Synergistic Analgesic Effect On Inflammatory Pain By Metformin And Electroacupuncture And Moxibustion And Its Purinergic Signaling Mechanisms

ObjectiveTo explore the synergistic effect of metformin on EA or moxibustion induced analgesia and find out how do purinergic signaling participate in the mechanisms.MethodsC57BL/6J mice were used as the study subjects.Screening mice with a baseline thermal radiation pain threshold of 10 s~16 s were randomly divided into control group and model groups.The chronic inflammatory pain models were induced by injecting 20 ?L of 0.5 mg/mL Complete Freund's adjuvant into the right hind paw.The pain threshold of mice was measured on the fourth day after modeling.If the pain threshold decreased by more than 50%,the model was successful.On the 4th day after model establishment,intervention like electroacupuncture,moxibustion,metformin acupoint injection,metformin + electroacupuncture,metformin + moxibustion were performed on the right “Zusanli” acupoint of mice;The mice in the intervention group were applied with electroacupuncture or moxibustion for 30 min at the right “Zusanli” acupoint of mice;The pain threshold of the right hind paw of each group was determined before modeling,on the 4th day after modeling(before intervention),0 min,30 min,60 min,90 min after the intervention by Plantar Test Apparatus(Hargreaves method,PL-200,Tai Meng,China).After blood was taken from the tip of the mouse,the blood glucose level was detected by blood glucose meter.The expression of AMPK mRNA in local acupoints was detected by real-time fluorescent quantitative PCR.The concentration of local adenosine in acupoints was detected by microdialysis combined with high performance liquid chromatography.Statistical analysis was performed by SPSS23.0 statistical software.Results 1.The analgesic effect of metformin on CFA model mice(1)Compared with the control group,the pain threshold of model group was decreased(P<0.05).Compared with the CFA group,the TWL of 4 mg metformin acupoint injection group was significantly increased,and the difference was statistically found at 30-120 min(P<0.05);TWL of 2 mg metformin group increased,and the difference was statistically significant at 60 min(P<0.05).(2)There was no significant difference in blood glucose measured before and after treatment between control group and CFA group(P>0.05).In metformin injection group,the blood glucose did not change significantly after metformin injection for 60 minutes(P>0.05).The blood glucose in gavage group decreased a lot after being intervened for 60 minutes(P<0.05).2.Effect of metformin combining with EA or moxibustion on analgesic effect of CFA model mice(1)Pain threshold of model group increased after metformin acupoint injection.TWL of mice in model group increased after EA treatment.EA was given after metformin injection,and the TWL was significantly improved compared with applying metformin alone or EA alone(P<0.05),and the difference was statistically significant(P<0.05).(2)Pain threshold of model group increased after metformin acupoint injection.TWL of mice in model group increased after moxibustion treatment.Moxibustion was given after metformin injection,and the TWL was significantly improved compared with applying metformin alone or moxibustion alone(P<0.05),and the difference was statistically significant(P<0.05).3.The expression of AMPK mRNA in Zusanli acupoint Compared with control group,the expression of AMPK mRNA in local acupoint of mice in CFA group was significantly decreased.The difference was extremely significant(P<0.01).Compared with CFA group,the expression of AMPK mRNA in local acupoint of mice in CFA+met,CFA+met+EA and CFA+met+Moxi group was significantly increased(P<0.01).4.The effect of metformin on concentration of adenosine in local acupoints Compared with control group,the local adenosine content of Zusanli acupoint in CFA group and the CFA with metformin group increased significantly(P <0.05),but compared with the CFA group,the concentration of adenosine at Zusanli acupoint in metformin group did not change significantly after metformin injection(P> 0.05).Conclusion 1.Dosage of 4 mg metformin acupoint injection is more effective and safer to relieve pain.2.Metformin could synergize the analgesic effect of EA or moxibustion on inflammatory pain.3.The expression of AMPK mRNA in local Zusanli acupoints might be involved in mediating the analgesic effect of metformin on inflammatory pain model mice;The expression of AMPK mRNA in local Zusanli acupoints might be involved in mediating the analgesia of combining metformin with EA or moxibustion.4.Metformin may not exert analgesic effect on inflammatory pain mice by increasing local adenosine concentration.