Translocator Protein (TSPO) Alleviates Neuropathic Pain by Activating Spinal Autophagy and Nuclear SIRT1/PGC-1α Signaling in a Rat L5 SNL Model

Abstract

Purpose: Recent studies showed promotion of astrocyte autophagy in the spinal cord would provide analgesic effects. Silent information regulator T1 (SIRT1) and α subunit of peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1α) are two master regulators of endogenous antioxidant defense and mitochondrial biogenesis. They play vital roles in both autophagy and neuropathic pain (NP). Our previous study showed that TSPO agonist Ro5-4864 elicited potent analgesic effects against NP, but the mechanisms remain unclear. This study aims to investigate the effects of TSPO agonist Ro5-4864 on autophagy and nuclear SIRT1/PGC-1α signaling in spinal dorsal horn.

Methods: A rat model of L5 spinal nerve ligation (SNL) was adopted. Rats were randomly assigned to the Sham group, the SNL group, the Ro (TSPO agonist Ro5-4864) group and the Ro+3-MA group. The behavior assessments were conducted at baseline, on Day 1, 3, 7 and 14 after SNL. The autophagy-related proteins (ATG7, Beclin1, LC3, and P62) in spinal dorsal horn were assayed and the nuclear SIRT1/PGC-1α and downstream factors were analyzed.

Results: Ro5-4864 alleviated the mechanical allodynia induced by SNL (P < 0.01 vs the SNL group), which could be totally abrogated by autophagy inhibitor 3-MA (P < 0.01 vs the Ro group). SNL induced elevated ATG7 (P < 0.01), Beclin1 (P < 0.01) and LC3-II/LC3-I (P < 0.01) contents and P62 accumulation (P < 0.01) on Day 7 and Day 14, which suggested an autophagy flux impairment. Ro5-4864 augmented ATG7 (P < 0.01), Beclin1 (P < 0.01) and LC3-II/LC3-I (P < 0.05) with decreased P62 (P < 0.01), which indicated a more fluent autophagic process. These effects were also totally abrogated by 3-MA (P < 0.01). Furthermore, Ro5-4864 activated the spinal nuclear SIRT1/PGC-1α signaling pathway.

Conclusion: TSPO improved both autophagy impairment and mitochondrial biogenesis, which may provide a new strategy for the treatment of NP.

Keywords: PGC-1α; SIRT1; TSPO; autophagy; neuropathic pain; silent information regulator T1; translocator protein; α subunit of peroxisome proliferator-activated receptor-γ coactivator-1.

Figure 1

The scheme of experiment protocol. A total of 120 rats were randomly assigned to four experimental groups (n = 30 in each group): (1) the sham group (Sham group); (2) the vehicle (control) group (SNL group); (3) the Ro group (2μg/10ul Ro5-4864, i.t. repeated for 3 days, on Day 3–5); (4) the Ro + 3-MA group (2μg/10ul Ro5-4864+4μg/10ul 3-MA, i.t. repeated for 3 days, on Day 3–5). Behavioral assessments were performed in all animals before surgery (day 0) and on Day 1, 3, 7 and 14 after SNL surgery or sham operation, separately. The ipsilateral L5 spinal cords were collected at baseline, Day 7, and Day 14 (n = 10 for each point of time in each group, among these 10 rats, 3 killed for immunofluorescence, 4 for Western blot, and 3 for electromicroscopic assessment).

Figure 2

Effects of TSPO agonist Ro5-4864 with or without autophagy inhibitor 3-MA on mechanical allodynia induced by SNL. Behavioral assessments of the four groups. **P < 0.01 compared with the Sham group at the same point in time. ^##P < 0.01 compared with the SNL (control) group at the same point in time. ^&&P < 0.01 compared with the Ro group at the same point in time. n = 10 in each group.

Figure 3

Single and Double staining of LC3 signals in spinal dorsal horn by immunofluorescence. (A) Representative immunofluorescent pictures of single staining of LC3 signals in spinal dorsal horn of the four groups on Day 7 and Day 14. (B) LC3 signals tensity in the spinal dorsal horn the four groups on Day 7 and Day 14. *P < 0.05 and **P < 0.01 compared with the Sham group at the same point in time. ^##P < 0.01 compared with the SNL (control) group at the same point in time. ^&P < 0.05 compared with the Ro group at the same point in time. (C) Representative immunofluorescent pictures of double staining of LC3 and TSPO signals in spinal dorsal horn of the four groups on Day 7 and Day 14.

Figure 4

Effects of TSPO agonist Ro5-4864 with or without autophagy inhibitor 3-MA on autophagic proteins. (A) Representative immunoblots for ATG7, P62, Beclin1, and LC3-I/LC3-II contents of the four groups on Day 7 and Day 14. (B–F) Illustration of the relative expression of ATG7/β-actin/Sham (B), P62/β-actin/Sham (C), Beclin1/β-actin/Sham (D), LC3-II /β-actin/Sham (E), and LC3-II/LC3-I/Sham (F) of the four groups. **P < 0.01 compared with the Sham group at the same point in time. ^#P < 0.05 and ^##P < 0.01 compared with the SNL (control) group at the same point in time. ^&&P < 0.01 compared with the Ro group at the same point in time. n = 4 for each point of time in each group.

Figure 5

Effects of Ro5-4864 on ultrastructure of spinal dorsal horn by TEM. (A) Representative electromicroscopic views of autophagosomes (red arrow), autolysosomes (yellow arrow) and lysosomes (blue arrow) in spinal dorsal horn of the four groups. Magnification, ×8,000 (B) Illustration of quantitative score of autophagosomes, autolysosomes and lysosomes on Day 14 of the four groups. *P < 0.05 and **P < 0.01 compared with the Sham group. ^##P < 0.01 compared with the SNL (control) group. ^&&P < 0.01 compared with the Ro group.

Figure 6

Effects of TSPO agonist Ro5-4864 on nuclear SIRT1/PGC-1α signaling. (A) Representative immunoblots for nuclear SIRT1, PGC-1α and Nrf2 contents of the Sham group, SNL group and Ro group on Day 7 and Day 14. (B–D) Illustration of the relative expression of nuclear SIRT1/lamin/Sham (B) PGC-1α/lamin/Sham (C) and Nrf2/lamin/Sham (D) of the Sham group, SNL group and Ro group. *P < 0.05 and **P < 0.01 compared with the Sham group at the same point in time. ^##P < 0.01 compared with the SNL (control) group at the same point in time. (E Representative immunoblots for cytoplasmic HO-1 contents of the Sham group, SNL group and Ro group on Day 7 and Day 14. (F) Illustration of the relative expression of cytoplasmic HO-1/β-actin/Sham. *P < 0.05 and **P < 0.01 compared with the Sham group at the same point in time. ^##P < 0.01 compared with the SNL (control) group at the same point in time. n = 4 for each point of time in each group.