Fig 1.
Gal3 expression in DRGs is increased after SNL.
(A) The mRNA level of gal3 is increased in DRGs of rats subjected to L5 SNL. Total RNA was extracted from the fifth lumbar dorsal horn sections and was subjected to real-time PCR to analyze the relative expression level of gal3 in each sample. Each sample was analyzed in triplicate. The 2-ΔΔCt method was used to quantify the relative levels of gal3. β-actin was used as reference for mRNA. n = 10. *p< 0.05 vs sham, # p< 0.05 vs SNL group. (B and C) Western blot analysis of gal3 protein expression in the fifth lumbar dorsal horn sections in each sample.*p< 0.05 vs sham, # p< 0.05 vs SNL group.
Fig 2.
Gal3expression in microglia is increased after SNL.
(A) Primary spinal microglial cells were isolated from sham-, SNL-, and MCP-treated rats at the indicated time, and relative gal3 mRNA levels were analyzed by real-time PCR according to the methods described above. n = 10. *p< 0.05 vs sham, # p< 0.05 vs SNL group. (B and C) Primary spinal microglial cells were isolated from sham-, SNL-, and MCP-treated rats at the indicated time, and relative gal3protein levels were analyzed usingwestern blot. n = 10. *p< 0.05 vs sham, # p< 0.05 vs SNL group. (D) Double immunofluorescence labelling for gal3 (green) and cell-type markers (Iba-1, microglia marker, red) in fifth lumbar dorsal horn sections 10 d after peripheral nerve injury. Scale bar = 50 μm.
Fig 3.
MCP inhibits SNL-induced activation of autophagy in spinal microglia.
(A and B) Primary spinal microglial cells were isolatedfrom sham-, SNL-, and MCP-treated rats at day 10, and the autophagosome formation was visualized by assaying LC3 green puncta. Punctate staining is indicative for the redistribution of LC3 to autophagosomes. The average number of LC3 green puncta per cell with standard deviation for each group is presented. *p< 0.05 vs sham, # p< 0.05 vs SNL group. (C and D) Primary spinal microglial cells were isolated from sham-, SNL-, and MCP-treated rats at the indicated time, and LC3B protein levels were assayed using western blot analysis. p< 0.05 vs sham, # p< 0.05 vs SNL group. (E and F) Primary spinal microglial cells were isolated from sham-, SNL-, and MCP-treated rats at day 10, and the protein levels of p62 were assayed using western blot analysis. p< 0.05 vs sham, # p< 0.05 vs SNL group. (G-I) Microglial cells were isolated from sham-, SNL-, and MCP-treated rats at day 10, and the expression level of gal3 and autophagy activation was assayed using double-label immunofluorescence analysis. Scale bar = 50 μm.
Fig 4.
MCP inhibits LPS-induced activation of autophagy in microglia.
(A and B) Mixed microglial cultures isolated from sham rats were treated with LPS (0.5ng/μl) and MCP (1 μg/μl), and the autophagosome formation was visualized by assaying LC3 green puncta. The average number of LC3 green puncta per cell with standard deviation for each group is presented. *p< 0.05 vs control, # p< 0.05 vs LPS group. Mixed microglial cultures isolated from sham rats were treated with LPS (0.5ng/μl) MCP (1 μg/μl) or Rapa (500 nM), and LC3B protein levels (C and D) or p62 protein levels (E and F) were assayed using western blot analysis. *p< 0.05 vs control, # p< 0.05 vs LPS group.
Fig 5.
MCP inhibits LPS-induced releases of proinflammatory cytokines by regulating autophagy in microglia.
Mixed microglial cultures isolated from sham rats were treated with LPS (0.5ng/μl), MCP (1 μg/μl) and Rapa (500 nM). ELISA analysis of TNF-α (A), IL-1β (B) and IL-6 (C) of protein levels were carried out using Elisa kit. Data are expressed as mean ± SD. *p< 0.05 vs control, # p< 0.05 vs MCP group.
Fig 6.
MCP results in a decreased mechanicaland cold hypersensitivity.
Mechanical (B) and cold (C) pain-related hypersensitivity developed after treatment with MCP (100 mg/kg/day) and Rapa (1 mg/kg/day) at the indicated time after surgery. n = 8. Data are expressed as mean ± SD. *p< 0.05 vs control, # p< 0.05 vs MCP group.