Antagonistic Effects Of Koumine On Neuropathic Pain And Its Relationship With The Neurosteroid Allopregnenolone In Spinal Cord

Neuropathic pain （NPP） is a pain syndrome caused by pathological changes orinjures in somatic sensation system. NPP is characterized by spontaneous pain,hyperalgesia and allodynia. The incidence of NPP is high. Millions of people havebeen suffering from NPP. NPP is severe and the mainstay is medical therapy. But thetherapeutic effect of current medications is unsatisfactory and inconstant with myriadside effects, underscoring the need for novel analgesics.To discover novel analgesics from botanicals and to further research the relatedessential scientific questions is an important way to find a novel drug for the relief ofNPP. Gelsemium, a small genus of the family Loganiaceae, is a centuried medicalplant. The main active constituents of Chinese Gelsemium are indole alkaloids.Koumine (molecular formula: C₂₀H₂₂N₂O; molecular weight:306.40) is the mostabundant and less toxic among these indole alkaloids, belonging to monoterpeneindole alkaloids. The chronic constriction injury （CCI） and spinal nerve ligation （SNL）models are two of the most extensively used animal models of painful peripheralmononeuropathy. Recently, we first observed that koumine possessed profound reliefof NPP by the indexes of thermal withdrawal threshold, mechanical withdrawalthreshold and spontaneous pain on CCI and SNL models. The effective dose is farbelow the toxic dose so that koumine maybe have the value in the treatment of NPP.However, the mechanism of koumine on analgesic effect has not been wellunderstood. The pathogenesis of periphery NPP involves periphery and centralmechanism, especially dorsal horn （DH） of spinal cord is the important regulatory site.For the past few years, a growing body of evidence indicates that neuroactive steroidsare involved in the modulation of NPP. Allopregnanolone, also called3α,5α-tetrahydroprogesterone （3α，5α-THP）, is one of the most important neuroactivesteroids. It is reported that upregulation of3α,5α-THP produces significant analgesiceffect, implying that spinal3α，5α-THP might be an important regulatory molecular ofNPP. Relevant data show that the analgesic effect of koumine on NPP might beassociated with spinal3α,5α-THP while the detailed relationship is unknown. Accordingly, this study is aimed to discuss the relationship between the analgesiceffect of koumine on NPP and its regulation of spinal allopregnanolone by multiplesubject methods on rat CCI model, hunting the key effector molecule of koumine; andthen to investigate the analgesic effect of koumine on NPP and its regulation of keyspinal allopregnanolone synthetase （3α-hydroxysteroid oxido-reductase,3α-HSD）,identifying the target of koumine. We expect to elucidate the spinal molecularmechanism of the analgesic effect of koumine on NPP, to provide the new target andnew way for the discovery of novel analgesic medicines for NPP, to help theunderstanding of central pathogenesis of NPP and to establish the rationale of newtherapeutic effect of koumine of NPP.The detailed data are listed here.1. Effect of koumine on CCI induced neuropathic pain in rat.The effect of koumine on CCI induced neuropathic pain （thermal hyperalgesia andmechanical allodynia） in rat was evaluated. Animals with qualified baseline painthreshold were assigned into koumine treated groups (7mg·kg^-1,1.4mg·kg^-1and0.28mg·kg^-1), positive control group (gabapentin40mg·kg^-1), vehicle negative controlgroup, and sham control group. Thermal and mechanical withdrawal latencies （TWLand MWL）of each hind paw were measured respectively. The postoperative painthreshold was measured on postoperative days3,5,7and10. Koumine (0.28,1.4,7mg·kg^-1), gabapentin (40mg·kg^-1), or vehicle was administered s.c. twice per day for7consecutive days beginning from postoperative day3. It was shown that kouminesignificantly reversed thermal hyperalgesia and mechanical allodynia. The analgesiceffect of koumine was dose-dependent and time-dependent. Koumine (7mg·kg^-1)exerted analgesic activity within2days after the first administration and reached themaximum protective effect on day7of treatment （%inhibition:86.2±2.0%for TWLand81.7±2.3%for MWL）. These data suggest that koumine might reverseneuropathic pain.2. Effect of koumine on neurosteroids level in rat spinal cord after CCI inducedneuropathic pain.The content of allopregnanolone in the L5-6spinal cord of CCI rats with kouminetreatment for7consecutive day was detected. Rats were assigned into kouminetreated groups (7mg·kg^-1,1.4mg·kg^-1and0.28mg·kg^-1), a vehicle control group, and a sham control group. It was shown that koumine elevated the level ofallopregnanolone in the L5-6spinal cord in a dose-related manner when kouminerelief NPP. These data suggest that the analgesic effect of koumine may be associatedwith the upregulation of