Galectin-3 Inhibition Is Associated with Neuropathic Pain Attenuation after Peripheral Nerve Injury

Abstract

Neuropathic pain remains a prevalent and persistent clinical problem because it is often poorly responsive to the currently used analgesics. It is very urgent to develop novel drugs to alleviate neuropathic pain. Galectin-3 (gal3) is a multifunctional protein belonging to the carbohydrate-ligand lectin family, which is expressed by different cells. Emerging studies showed that gal3 elicits a pro-inflammatory response by recruiting and activating lymphocytes, macrophages and microglia. In the study we investigated whether gal3 inhibition could suppress neuroinflammation and alleviate neuropathic pain following peripheral nerve injury. We found that L5 spinal nerve ligation (SNL) increases the expression of gal3 in dorsal root ganglions at the mRNA and protein level. Intrathecal administration of modified citrus pectin (MCP), a gal3 inhibitor, reduces gal3 expression in dorsal root ganglions. MCP treatment also inhibits SNL-induced gal3 expression in primary rat microglia. SNL results in an increased activation of autophagy that contributes to microglial activation and subsequent inflammatory response. Intrathecal administration of MCP significantly suppresses SNL-induced autophagy activation. MCP also inhibits lipopolysaccharide (LPS)-induced autophagy in cultured microglia in vitro. MCP further decreases LPS-induced expression of proinflammatory mediators including IL-1β, TNF-α and IL-6 by regulating autophagy. Intrathecal administration of MCP results in adecreased mechanical and cold hypersensitivity following SNL. These results demonstrated that gal3 inhibition is associated with the suppression of SNL-induced inflammatory process andneurophathic pain attenuation.

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.