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. 2021 Sep 8:9:714996.
doi: 10.3389/fcell.2021.714996. eCollection 2021.

Dexmedetomidine Relieves Neuropathic Pain in Rats With Chronic Constriction Injury via the Keap1-Nrf2 Pathway

Yatao Liu  1 , Wei Liu  2 , Xiao-Qing Wang  1 , Zhan-Hai Wan  1 , Yong-Qiang Liu  1 , Meng-Jie Zhang  1
Affiliations

Dexmedetomidine Relieves Neuropathic Pain in Rats With Chronic Constriction Injury via the Keap1-Nrf2 Pathway

Yatao Liu et al. Front Cell Dev Biol. .

Abstract

This study aimed to determine the role of dexmedetomidine (Dex) in neuropathic pain (NP) after chronic constriction injury (CCI) in a rat model as well as its underlying mechanism. First, a CCI rat model was established. After treatment with Dex, the severity of NP was ascertained by monitoring paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) at different time points. Immunohistochemical analysis was performed to determine the levels of Keap1 and Nrf2 in the spinal cord. Furthermore, the levels of Keap1-Nrf2-HO-1 pathway molecules, apoptotic proteins, and antioxidant genes in the spinal cord or isolated primary microglia were determined using quantitative polymerase chain reaction and western blotting. The release of proinflammatory cytokines was detected via enzyme-linked immunosorbent assay. To evaluate Dex-treated CCI-induced NP via the Keap1-Nrf2-HO-1 pathway, the rats were intrathecally injected with lentivirus to upregulate or downregulate the expression of Keap1. We found that Dex inhibited pathological changes and alleviated sciatic nerve pain as well as repressed inflammation, apoptosis, and redox disorders of the spinal cord in CCI rats. Keap1 protein expression was substantially downregulated, whereas Nrf2 and HO-1 expressions were significantly upregulated in the spinal cord after Dex administration. Additionally, Keap1 overexpression counteracted Dex-mediated inhibition of NP. Keap1 overexpression led to a decrease in Nrf2 and HO-1 levels as well as PWT and PWL but led to an aggravation of inflammation and antioxidant disorders and increased apoptosis. Keap1 silencing alleviated NP in rats with CCI, as evidenced by an increase in PWT and PWL. Keap1 depletion resulted in the alleviation of inflammation and spinal cord tissue injury in CCI rats. Collectively, these findings suggest that Dex inhibits the Keap1-Nrf2-HO-1-related antioxidant response, inflammation, and apoptosis, thereby alleviating NP in CCI rats.

