Liquiritin Alleviates Pain Through Inhibiting CXCL1/CXCR2 Signaling Pathway in Bone Cancer Pain Rat

Abstract

Bone cancer pain (BCP) is an intractable clinical problem, and lacked effective drugs for treating it. Recent research showed that several chemokines in the spinal cord are involved in the pathogenesis of BCP. In this study, the antinociceptive effects of liquiritin, which is an active component extracted from Glycyrrhizae Radix, were tested and the underlying mechanisms targeting spinal dorsal horn (SDH) were investigated. The BCP group displayed a significant decrease in the mechanical withdrawal threshold on days 6, 12, and 18 when compared with sham groups. Intrathecal administration of different doses of liquiritin alleviated mechanical allodynia in BCP rats. The results of immunofluorescent staining and western blotting showed that liquiritin inhibited BCP-induced activation of astrocytes in the spinal cord. Moreover, intrathecal administration of liquiritin effectively inhibited the activation of CXCL1/CXCR2 signaling pathway and production of IL-1β and IL-17 in BCP rats. In astroglial-enriched cultures, Lipopolysaccharides (LPS) elicited the release of chemokine CXCL1, and the release was decreased in a dose-dependent manner by liquiritin. In primary neurons, liquiritin indirectly reduced the increase of CXCR2 by astroglial-enriched-conditioned medium but not directly on the CXCR2 target site. These results suggested that liquiritin effectively attenuated BCP in rats by inhibiting the activation of spinal astrocytic CXCL1 and neuronal CXCR2 pathway. These findings provided evidence regarding the the antinociceptive effect of liquiritin on BCP.

Keywords: CXCL1; CXCR2; bone cancer pain; glia-neuron interaction; liquiritin; spinal cord.

Figure 1

The chemical structure of Liquiritin (LQ, C₂₁H₂₂O₉). The chemical class is flavonoids.

Figure 2

Mechanical allodynia induced by bone cancer and the effects of intrathecally administered Liquiritin (LQ) on motor performance of naïve rats in rotarod test. After operation, the mechanical pain domain has no obvious change in sham group. In contrast, in bone cancer pain (BCP) group, paw withdrawal thresholds (PWT) began to decrease on day 6 until day 18 (F_2,21 = 22.94, ^***P < 0.001 vs. control; n = 8, two way ANOVA, 2A). Compared with baseline response, intrathecal injection of Liquiritin (LQ) (20, 100, 500, and 1,000 ug/kg) for 7 days did not affect the motor performance (F_4,35 = 0.5979, P > 0.05 vs. control; n = 8, two way ANOVA, 2B). The results were expressed as percentage of each rat’s own baseline value.

Figure 3

Effects of intrathecal Liquiritin (LQ) administration on bone cancer induced mechanical allodynia. (A) Intrathecal injection of LQ dose-dependently alleviated the mechanical allodynia in bone cancer pain (BCP) rats and this effect was still observed 6 days after drug withdrawal on day 18 (F_5,42 = 21.4, ^**P < 0.01, ^***P < 0.001 vs. BCP + Veh group, ^##P < 0.01, ^###P < 0.001 vs. BCP + 20 μg/kg group; n = 8, two way ANOVA, 3A). The dose-effect or log (dose)-effect curves for the analgesic effects of intrathecally administered LQ were shown in (B, C). The ED50 of LQ on bone cancer-induced mechanical allodynia was 46.85 μg/kg. (D) Intrathecal injection of single-dose of LQ on postoperative day (POD) 18 i.t. still alleviated paw withdrawal thresholds (PWT). The effects reached peak at 3 h after injection and then gradually faded away, where only the 20 ug/kg works at 3 h after injection. (F_5,42 = 105.8, ^**P < 0.01, ^***P < 0.001 vs. BCP + Veh group; n = 8, two way ANOVA).

