Fig 1: Autophagy flux is impaired during the maintenance of neuropathic pain. (A) The thresholds of mechanical allodynia (left) and thermal hyperalgesia (right) after spinal nerve ligation (SNL) were performed before catheter implantation (baseline value, BL), and on days 3, 7, 14 and 28 postsurgery. Each test was repeated 3 times. Data are presented as the mean ± SD. **p < 0.01 vs sham; n = 6 mice/group. (B) Reactive oxygen species (ROS) levels in the dorsal horn (DH) of the spinal cords reflected by 8-hydroxydeoxyguanosine (8-OHdG) immunofluorescence intensity at different time points after SNL. Bar: 50 μm. (C) Statistical results of intensity of 8-OHdG staining in Figure 1B. Data are presented as the mean ± SD. **p < 0.01 vs sham; n = 3 mice/group, 6 slices per mouse were employed. (D) Pro-inflammatory factor levels of IL1B, TNF, CCL7, and MMP2 in spinal cords from a sham 14 d group and SNL 14 d group. **P < 0.01 vs sham; n = 4 mice/group. (E) Double immunofluorescence staining of 8-OHdG (green) with neuron marker RBFOX3/NeuN (red, upper) or astrocyte marker glial fibrillary acidic protein (GFAP, red, down) in the dorsal horn of the spinal cords, SNL 14 d (SNL postoperative 14 d), respectively, n = 4 mice/group, 6 slices per mice were employed. Bar: 50 μm. (F) Western blots of autophagy proteins, including LC3, ATG5, and SQSTM1. ACTB was used as loading control. Data are presented as the mean ± SD (N = 3). *p < 0.05, **p < 0.01 vs Sham; ns: no significance. n = 3 mice/group. (G) The mRNA level of Sqstm1 in spinal cords on day 7, 14 and 28 after SNL. Data are presented as the mean ± SD. ns: no significance; n = 4 mice/group. (H) Representative immunofluorescence images of spinal dorsal cord staining LC3 or SQSTM1 from sham 14 d and SNL 14 d groups. n = 3 mice/group, 6 slices per mouse were employed. Data are presented as the mean ± SD, **p < 0.01 vs Sham
Fig 2: Impaired autophagy increases the levels of neuroinflammatory factors by activating the TRAF6-MAPK8-NFKB signaling pathways. (A) Western blot analysis of p-MAPK8, p-MAPK14, p-MAPK1, and p-RELA in primary astrocytes after treatment with autophagy inhibitor Atg7 siRNA and astrocyte activators IL1B or TNF. (B-E) The statistical results of p-MAPK8 (B), p-RELA (C), p-MAPK14 (D) and p-MAPK1 (E) in Figure 5A. Data are presented as the mean ± SD (N = 3). *p < 0.05, **p < 0.01; ns: no significance. (F,G) Effects of inhibitors (in-) of MAPK8 (SP600125), MAPK14 (SB203580), MAPK1 (U0126), and RELA (JSH-23) on CCL7 (F) and MMP2 (G) release from autophagy impaired astrocytes. Data are presented as the mean ± SD (N = 3). **p < 0.01; ns: no significance. (H) Immunoprecipitation results of K63 ubiquitin proteins and TRAF6 in cultured primary astrocytes after treatment with or without autophagy inhibitor Atg7 siRNA and astrocyte activators IL1B or TNF. (I) Effect of TRAF6 inhibition on levels of p-MAPK8 and p-RELA in autophagy impaired astrocytes after treatment with astrocyte activators IL1B or TNF. (J) The statistical results of ATRAF6, p-MAPK8, and p-RELA in Figure 5I. Data are presented as the mean ± SD (N = 3). **p < 0.01 compared with control (Ctrl) siRNA group. ##p < 0.01 compared with the Atg7 siRNA treated group. (K) Immunoprecipitation results of K63 ubiquitin proteins and TRAF6 in spinal cords after intrathecal injection of autophagy inhibitor 3-MA or activator rapamycin (Rapa) during the maintenance phase of neuropathic pain. (L) Western blots and statistical results of TRAF6 in spinal cord after intrathecal injection of autophagy inhibitor 3-MA or activator rapamycin (Rapa). Data are presented as the mean ± SD (N = 3). n = 4 mice/group. *p < 0.05, **p < 0.01 compared with DMSO group
Fig 3: The proposed basic mechanism of the effect of edoxaban (EDX) on tumor growth and apoptosis mediated by factor Xa-PAR2. ( A ) In the absence of EDX, tissue factor (TF) expressed in inoculated Colon26 cells activates blood coagulation and inflammation in host mice, and generated factor Xa activates PAR2, whereas generated thrombin possibly activates PAR1/PAR4 on Colon26 cells and various stroma cells in the tumor microenvironment. PAR2 activation stimulates TF expression, STAT3-mediated IL-6, PAI-1, and MMP-2 production in peri-cancer cells, and RAS/PI3K/SyK/Ki67/cyclin D1 signaling pathways in tumor cells ( black upward arrows ). These affect tumor growth. In addition, thrombin-mediated PAR1 and PAR4 pathways induce p53 expression, leading to apoptosis, which may be regulated by the PAR2-dependent system. ( B ) EDX administration to mice suppresses factor Xa-mediated PAR2 activation, reducing PAR2 levels, followed by a decrease in plasma TF, IL-6, PAI-1, and MMP-2 levels and RAS/PI3K/SyK/Ki67/cyclin D1 signaling pathways ( white downward arrows ), leading to tumor growth suppression. Furthermore, suppression of PAR2 pathway activation by EDX may reduce the regulatory system against the PAR1/PAR4 pathway and may increase the p53-dependent apoptosis ( black upward arrows ).
Fig 4: Impact of maternal diets deficient in folic acid (FADD) or choline (ChDD) and the control diet (CD) during pregnancy and lactation on neuronal levels of matrix metalloprotease (MMP)-2 in ischemic brain tissue of female and male offspring one month after ischemic stroke. Representative images of MMP-2 immunofluorescence colocalization with neuronal nuclei (NeuN) and 4′,6-diamidino-2-phenylindole (DAPI) (A) and semi-quantitative quantification of MMP-2 levels within ischemic region of brain tissue (B). Mean ± SEM of 3 to 4 animals per group. * p < 0.05, Tukey’s pairwise post hoc analysis between male CD and ChDD offspring (one-way ANOVA analysis between males and significant maternal diet main effect). Magnification 200X, scale bar 50 µm.
Fig 5: MN/SC-mediated EV delivery and improvement of the dermis microenvironment in vivo. a Timeline of the in vivo experiment in aged C57BL/6 mice with MN@EV/SC. b Histological analysis of skin tissues treated with different patches via H&E staining (upper), and MT staining (lower). Scale bar, 500 µm. Black arrows represent epidermis or dermis (n = 5). c Quantification of dermal thickness from skin sections. d MMP-2 concentration in mice skin homogenates. e Quantification of collagen-occupied area in the dermis. f Collagen volume fraction of skin sections. g Representative immunofluorescent staining images of skin tissues for ER-TR7, p16, p21, and DHE. DAPI (blue) were used for visualizing cell nuclei. Scale bar, 100 µm. Data are expressed as the mean ± SEM. N.S. = not significant, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001
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