Endocannabinoid-IL-4 Positive Feedback Is The Key Mechanism Of Modulating The Analgesic Effect Of Combined-acupoint Electroacupuncture

Chronic pain,especially neuropathic pain,is a serious challenge to human beings,bringing physical and mental suffering.Pharmacological measures are still the dominant treatment.However,side effects of drugs have limited their application.Acupuncture and its related techniques like electroacupuncture（EA）have been widely applied in clinical for their sufficient analgesic effect and fewer side effects.But more work need to be done in optimizing the treatment scheme and clarifying the analgesic mechanism.Previous studies have shown that various EA treatment schemes based on different acupoints provided analgesic effects on neuropathic pain.But very few studies compared the analgesic effects among different EA schemes directly,which makes standardizing the scheme difficult.Endogenous analgesic substances and anti-inflammatory cytokines have been reported to participate in EA analgesia.However,whether their function is related to the specific acupoints remains unclear.And whether there is interaction between endogenous analgesic substance and cytokines is to be answered.In the current study,we employed chronic constriction of sciatic nerve injury（CCI）model,tried to find the optimized EA scheme by comparing the analgesic effect of single-acupoint and combined-acupoint EA treatment.We defined the key role of CB2 receptor and IL-4 in EA analgesia with molecular biological,behavioral,pharmacological and morphological techniques,and further revealed its mechanism by precisely regulate cannabinoid CB2 receptor on transgenic mice.Part 1:The different analgesic effects of single and combined-acupoint EA on neuropathic pain[Objectives]The study was done to compare the analgesic effects of single-acupoint EA and combined-acupoint EA,and explore whether the difference of analgesic effect is determined by different physical stimulation parameters.[Methods]1.Forty adult male SD rats were randomly divided into Control group,CCI group,Single group and Combined group.Sham operation was done in Control group,while CCI model was established in the other 3 groups.After CCI model established,different treatments were applied to rats for 2 weeks:immobilization for Control and CCI group;single-acupoint EA for Single group;combined-acupoint EA for Combined group.Paw withdrawal threshold（PWT）was measured before CCI surgery,before 1^st treatment,and after 2^nd,4^th,6^th 9^th,11^thand 13^thday’s treatment.2.Forty adult male SD rats were randomly divided into CCI,Single,High-Intensity,Combined and Sham-Combined group.All the rats were CCI modeled.Experimental treatments were consecutively performed for 2 weeks.The treatments to CCI,Single and Combined group were the same as the previous experiment.Single-acupoint EA with high intensity stimulation was applied to High-Intensity group,and Sham-Combined group was stimulated at sham acupoints.PWT was measured before CCI surgery,before 1^st treatment,and after 2^nd,4^th,6^th 9^th,11^thand 13^thday’s treatment.[Results]1.CCI modeling decreased PWT significantly（P<0.01 vs Control）;both single and combined-acupoint EA treatments suppressed CCI-induced pain by significantly elevating PWT（P<0.05,P<0.01 vs CCI,respectively）;the analgesic effect of combined-acupoint EA is superior to that of Single group（P<0.05）.2.The PWT of High-Intensity group was significantly higher than that of CCI group（P<0.05）,but still lower than Combined group（P<0.05）;PWT of Sham-Combined group was not superior to CCI group,even though the stimulating range was expanded.[Conclusion]The analgesic effect of combined-acupoint EA treatment is superior to single-acupoint EA,and this effect is not related to the increase of stimulation intensity or range.Part 2:The molecular mechanism of superior analgesic effect in combined-acupoint EA[Objectives]The study was done to compare the molecular mechanism of the difference in analgesic effect between single and combined-acupoint EA,by evaluating the expression of analgesic related molecules.