[Effect of autophagy on N-methy-D-aspartate receptor and hyperalgesia in a rat model of neuropathic pain].

Objective: To investigate the effects of autophagy on N-methy-D-aspartate (NMDA) receptor and its subunit NR2B and behavioral test in a rat model of neuropathic pain (NP).
Methods: Male Sprague-Dawley (SD) rats were divided into sham group, NP group, autophagy inhibitor 3-methyladenine (3-MA) pretreatment group (3-MA+NP group) and autophagy inducer rapamyein (Rap) group (Rap+NP group) by random number table with 22 rats in each group. NP animal model was reproduced by ligating sciatic nerve, while sciatic nerve of the rats in the sham group were only exposed but not ligated. The rats in two pretreatment groups were intraperitoneally challenged with 3-MA 15 mg/kg or Rap 10 mg/kg injection 1 hour before operation. Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured before and 1, 3, 7, 14 days after operation in each group. Spinal cord tissues were harvested at 1 day and 7 days after operation for autophagosome observation by electron microscope. The expressions of autophagy protein microtubule-associated protein 1 light chain 3-II (LC3-II), Beclin1, and NMDA, NR2B were determined by Western Blot. The positive expression of LC3 was detected by immunofluorescence.
Results: Compared with sham group, the MWT and TWL of rats in NP group were decreased gradually with the prolongation of operation time, the number of autophagosome, the expressions of LC3-II, Beclin1, NMDA, NR2B, and the positive expression of LC3 in spinal cord were significantly increased at 1 day after operation and till 7 days, which indicated that NP led to hyperpathia and autophagy activation. Compared with NP group, MWT was significantly further decreased, TWL was further shortened, the number of autophagosome was decreased, the expressions of LC3-II and Beclin1 in spinal cord were decreased, and NMDA and NR2B expressions were further increased after 3-MA pretreatment, with significant differences at 1 day after operation [MWT (g): 29.4±2.4 vs. 42.5±6.6, TWL (s): 7.2±1.0 vs. 8.8±1.1, LC3-II/β-actin: 0.38±0.03 vs. 0.52±0.07, Beclin1/β-actin: 0.29±0.06 vs. 0.59±0.05, NMDA/β-actin: 0.62±0.06 vs. 0.50±0.06, NR2B/β-actin: 0.57±0.03 vs. 0.46±0.03, all P < 0.05]. Immunofluorescence staining confirmed that the positive expression of LC3 was significantly decreased. Rap pretreatment could increase MWT, TWL and the number of autophagosome, increase LC3-II and Beclin1 expressions in spinal cord, and decrease NMDA and NR2B expressions in NP rats, and significant differences at 1 day after operation were found as compared with those in NP group [MWT (g): 49.4±4.4 vs. 42.5±6.6, TWL (s): 10.5±1.2 vs. 8.8±1.1, LC3-II/β-actin: 0.67±0.09 vs. 0.52±0.07, Beclin1/β-actin: 0.71±0.08 vs. 0.59±0.05, NMDA/β-actin: 0.40±0.05 vs. 0.50±0.06, NR2B/β-actin: 0.34±0.04 vs. 0.46±0.03, all P < 0.05], and immunofluorescence showed that the positive expression of LC3 was increased and lasted for 7 days. It indicated that Rap could increase the activity of autophagy, alleviate the occurrence of hyperalgesia, and reduce the expressions of NMDA receptor and its NR2B subunit.
Conclusions: NP could regulate the variety of NMDA/NR2B and hyperalgesia via increasing autophagy.