Netrin-1 Affects Oxidative Stress and Neurite Outgrowth in Primary Cortical Neurons Post OGD/R via the ERK Signaling Pathway
Objective To investigate the mechanism by which Netrin-1 improves oxidative stress and promotes neurite outgrowth following oxygen-glucose deprivation/reoxygenation(OGD/R)in primary cortical neurons.Methods Primary cortical neurons of rats were randomly assigned to the following groups:control group(Control group),oxygen-glucose deprivation/reoxygenation model group(OGD/R group),OGD/R+overexpression of Netrin-1 group(OGD/R+LV-Ntn1 group),OGD/R+Netrin-1 interference group(OGD/R+LV-sh_Ntn1 group),and OGD/R+overexpression of Netrin-1+ERK1/2 inhibitor group(OGD/R+LV-Ntn1+U0126 group).After establishing the OGD/R model,cell viability was assessed using the CCK-8 assay,cell toxicity was evaluated via lactate dehydrogenase(LDH)assay,oxidative stress levels were measured by malondialdehyde(MDA),superoxide dismutase(SOD),glutathi-one peroxidase(GPx),and the ratio of reduced glutathione(GSH)to oxidized glutathione(GSSG),im-munofluorescence staining was employed to examine neurite morphology,qPCR was used to measure mRNA expression levels of PSD95 and GAP43,Western-blot was performed to analyze protein expression levels of ERK,PSD95,and GAP43.Results Compared to the Control group,the OGD/R model significantly upregulated the expression levels of the ERK pathway(P＜0.01),inhibited the cell viability of primary cortical neurons(P＜0.001),increased LDH release(P＜0.001),significantly elevated MDA levels(P＜0.001),reduced SOD activity,GPx activity,and the ratio of GSH/GSSG(P＜0.001),markedly decreased the length of the longest neurite and the number of primary neurites(P＜0.001),and decreased the expression levels of PSD95 and GAP43 mRNA and proteins.In comparison to the OGD/R group,overexpression of Netrin-1 significantly upregulated the expression levels of the ERK pathway(P＜0.001),enhanced the cell viability of primary cortical neurons(P＜0.001),decreased LDH release(P＜0.001),significantly reduced MDA levels(P＜0.001),increased SOD activity,GPx activity,and the ratio of GSH/GSSG(P＜0.001),increased the length of the longest neurite and the number of primary neurites(P＜0.001),upregulated the expression levels of PSD95 and GAP43 mRNA and proteins(P＜0.001).Compared to the Netrin-1 overexpression group,U0126 reversed the effects of Netrin-1 on the above indicators in rat primary cortical neurons(P＜0.05).Conclusion Netrin-1 can improve oxida-tive stress and promote neurite outgrowth in rat primary cortical neurons following OGD/R through the ERK signaling pathway.
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