Korean J Pain.  2021 Oct;34(4):405-416. 10.3344/kjp.2021.34.4.405.

Sec-O-glucosylhamaudol mitigates inflammatory processes and autophagy via p38/JNK MAPK signaling in a rat neuropathic pain model

• Oh SH
• Kim SW
• Kim DJ
• Kim SH
• Lim KJ
• Lee K
• Jung KT

• KMID: 2520748
• DOI: http://doi.org/10.3344/kjp.2021.34.4.405

Abstract

Background
This study investigated the effect of intrathecal Sec-O-glucosylhamaudol (SOG) on the p38/c-Jun N-terminal kinase (JNK) signaling pathways, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-related inflammatory responses, and autophagy in a spinal nerve ligation (SNL)-induced neuropathic pain model.
Methods
The continuous administration of intrathecal SOG via an osmotic pump was performed on male Sprague–Dawley rats (n = 50) with SNL-induced neuropathic pain. Rats were randomized into four groups after the 7th day following SNL and treated for 2 weeks as follows (each n = 10): Group S, sham-operated; Group D, 70% dimethylsulfoxide; Group SOG96, SOG at 96 μg/day; and Group SOG192, SOG at 192 μg/day. The paw withdrawal threshold (PWT) test was performed to assess neuropathic pain. Western blotting of the spinal cord (L5) was performed to measure changes in the expression of signaling pathway components, cytokines, and autophagy. Additional studies with naloxone challenge (n = 10) and cells were carried out to evaluate the potential mechanisms underlying the effects of SOG.
Results
Continuous intrathecal SOG administration increased the PWT with p38/JNK mitogen-activated protein kinase (MAPK) pathway and NF-κB signaling pathway inhibition, which induced a reduction in proinflammatory cytokines with the concomitant downregulation of autophagy.
Conclusions
SOG alleviates mechanical allodynia, and its mechanism is thought to be related to the regulation of p38/JNK MAPK and NF-κB signaling pathways, associated with autophagy during neuroinflammatory processes after SNL.

Keyword

Analgesia; Autophagy; Biological Products; Cytokines; Hyperalgesia; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Neuralgia; NFkappa B; Pain

Figure

• 

  Fig. 1 Flow diagram illustrating the study protocol. Group S: sham-operated, Group D: control group treated with 70% dimethylsulfoxide (DMSO) after 7th day following spinal nerve ligation (SNL), Group SOG96: administered Sec-O-glucosylhamaudol (SOG) at 96 μg/day for 2 weeks using an osmotic pump after the 7th day following SNL, Group SOG192: administered SOG at 192 μg/day for 2 weeks using an osmotic pump after the 7th day following SNL, Group SOG192-N: administered SOG at 192 μg/day for 2 weeks using an osmotic pump after the 7th day following SNL and subjected to the naloxone challenge test.

• 

  Fig. 2 Paw withdrawal threshold (PWT) during observational periods. The PWT was observed on the 7th, 14th, and 21st day after spinal nerve ligation (SNL). On the 7th day after SNL, mechanical allodynia was confirmed in all rats based on a significant decrease in PWT compared to that in group S (P < 0.001). There were no significant differences among experimental groups including groups D, SOG96, and SOG192. The rats treated with continuous administration of Sec-O-glucosylhamaudol (SOG) intrathecally (group SOG96 and group SOG192) showed a higher PWT than those in the control group (group D) on the 14th and 21st day after SNL (P < 0.001). There were no significant differences in the PWT between groups SOG96 and SOG192 during the observational periods (P = 0.728 on 7th day; P = 0.089 on 21st day). Group S: sham-operated, Group D: control group treated with 70% dimethylsulfoxide after 7th day following SNL, Group SOG96: administered SOG at 96 μg/day for 2 weeks using an osmotic pump after the 7th day following SNL, Group SOG192: administered SOG at 192 μg/day for 2 weeks using an osmotic pump after the 7th day following SNL. *P < 0.05 compared with group D.

• 

  Fig. 3 Naloxone challenge test on the 14th and 21st day. The paw withdrawal threshold (PWT) after spinal nerve ligation (SNL) did not change after the intraperitoneal administration of naloxone (2 mg/kg) either at 30 or 60 minutes. No signs of opioid withdrawal were observed until 60 minutes. Group SOG192-N: administered Sec-O-glucosylhamaudol at 192 μg/day for 2 weeks using an osmotic pump after the 7th day following SNL and subjected to the naloxone challenge test.

• 

  Fig. 4 Expression of mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinase (JNK), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and proinflammatory cytokines in the ipsilateral dorsal horn of the L5 spinal cord after spinal nerve ligation (SNL) on the 21st day (A). The expression levels of p38 MAPK, phosphorylated JNK-I/II, and p65 NF-κB were increased after SNL and markedly decreased by treatment with Sec-O-glucosylhamaudol (SOG). The production of proinflammatory cytokines was increased after SNL. SOG markedly decreased the expression levels of IL-1 and TNF-α, but there was no significant decrease in the levels of IL-6. Autophagic activation in the ipsilateral dorsal horn of the L5 spinal cord on the 21st day after SNL is shown (B). Upregulation of autophagy by SNL was confirmed by the increased expression levels of microtubule-associated protein 1 light chain 3 (LC3)-I/II and Beclin 1 with a concomitant decrease in p62, and autophagy processes were downregulated after SOG treatment. Values were normalized to β-actin levels. The error bars indicate mean ± standard deviation. IL-1: interleukin-1, IL-6: interleukin-6, TNF-α: tumor necrosis factor-alpha, Group S: sham-operated, Group D: control group treated with 70% dimethylsulfoxide after the 7th day following SNL, Group SOG96: administered SOG at 96 μg/day for 2 weeks using an osmotic pump after the 7th day following SNL, Group SOG192: administered SOG at 192 μg/day for 2 weeks using an osmotic pump after the 7th day following SNL. aP < 0.05 vs. Group S. bP < 0.01 vs. Group S. cP < 0.05 vs. Group D. dP < 0.01 vs. Group D.

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  Fig. 5 Expression of mitogen-activated protein kinase (MAPK)/c-Jun N-terminal kinase (JNK), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and proinflammatory cytokines in capsaicin-exposed R-DRG-505 cells. Capsaicin treatment induced an increase in the expression levels of p38/JNK MAPK, p65 NF-κB, IL-1β, IL-6, and TNF-α (A) with the concomitant upregulation of autophagy (B), which were reversed by Sec-O-glucosylhamaudol (SOG) treatment. Pharmacological p38/JNK MAPK inhibitors resulted in the consequent inhibition of p65 with decreased expression of proinflammatory cytokines and the downregulation of autophagy. Values were normalized to β-actin levels. The error bars indicate mean ± standard deviation. SB203580: p38 inhibitor, SP600125: JNK inhibitor, IL-1: interleukin-1, IL-6: interleukin-6, TNF-α: tumor necrosis factor-alpha, LC3: microtubule-associated protein 1 light chain 3. aP < 0.05 vs. Capsaicin 0 μM. bP < 0.01 vs. Capsaicin 0 μM. cP < 0.05 vs. Capsaicin 400 μM. dP < 0.01 vs. Capsaicin 400 μM.

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