Study On The Analgesic And Antipruritic Mechanisms Of Saponins From Stauntonia Chinensis And Its Active Components By Modulating Acid-sensing Ion Channels

Stuntonia chinensis is a traditional medicine with analgesic effects.Injection and tablets made from Stuntonia chinensis have been widely used in clinic.It has been found that pentacyclic triterpenoid saponin（PTS）could inhibit capsaicin-induced TRPV1 receptor current,which indicates that the modulation of TRPV1 channel by Stuntonia chinensis may be the cause for its analgesic effect.Acid sensing ion channels（ASICs）,which are similar to TRPV1,are also one of the important pathways of pain signal conduction.ASICs not only participate in the process of pain conduction,but participate in the signal conduction of taste and itch.Therefore,we speculate that PTS may also be able to modulate ASICs.In this study,ASICs were targeted to explore the mechanism of PTS in pain and itch conduction,and what active components of PTS play a key role in the mechanism.The whole-cell patch clamp recordings were performed to observe different concentrations of PTS against p H5.0-induced ASICs current.The results showed that the concentration of 20μg/m L,100μg/m L and 500μg/m L PTS could inhibit the peak current of ASICs,and and the inhibition of 500μg/ml PTS was significant.In animal pain model,PTS(20 mg·kg^-1)has analgesic effect on the glacial acetic acid-induced acute inflammatory pain,and the activator 2-guanidine-4-methylquinazolin（GMQ）（10 m M）of ASICs could antagonize some of the analgesic effects of PTS;it also has analgesic effect on the Complete Freund’s Adjuvant（CFA）-induced chronic inflammatory pain.By immunofluorescence,it was found that PTS can down-regulate the expression of ASIC3 protein,the results indicate that PTS can produce analgesic effect by modulating ASICs.In AEW（acetone/diethylether and water）-induced dry skin disease model,PTS(20 mg·kg^-1)could significantly reduce the number of scratching,relieve chronic itching symptoms,and reduce the expression of inflammatory factors（IL-1β,IL-6,TNF-α）in animal serum.As mentioned above,PTS can block the conduction of pain and itch signals by modulating ASICs current in DRG neurons.In order to elucidate the key active components of PTS modulating ASICs.Eight monomers with known chemical structure were isolated from the PTS.Reverse screening method was adopted to observe the combined effects of eight components,and then remove one component one by one.By comparing the pharmacological effects of seven component combinations and the original eight component combinations,the key active components that can reverse the pharmacological effects of the original eight component combinations were screened out.The whole cell patch clamp technique was used to observe the effects of different components of PTS on ASICs current.The results showed that compared with the pharmacological effects of the eight monomer combinations,the combination of the remaining seven monomer combinations lost the inhibition of ASICs current after removing YF-33 and YF-49respectively.Therefore,we speculate that YF-33 and YF-49 are the key active components of PTS to modulate ASICs current and play a key role in relieving pain and itching.The effects of different concentrations of YF-33 and YF-49 on ASICs current were also observed by whole cell patch clamp technique.The results showed that YF-33 and YF-49 could inhibit ASICs currents in a concentration dependent-manner,and their half maximal inhibitory concentration were 0.15036±0.0342μM,0.18051±0.0631μM.In animal pain model experiment,YF-33（15μM）and YF-49（18μM）had significant analgesic effect on glacial acetic acid-induced acute inflammatory pain,and GMQ could antagonize some of the analgesic effects of PTS.In AEW-induced dry skin disease model,YF-33（15μM）and YF-49（18μM）could significantly reduce the number of scratching,reduce the expression of inflammatory factors（IL-1β,IL-6,TNF-α）in animal serum.As mentioned above,YF-33 and YF-49achieved analgesic and antipruritic effects by modulating ASICs.According to experimental results mentioned above,the PTS can inhibit the ASICs current in DRG neurons,down regulate the expression of ASIC3 protein,block the transmission of pain and itch signals,and then play an analgesic and antipruritic role.Among them,YF-33 and YF-49 should be the key active components of PTS by modulating ASICs.