| Pain is an uncomfortable feeling and emotional experience that is associated with potential or actual tissue damage.Chronic pain is defined as pain that persists or continues intermittently for more than 3 months.Especially neuropathic pain,has a long course and is prone to various complications.In addition,patients are more prone to suffer from insomnia,anxiety,depression and other sub-health problems,which seriously affect the quality of life.However,most of the drugs used to treat chronic pain have different degrees of side effects,and more than half of the patients are dissatisfied with the analgesic effect of the drugs.Asarum was first published in Shennong Herbal Classic in the late Eastern Han Dynasty.It is a commonly used traditional Chinese medicine and widely used in the treatment of headache,toothache,nose,trigeminal neuralgia and rheumatic pain.Studies have reported that the volatile oil components of Asarum and the water decoction have good analgesic effects.Transient Receptor Potential Vanilloid 1(TRPV1),as a member of TRP family,is defined as a pain gating molecule.TRPV1 is one of the effective targets for the treatment of chronic pain.It plays an important role in the occurrence and development of neuropathic pain.In order to further evaluate the analgesic effect of Asarum,the analgesic activity of water extract of Asarum(WA)and Asarinin was systematically evaluated by various acute and chronic pain behaviors.The mechanism of WA analgesia was discussed based on the expression and function changes of TRPV1 in the mouse model of chronic constriction injury(CCI).Objective:Several acute and chronic pain models were used to evaluate the analgesic activity of WA and Asarinin.Based on the expression and function of TRPV1,the analgesic mechanism of WA was studied at the level of neuropathic pain model.Methods:(1)The tail soaking test was used to evaluate the effect of WA on pain behavior induced by noxious heat stimulation,and to determine the optimal time and concentration of analgesia.Six to eight weeks old male C57BL/6 mice were randomly selected.Eight mice in each group were treated with WA(32 mg/kg)by gavage.The tail flick time of the experiment of soaking in water bath at 48 for 0,1,2,3,4,5 hours after gavage was recorded to determine the optimal analgesic time.5.3 mg/kg,32mg/kg and 192 mg/kg WA were given by gavage respectively.The tail-flicking time of soaking experiment was recorded after 2 hours of gavage.and the optimal analgesic concentration was determined.(2)Thermal irradiation and VonFrey experiment were used to evaluate the effects of WA on pain induced by noxious heat stimulation and mechanical stimulation.Sixteen mice were randomly divided into Control group(distilled water)and WA group(32 mg/kg).Then the mechanical withdrawal threshold(MWT)and the thermal withdrawal latency(TWL)of mice were measured after 2 hours of intragastric administration of saline and WA,respectively.(3)To evaluate the effect of WA on pain induced by capsaicin and formaldehyde Acute pain test of capsaicin:Control group(distilled water),WA group(32mg/kg);1%formalin pain test:Control group(distilled water),WA group(32mg/kg),6 male mice were randomly selected in each group.Two hours later,capsaicin(500μM/10μL/mouse)and 1%formalin(10μL/mouse)were injected into the right paw of mice.Video recording was performed for 30 minutes in the capsaicin injection group and for 1 hour in the formalin group,and the times of raising feet and licking feet were counted.(4)To evaluate the effect of WA on pain sensitization induced by neuropathic pain.Thirteen male mice in each group were randomly divided into six groups.They were as follows:blank(naive)group,sham operation group,CCI group,WA-L group,WA-H group,gabapentin(GBPT)group.After 5 days of establishing CCI model,obvious pain behavior was observed and drug administration was started:blank group,sham operation group and CCI group were given ultrapure water;WA-L group was given 32mg/kg/day WA by gavage,WA-H group was given 96mg/kg/day WA by gavage,GBPT group was given 0.2 g/kg/day plus bar pentin by gavage once a day for 9 days.The body weight of six groups of mice was measured The mechanical withdrawal threshold and the thermal xwithdrawal latency were measured at 0,1,3,5,7,9,11 and 13 days after operation.(5)Based on the function and expression activity of TRPV1,the mechanism of WA inhibiting pain sensitization induced by neuropathic pain was explored.On the 14th day,the DRG of L4-L6 and sacrum of mice were taken 2 hours after gastric administration.The physiological activity of TRPV1 of DRG neurons in each group was observed by calcium ion imaging.The lumbar L4-L6 and sacral DRG of mice were taken and the expression of TRPV1 in each group was observed by immunofluorescence staining and real-time fluorescence quantitative-polymerase chain reaction.