Keywords: apoptosis; chronic constriction injury; inflammation; neuropathic pain; pain behavior; redox equilibrium.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Dexmedetomidine (Dex) mitigated chronic constriction injury (CCI)-induced neuropathic pain. (A) Paw withdrawal threshold (PWT) (g) at 0, 1, 3, 7, and 14 days after operation. (B) Paw withdrawal latency (PWL) (s) at 0, 1, 3, 7, and 14 days after operation. (C) IL-1β, IL-6, and TNF-α expression levels were determined using enzyme-linked immunosorbent assay. (D) mRNA levels of caspase-9, Bax, and Bak in spinal cord tissues were determined using quantitative polymerase chain reaction. (E) Protein levels of caspase-9, Bax, and Bak in spinal cord tissues were determined using western blotting. **P < 0.01, ***P < 0.001 vs. Sham group; ##P < 0.01 vs. CCI group.
FIGURE 2
FIGURE 2
Dexmedetomidine promoted antioxidant capacity in CCI-induced rats. (A) Expression levels of antioxidant genes were determined using quantitative polymerase chain reaction. (B) Analysis of glutathione (GSH) levels. (C) A superoxide dismutase (SOD) assay kit was used to determine SOD activity. (D) Determination of catalase (CAT) activity in spinal cord tissues. *P < 0.05, **P < 0.01 vs. Sham group; #P < 0.05 vs. CCI group.
FIGURE 3
FIGURE 3
Role of Dex in Keap1–Nrf2 pathway expression and activation in CCI-induced rats. (A,B) mRNA expression levels of Keap1 and Nrf2 were measured using quantitative polymerase chain reaction (qPCR). (C) mRNA expression levels of HO-1 were measured using qPCR. (D) Isolated microglia were transfected with ARE luciferase vector, followed by determination of luciferase activity in the cells at 36 h after transfection. (E) Protein expression levels of Nrf2, Keap1, and HO-1 were measured using western blotting. (F) Immunohistochemical staining of Keap1 and Nrf2 expression in spinal cord tissues. Scale bar, 50 μm. *P < 0.05, **P < 0.01 vs. Sham group; #P < 0.05, ##P < 0.01 vs. CCI group.
FIGURE 4
FIGURE 4
Overexpression of Keap1 in Dex-treated CCI-induced rats intrathecally injected with lentiviral-NC or lentiviral-Keap1 for 1 week. Rats were then subjected to Dex treatment and CCI surgery. (A,B) mRNA expression levels of Keap1 and Nrf2 was measured using qPCR. (C) mRNA expression level of HO-1 in the spinal cord was detected using qPCR. (D) Isolated microglia were transfected with ARE luciferase vectors, and luciferase activity was determined in the cells at 36 h after transfection. (E) Protein expression levels of Nrf2, Keap1, and HO-1 were measured using WB. (F) Immunohistochemical staining of Keap1 and Nrf2 expression in spinal cord tissues. Scale bar, 50 μm. *P < 0.05, **P < 0.01 vs. CCI group; #P < 0.05, ##P < 0.01 vs. CCI+Dex group.
FIGURE 5
FIGURE 5
Effect of Keap1 overexpression on Dex-alleviated neuropathic pain in CCI-induced rats intrathecally injected with lentiviral-NC/lentiviral-Keap1 for 1 week. Rats were then subjected to Dex treatment and CCI surgery. (A) Paw withdrawal threshold (g) at 0, 1, 3, 7, and 14 days after operation. (B) Paw withdrawal latency (s) at 0, 1, 3, 7, and 14 days after operation. (C) IL-1β, IL-6, and TNF-α expression levels were measured in spinal cord tissues using ELISA. (D) mRNA expression levels of caspase-9, Bax, and Bak were determined in spinal cord tissues using quantitative polymerase chain reaction. (E) Protein expression levels of caspase-9, Bax, and Bak in spinal cord tissues were determined using western blotting. *P < 0.05, **P < 0.01 vs. CCI group; #P < 0.05, ##P < 0.01 vs. CCI+Dex group.
FIGURE 6
FIGURE 6
Effect of Keap1 overexpression on Dex-alleviated antioxidant capacity in spinal cord tissues of rats. Rats were intrathecally injected with lentiviral-NC or lentiviral-Keap1 for 1 week and then subjected to Dex treatment and CCI surgery. (A) Levels of antioxidant genes in the spinal cord were determined using quantitative polymerase chain reaction. (B) Analysis of glutathione levels in spinal cord tissue. (C) Superoxide dismutase activity in spinal cord tissues was examined using a SOD assay kit. (D) Analysis of catalase activity. *P < 0.05, **P < 0.01 vs. CCI group; #P < 0.05, ##P < 0.01 vs. CCI+Dex group.
FIGURE 7
FIGURE 7
Keap1 knockdown in CCI-induced rats intrathecally injected with lentiviral-shNC or lentiviral-shKeap1 for 1 week. Rats were then subjected to CCI surgery. (A,B) Expression levels of Keap1 and Nrf2 in the spinal cord were measured using qPCR. (C) mRNA expression level of HO-1 in the spinal cord was assayed using qPCR. (D) Isolated microglia were transfected with ARE luciferase vector, and luciferase activity was measured at 36 h after transfection. (E) Protein expression levels of Keap1, Nrf2, and HO-1 were determined using western blotting. (F) Immunohistochemical staining for Keap1 and Nrf2 expression. Scale bar, 50 μm. **P < 0.01 vs. Sham group; ##P < 0.01 vs. CCI+shNC group.
FIGURE 8
FIGURE 8
Effect of Keap1 overexpression on Dex-alleviated neuropathic pain in CCI-induced rats intrathecally injected with lentiviral-shNC/lentiviral-shKeap1 for 1 week. Rats were then subjected to CCI surgery. (A) Paw withdrawal threshold (g) at 0, 1, 3, 7, and 14 days after operation. (B) Paw withdrawal latency (s) at 0, 1, 3, 7, and 14 days after operation. (C) Expression levels of IL-1β, IL-6, and TNF-α was identified in the spinal cord using enzyme-linked immunosorbent assay. (D) mRNA expression levels of apoptotic factors caspase-9, Bax, and Bak in spinal cord tissues were determined using quantitative polymerase chain reaction. (E) Protein expression levels of caspase-9, Bax, and Bak in spinal cord tissues were determined using western blotting. **P < 0.01, ***P < 0.001 vs. Sham group; ##P < 0.01 vs. CCI+shNC group.
FIGURE 9
FIGURE 9
Effect of Keap1 silencing on CCI-repressed antioxidant capacity in spinal cord tissues of rats. Rats were intrathecally injected with lentiviral-shNC or lentiviral-shKeap1 for 1 week and then subjected to CCI surgery. (A) Levels of antioxidant genes in the spinal cord were determined via quantitative polymerase chain reaction. (B) Analysis of glutathione levels. (C) A superoxide dismutase assay kit was used to examine SOD activity. (D) Determination of catalase activity. *P < 0.05, **P < 0.01 vs. Sham group; #P < 0.05, ##P < 0.01 vs. CCI+shNC group.
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