Protective Effects And Mechanisms Of Corilagin On Tert-butyl Hydroperoxide-induced Oxidative Stress In SH-SY5Y And N9 Cell Lines

IntroductionOxidative stress plays important roles in aging and neurodegenerative disorders. With a high content of oxidizable substrates such as polyunsaturated fatty acids, poor catalase activity and low iron-binding capacity, brain tissue may be prone to ROS damage. It has been found that inflammation the most important cause.It has been proven that inflammation induced the death of neuron, which accelerated the inflammation in turn. Microglia, the macrophage-like cells, has been implicated as mediators of inflammation in the central nervous system. ROS-mediated stress in microglia could result in cellular injuries and evoke neuronal injury preferentially. Activated microglia produce inflammatory mediators including nitric oxide, reactive oxygen species (ROS), and cytokines, such as tumor necrosis factor-a, which are associated with several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis.Therefore, scavenging free radicals, preventing ROS induced toxicities and inhibiting the microglia over-activation might be some reasonable therapeutic strategies for the treatment of neurodegenerative diseases. Phenolic compounds have strong radical scavenging abilities, but they usually have some severe adverse effects. So, finding natural substances with neuroprotective potential that can protect cells from oxidative damage has drawn considerable attention.Corilagin (beta-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-D-glucose), a novel member of the tannin family which has been discovered in many medicinal plants such as Phyllanthus species etc., has been proven to possess a multiformity of pharmacological activity, including antitumor, antioxidative, antiatherogenic, thrombolytic, antihypertensive, hepatoprotective, antiviral, antibacterial and anti-inflammatory effects. Although its antioxidant and anti-inflammatory properties were confirmed, there are no reports concerning the neuroprotective effects available. In addition, reports were almost focusing on the extract containing corilagin. Therefore corilagin, especially the purified substance, deserves an in-depth and further exploitation.ObjectiveThe present study was aimed to investigate the potential protective effects of corilagin on cell oxidative stress models of human neuroblastoma SH-SY5Y cells and N9 murine microglia cells which induced by tert-butyl hydroperoxide (TBHP) and elucidate the related mechanisms, which were undertaken to supply the experimental foundations for the potential clinical application of corilagin as a kinds of antioxidative and anti-inflammatory drugs in treatment of neurodegenerative diseases.MethodsFirst of all, cytotoxicities of corilagin on SH-SY5Y and N9 cells determined by SRB assay and oxidative stress models were induced by TBHP in SH-SY5Y and N9 cells. With the MTT and LDH release tests and observation of the morphological changes, the protective effects of corilagin were evaluated.Secondly, the ROS levels and mitochondrial membrane potential were analyzed using the fluorescent probe DCFH-DA and Rh-123, respectively. In addition, the levels of MDA, T-AOC, T-SOD and GSH were measured by biochemistry assay to evaluate the influence of antioxidative abilities of corilagin on SH-SY5Y and N9 cells.Thirdly, with AO/EB fluorescent staining used to observe apoptosis, agarose gel electrophoresis to detect DNA fragmentation and flow cytometry (FCM) to detect the cell sub-G1 peaks, the influence of corilagin on cell apoptosis induced by TBHP were observed. In order to make clear the relative mechanisms of antiapoptosic effects of corilagin, chromometry was used to measure the activation of Caspase-3 and western blotting analysis used to observe the effects of corilagin on the signaling molecules Caspase-3, Caspase-9, Bcl-2 and Bax, which are relevant with cell apoptosis.Fourthly, to investigate whether corilagin medicated in the inflammation of N9 cells activated by TBHP, Griess assay and absorption spectrometry were used for NO levels and NOS activities determination respectively; the expression of iNOS mRNA assessed by means of analysis of relative gene expression data using real-time quantitative PCR; ELISA assay used for TNF-α; In-situ immuno-flusent staining applied for the observation of the NF-KB mechanisms.Statistic analysisQuantitative data were presented as mean±standard deviation (S.D.). The statistical analysis between various experimental results was performed using one-way ANOVA, while those between two groups were analyzed by t-test. P<0.05 was considered statistically significant.Results(1) Protective Effects Corilagin exhibits cytotoxicities on SH-SY5Y and N9 at high concentrations with IC50 values of 389.64±29.83 (μM, 24 h), 202.80±17.64 (μM, 48 h) for SH-SY5Y and 179.72±46.54, 61.64±6.80 for N9 cells. Challenge with TBHP for 24 h resulted in the morphological changes: neuritis of most SH-SY5Y cells shortened, broken, or lost, cell bodies crimpled, N9 cells shrinkage, even floatation, and some of which were lysed or replaced by debris. TBHP inhibited SH-SY5Y and N9 cells viability in a time-and-dose-depended manner, the inhibition about 50% with TBHP (40μM, 24 h) for SH-SY5Y, as for N9 cells, and 600μM. Compared with the TBHP group, morphologies of cells mitigated with preincubation of corilagin 24 h prior to the exposure of TBHP, and cell viability increased, LDH leakage lowered significantly and dose-dependently. The results indicated corilagin could attenuate TBHP-induced oxidative stress in SH-SY5Y and N9 cells.(2) Antioxidative Effects Treatment with TBHP resulted in an increase of DCF fluorescence and decrease of Rh-123 fluorescence, which suggested the accumulation of intercellular ROS, damage of michondrial membrane which resulted in the loss of MMP. Compared with TBHP group, pretreatment of corilagin decreased the DCF fluorescence and inversed the MMP reduction, which confirmed that corilagin, may protect oxidative damage through ROS scavenging and MMP stabilization. In antioxidative characteristic measurements, treatment with TBHP resulted in MDA increased, and T-AOC, SOD, GSH levels lowed. In contrast, pretreatment with corilagin ameliorate these items. Combined with the previous part, the results confirmed that corilagin can protect the oxidative damage with its antioxidative activity, and the relevant mechanisms may involve mitochondrial protection.(3) Anti-apoptosis Apoptosis induced by TBHP was observed, which inhibited by pretreatment of corilagin. While SH-SY5Y cells preincubated with corilagin before TBHP exposure, caspase-3 enzyme activity inhibited. Expressions of caspase-3 and caspase-9 were significantly down-regulated and Bcl-2 up-regulated by corilagin without the obviously influence on Bax. The results suggest that corilagin could inhibited the apoptosis which induced by TBHP, in mitochondrial-depended pathway, with the regulation of Caspases and Bcl-2.(4) Anti-inflammation TBHP incubation resulted in the increase of NO and TNF-αsecretion, as well as iNOS activity. With the NO scavenging activity, intervention with corilagin could decrease the secretion of NO and TNF-αsignificantly, without obviously effect on iNOS activity. Furthermore, the expression of iNOSmRNA of cells preincubated with corilagin was significantly down-regulated; NF-KB activation and translocation were attenuated. The results collectively showed that corilagin could inhibit the inflammatory factors, such as NO and TNF-α, corresponding with the down-regulation of iNOSmRNA, besides the NO scavenging itself in vitro. The mechanisms in which corilagin prevent the impairment of oxidative stress may involve NF-κB pathway.ConclusionCorilagin could prevent the impairment of oxidative stress challenged with TBHP on SH-SY5Y cells and N9 cells. The effects may relate to its antioxidative, antiapopototic and anti-inflammatory activities with the mechanisms involved in ROS scavenging, inhibiting apoptosis which induced in mitochondrial-dependent throughway, and anti-inflammation on the microglia activated by oxidative stress.In a word, corilagin presents the potential protection on neuron and microglia cell. Hence, it would be considered to be a potential choice of prevention and cure to NDD.