Role Of HSP70 In Neuronal Oxidative Stress And The Neuroprotective Effects Of Polypeptide From Chlamys Farreri (PCF)

Oxidative stress has long been linked to cell death in many neurodegenerative conditions. Oxidative damages mediated by reactive oxygen species (ROS) could trigger cellular stress responses at the same time. Heat shock protein70 (HSP70) is a highly conserved stress proteins family expressed under pathological conditions. HSP70(HSP72) and glucose-regulated protein 78 (GRP78) are two HSP70 members respectively located in cytoplasm and endoplasmic reticulum. HSP70 (HSP72) mainly function as protein chaperones and some studies have shown its anti-apoptotic and anti-DNA damage effects. Mitogen-activated protein kinase (MAPK) and growth arrest and DNA damage inducible protein (GADD) are two stress response families which could be activated by ROS and highly related to cell apoptosis and DNA damage, however, whether the function of HSP70 in protection neurons against oxidative stress is related to MAPK and GADD is still unknown. GRP78 is the molecular chaperone mark of endoplasmic reticular stress (ERS) and severe ERS could initiate apoptosis. Whether and how ERS characterized by increased expression of GRP78 participated in the neuronal injury mediated by oxidatve stress need further study. Treatment with antioxidants is a promising approach for inhibiting neuronal oxidative damage. Owing to severe adverse effects, many synthetic antioxidants is restricted to clinical usage, therefore, searching for natural antioxidants with neuroprotective potential may provide new insight into therapeutic strategies. Until now the antioxidative effects of marine active products are rarely studied. The previous studies have demonstrated the neuroprotective effect of polypeptide from Chlamys farreri (PCF), a natural marine antioxidant, in an rat middle cerebral artery occlusion (MCAO) and reperfusion model and the molecular mechanisms need to verify. In the present study, MTT, electronspinresonance (ESR), transmission electric micscopy, fluorescence staining, western blot, RT-PCR, enzyme and biochemical assays are performed to investigate the role of HSP70 and GRP78 marked ERS in neuronal oxidative stress and the neuroprotective mechanisms of PCF. The study consist of three parts:first, the neuroblastoma SH-SY5Y cells are used as an in vitro model to established hydrogen peroxide (H2O2)-induced neuronal oxidative damage. After observing the time-course expression of HSP70, MAPK, GADD153 and GADD45βin H2O2-treated cells, the cells are pretreated with GGA (inducer of HSP70 expression) or quercetin (inhibitor of HSP70 expression) before H2O2-incubution and the difference in the expression of MAPK, GADD153 and GADD45βare assesed to explore the role of HSP70 in neuronal oxidative injury. Second, the H2O2-induced endoplasmic reticular ultrastructure changes and the expression of GRP78/Bip and GADD153/CHOP are studied to verify the ERS. Then NAC (ROS scavenger) and 4-PBA (an chemical chaperones and inhibitor of ERS) are pretreated to the medium before H2O2-incubution and cell viability, apoptosis, ROS production and the expression of GRP78, CHOP, JNK and are investigated to explore the role of ERS in neuronal oxidative stress. In the last part the antioxidative proterty of PCF on ROS production, lipid peroxidation, DNA oxidative damage and the activity of endogenous antioxidant defense components are assesed and the anti-apoptotic mechanisms of PCF on the expression of MAPK, GRP78, CHOP/GADD153, HSP70 and Bcl-2 are assesedThe results are as following:①More ROS production, increased expression of HSP70, GADD153/CHOPmRNA and GADD45βmRNA, activation of ERK1/2 and JNK accompanied by decreased cell viability in a time-dependent manner were observed in H2O2-treated cell compared to control non-treated cells; Compared to H2O2-alone-treated cells, GGA pretreatment induced high levels of HSP70 expression with DNA oxidative damage, apoptosis, phosphorylations of ERK1/2 and JNK and the expression of GADD153mRNA inhibition;②Endoplasmic reticulum dilation and increased expression of GRP78 and CHOP in a time-dependent manner were observed in H2O2-alone-treated cells, while NAC and 4-PBA pretreated cells showed more cell viability, less apoptosis and decreased expression of GRP78 and CHOP, less activation of JNK and increased level of Bcl-2 than H2O2-alone-treated cells;③PCF treatment inhibited H2O2-induced cell death, ROS production, apoptosis, LPO and DNA damage, meanwhile promoted endogenous antioxidant defense components including glutathione peroxidase, catalase, superoxide dismutase, glutathione and total antioxidative capacity. PCF significantly blocked H2O2-induced phosphorylation of c-Jun N-terminal kinase (JNK) of the MAPK family and the expression of GRP78 and CHOP. The levels of HSP70 and Bcl-2 were increased in PCF-pretreated cells.These results showed that:①The anti-apoptotic function and anti-DNA damage of HSP70 is related to the inhibition of phosphorylation of ERK1/2 and JNK and the regulation of GADD45βmRNA and GADD153 mRNA expression;②Oxidative stress could trigger ERS, GRP78/CHOP/JNK/Bcl-2 participated in SH-SY5Y apoptosis mediated by H2O2-induced ERS;③The antioxidative and anti-aopototic properties contribute to the neuroprotective effects of PCF. PCF could upregulate HSP70 and inhibit ERS and CHOP/JNK/Bcl-2 may be the downstream target of PCF.In conclusion, this study mainly focuse on the molecular role of HSP70 and GRP78 mediated stress responses in neuronal oxidative stress injury and the neuroprotective effects of PCF. This study provided useful information for developing effective antioxidant with neuroprotective function. The highlight of innovation of the study are:①Time-course changes of ROS in H2O2-induced SH-SY5Y cells were measured by ESR;②GADD45βand GADD153 are the downstream signal for HSP70 participating in neuronal oxidative DNA damage and repair;③H2O2 could activate ERS and lead to SH-SY5Y cell apoptosis by CHOP/JNK pathways;④The study have first provided evidence that the neuroprotective effect of marine antioxidant PCF were mediated, at least, through scavenging oxygen free radicals, reinforcement of endogenous antioxidant defense, prevention of oxidation of macromolecules, lipids, and DNA, upregulating HSP70, and blocking the ERS and activation of downstream CHOP/JNK1/2. PCF could inhibit JNK pathway and thus inhibit SH-SY5Y cell apoptosis. In the future, further study as following should be performed:①Special stable overexpression and downexpression of HSP70 neuronal cell model should be developed to overexpress or knockdown HSP70 then to investigate the functions of HSP70;②Interaction of HSP70 and MAPK and GADD need be further verified;③the molecular targets for ERS induced apoptosis mediated by H2O2, the pharmacokinetics of PCF in neurodegeneration therapies and the molecular mechanisms of PCF on the signaling pathways of ROS-mediated primary neuronal cell growth and apoptosis.