Fig 2: LCN2 shRNA-AAV treatment down-regulated the expression of proinflammatory cytokines following RI/R injury and exhibited a neuroprotective effect on the retinae. A: Schematic diagram of experimental workflow. B, C: ELISA showed the protein level of TNF and IL-1β in rat retinae following LCN2 shRNA-AAV treatment, compared to the RI/R group (n = 6). D: Immunofluorescence images showed the number of RBPMS-positive RGCs in rat retinae following LCN2 shRNA-AAV treatment, compared to the RI/R group. E: Bar graph depicted the number of RBPMS-positive RGCs per mm2 in each group (n = 6). F: TUNEL tests showed the apoptosis in rat retinae following following LCN2 shRNA-AAV treatment, compared to the RI/R group. G: Bar graph depicted the TUNEL positive cells in each group (n = 6). H: Visual function following RI/R with LCN2 shRNA-AAV treatment were measured by f-VEP test. I, J: Bar graphs depicted the amplitude of N2-P2 and the latency of P2 in each group (n = 6). Data in B, C, E, G, I, J were represented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s.: no significance, (compared with the control group/the RI/R group using Student t-test). Bar = 100 μm in Db-De, Bar = 50 μm in Fa-Fi. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer.

Fig 3: Superior effects of cHGF over FK506 in prolonging allograft survival and achieving functional recovery in the allo-OLT rat model. (Ai, ii) Survival curve (i) and the MST/days (ii) in allo-OLT rat model (black, n = 24) following oral treatment with FK506 (blue, 0.2 mg/kg/day, equal to the clinical dosage, n = 24) and comparison of the model rats to Syn rats (gray, n = 10). (Bi, ii) Survival curve of the rats that received cHGF (red, n = 24) compared with that of the allo-OLT (black, n = 24) and Syn rats (gray, n = 10) (i) and quantification of the MST/days (ii). (Ci–iii) Allo-OLT model rats received FK506 (blue, n = 24) or cHGF (red, n = 24) in an early time period (i) and later time period (ii), and the later time period of MST was analyzed (iii). (Di, ii) IHC staining (brown) in subgroups (i, boxes) of IL-2 in liver allografts of the FK506 (blue-striped bar), cHGF groups (red-striped bar), the allo-OLT (black) and Syn (gray), and quantitative analysis (ii), n = 6. (Ei–iii) The images of IHC staining (brown) for the proinflammatory cytokines IL-12, TNF-ɑ, IFN-γ (i, boxes), and the anti-inflammatory cytokine IL-4 (iii, boxes) in subgroups of liver allografts and comparative analysis (ii), with the analyzed data for the Syn (gray bars) and allo-OLT (black bars) groups (ii) from Supplementary Figure S3E —i, ii, boxes, n = 6). (F)i–iii) IHC staining for the functional hepatic proteins AST (i), ALB (ii), and ALP (iii) in liver allografts (boxes) of the different subgroups and quantitative analysis of those groups [Syn (gray); allo-OLT (black); cHGF (red latticed bar), and FK506 (blue latticed bar), n = 6). Staining for ALP and quantitative analysis showed that FK506 had a stronger effect on ALP levels (iii). The analysis also included the staining data for the Syn (gray bars) and allo-OLT (black bars) groups presented in Supplementary Figure S3F ; n = 6; the boxes represent one-sixth of the whole stained field. Original magnification, ×400; scale bar =200 μm. The data are representative of at least three independent experiments and presented as the means ± SDs. *p < 0.05 vs. FK506; #p < 0.05 vs. cHGF, and ***p < 0.0001 vs. the allo-OLT group (Student’s t-test, two-way ANOVA); ns represents no significance.

Fig 4: Effects of SOF, EDV, and their combination on IL-1β and TNF-α expression. The levels of either IL-1β (A) or TNF-a (C) in the kidneys of sham, UUO, UUO + SOF, UUO + EDV, and UUO + SOF + EDV groups were determined by ELISA. From the kidney tissues of sham, UUO, UUO + SOF, UUO + EDV, and UUO + SOF + EDV groups, total RNA was extracted. The mRNA transcription of TNF-α (B) was detected using real-time PCR analysis. The mean fold induction of TNF-a’s mRNA, which was normalized to GAPDH, is displayed. Data are expressed as mean ± standard error of the mean (n = 4). Multiple comparisons were conducted using a one-way analysis of variance followed by Tukey-Kramer as a post hoc test for parametric data and Kruskal-Wallis test for non-parametric data; a: versus sham control group; b: versus UUO group; c: versus UUO + SOF + EDV group at P-value < 0.05

Fig 5: Different concentrations of progesterone activated the PI3K/AKT signaling pathway and inhibited the inflammatory response in preeclampsia rats. In the Normal, Normal+10-8mol/L progesterone, Normal+10-6mol/L progesterone, Normal+10-4mol/L progesterone, preeclampsia, preeclampsia+10-8mol/L progesterone, preeclampsia+10-6mol/L progesterone, preeclampsia+10-4mol/L progesterone groups. (A) AKT, p-AKT, PI3K, and p-PI3K expression in placenta tissues. (B) The expression of pro-inflammatory factors TNF-α and IL-1β and anti-inflammatory factors IL-4, IL-10, and IL-13 in the placenta tissues. Pro: progesterone; PE: preeclampsia; IL: interleukin; TNF: tumor necrosis factor; p-PI3K: phosphorylation of phosphoinositide 3-kinase; PI3K: phosphorylation of phosphoinositide 3-kinase; AKT: protein kinase B; p-AKT: phosphorylation of protein kinase B. *P <0.05 vs Normal group; #P <0.05 vs preeclampsia group.