Effects Of Koumine On Neuropathic Pain And Its Possible Mechanism On Glia Cells And TSPO In Spinal Cord

Currently, to explore the pathogenesis, find new effective drugs and its targets are the major studying orientations for neuropathic pain(NPP). To discover novel analgesics from medical plants and to further research the related essential scientific questions is an important way to find a novel drug for NPP. Koumine(molecular formula: C20H22N2O; molecular weight: 306.40), belonging to monoterpene indole alkaloids, is the most abundant and less toxic compound among the indole alkaloids, isolated from medical plant Gelsemium elegans. Our previous study has shown that koumine has a significant analgesic action on neuropathic pain with high efficiency and low toxicity, The spinal cord is an important part for the analgesic action of koumine. However, the mechanism of koumine on analgesic effect has not been well understood. Recently, several literatures have reported that glia cells in spinal cord play an important role in NPP by relaeasing a series of neuroactive substances and proinflammatory cytokines. Translocator protein(18 k Da; TSPO), previously known as the peripheral-type benzodiazepine receptor(PBR), is an 18 k Da protein consisting of 169 amino-acids. It primarily localized in the outer mitochondrial membrane. In nerve tissue, it mainly expressed in glia cells. TSPO is expected to be an therapeutic target for neurological and psychiatric disorders. Based on the literatures and our previous work, we aimed to explore the effect of koumine on NPP and its role on regulation the activation of glia in spinal cord; analyze the relationship between anti-NPP effect and expression level of TSPO; investigate the regulation mechanism of koumine to TSPO from binding affinity and function aspect, in order to explain the molecular mechanisms of the spinal cord for the anti-NPP effect of koumine, and preliminary analysis its targets.Research Contents1. Relationship between the effect of koumine on neuropathic pain and glia cells activation. Establishment of chronic constrictive injury(CCI) induced neuropathhic pain model, the rat paw withdrawal threshold after CCI was measured with von Frey filaments. Immunofluorescent histochemistry was used to detect the expressions of spinal astrocytic GFAP, microglial, neuron and TSPO. Animals with qualified baseline pain threshold were assigned into naive control group, sham control group and CCI model group. Rats were killed after mechanical allodynia was measured on postoperative days 3, 6, 9, Respectively. The lumber L5-6 segments of spinal cord were dissected for immunofluorescent histochemistry assay. Immunohistochemistry showed that microglia have activated in spinal dorsal horn when CCI model was established(postoperative day 3), reached a peak on postoperative day 6, then decreased on postoperative day 9, but remained at the high level; The expression of astrocyte significant increased on postoperative day 6, then further enhanced on postoperative day 9; The expression TSPO were significantly upregulated on postoperative day 3, and sustained increase following the decrease of mechanical threshold. Double immunofluorescent labeling shows TSPO have a high co-expression with microglia and astrocyte. There was no significant changes in the expression of the neurons. These findings indicating that spinal microglia, astrocytes and TSPO are involved in the development and maintenance of neuropathic pain behaviors in rats.2. Relationship between the effect of koumine on neuropathic pain and expression level of TSPO Observe the the analgesic effect on CCI rats after treatment the koumine for 7 consecutive days, then observe the expression levels of astrocyte, microglial, neuron and TSPO of spinal cord. Rats were assigned into koumine treated groups(7 mg/kg, and 0.28 mg/kg) and vehicle control group. Koumine(0.28 and 7 mg?kg-1), vehicle was administered s.c. at the volume of 0.25 m L/100 g once per day for 7 consecutive days beginning from postoperative day 3. Rats were killed by decapitation after drug administration on the morning of postoperative days 9, the lumber L5-6 segments of spinal cord were dissected for immunofluorescent histochemistry assay. The results shown the mechanical withdrawal threshold was significantly increased in rats after continued administration of koumine for 7 