Effects Of Koumine On Astrocyte Activation And Its Relationship With Autophagy

Spinal dorsal horn is an important region for neuropathic pain modulation.Activated spinal glial cells,mainly the activation of astrocytes play an important role on the maintenance of neuropathic pain,which has become a hot research in recent years at home and abroad.Activated spinal glial cells can release a series of neuroactive substances and proinflammatory cytokines,which is an important mechanism of algesia.Koumine is the active ingredient of indole alkaloids in Gelsemium elegans Benth.,which is a traditional medicinal plant.Koumine is the most abundant composition and exerts relatively low toxicity.Previous study suggested that koumine possesses the potential analgesic property on neuropathic pain.Koumine can inhibit spinal astrocyte activation,but its specific mechanism is expected to be clarified.Autophagy is the main pathway for the degradation of proteins and organelles.The relationship between autophagy and inflammation has been reported in the literature,focusing primarily on the regulation of autophagy on inflammation.Suppressed autophagic activity(autophagic vesicle accumulation)and inflammatory response has been observed during neuropathic pain.In view of this,this study further elucidated the analgesic effect of koumine on the chronic constriction injury(CCI)rat model.On this basis,on the one hand,the changes of autophagy on spinal cord and the effects of koumine on it were detected using CCI model in rats.On the other hand,in the primary astrocyte activation model,we investigated whether the inhibitory effect of koumine on astrocyte activation is related to autophagy.The main contents are as follows.1 Effect of koumine on astrocytes activation1.1 Analgesic Effect of koumine on CCI model in rats and the effect on astrocytes activation in rat spinal cord on CCI modelAnalgesic effect of koumine on CCI neuropathy was observed by measuring mechanical withdrawal threshold(MWT)on CCI rat model.Experimental animals were randomly divided into sham operation group,model group and koumine-treated group(0.28 mg/kg and 7.0 mg/kg).Koumine or saline was administered subcutaneously once a day for 7 days beginning from postoperative day 3.Pain thresholds were measured before surgery,on postoperative day 3(pre-dosing)and 1 h after each administration(post-dosing).The results showed that koumine has significant anti-pain effect in CCI rats.After behavioral test was finished,the spinal cord of lumber segment 4 to 6 was harvested,the astrocyte-specific marker GFAP was detected by western blot to evaluate the effect of koumine on the activation of astrocytes in spinal cord.The results showed that the protein expression of GFAP was notably enhanced in the CCI model group in contrast to the Sham operation group.The increasing expression of GFAP was significantly suppressed by koumine.Koumine could significantly inhibit the activation of spinal astrocytes.Koumine inhibits the expression of GFAP in the rat spinal cord,which negatively correlates the analgesic effect of koumine on CCI rats.1.2 Effect of koumine on LPS-induced astrocytes activationTo investigate the effect of koumine on astrocyte activation,the astrocyte specific marker GFAP was detected by western blot in primary astrocytes.Astrocytes were divided into control group,LPS(1 ?g/ml)-induced model group and koumine-treated with different concentration groups(25 ?M,50 ?M,100 ?M).The results showed that GFAP expression was increased in 1 ?g/ml LPS-induced model group compared with the control group,while 100 ?M koumine could significantly inhibit the level of GFAP,which indicated that koumine could inhibit the activation of astrocytes.2 Effect of koumine on autophagy of astrocytes2.1 Regulatory effects of koumine on autophagy of spinal cord astrocytes in CCI ratsAfter behavioral test was finished,the spinal cord of lumber segment 4 to 6 washarvested after perfusion fixation.The astrocyte marker GFAP and autophagic marker LC3 were labeled by immunofluorescence histochemistry.The results showed that most of the LC3 could co-express with GFAP,indicating that the autophagy of the spinal dorsal horn occurred in astrocytes.There was no significant difference in the fluorescence intensity of GFAP and LC3 between the ipsilateral and the contralateral spinal dorsal horn in the Sham group,the expression of GFAP and LC3 was enhanced in ipsilateral spinal dorsal horn of the CCI model group in contrast to the contralateral spinal dorsal horn.The spinal astrocytes was activated in CCI rats,at the same time,the autophagy of spinal astrocytes is activated.Koumine can reduce the fluorescence intensity of GFAP and LC3.The expression of autophagy-related proteins Beclin-1,LC3 ?,LC3 ? and p62 were detected by western blot to observe the effects of koumine on the autophagy activity of spinal cord after the end of the behavioral experiment.Compared with the sham operation group rats,the expression of p62 in the CCI model group rats was significantly increased,suggesting that autophagic flow was obstructed during neuropathic pain.While high dose of koumine could down-regulate the expression of p62.Koumine reduces the level of spinal p62 in rats,which negatively correlates the analgesic effect of koumine on CCI rats.2.2 Effect of koumine on autophagy of LPS-induced activated astrocytesThe expression of autophagy-related proteins LC3?,LC3?and p62 were detected by western blot in primary astrocytes to investigate the effect of koumine on the autophagic activity of astrocytes.Astrocytes were divided into control group,LPS(1?g/ml)-induced model group and koumine-treated with different concentration groups(25 ?M,50 ?M,100 ?M).The data showed that the expression of LC3 ? in 1 ?g/ml LPS group was significantly increased compared with the control group,and the level of p62 has increased trend.The expression of LC3? and p62 was down-regulated by koumine,which indicated that koumine could enhance the autophagic activity of activated astrocytes.3 Relationship between the inhibitory effect of koumine on astrocyte activation and the autophagyThe expression of astrocyte-specific markers GFAP and autophagy-related proteins LC3 ?,LC3 ? and p62 were detected at the same time by western blot and immunofluorescence cytochemistry method in primary astrocytes.Astrocytes were divided into four groups: control group,LPS(1 ?g/ml)-induced model group,koumine treatment group(100 ?M)and koumine + autophagy antagonist(Chloroquine)group.Compared with the control group,the expression of GFAP ? LC3 ? and p62 were increased in 1 ?g/ml LPS-induced model group.Koumine can reduce the expression of these proteins,while the autophagy inhibitor chloroquine could partially reverse this effect.In summary,koumine can significantly attenuate spinal astrocytes activation,and its effect may be related to the enhancement of autophagic flow in astrocytes.