Figure 2

Different effects of Nec-1 and z-VAD on brain injury after ICH. Nec-1 (the specific inhibitor of necroptosis) and z-VAD (a caspase inhibitor) were used to explore whether necroptosis contributes to brain injury after ICH. (a) The necroptosis in cells were detected by PI staining and apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. Nec-1 reduced the PI+ cells, whereas it had no significant effects on TUNEL+ cells (apoptosis), and z-VAD only downregulated the TUNEL+ cells. Additionally, combination of Nec-1 and z-VAD obviously both reduced the PI+ cells and TUNEL+ cells. Scale bar=100 μm. (b) Corresponding bar graph revealed relative levels of PI/TUNEL+ cells. **P<0.0001 (both in PI and TUNEL) versus Sham group; NS, not significant difference (P=0.0909 in PI and P=0.0857 in TUNEL) versus ICH group; ^&&P<0.0001 in PI and NS (P=0.4233) in TUNEL versus vehicle group; NS (P=0.4233) in PI and ^##P<0.0001 in TUNEL versus vehicle group; ^$$P<0.0001 in PI and NS (P=0.0663) in TUNEL versus z-VAD group; all were unpaired t-test, n=6. (c) IP showed that Nec-1 reduced interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8, indicating that the formation of necrosome was inhibited. Input, 5% of extract before IP. (d) Corresponding bar graph revealed relative levels of association. **P=0.0097 in RIP1, **P=0.0099 in RIP3, **P=0.0010 in MLKL and **P=0.0022 in caspase-8 versus Sham group; NS, not significant difference (P=0.3077 in RIP1, P=0.7942 in RIP3, P=0.9047 in MLKL and P=0.4930 in caspase-8) versus ICH group; ^&P=0.0102 in RIP1, ^&P=0.0493 in RIP3, ^&&P=0.0074 in MLKL and NS (P=0.9349) in caspase-8 versus vehicle group; NS (P=0.8658) in RIP1, NS (P=0.6857) in RIP3, ^#P=0.0205 in MLKL and ^#P=0.0281 in caspase-8 versus vehicle group; ^$$P=0.0065 in RIP1, ^$P=0.0499 in RIP3, ^$P=0.0403 in MLKL and NS (P=0.6746) in caspase-8 versus z-VAD group; all were unpaired t-test, n=3. (e) Expression of albumin, which is regarded as the index of BBB injury, was increased after ICH, whereas it could be significantly decreased when treated with Nec-1 and/or z-VAD. (f) Corresponding bar graph revealed relative levels of albumin. *P=0.0200 versus Sham group; NS, not significant difference (P=0.9779) versus ICH group; ^&P=0.0461 versus vehicle group; ^#P=0.0496 versus vehicle group; ^$P=0.0126 versus z-VAD group; all were unpaired t-test, n=6. (g) Compared with ICH group, the brain water content was partially attenuated in Nec-1 and/or z-VAD group, **P<0.0001 (both in Ipsi-CX and Ipsi-BG) versus Sham group; NS, not significant difference (P=0.9022 in Ipsi-CX and P=0.9660 in Ipsi-BG) versus ICH group; ^&P=0.0109 in Ipsi-CX and ^&&P=0.0077 in Ipsi-BG versus vehicle group; ^#P=0.0162 in Ipsi-CX and NS (P=0.0727) in Ipsi-BG versus vehicle group; ^$$P=0.0015 in Ipsi-CX and ^$$P=0.0039 in Ipsi-BG versus z-VAD group; all were unpaired t-test, n=6. (h) The levels of TNF-α in the CSF were measured by ELISA and reduced by Nec-1 treatment. **P<0.0001 versus Sham group, NS, not significant difference (P=0.7897) versus ICH group; ^&&P<0.0001 versus vehicle group, ^##P=0.0008 versus vehicle group, ^$$P<0.0001 versus z-VAD group; all were unpaired t-test, n=6. All data are expressed as means±S.E.M. and mean value for Sham group was normalized to 1.0.