[Methods]1.Twenty-four adult male SD rats were divided into Control,CCI,Single and Combined group.The modeling and treatments were conducted as what Part 1 illustrated.Animals were sacrificed at day 6 after treatment,and spinal cord tissue were collected.The quantity of Endorphin,Enkephalin,MOR,DOR were examined with Enzyme linked immunosorbent assay（ELISA）;MOR and DOR level were also tested with Western Blot method.2.The quantity of endocannabinoids（eCB）molecules:AEA,2-AG,and the receptors cannabinoid receptor 1（CB1R）,cannabinoid receptor 2（CB2R）were measured with ELISA;the level of CB1R and CB2R were also examined with Western Blots.3.The quantity of IL-1β,IL-6,TNF-α,IL-4 and IL-10 were measured with ELISA.4.Thirty-two adult male SD rats were randomly divided into CCI,Single,Combined and Combined+CBR Inhibitor group,and all rats were conducted with CCI surgery.Treatment in CCI,Single and Combined group were the same as what Part 1 experiments illustrated;the EA treatment of Combined+CBR Inhibitor group was the same as the Combined group;the treatments lasted for 6 consecutive days.Non-selective opioid antagonist naloxone were administrated to all animals intraperitoneally before each day’s EA;CB1R and CB2R antagonists were applied intrathecally before EA treatments of 1^st,3^rd,5^th day in Combined+CBR group.PWT were measured before CCI surgery,before 1^sttreatment,and after 2^nd,4^th,6^th day’s treatment.[Results]1.ELISA results indicated the level of Endorphin,Enkephalin,as well as MOR and DOR were increased in Single and Combined group（P<0.01 vs Control）;which is in accordance with the Western Blot results of MOR and DOR.2.ELISA result indicated combined-acupoint EA in Combined group increased the quantity of AEA,2-AG,CB1R and CB2R（P<0.05,P<0.05,P<0.01,P<0.05 vs Control,respectively）,and this effect was absent in Single group.Western blot assay of CB1R and CB2R confirmed the findings.3.The release of IL-1β、IL-6、TNF-αwere significantly increased in CCI group（P<0.05,P<0.01,P<0.01 vs Control,respectively）,both single and combined-acupoint EA suppressed this effect.Only combined-acupoint EA increased anti-inflammatory cytokines’release,include IL-4 and IL-10（P<0.01 vs Control）.4.Naloxone totally suppressed the analgesic effect of single-acupoint EA,but only partially suppressed the analgesic effect in Combined group;administration of cannabinoid receptor antagonists and naloxone together totally reversed combined-acupoint EA’s analgesic effect.[Conclusion]After 6 day’s treatment,both single and combined-acupoint EA activate endogenous opioid system in spinal cord,but only combined-acupoint EA activates eCB system and promotes anti-inflammatory cytokine release.Part 3:The“eCB-IL-4”positive feedback in spinal cord is the key mechanism of combined-acupoint EA analgesia[Objectives]The study was done to investigate how eCB system and anti-inflammatory cytokines participate combined-acupoint EA analgesia,and clarify the mechanism.[Mechods]1.Forty adult male SD rats were divide into CCI+Vehicle,EA+Vehicle,EA+AM281,EA+AM630 group randomly.All rats were conducted with CCI surgery.Immobilization treatment was applied on CCI+Vehicle group,while combined-acupoint EA was applied to the other 3 groups for 6 consecutive days.On 1^st,3^rd and 5^th day of EA treatment,CB1R antagonist AM281 and CB2R antagonist AM630 were administrated intrathecally to EA+AM281 and EA+AM630 group,respectively;Vehicle was applied to CCI+Vehicle and EA+Vehicle group at the same time.PWT was measured before CCI surgery,before1^st treatment,and after 2^nd,4^th,6^th day’s treatment;animals were sacrificed after last treatment to obtain spinal cord tissue for IL-4,IL-10 ELISA test.2.Forty adult male SD rats were randomly divide into CCI+Ig G,CCI+IL4ab,EA+Ig G and EA+IL4ab group,all rats were CCI modeled.Combined-acupoint EA was applied to the EA+Ig G and EA+IL4ab group for 6 consecutive days;immobilization treatment was applied on CCI+Ig G and CCI+IL4ab group.On 1^st,3^rd