Figure 3

Knockdown of RIP1 reduced necroptosis in brain tissues after ICH. Knockdown and overexpression of RIP1 were used to study the role of RIP1-mediated necroptosis after ICH in rats. (a) PI+ cells decreased in the Si-RIP1 group (knockdown), whereas they increased in the Ad-RIP1 group (overexpression). Arrows point to PI+ cells. Scale bar=200 μm. (b) Related with (a), it revealed relative levels of PI+ cells. **P<0.0001 versus Sham group; NS, not significant difference (P=0.8453) versus ICH group; ^&&P<0.0001 versus Si-NC group; NS, not significant difference (P=0.9211) versus ICH group; ^##P<0.0001 versus Ad-GFP group; all were unpaired t-test, n=6. (c) IP demonstrated that interactions of RIP1 and RIP3, RIP1 and MLKL, and RIP1 and caspase-8 were significantly inhibited in the RIP1-knockdown group, whereas they increased in the overexpression group. Input, 5% of extract before IP. Quantitative analysis of IP was shown in (d), *P=0.0177 in RIP1, *P=0.0261 in RIP3, **P=0.0072 in MLKL and *P=0.0209 in caspase-8 versus Sham group; NS, not significant difference (P=0.7939 in RIP1, P=0.6515 in RIP3, P=0.4640 in MLKL and P=0.8812 in caspase-8) versus ICH group; ^&P=0.0254 in RIP1, ^&P=0.0184 in RIP3, ^&&P=0.0019 in MLKL and ^&&P=0.0010 in caspase-8 versus Si-NC group; NS (P=0.7254) in RIP1, NS (P=0.5590) in RIP3, NS (P=0.3154) in MLKL and NS (P=0.6284) in caspase-8 versus ICH group; ^#P=0.0164 in RIP1, ^#P=0.0138 in RIP3, ^#P=0.0367 in MLKL and ^##P=0.0083 in caspase-8 versus Ad-GFP group; all were unpaired t-test, n=3. (e) Expression of albumin was elevated in the Ad-RIP1 group, whereas it was opposite in the Si-RIP1 group. (f) Bar graph related to (e). **P=0.0007 versus Sham group; NS, not significant difference (P=0.8780) versus ICH group; ^&P=0.0493 versus Si-NC group; NS, not significant difference (P=0.9361) versus ICH group; ^#P=0.0470 versus Ad-GFP group; all were unpaired t-test, n=3. (g) Brain water content decreased in the Si-RIP1 group, whereas it was opposite in the Ad-RIP1 group,**P<0.0001 (both in Ipsi-CX and Ipsi-BG) versus Sham group; NS, not significant difference (P=0.4041 in Ipsi-CX and P=0.5003 in Ipsi-BG) versus ICH group; ^&&P<0.0001 in Ipsi-CX and ^&P=0.0209 in Ipsi-BG versus Si-NC group; NS, not significant difference (P=0.5276 in Ipsi-CX and P=0.6015 in Ipsi-BG) versus ICH group; ^##P<0.0001 (both in Ipsi-CX and Ipsi-BG) versus Ad-GFP group; all were unpaired t-test, n=6. (h) The levels of TNF-α in the CSF were reduced in RIP1-knockdown group, whereas the effect was diametric in the RIP1 overexpression group. **P<0.0001 versus Sham group; NS, not significant difference (P=0.5570) versus ICH group; ^&&P=0.0002 versus Si-NC group; NS, not significant difference (P=0.9151) versus ICH group; ^##P<0.0001 versus Ad-GFP group; all were unpaired t-test, n=6. All data are expressed as means±S.E.M., and mean values for Sham group were normalized to 1.0. Ad-GFP, adenovirus with GFP; Ad- RIP1, adenovirus with RIP1; Si-NC, Si-negative control.