Fig 2: RI-962 ameliorates TNFα-induced SIRS.C57BL/6 female mice (n = 14) were treated with vehicle, RI-962 (40 mg/kg), or GSK3145095 (40 mg/kg) via intraperitoneal injection for 15 min followed by the tail vein injection of mouse TNFα (300 μg/kg). At 6 h after TNFα injection, four mice in each group were killed at random, and the serums, hearts, livers, spleens, lungs, and kidneys were collected for analysis. a Survival rates of the mice treated with vehicle (n = 10), RI-962 (n = 10), and GSK3145095 (n = 10) after TNFα injection. ***p-value < 0.001 by Gehan-Breslow-Wilcoxon test. b Body temperature loss in the mice treated with vehicle (n = 14), RI-962 (n = 14), and GSK3145095 (n = 14) after TNFα injection. Data are presented as mean ± standard deviation, *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001 by two-tailed unpaired Student’s t-test. c, d The mice (n = 4) were killed at 6 h after TNFα administration, and the serum concentrations of IL-1β and IL-6 were measured using ELISA kits. Data are presented as mean ± standard deviation, **p-value < 0.01, ***p-value < 0.001 by two-tailed unpaired Student’s t-test. e The mice (n = 4) were killed at 6 h after TNFα administration, and the liver and kidney tissues were collected for analysis. Representative images of the histological analyses of the liver and kidney tissues by H&E staining (scale bar = 100 μm). Magnified views of the boxed regions for each image are shown below (scale bar = 20 μm). The black arrow represents portal area inflammatory cell infiltration, and the blue arrow represents glomerular neutrophil infiltration. f Liver proteins were tested by western blot to detect RIPK1, pRIPK1, RIPK3, pRIPK3, MLKL, and pMLKL with corresponding antibodies. β-actin was used as an internal control. Western blot represents three mice from each group. Molecular weight (Mw) markers are shown at right. Data were obtained from two independent experiments. Source data are provided as a Source Data file.

Fig 3: RIPA-56 alleviated nephropathy in db/db mice fed an HFD. a Flow chart of RIPA-56 treatment in db/db HFD animal model. RIPA-56 was used to pre-treat the wt/wt and db/db RD mice at 13 weeks old for 7 days. Then mice were fed an HFD mixed with RIPA-56 for 36 consecutive days. b Body weight, fasting blood glucose, BUN and ACR were compared in db-HFD mice treated with or without RIPA-56 at day 0 and after 14 days (n = 6). *p < 0.05, ***p < 0.001, and ****p < 0.0001 compared to db-HFD group (D14). c PAS staining of different groups of mice. Glomeruli and tubulointerstitium are displayed separately. Black arrows indicate tubular vacuolar degeneration. Scale bar, 50 μm. d Statistic analysis of mesangial matrix expansion and tubular vacuolar degeneration score in different groups of mice. *p < 0.05, **p < 0.01 compared to db-HFD group; ##p < 0.01 compared to wt-HFD group. e Representative Western blots showing protein expression of NGAL in db-HFD and db-HFD+R mouse kidneys. *p < 0.05 compared to db-HFD. f Immunohistochemistry staining of p-MLKL in mouse kidney specimens from indicated groups. Scale bar, 50 μm. g Representative Western blots showing protein expression of p-RIPK1/RIPK1, p-MLKL/MLKL in db-HFD and db-HFD+R mouse kidneys. h The relative protein levels of p-RIPK1/RIPK1, p-MLKL/MLKL to β-actin in different groups (n = 3). *p < 0.05 **p < 0.01 compared to db-HFD.

Fig 4: Representative RIP1 inhibitors.

Fig 5: Reduced protein level of RIP1 is observed in HDAC3 deficient macrophage. A–C Western blot analysis of RIP1 in Neuro-2a cells transfected with CTSB. D–G Western blot analysis of RIP1 in control and HDAC3 deificient RAW264.7 and BMDMs. H–M Western blot analysis of RIP1 in Vector and Hdac3−/− RAW264.7 stimulated with Leupeptin (25 μM), CA-074 (10 μM) or MG132 (10 μM) for 12 h. N–Q Western blot analysis of RIP1 in LysmCre and LysmCreHdac3f/f BMDMs stimulated with Leupeptin (100 μM) or CA-074 (40 μM) for 12 h. R, S Western blot analysis of RIP1 in Vector, Hdac1−/−, Hdac2−/−, Hdac3−/−, Hdac8−/− RAW264.7 cells. Data are representative of three independent experiments and showed as mean ± SEM. *P < 0.05 and **P < 0.01