and 5^th day of EA treatment,IL-4 neutralizing antibody was intrathecally injected to CCI+IL4ab and EA+IL4ab group;control Ig G was intrathecally injected to CCI+Ig G and EA+Ig G group.PWT was measured before CCI surgery,before 1^st treatment,and after 2^nd,4^th,6^th day’s treatment;animals were sacrificed after 6^th day’s treatment to obtain spinal cord tissue for AEA,2-AG ELISA test and CB2R Western Blot analysis.3.Fifty-four adult male SD rats were randomly divided into Control（6 rats）,CCI（24rats）and EA（24 rats）group.Rats in Control group underwent sham operation and rats in CCI and EA group underwent CCI surgery.After model had been established,rats were treated with immobilization or combined-acupoint EA for 2 weeks.6 animals in CCI and EA groups were sacrificed after 1^st,3^rd,6^th and 13^th treatment respectively to obtain spinal cord tissue,then AEA,2-AG,IL-4 levels were tested with ELISA;CB2R expression was examined by Western Blot.All data was compared with the Control group,in which spinal tissue were collected after 13^th treatment.[Results]1.Both AM281 and AM630 suppressed EA’s analgesic effect,specifically,the PWTs of these EA+AM281 and EA+AM630 group were significantly lower than EA+Vehicle group（P<0.05）;AM630 also suppressed the release of IL-4（P<0.05 vs EA+Vehicle）.2.IL-4 neutralizing antibody suppressed EA’s analgesic effect,as the PWT was lower in EA+IL4ab group than EA+Ig G group（P<0.05）;the antibody also decreased the expression of CB2R,AEA and 2-AG（P<0.05,P<0.01,P<0.05 vs EA+Ig G,respectively）.3.ELISA tests indicated eCBs and anti-inflammatory cytokines increased significantly in EA group,but AEA and 2-AG reacted at 1^st day of treatment（P<0.05 vs other groups）,while IL-4 elevated at 6^th day（P<0.01 vs other groups）;Western Blot result demonstrated CB2R expression was significantly increased（P<0.01 vs other groups）since 3^rd day’s treatment.[Conclusion]Combined-acupoint EA treatment up-regulates the level of eCBs in spinal cord,which then promotes the release of IL-4 to exert analgesia;in the other way round,IL-4also promotes the release of eCBs and the up-regulation of CB2R expression.eCB and IL-4 form a positive feedback in the mechanism of analgesic effect.The release of eCB ligands is the initiating factor of the positive feedback.Part 4:The mechanism of the spinal microglia in the“eCB-IL-4”positive feedback in combined-acupoint EA’s analgesia[Objectives]The study was done to determine the location of CB2R during EA induced analgesia,and to explore the regulation of microglia function by the“eCB-IL-4”positive feedback.[Mechods]1.Thirty-six adult male SD rats were randomly divided into Control（4 rats）,CCI（16rats）and EA（16 rats）group.Modeling and treatment methods were the same as what used in the Part 3 experiments.Four animals in CCI and EA groups were sacrificed after 1^st,3^rd,6^th and 13^th treatment respectively to obtain spinal cord frozen slices,then Iba1（microglia marker）and CB2R,as well as Neu N（neuron marker）and CB2R,were co-stained to identify the location of CB2R in spinal cord.All data was compared with Control group,in which spinal tissue were collected after 13^th treatment.2.（1）Sixteen adult male SD rats were divided into Control,CCI,Single and Combined group,the modeling and treatments were the same as what used in the Part 1experiments.Rats were sacrificed after 6 days of treatment,the spinal cord were made frozen slices for immunofluorescence staining.Iba1 and CD86（M1 polarized microglia marker）,as well as Iba1 and CD206（M2 polarized microglia marker）,were co-stained.（2）Sixteen adult male SD rats were randomly divided into CCI+Vehicle,EA+Vehicle,EA+AM281 and EA+AM630 group,the modeling and treatments were the same as what used in the Part 3 experiment.The spinal cords were obtained from each group after 6^thday’s treatment,and were frozen sliced.Iba1-CD206 were then co-stained.