Objective To investigate the effects of intrathecal injection of koumine on behavior and activation of glial cell in spinal dorsal horn of mice with bone cancer pain (BCP).
Methods Experiment 1: Thirty-six male SPF C57BL/6 mice, aged 4-6 weeks, weighing 18-22 g, were selected. The mice were randomly divided into two groups: sham operation group 1(group S1) and group BCP1, 18 mice in each group. In group S1, PBS solution 10 μl was injected into the bone marrow cavity of right distal femur. In group BCP1, PBS solution 10 μl containing 2 × 105 Lewis lung cancer cells was injected into the bone marrow cavity of right femur to establish BCP mouse model. Six mice in group S1 and group BCP1 were randomly selected to determine the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) 1 day before modeling, 4, 7, 10, 14, and 21 days after modeling. Six mice were sacrificed randomly after the behavioral tests completed 7, 14 and 21 days after modeling, and the expressions of astrocyte marker protein (GFAP) and microglial marker protein (Iba-1) in the spinal cord were detected by Western blot. The right femur of the mice was taken for HE staining. Experiment 2: Fifty-four male SPF C57BL/6 mice, aged 4-6 weeks, weighing 18-22 g, were selected. The mice were randomly divided into three groups: sham operation group 2 (group S2), group BCP2 and BCP+koumine group (group K), 18 mice in each group. PBS solution 10 μl was injected into the bone marrow cavity of group S2, and PBS solution 10 μl containing 2 × 105 Lewis lung cancer cells was injected into the bone marrow cavity of right distal femur to establish BCP mouse model in group BCP2. In group K, 20% DMSO solution 5 μl containing koumine 30 μg was slowly injected into the L4-5 or L5-6 14 days after establishment of BCP mouse model, and 6 mice were randomly selected to determine MWT and TWL 1 hour before administration, 1, 2, and 3 hours after administration. Twelve mice were randomly sacrificed 1 hour after administration. The expressions of GFAP and Iba-1 at spinal cord were detected by Western blot, and the concentrations of IL-6, IL-1 β and TNF-α at spinal cord were detected by ELISA.
Results Experiment 1: compared with group S1, the MWT of group BCP1 was significantly decreased (P < 0.05), TWL was significantly shortened 7, 10, 14, and 21 days after modeling (P < 0.05), the expressions of GFAP and Iba-1 proteins in the spinal cord were significantly increased 7, 14, and 21 days after modeling (P < 0.05). The results of HE staining indicated that there was no bone destruction, and the bone marrow cells in the bone marrow cavity were normal in group S1. The bone was destroyed, and a large number of Lewis lung cancer cells infiltrated in the trabecular bone, bone marrow cavity and surrounding soft tissues in group BCP1. Experiment 2: compared with group S2, the MWT was significantly decreased, and the TWL was significantly shortened 1 hour before administration, 1, 2, and 3 hours after administration (P < 0.05), the expressions of GFAP and Iba-1 proteins in the spinal cord were significantly increased 1 hour after administration (P < 0.05), and the concentrations of IL-6, IL-1β and TNF-α were significantly increased 1 hour after administration in group BCP2(P < 0.05); the MWT were significantly decreased, and the TWL were significantly shortened 1 hour before administration and 3 hours after administration in group K (P < 0.05). Compared with group BCP2 , the MWT was significantly increased, and the TWL was significantly prolonged 1 and 2 hours after administration (P < 0.05), the expressions of GFAP and Iba-1 proteins in the spinal cord were significantly decreased 1 hour after administration (P < 0.05), and the concentrations of IL-6, IL-1β and TNF-α in the spinal cord were significantly decreased 1 hour after administration in group K (P < 0.05). There was no significantly differences in the expressions of GFAP, Iba-1 proteins and the concentrations of IL-6, IL-1β and TNF-α in the spinal cord 1 hour after administration between groups S2 and K.
Conclusion Intrathecal injection of koumine can relieve pain in BCP mice, reduce neuroinflammation by inhibiting the activation of astrocytes and microglia. |
