Preliminary Study On ROS Mediated Apoptosis In Hindgut Of Urechis Unicinctus Exposed To Sulfide

Excessive sulfide can inhibit the activity of cytochrome c oxidase, block the electron transport in the mitochondria, lead to the increase and accumulation of reactive oxygen species (ROS), and injure the lipid, protein, nucleic acid in the cells. Superoxide dismutase (SOD) can catalyze the superoxide anion into hydrogen peroxide and oxygen, then hydrogen peroxide is further decomposed into water and oxygen by catalase, thus the damage of ROS is reduced. However, when the catalytic activity of SOD is not able to eliminate the accumulation of ROS in cells, excessive ROS will result in change of the mitochondrial permeability, cytochrome c of mitochondriais released into the cytoplasm and activates Caspase-3. The activated Caspase-3 will induce the cellular apoptosis by degrading a kind of DNA repair enzyme which making the damaged DNA cannot be required. Urechis unicinctus, a species of Echiura, inhabits a U-shape burrow of the coastal sediment, and usually encounters sulfide-rich environment in the burrow during the low tide. In this paper, SOD activity and ROS content in hindgut of U. unicinctus exposed to sulfide in lab was detected by methods of autoxidation of pyrogallol and DCFH probe. Furthermore, changes of Caspase-3 content were detected by Western Bloting in the hindgut of U. unicinctus with the extension of sulfide exposure time. The main results were as follows:SOD activity in hindgut of U. unicinctus exposed to 50 μmol/L sulfide for 24 h reached the highest which was 1.3-fold higher than that of the control, then it gradually decreased and reached the lowest at 72 h, which was only 29% of the control. However, a significant increase of the SOD activity was presented in the hindgut of U. unicinctus exposed to 150 μmol/L sulfide for 6 h, and it was 1.6- fold higher than that of the control, then gradually decreased and reached the lowest at 72 h. only 61% of the control. It was suggested that sulfide can cause the increase of SOD activity in the hindgut of U. unicinctus exposed to sulfide in a short time, then the activity decreases.The content of ROS in the hindgut of U. unicinctus exposed to sulfide for 6 h was significantly increase (p<0.05), and the trend was maintained until the end of the experiment. However, the content of ROS in 50 μmol/L sulfide group was always lower than that of 150 μmol/L group at the same time. For example, it was 3.8-fold (50 μmol/L) and 4.5-fold (150 μmol/L) higher than that of the control at 72 h, respectively. It was suggested that the accumulation of ROS occurs in the hindgut of U. unicinctus exposed to sulfide,In this study, a full length cDNA of Caspase-3 from U. unicinctus was isolated with rapid amplification of cDNA end method (RACE). A prokaryotic recombinant expression vector pET28a-caspase-3 was constructed and transformed into the competent cells of E. coli BL 21 (DE 3) to obtain the recombinant Caspase-3 protein, and then the protein was purified and prepared polyclonal antibody. Furthermore, the protein expression of Caspase-3 was determined by Western Blotting in the hindgut of U. unicinctus after sulfide stress. The result showed that no significant difference of Caspase-3 content was found in the hindgut of U. unicinctus when exposed at 50 μmol/L sulfide. However, a significant increase of the Caspase-3 amount was presented in the hindgut of U. unicinctus exposed to 150 μmol/L sulfide for 24 h, and it was 1.8-fold higher than that of the control at 72 h. Our finding suggested that survive of the hindgut cells will be not affected obviously at 50 μmol/L sulfide, while the sulfide of 150 μmol/L may cause death of the hindgut cells when U. unicinctus exposed to sulfide for a long time.In summary, a negative correlation between the ROS content and the SOD activity was presented in the hindgut of U. unicinctus exposed to sulfide, inferring that SOD in the hindgut of U. unicinctus is able to scavenge the ROS to some extent. Furthermore, combined with the results of Caspase-3 content, it is suggested that the accumulation of ROS in the hindgut of U. unicinctus exposed to 50 μmol/L sulfide does not cause obviously the death of cells; while, apoptosis may be led in the hindgut of U. unicinctus exposed to 150 μmol/L sulfide determined with a significantly increase of Caspase-3 amount.