 cells with formaldehyde (60, 120, and 240μmol/L) for 24 h, concertration-dependently, inhibited the expression of Bcl-2 protein and promoted the release of Cyt-c. Our data suggested that formaldehyde induces cell apoptosis and neurotoxicity by triggering the mitochondrial pathways of apoptosis.After treated with 120μmol/L formaldehyde for 9 h, the level of intracellular ROS in PC12 cells was increased. When PC12 cells were treated with 60, 120, and 240μmol/L of formaldehyde for 24 h the expression and activity of PON-1 in cells were decreased. These data implied that the neurotoxicity of formaldehyde involves the inhibited expression and activity of PON-1, which enhances the accumulation of intracellular ROS.2. H₂S protects PC12cells against formaldehyde-induced cytotoxicity and the underlying mechanismsPretreatment with 200μmol/L of H₂S for 30 min markedly increased the viability of PC12 cell treated with 120 or 240μmol/L of formaldehyde for 24 h. Pretreatment with H₂S, at the concerntration of 200μmol/L and 400μmol/L, for 30 min significantly increased the viability of PC12 cell treated with 120μmol/L of formaldehyde for 24 h. Pretreatment with 200μmol/L of H₂S for 30 min markedly suppressed the increased LDH release, deficted morphological changs, and enhanced apoptosis by treatment with 120μmol/L formaldehyde for 24 h. These findings suggested that H₂S protects PC12 cells against formaldehyde-induced neurotoxicity.Pretreatment of PC12 cells with H₂S (200μmol/L) for 30 min weaked singificantly the loss of MMP induced by exposure to 120μmol/L of formaldehyde for 9 h and suppressed the reduction of Bcl-2 protein expression and promotion of Cyt-c release induced by 24 h of treatment with 120μmol/L formaldehyde, suggesting that H₂S prevents formaldehyde-triggered the mitochondrial pathways of apoptosis.Pretreatment of PC12 cells with H₂S (200μmol/L) for 30 min singificantly weaked the accumulation of ROS induced by exposure to 120μmol/L of formaldehyde for 9 h and attenuated the inhibited expression and activity of PON-1 by treatment with 120μmol/L of formaldehyde for 24 h. 200μmol/L of H₂S for 24 h enhanced the activity of PON-1 in PC12 cells. These data indicated that H₂S blocks formaldehyde-induced the accumulation of intracellular ROS and the inhibition of PON-1 expression and activity and upregulates PON-1 activity.Pretreatment of PC12 cells with 200μmol/L of 2-Hydroxy-4- methylquinoline (2-HQ), a potent inhibitor of PON-1, significantly prevented the protective action of H₂S (200μmol/L) against the accumulation of intracellular ROS, cytotoxicity, and apoptosis induced by exposure to formaldehyde (120μmol/L), which implied that H₂S protects PC12 cells against the neurotoxicity of formaldehyde through inhibition of formaldehyde-induced derease in expression and activity of PON-1 and enhancment of PON-1 activity, which blocks formaldehyde-triggered accumulation of intracellular ROS, resulting in preventment of the mitochondrial pathways of apoptosis .【Conclusion】1. Formaldehyde causes neurotoxicity to PC12 cells by inhibition of PON-1 expression and activity, which enhances the accumulation of intracellular ROS, resulting in activation of the mitochondrial pathways of apoptosis.2. H₂S antagonizes formaldehyde-induced toxicity to PC12 cells and the underlying mechanism is associated with enhancing PON-1 activity and attenuating the reduced expression and activity of PON-1 by formaldehyde, which deraeses in the level of intracellular ROS, resulting in suppression of formaldehyde-triggered mitochondrial pathways of apoptosis.