Fig 2: miR‐106b inhibits RIPK3 and phosphorylation of AKT through inhibiting JMJD3 to restrain mouse skin wound healing. A, The expression of miR‐106b, JMJD3 and RIPK3 in mice determined by RT‐qPCR. B, Skin wound‐healing area on days 1, 4, 7 and 14. C, Photographs of wound healing in mice on day 7 post‐injury. D, Epithelial tissue morphology and angiogenesis observed by H&E staining on day 7. E, Collagen content determined by Masson staining. F, Expression of JMJD3, RIPK3, phosphorylated AKT and AKT normalized to β‐actin measured by Western blot assay on day 7. G, Expression of collagen I and chemokine KC normalized to β‐actin in keratinocytes measured by Western blot assay on day 7. H, Expression of pro‐inflammatory proteins (IL‐1β and TNF‐α) and angiogenesis proteins (VEGF and TGF‐β1) normalized to β‐actin measured by Western blot assay on day 7. * P < .05 vs the PBS group; # P < .05 vs the EV‐miR‐NC + oe‐NC group; & P < .05 vs the EV‐miR‐106b + oe‐NC group; NS, no significant difference. Data were expressed as mean ± standard deviation, and comparison between two groups was performed by unpaired t test (n = 10). Wound‐healing area at different time‐points was analysed by repeated‐measures ANOVA with the Bonferroni post hoc test

Fig 3: Suppressive effects of Nec-1 on necroptosis in a mouse model of LPS-treated ALI. (A) Western blotting of lysates from mouse lung tissues was used to estimate the expression levels of necroptosis-associated proteins (RIP1 and RIP3). (B) Semi-quantification of the protein expression levels of RIP1 and RIP3. Representative images of at least three independent experiments are shown. (C and D) Estimation of the degree of apoptosis in primary lung cells, as determined by flow cytometry after staining with Annexin V and PI. (E) Ultrastructure of alveolar epithelial cells in the indicated mouse models (green arrow, lamellar body; red arrow, microvilli). Data are presented as the mean ± SEM from at least three independent experiments (n=5 mice/group). *P<0.05, as indicated. ALI, acute lung injury; LPS, lipopolysaccharide; Nec-1, necrostatin-1; PI, propidium iodide; RIP, receptor-interacting serine/threonine-protein kinase.

Fig 4: Putative mechanism of necroptosis and relative inflammatory responses during the development of CCI. In the CCI model, CCI stimulates TNF-α production and TNFR1 signaling, and activation of the downstream RIP1/RIP3-MLKL signaling pathway, which triggers necroptotic death and release of HMGB1 from the nucleus to the cytoplasm, further contributing to additional inflammatory factors. Nec-1 and Melatonin administration decreased necroptosis and extracellular HMGB1 levels, which decreased inflammation induced by RAGE expression and the detrimental effect of HMGB1 signaling in CCI. Thus, Nec-1 and melatonin inhibit the inflammatory responses and downstream NF-κB, NLRP3, IL-6 and IL-1β, contributing to neuroprotection. A20 also plays a role in this process and deficient expression of A20 could aggravate CCI-induced necroptosis. In addition, silencing A20 could impair the anti-necroptotic effect of Nec-1 and melatonin.

Fig 5: Histological assessment of the liver. Representative photos are shown from each group. Mice were treated with each reagent for 2 weeks and sacrificed at 10 weeks (10W) or 48 weeks (48W) of age. Cnt, control mice: KO, PTEN KO mice. D, DMSO: G, GSK872; F, ferrostatin-1. (a) Hematoxylin staining (H&E, bar 100 μm); H&E (M) indicates magnified photographs (bar 100 μm). Oil red O staining (bar 40 μm), PI staining (white arrowheads, bar 100 μm), and immunohistochemical staining of RIPK3 (black arrowheads, bar 20 μm) and TUNEL (black arrowheads, bar 100 μm) were performed. (b) NAFLD activity score. Few inflammatory foci and little hepatocyte ballooning were observed in the PTEN KO mice at 10 weeks of age. # KO10W-D vs. KO48W-D (each factor was statistically significant, p < 0.05). S, steatosis; I, inflammatory cell foci, B, ballooning of hepatocytes. (c) The number of PI-positive cells. * Cnt10W-D vs. KO10W-D (statistically significant, p < 0.05); # vs. KO10W-D (statistically significant, p < 0.05); n.s, not significant. (d) Immunofluorescent staining for albumin (Green), PI (Red), and Hoechst33342 (Blue). Bar 50 μm.