Study On The Electrophysiological Characteristics Of Mast Cells And The Antipruritic Mechanism Of Cimifugin In Xiaofengsan

The formation of itching is related to the mediators released from mast cells.Lots of ion channels changes during the progress of mast cell degranulation.These channels include calcium channel,potassium channel,chlorine channel,TRP channel and P2X channel.Ant-Ig E,ATP and the compound 48/80 are commonly used as mast cell activators.They could induce mast cells degranulation and inflammatory mediators production,which may be related with the increasing of intracellular calcium concentration.In our research,using tissue section staining,we observed that Mast cell degranulation,which play an important role in itch by compound 48/80 induced;Using whole-cell patch clamp recording method,we detected the changes of whole-cell current generated by compound 48/80 in mouse mast cell tumor P815 cells under different external fluid environments.The results showed that the change value of inward current induced by compound 48/80 in high K~+solution was greater than that of normal solution.In the low Cl~-solution,the changes of inward current and outward current induced by compound 48/80 were both greater than that of normal solution.We also found that the change of whole-cell currents induced by compound 48/80 was related to endogenous calcium ions.In addition,the inward and outward currents induced by compound 48/80 were blocked by the calcium-activated medium conductance potassium channel(Kca3.1)antagonist(Clotrimazole)and the calcium-activated chloride channel(CACC)antagonist(DIDS),respectively.Thus,these results indicated that the presence of Kca3.1 and CACC in mouse mast cell tumor P815 cells.We also recorded the whole-cell current in mouse peritoneal mast cells induced by compound 48/80 under the low Cl~-solution and contain inhabitors solution.The results demonstrated that the outward current induced by compound 48/80 under the low Cl~-solution and contain inhabitors solution was smaller than that of normal external fluid.We confirmed that the presence of calcium-activated chloride channels in mouse peritoneal mast cells.And we confirmed that the CACC on peritoneal mast cells of mice activated by compound 48/80 was correlated with intracellular calcium.Meanwhile,we also found that mouse peritoneal mast cells could be activated by extracellular high concentration ATP(5m M ATP),which could be blocked by P2X7 antagonist(AZ10606120).Results from these results,we concluded that P2X7 channel exists in mouse peritoneal mast cells.In addition,mouse peritoneal mast cells could also be activated under the acidic condition.Acid could induce the increasing of outward currents.In the research on the antipruritic mechanism of Xiaofengsan,we found that Xiaofengsan has an inhibitory effect on the inward current of P2X7 channels on mouse peritoneal mast cells activated by extracellular ATP.And cimicifuga is one of effective components in suppressing P2X7 channel.In addition,we examined the antipruritic mechanism of several components which isolated from Xiaofengsan by using calcium imaging technology.The effective compounds include matrine,osthole and cimicifugin,and the mast cell activators include compound 48/80,Ant-Ig E and different concentrations of ATP.We only found that the increasing of calcium ion concentration induced by 100?M ATP could be inhibited by matrine.