Effects Of Flavonoids From Dragon's Blood On TTX-R Sodium Currents

Dragon's Blood is a traditional natural anti-inflammatory and analgesic drug extracted from tree-stem of Dracaena cochinchinensis(Lour.)S.C.Chen,which has good effects of anti-inflammatory,anti-bacterial,anti-oxidative and analgesic in clinic.Further studies using electrophysiological recordings have revealed that cochinchinenin A(CA),cochinchinenin B(CB),and loureirin B(LB)are the three main active components of Dragon's blood and potentially mediate Dragon's blood's analgesic effect by inhibiting TTX-R sodium channel and TRPV1 channel in the primary sensory neurons.The three flavonoids of Dragon's blood can inhibit TTX-R sodium currents in a concentration-dependent manner.In previous studies,to acquire similar pharmacological effects as Dragon's blood,higher dose of single component is required,while the dose of the combined use of the three main effective components mentioned above is much lower,indicating that there might exist strong interaction among them.To clearly identify the underlying interaction,different combinations of the three main components were applied on TTX-R sodium currents in DRG cells,so as to evaluate their interaction parameters by Gerco model.The results demonstrate that the antagonistic effect could be observed in combination I(CA and CB)as well as combination II(CA and LB),and additive effect was shown in combination III(CB and LB),while synergistic effect was significantly shown in combination IV(CA,CB,and LB).Receptor theory has confirmed that when CA or CB is applied alone on TTX-R sodium current in DRG cells,they conforms to the receptor occupation theory,and the receptor rate theory,respectively.The different modes of action on TTX-R sodium channel,different rate constants of combination and different rate constants of dissociation directly induce the antagonistic interaction between CA and CB.However,it is still impossible to rationally explain the phenomenon that there is a strong synergistic interactiony between the three components by using the receptor theory alone.In this case,modulation mechanism of LB in DRG cells is very likely to be the key to the emergence of synergistic phenomenon.In this study,we designed experiments to explore the mode of action of LB on TTX-R sodium currents in rat DRG cells,the mechanism of how LB modulate TTX-R sodium currents in DRG cells,and to verify the effect of four combinations on peak values of TTX-R sodium current with time,attempting to explain the synergistic effect of CA,CB and LB on modulating TTX-R sodium channel in DRG cells,and provide a reference for the pharmacological mechanism for research and development of LB.In the experiment to explore the mode of action of LB on TTX-R sodium currents,only occupation theory could be chosen to explain the time dependence of the inhibitory effect induced by LB on TTX-R sodium currents.The results show that the model of the occupation theory only fit well with the initial part of the curve but it could not describe the second part of the curve,which indicated that the increasing inhibitory effect of LB on the TTX-R sodium currents with the administration time had other reasons besides it directly acting on TTX-R sodium channel.Electrophysiological patch clamp technique and molecular biology methods were used to explore the mode of action of the LB to modulate the TTX-R sodium currents.After incubation of DRG cells with LB,the levels of AC,cAMP and PKA were significantly higher than those of the control group.After incubation of DRG cells with H-89 for 5 min,the peak values of TTX-R sodium current in rat DRG cells treated with1?mol/L H-89 and 0.64 mmol/L L B solution was not significantly changed compared with the control group.These studies demonstrated that LB can modulate the TTX-R sodium channel in DRG neurons via AC/cAMP/PKA pathway involving the activation of AC and PKA.In the experiment of investigating the effect of four combination drugs on peak values of TTX-R sodium current with time,the combination of CA,CB and LB has the strongest inhibitory effect,and its maximum inhibition rate is(85.63±2.76%).Using mathematical model to fit the whole-cell patch clamp experimental data,it is found that the single exponential equation can fit well all the experimental data of the combination I(R~2>0.99),the double exponential equation can fit well all of the curve for the combination II,III,IV(R~2>0.99).Which suggest that,except for combination I,the other three combinations have two-phase properties.These three combinations can inhibit the peak values of TTX-R sodium current by binding to a site of sodium channel protein in the cell membrane at the beginning of the reaction.And at the same time,due to the presence of LB,it could inhibit the peak values of TTX-R sodium current through the cAMP/PKA pathway,so the inhibition rate of the peak values of TTX-R sodium current could still be increased by the three combination containing LB.In summary,the combination of LB,CA and CB has a strong inhibitory effect on TTX-R sodium current at low concentration,which is caused by the strong synergistic effect among the three flavonoids.LB participates in the cAMP/PKA pathway when acting on DRG cells,which is the key to the strong synergistic effect of the three flavonoids.