The Inhibiting Effect Of Koumine On Astrocyte Reactivation

Chronic pain is common and complex and has a large impact on individuals and society according to its high prevalence, long duration and difficult treatment. However, effective treatment for chronic pain is still lacking. It is urgent to develop new durgs and find new drug targets for therapy of chronic pain. Dorsal horn of spinal cord is an important region for chronic pain modulation. In recent years, spinal glias have emerged as a promising target against chronic pain. Activated glial cells in the spinal cord play an important role in mediating enhanced pain states by releasing proinflammatory cytokines and other substances thought to facilitate pain transmission, which may be an important mechanism of the chronic pain occurs. Koumine, one of the main alkaloidal constituents of Gelsemium elegans, has a significant effect on both inflammatory and neuropathic pain. However, its analgesic mechanism is still needs to be further investigated. In this study, we investigated the analgesic effect of koumine on the type II collagen induced arthritis(CIA) rat model by measuring the mechanical withdrawal threshold(MWT). Based on that, the inhibiting effect of koumine on astrocyte reactication were expored by detecting the specific marker GFAP of astrocyte, then, the levels of proinflammatory cytokines IL-1? and TNF-? were detected by ELISA. At last, the levels of astrocyte specific marker GFAP were detected by immunofluorescence histochemistry and western blot in cultured astrocytes.The main contents are listed as follows. 1 Analgesic effect of koumine on CIA model in ratsAnalgesic effect of koumine on arthritis was measured by mechanical withdrawal threshold on rat CIA model. Experiment animals were randomly assigned into control group, model group and koumine-treated groups(0.6 mg/kg and 15 mg/kg). Koumine or normal saline was administered intragastrically once per day for 10 consecutive days from day 21 of initial immunization. Data showed that mechanical withdrawal threshold of 15 mg/kg koumine-treated group was significantly increased; mechanical withdrawal threshold of 0.6 mg/kg koumine-treated group was no significant changes. 2 Effect of koumine on astrocyte reactivation in rat spinal dorsal horn on CIA modelTo evaluate the effect of koumine on astrocyte reactivation, the lumbar segments of spinal dorsal were dissected after behavioral test was finished. GFAP, the specific marker of astrocyte, was detected by immunofluorescence histochemistry. Data showed that in rat spinal dorsal horn of CIA model group, immunofluorescence intensity of GFAP was increased, the number of positive astrocytes was increased, and astrocytes were activated. Immunofluorescence intensity of GFAP was decreased in koumine-treated groups inferring that koumine can inhibit the astrocyte reactivation. 3 Effect of koumine on the level of proinflammatory cytokine in rat spinal cord on CIA modelAfter behavioral test was finished, the spinal cord was dissected. The levels of proinflammatory cytokines IL-1? and TNF-? were determined by ELISA. Data showed that the levels of IL-1? and TNF-? were increased without significance in rat lumbar spinal cord with CIA. Koumine decreased the levels of IL-1? and TNF-?. It is suggests that effect of anti chronic inflammatory pain on koumine may be related to the inhibition of astrocyte reactivation in lumber spinal cord. 4 Effect of koumine on LPS-induced reactivation of astrocytesTo analyze the effect of AC5216 and koumine on astrocyte reactivation, levels of GFAP were detected by immunofluorescence cytochemistry and western blot in cultured astrocytes. Astrocytes were assigned into control group, model group of LPS(1 ?g/ml), AC5216-treated groups(10 nM, 100 nM and 1000 nM) and koumine-treated groups(25 ?M, 50 ?M, 100 ?M and 200 ?M). Data showed that expression of GFAP was enhanced in 1 ?g/ml LPS group, and expression of GFAP was down regulation by AC5216(100 nM, 1000 nM) and koumine(100?M, 200 ?M). It is suggest that AC5216 and koumine could inhibit astrocyte reactivation.In summary, koumine inhibits astrocyte reactivation. To inhibit spinal astrocyte reactivation and to down regulate the level of spinal proinflammatory cytokines may be one of mechanism of analgesic action of koumine on chronic inflammatory pain.