Oleanolic acid administration alleviates neuropathic pain after a peripheral nerve injury by regulating microglia polarization-mediated neuroinflammation

Abstract

Neuropathic pain caused by a peripheral nerve injury constitutes a great challenge in clinical treatments due to the unsatisfactory efficacy of the current strategy. Microglial activation-mediated neuroinflammation is a major characteristic of neuropathic pain. Oleanolic acid is a natural triterpenoid in food and medical plants, and fulfills pleiotropic functions in inflammatory diseases. Nevertheless, its role in neuropathic pain remains poorly elucidated. In the current study, oleanolic acid dose-dependently suppressed LPS-evoked IBA-1 expression (a microglial marker) without cytotoxicity to microglia, suggesting the inhibitory efficacy of oleanolic acid in microglial activation. Moreover, oleanolic acid incubation offset LPS-induced increases in the iNOS transcript and NO releases from microglia, concomitant with the decreases in pro-inflammatory cytokine transcripts and production including IL-6, IL-1β, and TNF-α. Simultaneously, oleanolic acid shifted the microglial polarization from the M1 phenotype to the M2 phenotype upon LPS conditions by suppressing LPS-induced M1 marker CD16, CD86 transcripts, and enhancing the M2 marker Arg-1 mRNA and anti-inflammatory IL-10 levels. In addition, the LPS-induced activation of TLR4-NF-κB signaling was suppressed in the microglia after the oleanolic acid treatment. Restoring this signaling by the TLR4 plasmid transfection overturned the suppressive effects of oleanolic acid on microglial polarization-evoked inflammation. In vivo, oleanolic acid injection alleviated allodynia and hyperalgesia in SNL-induced neuropathic pain mice. Concomitantly, oleanolic acid facilitated microglial polarization to M2, accompanied by inhibition in inflammatory cytokine levels and activation of TLR4-NF-κB signaling. Collectively, these findings confirm that oleanolic acid may ameliorate neuropathic pain by promoting microglial polarization from pro-inflammatory M1 to anti-inflammatory M2 phenotype via the TLR4-NF-κB pathway, thereby indicating its usefulness as therapeutic intervention in neuropathic pain.

Fig. 1. Effects of oleanolic acid on microglial activation upon LPS exposure. (A) Biochemical structure and spatial conformation of oleanolic acid. (B) BV2 microglia cells were treated with oleanolic acid (OA) ranging from 1 μM to 80 μM for 24 h. Cell viability was then evaluated by MTT assay. (C) Cells were administrated with various doses of oleanolic acid, prior to LPS exposure for 12 h. Then, the mRNA levels of IBA-1 were determined by qRT-PCR. (D) After stimulation with oleanolic acid (40 μM) and/or LPS for 12 h, the corresponding effects on the protein expression of IBA-1 were detected. *P < 0.05 vs. control group, ^#P < 0.05 vs. LPS group.

Fig. 2. Oleanolic acid suppressed microglial inflammatory response under LPS conditions. (A) After treatment with various doses of oleanolic acid, cells were exposed to LPS. Then, the mRNA levels of iNOS were analyzed. (B) The contents of NO production were detected. (C) The subsequent effects on the mRNA levels of IL-6 were determined. (D and E) Cells treated with 40 μM of oleanolic acid were stimulated with LPS for 12 h. Then, qRT-PCR assay was performed to detect the mRNA levels of IL-1β (D) and TNF-α (E). (F–H) ELISA analysis was conducted to measure the concentrations of IL-6 (F), IL-1β (G), and TNF-α (H) in supernatants from microglia under oleanolic acid and/or LPS conditions. *P < 0.05 vs. control group, ^#P < 0.05 vs. LPS group.

Fig. 3. Treatment with oleanolic acid affected microglial polarization. (A and B) After pretreatment with oleanolic acid for 10 h, cells were exposed to LPS. Then, the mRNA levels of M1 phenotype marker CD16 (A) and CD86 (B) were determined by qRT-PCR. (C and D) The subsequent effects on the M2 phenotype marker Arg-1 transcript (C) and IL-10 concentration (D) were assessed. *P < 0.05, ^#P < 0.05.

Fig. 4. Activation of the TLR4-NF-κB pathway was involved in oleanolic acid-mediated microglial inflammation. (A and B) The protein expression levels of TLR4 and p-p65 NF-κB were analyzed in microglia exposed to oleanolic acid and LPS. (C and D) Cells were transfected with recombinant TLR4 vectors and the subsequent protein levels of TLR4 and p-p65 NF-κB were determined. (E–G) The mRNA levels of CD16 (E), CD86 (F), and Arg-1 (G) were analyzed by qRT-PCR. (H–J) ELISA assay was carried out to evaluate the releases of IL-6 (H), IL-1β (I), and TNF-α (J) in the supernatants. *P < 0.05 vs. control group, ^#P < 0.05 vs. LPS group, ^$P < 0.05 vs. LPS + OA group.

Fig. 5. Injection with oleanolic acid ameliorated allodynia and hyperalgesia in SNL-mimicked neuropathic pain models. (A) Mice were administrated with oleanolic acid (OA; 2, 5, and 10 mg kg⁻¹) after SNL for 5 consecutive days. The mechanical allodynia on 1, 3, 5, and 7 d post-SNL was evaluated by Von Frey test. (B) The heat hyperalgesia was also performed to assess the pain behavior. *P < 0.05 vs. control group, ^#P < 0.05 vs. SNL group.

Fig. 6. Oleanolic acid administration alleviated neuroinflammation and microglial polarization in mice. (A) Mice were injected with the indicated doses of oleanolic acid starting from 1 h after SNL surgery. The protein levels of microglial marker IBA-1 were analyzed in spinal cord tissues of mice at 7 d post-SNL. (B and C) The mRNA levels of CD86 (B) and Arg-1 (C) were detected by qRT-PCR. (D–I) The subsequent effects on IL-6 (D), IL-1β (E), TNF-α (F), and IL-10 (G) concentrations, TLR4 pathway activation (H and I) were also measured. *P < 0.05 vs. control group, ^#P < 0.05 vs. SNL group.