Figure 4

Effect of intrathecal administration Liquiritin (LQ) on glial activation in the spinal dorsal horn of bone cancer pain (BCP) rats on postoperative day (POD) 12. (A–E) BCP induced a remarkable microglial activation, which was indicated by Iba1 upregulation in the ipsilateral spinal dorsal horn on POD 12. Intrathecal LQ administration did not inhibit the immunodensities of Iba1 in the ipsilateral spinal dorsal horn after BCP. Scale bar 100 μm; mean immunofluorescence intensity (F) and western blot (G) of Iba1 expressions after different treatments (F_4,15 = 18.54, ^***P < 0.001 vs. sham + Veh group, P > 0.05 vs. BCP + Veh group; F_4,15 = 19.82, ^***P < 0.001 vs. sham + Veh group, P > 0.05 vs. BCP + Veh group; n = 4, one way ANOVA, 4F, G ). (H–L) BCP induced activation of astrocytes remarkably, which was indicated by GFAP upregulation in the ipsilateral spinal dorsal horn on POD 12. Intrathecal LQ administration inhibited immunodensities of GFAP in the ipsilateral spinal dorsal horn after BCP. Scale bar 100 μm; mean immunofluorescence intensity (M) and western blot (N) of GFAP expressions after different treatments (F_4,15 = 37.49, ^***P < 0.001 vs. sham + Veh group; ^#P < 0.05, ^###P < 0.001 vs. BCP + Veh group; F_4,15 = 25.18, ^***P < 0.001 vs. sham + Veh group;^#P < 0.05, ^###P < 0.001 vs. BCP + Veh group; n = 4, one way ANOVA, 4M, N ).

Figure 5

Effects of intrathecal injection of Liquiritin (LQ) on bone cancer pain (BCP)-induced CXCL1 upregulation in spinal astrocytes. (A–E) BCP induced a remarkable upregulation of CXCL1 in the ipsilateral spinal dorsal horn. Intrathecal LQ administration inhibited BCP-induced increase in CXCL1 immunoreactivity in the spinal cord. Scale bar 100 μm; mean immunofluorescence intensity (F) and western blot (G) of CXCL1 expressions after different treatments (F_4,15 = 82.27, ^***P < 0.001 vs. sham + Veh group; ^#P < 0.05,^###P < 0.001 vs. BCP + Veh group; F_4,15 = 79.38, ^***P < 0.001 vs. sham + Veh group; ^##P < 0.01, ^###P < 0.001 vs. BCP + Veh group; n = 4, one way ANOVA, F, G). (H–J) Immunostaining images demonstrated CXCL1(green) was predominantly co-localized with GFAP (red) as shown by overlapped staining (the rightmost panel, yellow). Scale bar, 100 μm.

Figure 6

Effects of intrathecal injection of Liquiritin (LQ) on bone cancer pain (BCP)-induced CXCR2 upregulation in the spinal neurons. (A–E) BCP induced a remarkable upregulation of CXCR2 in the ipsilateral spinal dorsal horn. Intrathecal LQ administration inhibited BCP-induced increase in CXCR2 immunoreactivity in the spinal cord. Scale bar 100 μm; mean immunofluorescence intensity (F) and western blot (G) of CXCR2 expressions after different treatments (F_4,15 = 66.22, ^***P < 0.001 vs. sham + Veh group; ^##P < 0.01, ^###P < 0.001, vs. BCP + Veh group; F_4,15 = 72.94, ^***P < 0.001 vs. sham + Veh group; ^###P < 0.001 vs. BCP + Veh group; n = 4, one way ANOVA, 6F, G). (H–J) Immunostaining images demonstrated CXCR2 (green) was predominantly co-localized with NeuN (red) as shown by overlapped staining (the rightmost panel, yellow). Scale bar, 100 μm.

