Melatonin MT2 receptor agonist IIK-7 produces antinociception by modulation of ROS and suppression of spinal microglial activation in neuropathic pain rats

Abstract

Background: In recent years, several melatonin (MLT) receptor agonists have been approved by FDA for the treatment of sleep disorders and depression. Very few studies have shed light on their efficacy against neuropathic pain (NP). IIK-7 is an MT-2 agonist known to promote sleep. Whether IIK-7 suppresses NP has not been reported, and the signaling profile is unknown.

Objective: To investigate the effect of melatonin type 2 receptor agonist IIK-7 on partial sciatic nerve transection-induced NP in rats and elucidate the underlying molecular mechanisms.

Methods: NP was induced by the PSNT in the left leg of adult male Wistar rats. On post-transection day 7, rats were implanted with intrathecal (i.t) catheter connected to an infusion pump and divided in to four groups: sham-operated/vehicle, PSNT/vehicle, PSNT/0.5 μg/hr IIK-7 and PSNT/0.5 μg IIK-7/1 μg 4-p/hr. To test the MT-2 dependence on IIK-7 activity, the animals were implanted with a single i.t catheter and injected MT-2 antagonist 4-Phenyl-2-propionamidotetralin (4-p) 20 mins prior to IIK-7 injection on day 7 after PSNT. The antinociceptive response was measured using a mechanical paw withdrawal threshold. Activation of microglial cells and the expression of NP-associated proteins in the spinal cord dorsal horn was assessed by immunofluorescence assay (IFA) and Western blotting (WB). Reactive oxygen species (ROS) scavenging ability of IIK-7 was evaluated by using bone marrow-derived macrophages (BMDM).

Results: Treatment with the MT-2 agonist IIK-7 significantly alleviated PSNT-induced mechanical allodynia and glial activation along with the inhibition of P44/42 MAPK, HMGB-1, STAT3, iNOS and casp-3 proteins.

Conclusion: IIK-7 attenuates NP through the suppression of glial activation and suppression of proteins involved in inflammation and apoptosis. MT-2 receptor agonists may establish a promising and unique therapeutic approach for the treatment of NP.

Keywords: IIK-7; MT-2 receptor; allodynia; neuropathic pain.

Figure 1

Continuous intrathecal infusion of melatonin type-2 (MT-2) agonist IIK-7 attenuated the development of nerve-transection-induced neuropathic pain (n=6). Effects of intrathecal infusion of 0.5 μg IIK-7/1 μL/hr, 0.5 μg IIK-7/1 μg +4-Phenyl-2-propionamidotetralin (4-p)/1μl/hr and vehicle/1μL/hr on the mechanical paw withdrawal threshold in ipsilateral paw of partial sciatic nerve-transected rats and sham. PSNT, partial sciatic nerve transection; IIK-7, N-Butanoyl 2-(9-methoxy-6H-iso-indolo[2,1-a]indol-11-yl)-ethan-amine; 4-p, 4-Phenyl-2-propionamidotetralin.

Figure 2

Pre-treatment of IIK-7 antagonist 4-p 20 mins prior to IIK-7 administration did not result in significant blockade of IIK-7-induced anti-nociception in nerve-transection-induced neuropathic pain (n=6) . Effects of intrathecal single injection of vehicle 10 μL, 5 μg IIK-7 in 10 μL vehicle, pretreatment with 10 μg 4-p in vehicle 10 μL followed by the single injection of 5 μg IIK-7, on the mechanical paw withdrawal threshold in ipsilateral paw of partial sciatic nerve-transected rats and sham group; n=6 rats per group. Abbreviations: PSNT, partial sciatic nerve transection; IIK-7, N-Butanoyl 2-(9-methoxy-6H-iso-indolo[2,1-a]indol-11-yl)-ethan-amine; 4-p, 4-Phenyl-2-propionamidotetralin.

Figure 3

Continuous infusion of IIK-7 attenuated PSNT-induced activation of HMGB-1, pERK1/2, iNOS, STAT3 and caspase-3 in vivo. Representative immunoblots are HMGB-1, P44/42 MAPK, iNOS, STAT 3 and casp-3 from the left dorsal quadrant portion of the lumbar spinal cord lysate of sham surgery or PSNT rats continuously infused with either vehicle or IIK-7 for 7 days using an osmotic pump. Abbreviations: IIK-7, N-Butanoyl 2-(9-methoxy-6H-iso-indolo[2,1-a]indol-11-yl)-ethan-amine; P44/42 MAPK, P44/42 mitogen-activated protein kinase; iNOS, Inducible nitric oxide synthase; STAT3, Signal transducer and activator of transcription 3; CASP-3, caspase3; HMGB-1, High mobility group box 1.