allopregnanolone in the spinal cord, which might be the keyeffector molecule of koumine on the relief of NPP.The above data show that koumine may relief CCI-induced periphery NPP, andincrease the level of allopregnanolone in the L5-6spinal cord in a dose-related manner.The key synthetase of allopregnanolone,3α-HSD, catalyze dihydroprogesterone into3α,5α-THP. In order to elucidate the mechanism of koumine upregulatingallopregnanolone in rat spinal cord, we further study the relationship between theanalgesic effect on NPP and the regulation of3α-HSD on the spinal cord by theenzyme structure （enzyme expression） and function （enzyme activity）.3. Effect of koumine on3α-HSD immunohistochemical staining in DH of rat L5-6spinal cord after CCI induced neuropathic pain.The spinal distribution of3α-HSD of CCI rats with koumine treatment wasobserved by fluorescence-immunohistochemisty. Rats were assigned into kouminetreated groups (7mg·kg^-1 and0.28mg·kg^-1), a vehicle control group, a sham controlgroup and Na ve group. Koumine (0.28and7mg·kg^-1), vehicle was administered s.c.at the volume of0.25mL/100g twice per day for7consecutive days beginning frompostoperative day3. Rats were killed by decapitation1h after drug administration onthe morning of postoperative days4,6and9and on postoperative day13. The lumbersegments （L5-6） of the spinal cord were dissected and tested byfluorescence-immunohistochemisty. It was shown that the immunostaining of3α-HSD gradually and significantly increased in DH of spinal cord in CCI rats onpostoperative days6,9and13. On postoperative day9, koumine significantlyincreased the immunostaining of3α-HSD in DH of spinal cord of CCI rats in adose-dependent manner. The enzyme3α-HSD is widely distributed in DH of spinalcord and co-expresses with neuron, microglia and astrocyte.4. Effect of koumine on3α-HSD mRNA expressions in DH of rat L5-6spinal cordafter CCI induced neuropathic pain.The lumbar3α-HSD mRNA expression of CCI rats with koumine treatment wasstudied by realtime-PCR. The animal assignments and drug treatments were the same with the above. Spinal RNA was extracted and determined the integrality andconcentration. The level of3α-HSD mRNA was detected by realtime-PCR. It wasshown that the level of3α-HSD mRNA gradually and significantly increased in DHof spinal cord in CCI rats on postoperative days6,9and13. On postoperative days9and13, koumine significantly increased the level of3α-HSD mRNA in lumbar spinalcord of CCI rats in a dose-dependent manner. These results suggested that kouminemight increase3α-HSD mRNA expression to up regulate the expression of3α-HSDprotein.5. Effect of koumine on the3α-HSD catalytic activity in rat spinal cord after CCIinduced neuropathic pain.Effect of koumine on spinal3α-HSD activity in CCI rats was observed by enzymekinetics. Rat were assigned into koumine treated groups (7mg·kg^-1and0.28mg·kg^-1),a vehicle control group, a sham control group and Na ve group. Koumine (0.28and7mg·kg^-1), vehicle was administered s.c. twice per day for7consecutive daysbeginning from postoperative day3. Rats were killed by decapitation1h after drugadministration on the morning of postoperative day9. The lumber segments （L5-6） ofthe spinal cord were dissected, extracted and tested by enzyme activity analysis. Itwas shown that the enzyme activity of3α-HSD was significantly increased in CCIrats. Koumine further enhanced the activity of3α-HSD in the spinal cord of CCI ratsin a dose-dependent manner.6. Effect of medroxyprogesterone acetate （MPA） on the analgesic effect ofkoumine on mechanical allodynia in CCI rats.Effect of MPA, an inhibitor of3α-HSD, on the analgesic effect of koumine wasobserved by intrathecal injection. Catheters were inserted into the intrathecal space.Proper location of the catheter was confirmed by intrathecal injection of lidocaine.After recovery, periphery neuropathy was induced by CCI. Rats with mechanicalallodynia were assigned into DMSO+NS group, MPA(1.25mg·kg^-1)+NS group,DMSO+koumine group, MPA(0.25mg·kg^-1)+koumine group, MPA(0.5mg·kg^-1)+koumine group and MPA(1.25mg·kg^-1)+koumine group. On postoperativeday9of CCI, DMSO or MPA was intrathecally injected,0.5h later koumine (7mg·kg^-1) or physiological saline was administered s.c., another1h later MWL of hindpaws were detected. It was shown that s.c. koumine significantly relieved the mechanical allodynia, itrathecal injection of MPA dose-dependently reversed theanalgesic effect of koumine and had no effect on the MWL of CCI rats itself. Thesedata implied that the analgesic effect of koumine might associated with theupregulation of the activity of spinal3α-HSD.In summary, koumine may up regulate spinal3α-HSD, including the expressionand enzyme activity, and further to enhance the level of3α,5α-THP to produce theanalgesic effect on NPP. The enzyme3α-HSD may be the key target for koumine toregulate the level of spinal3α,5α-THP.