Figure 7

Effects of intrathecal injection of Liquiritin (LQ) on bone cancer pain (BCP)-induced upregulation of IL-1β, IL-17, and ccl2 levels in spinal dorsal horn were revealed by RT-PCR. (A–C) RT-PCR showed no remarkable differences regarding the levels of IL-1β, IL-17, and ccl2 between the sham + Veh group and sham + LQ 500 μg/kg group (F_5,18 = 138.6, P > 0.05 vs. sham +Veh group; F_5,18 = 41.14, P > 0.05 vs. sham +Veh group; F_5,18 = 284.8, P > 0.05 vs. sham +Veh group; n = 4, one way ANOVA, 7A–C). A significant upregulation of levels of IL-1β, and IL-17 as well as chemokine ccl2 were observed in BCP, and were increased by 3.80-, 2.11-, and 3.78-folds, respectively, on day 12 after injection of walker 256 cells (F_5,18 = 138.6, ^***P < 0.001 vs. sham +Veh group; F_5,18 = 41.14, ^***P < 0.001 vs. sham +Veh group; F_5,18 = 284.8, ^***P < 0.001 vs. sham +Veh group; n = 4, one way ANOVA, 7A–C). Only IL-1β mRNA was decreased in the LQ 20 μg/kg group (F_5,18 = 138.6, ^##P < 0.01 vs. BCP + Veh group; n = 4, one way ANOVA, 7A). (A, B) LQ 100 μg/kg decreased the expression of IL-1β and IL-17 mRNA to 73.41% and 78.83%, respectively, when compared with BCP group injected with vehicle (F_5,18 = 138.6, ^###P < 0.001 vs. BCP + Veh group, F_5,18 = 41.14, ^##P < 0.01 vs. BCP + Veh group; n = 4, one way ANOVA, 7A, B). (A, B) Under the management of LQ 500 μg/kg, the expression of IL-1β and IL-17 mRNA was decreased to 46.83% and 64.21% (F_5,18 = 138.6, ^###P < 0.001 vs. BCP + Veh group, F_5,18 = 41.14, ^###P < 0.001 vs. BCP + Veh group; n = 4, one way ANOVA, 7A, B). (C) No significant decrease was observed in the levels of ccl2 after the three concentrations of LQ administration following BCP surgery (F_5,18 = 284.8, ***P < 0.001 vs. sham +Veh group, P > 0.05 vs. BCP + Veh group; n = 4, one way ANOVA, 7C).

Figure 8

Liquiritin (LQ) reduces LPS-induced mRNA increase of CXCL1 expression in primary cultured astrocytes. (A) Astrocytes were treated with indicated concentrations (1, 50, 100, 150, and 200 μm) of LQ and assessed using MTT assay. The control cells were treated with DMSO (F_5,30 = 0.1567, P > 0.05 vs. control; n = 6, one way ANOVA, A). (B) LPS stimulated astrocytes were exposed to LQ (1, 50, 100, 150, and 200 μm) and assessed using MTT assay (F_6,35 = 19.61, ^***P < 0.001 vs. control; n = 6, one way ANOVA, 8B). (C–E) Double staining of CXCL1 with GFAP showed the CXCL1 expression by astrocytes. Scale bar = 50 μm (F) LPS (1 μg/ml) dramatically increased CXCL1 mRNA expression in primary astrocytes at 0.5, 1, 3 and 6 h (F_4,15 = 24, ^**P < 0.01,^***P < 0.001 vs. control; n = 4, one way ANOVA, F). (G, H) LPS-induced CXCL1 upregulation was decreased by treatment with LQ (F_4,15 = 25.2,^***P < 0.001 vs. naïve, ^#P < 0.05, ^##P < 0.01,^###P < 0.001 vs. control; F_4,15 = 83.49, ^***P < 0.001 vs. naïve, ^#P < 0.05, ^###P < 0.001 vs. control; n = 4, one way ANOVA, 8G, H). All data are presented as means ± SEM.

Figure 9

Liquiritin (LQ) indirectly reduces CXCR2 upregulation in primary neurons mediated by astrocyte-conditioned medium. (A–C) Double staining of CXCR2 with NeuN demonstrated the expression of CXCR2 by neurons. Scale bar = 50 mm. (D, E) LQ did not directly act on the increase in LPS-induced CXCR2 in primary neurons (F_5,18 = 35.21, P > 0.05, ^***P < 0.001, ^###P < 0.001 vs. control, F_5,18 = 40.77, P > 0.05, ^***P < 0.001, ^###P < 0.001 vs. control; n = 4, one way ANOVA, D, E ). (F, G) LQ indirectly reduced the increase of CXCR2 mediated by astrocyte-conditioned medium (F_4,15 = 63.6, ^***P < 0.001 vs. Naive, ^###P < 0.001 vs. control, F_4,15 = 26.58, ^***P < 0.001 vs. naïve, ^#P < 0.05,^###P < 0.001 vs. control; n = 4, one way ANOVA, F, G ). (H, I) The astrocyte-conditioned medium incubated with LQ showed no effect on the increase of CXCR2 when CXCL1 was inhibited (F_4,15 = 32.03, ^***P < 0.001, P > 0.05 vs. control, F_4,15 = 85.85,^***P < 0.001, P > 0.05 vs. control; n = 4, one way ANOVA, H, I ). All data are presented as means ± SEM. N.S., No statistical difference was found between two groups, P > 0.05.