脑损伤
-
Figure 3 ｜ Secreted neuroserpin ameliorates cell death in cultured cortical neurons
Neuroserpin is produced by several types of neurons and is a secretory protein with neuroprotective effects. We thus examined whether OGD influences the intracellular or secreted levels of neuroserpin in cortical neurons. Notably, neuroserpin level was not altered in the cell homogenate containing intracellular and membrane proteins, but cellular neuroserpin mRNA and extracellular neuroserpin protein were both markedly decreased by OGD (Figure 3A?D), suggesting substantial inhibition of gene expression and extracellular protein secretion of neuroserpin. Consistent with the in vitro results, the mRNA level of neuroserpin was also markedly downregulated in brain tissues of MCAO mice (Figure 3E). We speculated that the deprivation of secreted neuroserpin leads to weakened neuroprotection, thereby contributing to OGD/R-induced cellular damage. To test this hypothesis, we collected the conditioned medium from normal cortical neurons cultured in standard conditions. Instead of adding new culture medium to OGD-injured neurons, we added the conditioned medium containing secreted neuroserpin for the reperfusion phase (Figure 3F). Conditioned medium treatment markedly increased the cell viability of OGD/R-injured neurons. Moreover, when anti-neuroserpin antibody was used to neutralize neuroserpin in the conditioned medium, the protective effect of the conditioned medium was canceled (Figure 3G). These results suggest that secreted neuroserpin is an important factor for neuroprotection, and OGD substantially inhibits its secretion from the endogenous sources, thus worsening cell survival. Our results showed that extracellular neuroserpin alleviates cell damage. We thus next examined whether the addition of recombinant neuroserpin also has a protective effect. Neuroserpin (1–20 ng/mL) was pretreated to 7 DIV cortical neurons for 4 hours and was continuously present during the 4 hours of OGD phase but was absent during the reperfusion phase (Figure 3H). Neuroserpin effectively alleviated OGD/R-induced damage at 20 ng/mL(Figure 3I). We further used the live and dead cell assay to visualize cell viability. OGD/R triggered a pronounced decreased number of viable cells and an increased number of damaged cells, but the addition of neuroserpin prevented OGD-induced cell damage (Figure 3J and K). We further examined whether neuroserpin could reverse cell damage when applied after OGD (Figure 3L). Neuroserpin (50 and 100 ng/mL) markedly enhanced cell viability of the OGD/R-injured cells (Figure 3M). Consistent with the cell viability results, the numbers of TUNEL-positive apoptotic cells markedly increased after OGD/R, whereas neuroserpin (20 and 100 ng/mL) reversed the apoptosis level (Figure 3N and O). These results showed that complementing endogenous extracellular neuroserpin by adding exogenous protein improves cell survival against OGD/R.