(6)Asarinin is an indictor chemical constituent of Asarum To investigate the effects of Asarinin on pain induced by noxious heat stimulation and noxious mechanical stimulation.Six male mice in each group were divided into control group,Asarinin-L group(0.8mg/kg),Asarinin-M group(2.5mg/kg)and Asarinin-H group(7.5mg/kg).The MWT and TWL were measured half an hour after subcutaneous injection of 10μL/mouse into the right sole of mice.(7)To evaluate the effect of Asarinin on pain sensitization induced by inflammatory pain.They were divided into control group(Saline+1%ethanol)and Asarinin-L group(8mg/kg),Asarinin-M group(25mg/kg)and Asarinin-H group(75mg/kg),with 8 mice in each group.Inflammatory pain model induced by complete Freund’s adjuvant(CFA)was given orally on the second day.Ten times of stimulation of 0.4g and 0.07g were given to the right sole of mice.The proportions of raising feet were counted after 1 h and 2 h of administration,and TWL was measured.Results:(1)WA can inhibit the pain caused by noxious heat stimulation for up to 5 hours,and the analgesic effect is the best after 2 hours of administration.WA inhibits the pain caused by noxious heat stimulation in a dose-dependent manner,and the analgesic effect is most obvious at the concentration of 32mg/kg.(2)Thermal irradiation and VonFrey experiments showed that WA significantly inhibited noxious thermal and mechanical stimulation induced pain behavior.(3)Capsaicin-induced pain experiment and formalin-induced pain experiment showed that the pain behavior induced by capsaicin was inhibited in mice treated with WA,and the number and time of licking feet were significantly decreased,while the pain caused by formalin was not inhibited.(4)During the establishment of CCI model,the mechanical withdrawal threshold and the thermal withdrawal latency of mice showed that they decreased from the 3rd day after operation and reached the lowest value on the 5th day.However,the mechanical withdrawal threshold and the thermal withdrawal latency increased slowly from the 7th day after administration,reached the peak on the 9th day,and maintained a stable threshold on the 9th to 13th day,and entered the plateau stage.On the 13th day,the experimental results showed that naive group and sham group were basically the same,but there was no difference;the thresholds of CCI group were significantly decreased(P<0.001);the mechanical withdrawal threshold and the thermal withdrawal latency were significantly increased in WA-L group,WA-H group and GBPT group,compared with CCI group,the difference was significant(P<0.001).Moreover,WA-L had the highest threshold at 2 hours of intragastric administration,and had the most obvious analgesic effect.The difference between WA-L and CCI was significant at 6 hours,indicating that the analgesic effect of WA could reach 6 hours or more.(5)Calcium imaging results showed that both WA-L(6.4μg/mL)and WA-H(64μg/mL)could effectively inhibit the calcium influx induced by capsaicin,a TRPV1 agonist(P<0.01 or P<0.001).Calcium imaging results showed that after the establishment of CCI model,the proportion of DRG neurons responding to TRPV1 agonist capsaicin increased significantly(P<0.05);after administration,the proportion of DRG neurons responding to capsaicin in WA-H and GBPT groups was significantly decreased(P<0.05 or P<0.001).Although there was no significant difference in WA-L group and CCI group,there was a downward trend.(6)Immunofluorescence staining showed that the percentage of TRPV1+neurons increased significantly after CCI(P<0.05),and the percentage of TRPV1+ neurons decreased significantly in either WA-L group,WA-H group or GBPT group(P<0.05 or P<0.001)(7)Real-time quantitative PCR results showed that the expression of TRPV 1 was increased in CCI group(P<0.05),but decreased significantly in WA-L group,WA-H group and GBPT group(P<0.05 or P<0.001 or P<0.01).(8)Asarinin-M group(2.5mg/kg)could effectively inhibit the pain induced by noxious heat stimulation and mechanical stimulation.(9)TWL in the chronic inflammatory pain model induced by complete Freund’s adjuvant decreased significantly on the second day of modeling,but increased in each group 1 hour after administration,and the analgesic effect of Asarinin-M group(25mg/kg)was the best after 2 hours(p<0.01).Vonfrey experiment showed that the frequency of foot-lifting increased significantly on the 2nd day of modeling under 0.4g and 0.07g stimulation.After administration,the frequency of foot-lifting decreased gradually after 1 hour and reached the lowest level after 2 hours(P<0.05 or P<0.001 or P<0.01).Conclusion:Water extract of Asarum and its index component Asarinin have significant analgesic effect on many kinds of acute and chronic pain.Water extract of Asarum inhibits CCI-induced pain behavior by regulating the expression and activity of TRPV 1. |
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