Fig 1: The expression and cellular localization of STING in mPFC of CIBP rats. (A) Timeline of the treatment schedule. (B) The paw withdrawal threshold (PWT) measured mechanical allodynia (Left). The use of limbs was measured by limb use score (Right), n = 4. (C) The tracks and analysis of elevated plus-maze experiment. Representative activity traces of sham (a) and CIBP (b) in the elevated plus-maze experiment are shown. Percentage of open arm retention time was analyzed (c), which was used to measure the degree of anxiety. (D) HE staining of tibial on tumor inoculated side. The asterisks are used to mark the areas of bone destruction at day 21 in the CIBP rats. (E) The expression of STING at days 0, 7, 14, and 21 was determined using Western blots. GAPDH was detected as an internal control. (F) Representative immunofluorescence images of IBA-1 (green), GFAP (green), NEUN (green), and STING (red) in CIBP group rats at day 14. The nuclear was labeled with DAPI (blue), and magnification is shown in the fourth panel. The result showed that STING is mainly located in the microglia of mPFC. Scale bar = 50 μm. Data are expressed as mean ± S.E.M. n = 4, * p < 0.05, ** p < 0.01 vs. sham group. Full pictures of the Western blots are presented in Supplementary Materials.
Fig 2: Immune inflammation exerts a crucial role in bone cancer pain at D17. (A) PPI network constructed by the 147 DEmRNA-coding protein. (B) The node’s color scale indicates connectivity with other nodes, and the edge’s thickness indicates the interaction’s combined score. The clusters were the top seven subnetworks calculated by MCODE. (C) A functional enrichment network is constructed by nodes representing terms. (D,E) Astrocyte and microglia activated in the mPFC of BCP rats. Representative immunofluorescence images showing the morphologies of the astrocyte labeled with GFAP (D), microglia labeled with Iba1 (E) in the mPFC. (F) The pro-inflammatory cytokines upregulated in the mPFC of BCP rats. Data are expressed as mean ± SD and statistically analyzed by Student’s t-test. n = 5 per group. **p < 0.01, vs. Sham groups.
Fig 3: Selective inhibition of AMPK/PPARγ signaling in activated microglia cells abolished the effect of rh-APN on inhibiting neuroinflammation and promoting hematoma resolution 72 hours after GMH (A) Representative images of immunofluorescence staining showing fluorescent dye-labeled liposomes (A, green) were swallowed almost entirely in microglia D, E (Iba1+, red) rather than in Neurons (B) (NeuN+, red) and Astrocytes (C) (GFAP+, red) at 72 h after GMH. (B) Representative images of Western blot data showing the expression of p-AMPK, PPARγ and CD36, as well as CD68 and CD206 either with rh-APN treatment alone, rh-APN + Lipo-Dorsomorphin or rh-APN + Lipo-GW9662. (C) No changes observed in the expression of AdipoR1 with Lipo-Dorsomorphin and Lipo-GW9662 intervention. (D) Western blot analysis of p-AMPK to AMPK ratio showed p-AMPK increased in the rh-APN treatment group and decreased in Lipo-Dorsomorphin group. (E, F, H) Western blot data showed (E) PPARγ, (F) CD36 and (H) CD206 expression increased with rh-APN treatment but decreased in Lipo-Dorsomorphin and Lipo-GW9662 groups. (G) Western blot data showed that rh-APN significantly decreased CD68 expression, whereas Lipo-Dorsomorphin and Lipo-GW9662 reversed the inhibitory effect of rh-APN. (All samples of GMH + rh-APN in Western blot were from the same animals which were euthanized after short-term neurobehavioral tests). Values are expressed as mean ± SD. ANOVA, Dunnett. n = 6 for each group. *P < 0.05 compared to sham, #P < 0.05 compared to GMH + Vehicle, @P < 0.05 compared to GMH + rh-APN or GMH + rh-APN + Lipo-PBS.
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