Dexmedetomidine and Ketamine Attenuated Neuropathic Pain Related Behaviors via STING Pathway to Induce ER-Phagy

Abstract

Our previous work indicated that ER-phagy level had altered in spinal nerve ligation (SNL) rats. In this study, we investigated whether dexmedetomidine or ketamine exhibits anti-anxiety or anti-nociceptive effects via modulation of the spinal STING/TBK pathway to alter ER-phagy in SNL rats. We evaluated the analgesic and anti-anxiety effects of ketamine and dexmedetomidine in SNL rats. 2'3'-cGAMP (a STING pathway agonist) was administrated to investigate whether enhanced spinal STING pathway activation could inhibit dexmedetomidine or ketamine treatment effects in SNL rats. Analgesic effects were assessed with the mechanical withdrawal threshold (MWT) and anti-anxiety effects were measured via an open field test (OFT). Protein expression levels were evaluated by immunoblotting. Distribution and cellular localization of Grp78 (ER stress marker) were evaluated by confocal immunofluorescence. SNL induced mechanical hypersensitivity and anxiety in rats; dexmedetomidine and ketamine both provided analgesia and anti-anxiety effects in SNL rats. Furthermore, the STING pathway was involved in the modulation of ER stress and ER-phagy in SNL rats and dexmedetomidine and ketamine alleviated ER stress by inhibiting STING pathway to enhance ER-phagy. Thus, both ketamine and dexmedetomidine provided anti-anxiety and anti-nociceptive effects by alleviating ER stress through the inhibition of the STING/TBK pathway to modulate spinal ER-phagy in SNL rats.

Keywords: STING pathway; autophagy; dexmedetomidine; endoplasmic reticulum stress; ketamine; neuropathic pain.

Figure 1

Timeline of the experimental procedure. Abbreviations: D, dexmedetomidine administration; K, ketamine administration; G, 2’3’-cGAMP administration; O, spinal nerve ligation operation; TC, tissue collection; M, mechanical withdrawal threshold; OF, open field test.

Figure 2

Spinal nerve ligation activated the STING/TBK pathway. (A)Mechanical withdrawal threshold in sham vs. SNL groups (two-wayANOVA, P < 0.001). (B) Trace map and heat map ofthe open field test in sham vs. SNL groups. (C)Quantification of the open field test (one-way ANOVA, total distance:F = 16.781, P = 0.002; proportion of time spent in the center: F = 12.512, P = 0.005). (D) Western blotting in sham vs. SNL groups. (E) Quantification of immunoblotting in sham vs. SNL groups (one-way ANOVA, p-STING/STING: F = 16.75, P = 0.002; p-TBK/TBK: F = 10.786, P = 0.008; FAM134b/GAPDH: F = 19.265, P = 0.001; LC3/GAPDH: F = 10.756, P = 0.008; p62/GAPDH: F = 8.957, P = 0.014; Grp78/GAPDH: F = 10.897, P = 0.008). N = 6 rats per group, *P < 0.05 compared with the sham group; **P < 0.01 compared with sham group; ***P < 0.001 compared with sham group. Abbreviations: SNL, spinal nerve ligation; sham, sham operation of spinal nerve ligation; BL, baseline.

Figure 3

Co-localization of Grp78 with NeuN/GFAP/Iba in the ipsilateral spinal dorsal horn of SNL rats. The laminae I-III was illustrated by the blue zone in the ipsilateralL5 atlas. (A) Confocal immunofluorescence of Grp78 and NeuN (neuron marker) in laminae I-III of SNL rats at L5 level. (B) Confocal immunofluorescence of Grp78 and GFAP (astrocyte marker) in laminae I-III of SNL rats at L5 level. (C) Confocal immunofluorescence of Grp78 and Iba (microglia marker) in laminae I-III of SNL rats at L5 level. N = 6 rats per group. Scale bar = 50 μm.

