Study On The Analgesic Mechanism Of The Total Flavonoids Of Dragon’s Blood And The Combination Of It’s Components By Regulating Acid-Sensing Ion Channels

Dragon’s blood is a traditional medicine with good analgesic effect.Our previous study found that Dragon’s blood could inhibit voltage-gated sodium currents（VGSCs）and capsaicin-induced TRPV1 receptor currents,indicating that the regulation on voltage-gated sodium channel and TRPV1 channel of Dragon’s blood might be the cause of its analgesic pharmacological effect.Then we found that the total flavonoids of Dragon’s blood（TFD）also inhibited the VGSCs and capsaicin-induced TRPV1 currents.This inhibition was similar to dragon’s blood,suggesting that TFD might be the effective fraction of the dragon’s blood in regulating voltage-gated sodium channel and TRPV1 channel.Later studies revealed that the three flavonoids of TFD,cochinchinenin A（CA）,cochinchinenin B（CB）and loureirin B（LB）,also regulated VGSCs and capsaicin-induced TRPV1currents,and the combination of the three flavonoids had similar modulation of those two currents to TFD,suggesting that these three flavonoids might be the effective components of TFD to regulate these two channels.In the in vivo animal experiments,it was found that Dragon’s blood and TFD increased the heat-stimulated pain threshold in mice,reduced the heat-stimulated tail flick reaction time of mice and the number of writhing reactions caused by glacial acetic acid,which demonstrated the pharmacological effects of Dragon’s blood and TFD in relation to the above mechanisms.In the next experiment,we continued to explore the analgesic pharmacological mechanisms associated with TFD and its components.In addition to VGSCs and TRPV1 channels,acid-sensitive ion channel（ASIC）is also one of the important pathways for the transmission of pain signals,and new target for the pharmacology research of analgesic drug in recent years.Studies had shown that the isoflavone puerarin might produce analgesic effect by regulating ASIC.Therefore,this paper designed to study the combination of TFD and its components from three aspects:acid-sensitive ion channel,pain threshold test and ASIC protein expression to explore the relationship between the analgesic effects of TFD and ASIC.The effect of TFD on the ASICs in DRG neurons was investigated by whole-cell patch clamp technique.Results showed that TFD inhibited the ASICs current of DRG neurons（P<0.001）,and its half-effect inhibitory concentration（IC50）was（0.0013±0.0003）%,indicating that TFD could regulate ASIC currents in DRG neurons.In the pain model experiment,the analgesic activity of TFD was investigated by mouse formalin test and capsaicin pain test.The chronic inflammatory pain induced by complete Freund’s adjuvant（CFA）was injected into the left foot of the rats,and the analgesic effect of TFD was determined by heat-stimulated pain threshold of the administration site.The results showed that TFD significantly relieved pain caused by formalin and capsaicin,and also significantly relieved CFA-induced inflammatory pain.Previous studies had shown that ASICs were associated with inflammatory pain sensitization,and thus we speculated that the analgesic pharmacological effects of TFD were inseparable from the regulation of ASIC channels.Then explored the effects on the ASIC channels from the pharmacological effects of the components and combinations of TFD.The whole-cell patch clamp technique was used to record the effects of CA,CB and LB on ASICs in DRG neurons respectively and investigated whether these three components acted on the ASICs.The results showed that LB could suppress the transient ASICs currents,and its IC50 was 89.7±5.8μmol/L,while CA and CB had no effect on transient ASICs current.All of three components can suppress continuous ASICs currents with IC₅₀ of 100.4±4.3μmol/L,128±6.9μmol/L and 315.2±10.1μmol/L,respectively,indicating that these three components were the effective components of TFD in regulating ASICs of DRG neurons.In the rat model of acute inflammatory pain,the pharmacological effects of the three components and their combinations on inflammatory pain were explored by hot plate method,and the combination of the three components and ASIC activator（GMQ）were injected into the acute inflammatory model rats to explore whether the ASIC was associated with acute inflammatory pain.In the CFA-induced acute inflammatory pain test,CA and LB groups had significant analgesic effect,CB had no analgesic effect,and the combination had better analgesic effect than the components used alone at the same concentrations,which indicated that the three components had synergistic effects in relieving inflammatory pain.This analgesic effect could be inhibited by ASIC activators,which indicated that the combination of the three components produced analgesic effects by regulating ASIC channels.Previous studies had shown that ASICs were associated with inflammatory pain sensitization,Therefore,the dorsal root nerves from the chronic inflammatory pain model rats were labeled with ASIC3 protein and c-Fos protein by immunohistochemistry.The results showed that the expression of ASIC3 protein and c-Fos protein was decreased（P<0.001）,which indicated that the combination could down-regulate the expression of ASIC3 protein during inflammatory pain.From the above experimental results,it could be suggested that TFD interfered with the pain signal transmission by regulating the ASICs of DRG neurons,which resulted in its peripheral analgesic effect.The combination of CA,CB and LB is the effective material basis of the analgesic effects of the total flavonoid extracted from Dragon’s blood.The analgesic effect is produced by regulating the pain signal transmission through the ASIC3of DRG neurons.