consecutive days. The expression of microglia have no significant changes compared with vehicle control group, but the number of positive astrocytes significantly inhibited, at the same time, the increased TSPO expression significantly down-regulated. There was no significant changes in the expression of the neurons. Thus, we can conclude that the expression of TSPO and astrocytes significantly prevented by koumine on CCI operation, indicating the analgesic effect of koumine may be related to astrocytes and TSPO.3. The antagonism of PK11195 on anti-neuropathic pain effects of koumine The analgesic effect of a single dose of koumine in the CCI model was evaluated. On this basis, intrathecal implantation and administration PK11195, the specific antagonist of TSPO. Then observe the antagonism of PK11195 agianst the analgesic effect of koumine. Animals with qualified baseline pain threshold were assigned into koumine treated groups(7 mg/kg,1.4 mg/kg and 0.28 mg/kg), vehicle negative control group, and sham control group. Mechanical withdrawal latencies of each hind paw were measured 1 h after drug administration. The resultsshowed that CCI operate significantly decreased mechanical withdrawal latencyto mechanical stimulation. Repeated administration of koumine dose-dependently reversed mechanical allodynia in CCI with an ED50 of 5.83 mg/kg(95% confidence limit:3.53--13.36 mg/kg). Intrathecal injection 1μg PK11195 significantly depressed the effect of koumine(s.c.,7mg/kg)on behavioral signs of neuropathic pain. Intrathecal administration of TSPO antagonists PK11195 significantly prevented or delayed mechanical allodynia, indicate that koumine likely contributes to neuropathic pain by acting on TSPO directly and indirectly.4. Effect of koumine on TSPO bind to [3H]-PK11195 Radioligand Binding Assays was used to determine the Kd and Bmax(maximum number of receptor binding sites in the tissue preparation) values of TSPO with [3H]-PK11195. TSPO extraction of rat cerebral cortex and [3H]-PK11195 were used in the experiment. Analyses of saturation experiments for the binding parameters B max and Kd were performed, the result revealed that specific binding of [3H]-PK11195 to rat cerebral cortical with Kd=0.9337±0.1746 n M, Bmax=210.5±14.4 fmol/mg?prot, koumine could significant increase the Bmax. In competition assay, koumine was not found to compete with [3H]- PK11195, but increase the special binding of [3H]- PK11195 and TSPO, the result indicate that koumine may act as a positive allosteric modulator for TSPO.5. Effect of komine on the secretion of pregnenolone on C6 glioma cells. Measurement of pregnenolone production is used as a benchmark assay to denote TSPO functional activity. The effect of koumine on stimulate steroid biosynthesis in rat C6 glioma cells were explored. C6 cells were seeded in 96-well plates at a density of 10000 cells/well in a final volume of 100 μL,the addition of PK 11195, AC5216 to the C6 cells was made by the complete change of the simple salts medium to medium containing the appropriate concentration of PK 11195(40 μM), AC5216(40 μM) and koumine(8, 40, 200 μM), respectly. The amount of pregnenolone secreted into the medium was quantified by ELISA. After incubation with C6 glioma cells for 2 h, PK11195 and AC-5216 significantly increased the level of pregnenolone in the supernatant, consistent with previous reports. Similarly, C6 glioma cells were incubated with increasing concentrations of koumine, and a significant increase in pregnenolone production was also obtained. Koumine increased pregnenolone production in C6 glioma cells, this in vitro results implications that koumine could be a positive effector of TSPO.In summary, microglia and astrocyte activation in the spinal cord is observed in the development and maintenance of neuropathic pain, and the level of TSPO expression may be related to the glial expression. The anti-neuropathic pain effect of koumine is associated with a reduction of spinal astrocyte activation, suggesting that inhibition of spinal astrocytes is a possible mechanism for koumine contribute to neuropathic pain.The effect of koumine on neuropathic pain is closely related to activation of TSPO in spinal cord. Koumine may bind to the TSPO in a positive allosteric manner; and promote the formation of neurosteroids, suggesting that TSPO may be one of the targets for koumine on neuropathic pain.