芫花对热敏通道瞬时感受器电位香草素受体1的影响

Effect of Genkwa Flos on Transient Receptor Potential Vanilloid 1 of Thermosensitive Channel

目的:通过电生理全细胞膜片钳技术和动物行为学实验观察芫花对热敏通道瞬时变体电位香草素受体1(TRPV1)的影响,并探讨其机制。方法:以电生理全细胞膜片钳技术检测不同浓度中药芫花的75%乙醇提取物对瞬染人源TRPV1的HEK293细胞(h TRPV1/HEK293细胞)诱导产生的跨膜电流,运用TRPV1特异性拮抗剂辣椒平,观察其是否可以抑制中药芫花诱导产生的跨膜电流;行为学检测使用雄性C57BL/6小鼠30只,将其分为空白组6只,芫花高剂量组6只,芫花中剂量组6只和芫花低剂量组6只,布洛芬组6只。芫花治疗组以低、中、高(0. 195,0. 39,0. 78 g·kg-1·d-1) 3个剂量进行灌胃,1 h后,观察在(0±2)℃冷板和(55±1)℃热板中小鼠冷痛、热痛行为潜伏期的变化。结果:全细胞膜片钳实验显示在h TRPV1/HEK293细胞上芫花75%乙醇提取物可以激活TRPV1通道产生明显的跨膜电流,该跨膜电流与TRPV1激动剂辣椒素诱发的电流在电流密度和电流-电压关系各方面均类似;量效实验显示,与细胞外液对比,10 g·L-1芫花醇提物能够激活h TRPV1/HEK293细胞诱导其产生明显的跨膜电流(P <0. 01)。10 g·L-1芫花醇提物产生的跨膜电流> 3 g·L-1(P <0. 01)。TRPV1特异性拮抗剂辣椒平可以完全抑制10 g·L-1芫花醇提物诱导产生的跨膜电流;小鼠冷热板实验中,芫花延长小鼠的冷痛和热痛行为潜伏期,与剂量呈现一定的量效关系。冷板实验中,与空白组小鼠比较,芫花中剂量组小鼠冷痛行为潜伏期延长(P <0. 01),与空白组比较,芫花高剂量组冷痛时间也显著延长(P <0. 01),布洛芬组小鼠冷痛行为潜伏期较空白组延长(P <0. 01)。在热板实验中,与空白组比较,芫花高剂量组热痛行为潜伏期显著延长(P <0. 01),布洛芬组的热痛行为潜伏期明显延长(P <0. 05)。结论:芫花的75%乙醇提物中含有TRPV1的激动剂,芫花所具备的温性、止痛和抗炎功效可能是热敏通道TRPV1活化后产生的系列效应。

Objective:To study the effect of Genkwa Flos on the thermosensitive channel,transient receptor potential vanilloid 1(TRPV1)by electrophysiological whole cell patch clamp technique and animal behavior experiment,in order to explore its mechanism.Method:The whole-cell patch clamp technique was used to measure transmembrane currents induced by 75%ethanol extract from different concentrations of Genkwa Flos in HEK293 cells that expressed human TRPV1.TRPV1 specific antagonist capsaicin was used to observe whether it could inhibit the transmembrane current induced by Genkwa Flos.Totally 30 C57 BL/6 mice were taken for behavioral detection,and divided into blank group(6 mice),high-dose group(6 mice),medium-dose group(6 mice),low-dose group(6 mice)and ibuprofen positive control group(6 mice).Genkwa Flos treatment group was given low dose(0.195 g·kg-1·d-1),medium dose(0.39 g·kg-1·d-1)and high dose(0.78 g·kg-1·d-1)by gavage.One hour later,the changes of behavioral latency of cold pain and hot pain in mice were observed in cold plate at(0±2)℃and hot plate at(55±1)℃.Result:Whole cell patch clamp experiment showed that 75%ethanol extract of Genkwa Flos in hTRPV1/HEK293 cells could activate TRPV1 channel to generate obvious transmembrane current,which was similar with that generated by the known TRPV1 agonist capsaicin in current density and current-voltage relationship.The dose-effect experiments showed that compared with extracellular fluid,10 g·L-1 ethanol extract of Genkwa Flos could activate hTRPV1/HEK293 cells to induce significant transmembrane current(P<0.01).The transmembrane current generated by 10 g·L-1 ethanol extract of Genkwa Flos was more than 3 g·L-1(P<0.01).The TRPV1 specific antagonist capsaicin could completely inhibit the transmembrane current induced by 10 g·L-1 ethanol extract of Genkwa Flos.In the experiment of cold plate and hot plate in mice,there was a dose-effect relationship between the latencies of cold pain behavior and hot pain behavior in mice prolonged by Genkwa Flos.In the experiment of cold plate,compared with the blank group,the cold pain behavior latency of mice in the medium-dose group was significantly prolonged(P<0.01),and that of mice in the highdose group was significantly prolonged(P<0.01).Compared with the blank group,the cold pain behavior latency of mice in the ibuprofen positive control group was significantly prolonged(P<0.01).In the hot plate experiment,the incubation period of hot pain behavior in the high-dose group of Genkwa Flos was significantly longer than that in the blank group(P<0.01),while that in the ibuprofen positive control group was significantly longer than that in the blank group(P<0.05).Conclusion:More than one TRPV1 agonist was included in 75%ethanol extract of Genkwa Flos.The warm,analgesic and anti-inflammatory effects of Genkwa Flos may be a series of effects after activation of TRPV1.