Figure 4

Effectson pain behavior test following dexmedetomidine or ketamine administration. (A) MWT of sham and sham+D groups, sham: sham of SNL groups; sham+D: sham+dexmedetomidine administration group. (B) MWT of sham and sham-K groups, sham: sham of SNL groups; sham-K: sham+ketamine administration group. There were no significant changes in sham+D/sham+K compared to the sham group. (C) MWT of the sham and sham+cG group. There were no significant changes between the sham and sham+cG group (N = 4, two-way ANOVA, P > 0.05).

Figure 5

Dexmedetomidineprovided anti-anxiety effects by inhibiting the STING/TBKpathway. (A) MWT tests of control, SNL, SNL+D, and SNL+D+G groups (two-way ANOVA). (B) Quantification of open field of control, SNL, SNL+D, and SNL+D+G groups (one-way ANOVA, total distance: F = 13.260, P = 0.003; proportion of time spent in the center: F = 16.520, P = 0.006). (C) Trace map and heat map of control, SNL, SNL+D, and SNL+D+G groups. (D) Western blot of control, SNL, SNL+D, and SNL+D+G groups. (E) Western blot quantification of control, SNL, SNL+D, and SNL+D+G groups (one-way ANOVA, p-STING/GAPDH: F = 6.092, P = 0.006; p-TBK/GAPDH: F = 3.709, P = 0.034; FAM134b/GAPDH: F = 13.979, P = 0.007; LC3/GAPDH: F = 9.265, P = 0.001; p62/GAPDH: F = 4.268, P = 0.022; Grp78/GAPDH: F = 8.613, P = 0.001). N = 6 rats per group, *P < 0.05 compared with control; **P < 0.01 compared with control group;***P < 0.001 compared with control group;^#P < 0.05 compared with SNL;^##P < 0.01 compared with SNL group;^ΔP < 0.05 compared with SNL+D;^ΔΔP < 0.01 compared with SNL+D. Abbreviations: BL, baseline; Dex, dexmedetomidine; control group, rats received a sham operation and vehicle; SNL group, rats received the SNL procedure and vehicle; SNL+D group, rats received SNL, dexmedetomidine, and vehicle; SNL+D+G group, rats received SNL, dexmedetomidine, and 2’3’-cGAMP.

Figure 6

Ketamineprovided anti-anxiety effects by inhibiting the STING/TBKpathway. (A) MWT tests of control, SNL, SNL+K, and SNL+K+Ggroups (two-way ANOVA). (B) Quantification of open field ofcontrol, SNL, SNL + K, and SNL + K + G groups (one-way ANOVA, totaldistance: F = 16.034, P = 0.002; proportion of time spent in the center: F = 11.637, P = 0.005). (C) Trace map and heat map of control, SNL, SNL + K, and SNL + K + G groups. (D) Western blot of control, SNL, SNL + K, and SNL + K + G groups. (E) Western blot quantification of control, SNL, SNL + K, and SNL + K + G groups (one-way ANOVA, p-STING/GAPDH: F = 10.419, P = 0.001; p-TBK/GAPDH: F = 12.993, P = 0.003; FAM134b/GAPDH: F = 16.606, P = 0.005; LC3/GAPDH: F = 11.014, P = 0.005; p62/GAPDH: F = 4.047, P = 0.026; Grp78/GAPDH: F = 7.520, P = 0.002). N = 6 rats per group, *P < 0.05 compared with control; **P < 0.01 compared with control; ***P < 0.001 compared with control; ^#P < 0.05 compared with SNL; ^##P < 0.01 compared with SNL; ^ΔP < 0.05 compared with SNL+K; ^ΔΔP < 0.01 compared with SNL+K; ^ΔΔΔP < 0.001 compared with SNL+K. Abbreviations: BL, baseline; Ket, ketamine; control group, rats received a sham operation and vehicle; SNL group, rats received the SNL procedure andvehicle; SNL+K group, rats received SNL, ketamine, and vehicle; SNL+K+G group, rats received SNL, ketamine, and 2’3’-cGAMP.