Figure 4

IIK-7 reduces ROS production induced by TLR1/2 agonist PAM3CSK4. The bone-derived macrophages are either untreated (Blank) or treated with IIK-7 (25 ng) or stimulated with triacylated bacterial lipopeptide PAM3CSK4 (5 μg/mL) or pre-incubated IIK-7 for 30 mins followed by the addition of PAM3CSK4 (5 μg/mL). The cells were further incubated for 12 hrs and readings were noted at FL-1 channel using a flow cytometer after the addition of DCFHDA. ROS generation was measured in triplicate. The data shown are the mean ± SEM values of 3 individual experiments . **p<0.01 versus untreated control, ***p<0.001 versus untreated control. Abbreviations: IIK-7, N-Butanoyl 2-(9-methoxy-6H-iso-indolo[2,1-a]indol-11-yl)-ethan-amine; PAM3CSK4,Pam3CysSerLys4, TLR 1/2, Toll-like receptor 1/2.

Figure 5

IIK-7 treatment reduces PSNT-induced microglia activation in rat spinal cords. (A) On day 6 after drug treatment, spinal cord sections were fixed and single-labeled with fluorescein isothiocyanate-labeled anti-OX42 antibodies (green; microglia) and the nuclei stained with 4ʹ,6-diamidino-2-phenylindole (DAPI) (blue); then, images were captured and merged by fluorescence microscopy. The sections are from (A) Vehicle-injected PSNT rats, (B) IIK-7-injected rats. The nuclei (blue lane 1) and microglia (green lane 2) are merged in lane 3. (B) The microglial activation is quantified by counting the number of activated microglial cells and expressed relative to the value for the vehicle-injected group. Data are analyzed by Tukey test (**p<0.01). The pictures are representative of those seen in samples from 3 rats. Abbreviations: IIK-7, N-Butanoyl 2-(9-methoxy-6H-iso-indolo[2,1-a]indol-11-yl)-ethan-amine; PSNT, Partial sciatic nerve transection; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 6

IIK-7 treatment reduces PSNT-induced iNOS expression in rat spinal cords. (A) On day 6 after drug treatment, spinal cord sections were fixed and single-labeled with iNOS rabbit antibody followed by Alexa flour 594 antibody (red) and the nuclei stained with 4ʹ,6-diamidino-2-phenylindole (blue), then images were captured and merged by fluorescence microscopy. The sections are from (A) vehicle-injected PSNT rats, (B) IIK-7-injected rats. (B) The iNOS activation is quantified by counting the number of cells expressing iNOS relative to the value for the vehicle-injected group. Data are analyzed by Tukey test (*p<0.05). The pictures are representative of those seen in samples from 3 rats. Abbreviations: IIK-7, N-Butanoyl 2-(9-methoxy-6H-iso-indolo[2,1-a]indol-11-yl)-ethan-amine; DAPI, 4′,6-diamidino-2-phenylindole; iNOS, iNOS Inducible nitric oxide synthase.

Scheme 1

Schematic illustration of the potential mechanisms by which N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a]indol-11-yl)ethanamine (IIK-7) modulates pain. The extent of neuroinflammation in neuropathic pain depends on the two-way interactions between neurons and immune cells. According to current literature, microglia are the important cells that mediate neuronal inflammation at the spinal cord level after axotomy. Though microglial are known to be key cells involved in NP, their mechanisms of activation remain elusive. Current evidence indicates that reactive oxygen species (ROS) stimulate the activation. In the present study, we have shown, for the first time, that infusion of IIK-7 prevents microglial cell activation in partial sciatic nerve-transected rats. Simultaneously, IIK-7 treatment affects directly and/or indirectly intracellular pathways, which play a key role in regulating the immune response cascade: STAT3, CASP-3, HMGB-1 and iNOS proteins. These molecular actions of IIK-7 might be responsible for its analgesic effect during neuropathic pain and need future investigation. Abbreviations: IIK-7, N-Butanoyl 2-(9-methoxy-6H-iso-indolo[2,1-a]indol-11-yl)-ethan-amine; P44/42 MAPK, P44/42 mitogen-activated protein kinase; iNOS, inducible nitric oxide synthase; ROS, reactive oxygen species; STAT3, Signal transducer and activator of transcription 3; CASP-3, caspase3; HMGB-1, High mobility group box 1.