Figure 4

Conditioned medium-induced necroptosis in neurons in vitro. We used two in vitro ICH models to further explore the role of inflammatory factors, such as TNF-α, in inducing necroptosis: one is neurons treated with OxyHb directly, and the other is using supernatant of culture of microglia (stimulated with OxyHb in advance) as neurons’ conditioned medium, and treated with or without inhibitor of TNF-α. (a) The necroptosis and apoptosis of neurons in vitro were detected by PI and Annexin V double staining and flow cytometry analysis, respectively. PI−/Annexin V− represented survival neurons, PI+/Annexin V− represented necroptotic neurons, PI−/Annexin V+ represented apoptotic neurons, and PI+/Annexin V+ represented a mixed damage of neurons. The results of flow cytometry indicated a higher ratio (~27.8%) of apoptosis and a lower ratio (~11.4%) of necroptosis when neurons were stimulated with OxyHb. However, in conditioned medium treatment group, it was a higher percentage of necroptosis (~31.5%), whereas the ratio could be significantly reduced when treated with TNF-α inhibitor (~13.5%). (b) Related bar graph showed four different conditions of neurons in various groups; NS, not significant difference (P=0.4642) in PI+/Annexin V− cells and **P=0.0007 in PI−/Annexin V+ cells versus Control group; ^&&P=0.0001 in PI+/Annexin V− cells and NS, not significant difference (P=0.1498) in PI−/Annexin V+ cells versus Control group; ^##P=0.0004 in PI+/Annexin V− cells and NS, not significant difference (P=0.3401) in PI−/Annexin V+ cells versus Conditioned medium group; all were unpaired t-test, n=3. (c) IP revealed that when treated with conditioned medium, interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were remarkably increased. And these results were attenuated when pretreated with TNF-α inhibitor. (d) Consistent data analysis of IP. **P=0.0047 in p-Ser, **P<0.0001 in RIP3, **P<0.0001 in MLKL and **P=0.0002 in caspase-8 versus Control group; ^&&P<0.0001 in p-Ser, ^&&P=0.0006 in RIP3, ^&&P<0.0001 in MLKL and ^&&P<0.0001 in caspase-8 versus Control group; ^##P=0.0003 in p-Ser, ^##P=0.0018 in RIP3, ^##P=0.0012 in MLKL and ^##P=0.0002 in caspase-8 versus conditioned medium group. The mean values for the control group were normalized to 1.0, all were unpaired t-test, n=3. (e) Immunofluorescence analysis was performed with antibody for RIP1 /RIP3 (green), MLKL (red) and caspase-8 (purple) in cultured primary neurons under indicated treatment. Nuclei were fluorescently labeled with DAPI (blue). Representative images were shown. Arrows indicated the colocalization of RIP1-MLKL-caspase-8 and RIP3-MLKL-caspase. Scale bar=20 μm. (f) PI and Hoechst double staining was also used in detection of necroptosis. The results showed that neurons in conditioned medium had higher ratio of necroptosis (as arrows point to, PI+/Hoechst+ cells), which could be inhibited by TNF-α inhibitor. Scale bar=50 μm. (g) Numbers of PI+/Hoechst+cells. **P<0.0001 versus control group, ^&&P<0.0001 versus control group, ^##P<0.0001 versus conditioned medium group; all were unpaired t-test, n=6. All data are expressed as means±S.E.M.