（3）Sixteen adult male SD rats were randomly divided into CCI+Ig G,CCI+IL4ab,EA+Ig G and EA+IL4ab group,the modeling and treatments were the same as what used in the Part 3 experiment.The spinal cords were obtained from each group after 6^th day’s treatment,and were frozen sliced.Iba1-CD206 were then co-stained.3.Formulate interfering virus that can reduce the expression of CB2R in CD11b^Cremice,and its invalid control.Administrated the two viruses in the spinal cord of CD11b^Cremice and their homozygous wild type mice（WT）,respectively.Spinal cord slices were obtained after the viruses achieved its full performance,then examined whether lox P sequence in virus interacted with Cre enzyme in mice successfully.Thirty-six male CD11b^Cre mice were randomly divided into CCI+Scramble,CCI+Knockdown,EA+Scramble and EA+Knockdown group.Interfering virus was applied to CCI+Knockdown and EA+Knockdown group,control virus was applied to CCI+Scramble and EA+Scramble group.CCI surgery was performed 3 weeks after virus injection;combined-acupoint EA under isoflurane anesthesia were applied to EA groups for 6 days,and mice in CCI groups only received isoflurane anesthesia.PWT was measured before CCI surgery,before 1^st treatment,and after 2^nd,4^th,6^th day’s treatment.After all the treatments,mice were sacrificed to obtain spinal cord tissue.Three animals per group were used for Iba1-CB2R and Iba1-CD206 co-staining;6 samples per group were used for ELISA assay to measure the quantity of AEA,2-AG and IL-4.[Results]1.CB2R were expressed on both neurons and microglias.However,the neuronal CB2R was not changed by EA treatment,but the microglial CB2R expression was elevated.TheCB2R on microglia increased at 3^rd day after EA treatment（P<0.01 vs other groups）,and reached the peak at 6^th day.2.（1）Both single and combined-acupoint EA inhibited microglia polarization towards M1 phenotype,as the Iba1-CD86 co-staining rates in Single and Combined group were significantly lower than CCI group（P<0.01）;only combined-acupoint EA promoted microglia M2 phenotype polarization,as the Iba1-CD206 co-staining ratio in Combined group was significantly higher than other groups（P<0.01）;（2）CB2R antagonist AM630 inhibited microglia polarization to M2,the ratio of M2polarized microglia were decreased in EA+AM630 group（P<0.01 vs EA+Vehicle）;（3）IL-4 neutralizing antibody suppressed microglia M2 polarization,as the Iba1-CD206 co-staining ratio was decreased in EA+IL4ab group（P<0.01 vs EA+Ig G）.3.The lox P sequence carried by virus could interact with Cre enzyme in the mice.The analgesic effect of EA+Knockdown group was blocked by interfering virus,and the PWT was lower than that of EA+Scramble group（P<0.05）.The number of CB2R expressed on microglia in EA+Knockdown group was decreased（P<0.01 vs EA+Scramble）,the Iba1-CD206 co-staining ratio was also lower than that of EA+Scramble group（P<0.01）.The quantity of IL-4,AEA and 2-AG in spinal cord of EA+Knockdown group was significantly lower than EA+Scramble group（P<0.01,P<0.05,P<0.01,respectively）.[Conclusion]CB2R locates on spinal neurons and microglias.The expression of CB2R on microglia increases after combined-acupoint EA treatment.The combined-acupoint EA treatment also promotes microglia polarization to M2 phenotype,and this process could be regulated by CB2R and IL-4.Microglial CB2R participates in microglia M2 polarization,and is a key factor for the“eCB-IL-4”positive feedback in the combined-acupoint EA’s analgesic pathway.[Summary]Both single and combined-acupoint EA suppresses neuropathic pain induced by CCI.However,combined-acupoint EA exerted stronger analgesic effect.The enhanced analgesia was not due to the augmentation of intensity or expansion of physical stimulation range,but the activation of eCB analgesic system and the pro-release of anti-inflammatory cytokines at the spinal cord.The“eCB-IL-4”positive feedback is the key mechanism for achieving enhanced analgesic effect in combined-acupoint EA.eCBs released after EA exert on the activation of microglial CB2R,promote M2 polarization of microglia,and the IL-4 release.In the other way round,IL-4 could also activate the eCB system by increasing the level of eCB ligands and CB2R expression.