The Analgesic Mechanism Of Angelica Dahurica Based On Pain-related Ion Channel (TRPV1)

Objective:Angelica dahurica,belonging to the family Apiaceaei,is one of the most commonly used traditional Chinese medicines in clinical treatment.Angelica dahurica is widely used in the treatment of various types of pain,especially in the treatment of headache,toothache,abscess,furunculosis,and acne.Angelica dahurica can also be used with other traditional Chinese medicines to alleviate pain in all parts of the body.However,little is known about Angelica dahurica analgesic molecular mechanism underlying pain relief.Here,we investigated the analgesic effect of the Angelica dahurica extracts(ADE)in acute pain models and CFA-induced inflammatory pain mice model,and its mechanism based on the transient receptor potential vanilloid member 1(TRPV1)was also explored.This will provide new ideas for the research of the analgesic mechanism of Angelica dahurica and its clinical application.Methods:(1)Preparation of the Angelica dahurica extracts(ADE):The dry herb of Angelica dahurica(200g)were extracted with boiling water twice and filtered through gauze.Filtrates were then evaporated by rotary evaporation under vacuum at 60℃.Finally,the semi dry mass of ADE were prepared for subsequent experimental study.(2)The male mice(C57BL/6)about 8 weeks old were randomly divided into four groups(n=6).The mice in each group were given different doses of ADE(20/100/600 mg/kg)and distilled water respectively.The mice in each group were immersed in water bath at 48℃ before and 1,2,3,4 and 5 hours after administration.The latency of tail flick in each group was recorded within 5 hours.(3)Mice(C57BL/6)were randomly divided into control group(n=6)and ADE group(n=6),distilled water and ADE(100mg/kg)were administered 2 hours before experiment,and then through thermal radiation experiment,Von-Frey and cold stimulation experiment measured the the two groups mice of thermal withdrawal latency(TWL),mechanical withdrawal threshold(MWT)and tail-flick latency for cold stimulation.(4)Mice(C57BL/6)were randomly divided into control group(n=6)and ADE group(n=6).Distilled water and ADE(100 mg/kg)were given intragastrically 2 hours respectively before the experiment,then capsaicin(500μM,10μL/mouse)was injected subcutaneously into the right paw of two groups of mice to cause pain.The number of foot contractions and licking time of mice in 5 minutes were recorded and counted.Distilled water and ADE(100mg/kg)were given intragastrically for 2 hour respectively.Inflammatory pain was induced by subcutaneous injection of 5%formalin(10μL/mouse)into the right paw of two groups of mice.The number of foot contractions and the time of paw licking in one hour were recorded and counted.(5)Trpvl-/-mice and their wild-type homologous(WT,C57BL/6)mice were used to observe the difference of behavior before and after the same administration of distilled water and ADE(100mg/kg)by heat stimulation test and capsaicin-induced pain test,and to study the effect of ADE on pain threshold of Trpv1-/-mice.(6)The DRG neurons of mice(C57BL/6)were cultured and the effects of ADE on capsaicin sensitivity of DRG neurons of mice were observed by calcium ion imaging.(7)Sixty mice(C57BL/6)were randomly divided into four groups(n=15):chronic inflammatory pain mice model groups induced by complete Freund’s adjuvant(CFA):CFA model mice+Distilled Water;Diclofenac group:CFA model mice+Diclofenac(Diclofenac,10mg/kg/day);ADE-M group:CFA model mice+ADE-M(ADE,100 mg/kg/day);ADE-H group:CFA model mice+ADE-H(ADE,600mg/kg/day).During the 14 days from the day of modeling to the end of modeling,mice in each group were administered intragastrically every day with the same volume of distilled water or ADE or Diclofenac.(8)After the model was established,the mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)of four groups of mice were measured and analyzed by Von-Frey experiment and thermal radiation experiment.(9)On the 14th day after the model was established,DRG from the L4-6 of the affected side in mice was taken.Calcium imaging experiment was used to observe the different reactions of DRG neurons to capsaicin-induced Ca2+influx in mice of each group,in order to evaluate the changes of TRPV1 physiological activity in mice of each group.(10)The activity and expression of TRPV1 in DRG(L4-6 of the affected side)of mice in each group were detected and analyzed by immunofluorescence staining and Western BlottingResults:(1)One or two hours after administration of ADE(600mg/kg),the tail flick latency of mice was significantly different from that of control group,which indicated that ADE could reduce the sensitivity of mice to noxious heat stimulation.(2)After 2 hours of ADE(100mg/kg)administration,the sensitivity of mice to noxious heat and cold stimulation was significantly reduced.At the same time,ADE had no significant effect on acute pain behavior induced by noxious mechanical stimulation in mice.(3)ADE inhibited the pain induced by capsaicin and formalin in mice,which was manifested in the decrease of the number of foot contractions and licking time induced by capsaicin and formalin in ADE(100mg/kg)group.(4)The sensitivity of WT mice to noxious heat stimulation and capsaicin-induced pain was significantly different before and after administration of ADE(100mg/kg),but there was no significant difference between Trpvl-/-mice and administration of ADE(100mg/kg)mice before and after ADE(100mg/kg).(5)After treatment with ADE perfusion(610-6mg/L),the percentage of capsaicin response(Cap+ADE,P<0.001)and fluorescence intensity ratio(Cap+ADE,P<0.001)of DRG neurons decreased significantly,and the proportion of capsaicin response and fluorescence intensity ratio of DRG neurons increased again after re-elution.(6)Within 24 hours to 14 days after CFA injection,the mechanical hyperalgesia induced by CFA in mice treated with ADE(100/600 mg/kg)was significantly lower than that in mice of Con.The analgesic effect of ADE was better than that of Diclofenac.At the same time,compared with the TWL values of mice in Con group,the TWL values of mice in ADE-M group,ADE-H group and Diclofenac group increased significantly 2-14 days after CFA treatment.(7)Calcium imaging results showed that compared with Con group,the percentage of capsaicin-responsive cells in DRG of mice in Diclofenac,ADE-M and ADE-H groups decreased by 56.8%,47.3%and 48.7%respectively.The fluorescence intensity ratio of capsaicin response of DRG neurons in Diclofenac,ADE-M and ADE-H groups was significantly lower than that in Con group(P<0.001).(8)The results of immunofluorescence staining showed that:compared with Con group,the percentages of TRPV1+neurons in the DRG of mice in Diclofenac sodium,ADE-M and ADE-H groups were significantly reduced(Con,34.7%+1.5%;Diclofenac,28%+1.3%;ADE-M,20.5%+0.9%;ADE-H,26.7%+1.5%;P<0.001).(9)The relative quantification of TRPV1 protein in DRG of mice was detected by Western blotting.We found that the expression of TRPV1 in DRG of ADE-M group,ADE-H group and Diclofenac group decreased significantly compared with that of Con group,especially in ADE-M group(P<0.001).Conclusion:ADE can alleviate CFA-induced inflammatory pain in mice by inhibiting the activity and expression of TRPV1 in DRG neurons,which at least partly explains its analgesic effect and mechanism.ADE has significant analgesic effect,suggesting that it may become a new class of important drugs for inflammatory pain.