Study On The Relationship Between Anti - Oxidative Protective Neurons And DSSylation Reaction Of Geniposide

When the free radical level overcomes the antioxidant capacity in cells, it can cause intracelluar oxidative stress. Excessive free radicals induce protein oxidation, destroy the protein structure resulting in the loss of protein activity and function, furthermore, the cell apoptosis or necrosis. Such events are now known to cause various chronic diseases, including Alzheimer’s disease (AD) and other neurodegenerative disorders. The cell response to oxidative stress is to remove these oxidative-damage proteins in time. In eukaryotic cells, the mechanisms of the degradation of damaged proteins mainly include ubiquitin/proteasome system and autophagy/lysosomal system. Recently, it has been discovered that DSS1, a highly conserved protein in eukaryotes, is used as a labeland forms adducts with oxidative-damage proteins by an ATPase catalysis, which has not yet been identified. This reaction is called DSSylation. The DSS1-protein adducts are able to be further ubiquitinated and degraded via the ubiquitin/proteasome system. The DSS1 and DSSylation play important roles in the degradation process of oxidative-damage proteins to withstand oxidative stress damage and maintain normal physiological functions.Geniposide, the main active ingredient in Chinese medicine gardenia, has a role of antioxidation. Studies show that geniposide is able to regular the expression of Bcl-2 and heme oxygenase-1 (HO-1) by interfere with MAPK and PI3K signaling pathway to protect cells from apotosis. Our previous studies have found that geniposide improves the activity of the intracellular antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), increases the mRNA and protein expression levels of Bcl-2, reduces the expression levels of apoptosis related gene p53, Caspase3 and Caspase9 to protect SH-SY5Y cells against the formaldehyde oxidative damage. These studies above mainly focus on the effect of apoptosis signaling pathway and the antioxidant enzymes to reveal the antioxidant mechanism of geniposide. However, the role of geniposide for the removal of the oxidant-damage proteins has not been elucidated.Given the important role of DSS1 and DSSylation in the process of oxidative-damage proteins, here we aimed to investigate the relationship between the geniposide and DSSylation, offering a new direction to understand the antioxidant mechanism of geniposide. This study included two parts as follows:First, in order to study the protective effects of geniposide on nerve cells, the mouse neuroblastoma cells (N2a) were used to establish formaldehyde oxidative damage model; CCK- 8, microscope, flow cytometry, Western bolt were applied to detect the cell vitality, cell morphology, the levels of ROS and the expression levels of DSS1/DSS1-protein adducts, respectively.Second, to further study the interaction between geniposide and DSSylation in vitro, The human DSS1 and HSPA8 genes were cloned and the DSS1 and HSPA8 protein were purified with affinity chromatography,. Next, we built a DSSylation reaction system in vitro to identify whether the HSPA8 can catalyze the DSSylation reaction. In addition, we established N2a cell line of DSS1 overexpression by lentivirus transfection CMV-DSS1-Myc-DDKplasimid.The results of two parts were as follows:1. By CCK-8 cell viability assay and cell morphology observation, it was found that high concentration geniposide obviously improved the reduction of cell viability and recover N2a morphological changes caused by formaldehyde, but geniposide in low and middle concentrations had no such effect.2. The results of flow cytometry and Western bolt showed that under the oxidative stress condition, high concentration geniposide decreased the level of ROS and increased DSS1 and its adducts expression to promote the degradation of oxidative damage proteins, which might be one of the antioxidant protection ways of geniposide.3. The recombinant expression plasmids of pET151-DSS1 and pET151-HSPA8 were successfully constructed through the pET151/D-TOPO cloning technology. DSS1 and HSPA8 protein were isolated and purified by Ni metal chelate affinity chromatography.4. DSSylation reaction system in vitro was be established and HSPA8 was identified not to be involved in DSSylation reaction by Western blot.5. The recombinant plasmid of L309-CMV-DSS1-Myc-DDK was successfully established by PCR directional cloning technology and transfected into N2a via lentivirus. However, we failed to obtain stable N2a cell line of DSS1 overexpression due to the lower transfection efficiency.Conclusion:1. The high concentration (250μM) of geniposide resisted the reduction of N2a cell viability and the changes of cell morphology induced by formaldehyde.2. Geniposide significantly decrease the level of ROS under oxidative dress, while geniposide with high concentration (250μM) increased the expressionof DSS1/DSS1-protein adducts. It is indicated that geniposide promoted the degradation of oxidativedamaged proteins to play antioxidant and protective roles3. The mechanism of geniposide in response to DSSylation in vitro had not yet been clear, which needed to be further studied.