| Objective1.To determine the time that neuropathic pain occurred in diabetic rats after streptozotocin(STZ)injection,and confirm that the occurrence and maintenance of diabetic neuropathic pain(DNP)are related to PKC/NF-κB pathway,activation of B1 R in microglia,the increased expression of inflammatory cytokines and pain promoted neuropeptides in spinal dorsal horn(SDH).2.Study on the analgesic effect of EA at different frequencies in DNP model rats,and determine which frequency has better analgesic effect on DNP.3.The dominant frequency electroacupuncture was selected to study the molecular mechanism of EA for treating DNP,to determine whether EA inhibits PKC/NF-κB pathway,the activation of B1 R in microglia,and downregulates the expression of inflammatory cytokines and pain promoted neuropeptides in SDH to relieve DNP.MethodExperiment 1 Study on the mechanism of PKC/NF-κB pathway and Microglia B1 R activation at SDH involved in occurrence and maintenance of DNP.Male SD rats were randomly divided into vehicle group(n=20)and model group(n=96).Type 1 diabetes model was established by intraperitoneal injection of STZ.Fasting blood glucose(FBG),body weight(BW),paw withdrawal latency(PWL)and paw withdrawal threshold(PWT)were monitored at baseline,one week,two weeks,three weeks and four weeks after injecting STZ to determine the establishment of DNP model.Western blotting was used to detect the expression of p-PKC、NF-κB p65、B1R、CD11b、IL-1β and TNF-α protein in the SDH of rats.The m RNA levels of B1 R,IL-1 β and TNF-α in SDH were detected by RT-q PCR,and the positive co-expression of CD11b/B1 R at spinal and SP and CGRP at spinal were detected by immunofluorescence labeling.Experiment 2 Study on the analgesic effect of EA at different frequencies in DNP model rats.Male SD rats were randomly divided into vehicle group(n=8),DNP group(n=10),2Hz EA group(n=10),100 Hz EA group(n=10).Bilateral “Zusanli”(ST36)and“Kunlun”(BL60)were selected for EA treatment,the 2Hz EA group was treated with2 Hz EA,while the 100 Hz EA group was treated with 100 Hz EA,30 minutes for each time(the current was 1 m A in the first 15 min and 2 m A in the last 15 min).The EA treatment started from two weeks(15 days)after modeling,once a day for one week.The rats in the vehicle group and DNP group were given same calming procedure without EA treatment.Fasting blood glucose,body weight,paw withdrawal latency and paw withdrawal threshold were monitored were detected and recorded.Experiment 3 Study on the mechanism of electroacupuncture alleviates DNP by inhibitng activation of microglia B1 R at SDH.The rats were divided into vehicle group(n=16),DNP group(n=20)and DNP+EA group(n=20).After confirming that 2 Hz frequency was better than 100 Hz on treating DNP in research 2,2Hz electroacupuncture was selected to study the mechanism of electroacupuncture analgesia,the treatment of EA was the same as research 2.Western blotting was used to detect the p-PKC、NF-κB p65、B1R、CD11b、IL-1β、TNF-α protein expression in the SDH in rats.The m RNA levels of B1 R,IL-1 β and TNF-α in SDH were detected by RT-q PCR,and the positive co-expression of B1R/CD11 b at spinal and SP and CGRP at spinal were detected by immunofluorescence labeling.ResultsExperiment 1 Study on the mechanism of PKC/NF-κB pathway and Microglia B1 R activation at SDH involved in occurrence and maintenance of DNP.1.Observation of the BW,FBG,PWL and PWT of rats: Compared with the vehicle group,1 week,2 weeks,3 weeks and 4 weeks after STZ injection,the body weight of model group rats significantly decreased(P <0.01)and the fasting blood glucose significantly increased(P <0.01);Compared with the vehicle group,after 2 weeks,3weeks and 4 weeks of STZ injection,the PWL and PWT decreased significantly in the model group(P <0.01).2.Observation of the p-PKC、NF-κB p65、CD11b、B1R、IL-1β protein expression in SDH of rats :Compared with the Vehicle group,after 1 week of STZ injection,the expression of NF-κB p65,CD11 b,B1R and IL-1β in the SDH of the model group increased(P <0.01,P <0.01,P <0.05,P <0.01);Compared with the vehicle group,2and 3 weeks after STZ injection,the expression of p-PKC,NF-κB p65,CD11 b,B1R,TNF-α and IL-1β in the SDH of model group increased(P <0.01,P <0.01,P <0.01,P<0.05,P <0.01,P <0.01);Compared with the vehicle group,after 4 weeks of STZ injection,the expression of p-PKC,NF-κB p65,CD11 b and TNF-α in the spinal Dorsal Horn of the model group increased(P <0.01,P <0.01,P <0.05,P <0.01).3.Observation of B1 R,TNF-α,IL-1β m RNA levels in SDH of rats: Compared with the vehicle group,after 1 week of STZ injection,the m RNA levels of B1 R and IL-1βin SDH of the model group increased significantly(P <0.01);after 2 weeks of STZ injection,the m RNA levels of B1 R and TNF-α in SDH of the model group increased(P <0.05);after 3 weeks of STZ injection,the m RNA levels of B1 R,IL-1β and TNF-αin SDH of the model group increased(P <0.01,P <0.05,P <0.05);after 4 weeks of STZ injection,the m RNA levels of B1 R in SDH of the model group increased(P<0.05).4.Observation of the positive co-expression of B1R/CD11 b and the positive expression of SP,CGRP at spinal.Compared with the vehicle group,after 1 week,2weeks,3 weeks and 4 weeks of STZ injection,the positive expression of CD11 b,B1R,SP and CGRP in the SDH of the model group increased(P <0.01 or P <0.05).CD11 b of microglia was expressed in the whole SDH of rat,B1 R,SP and CGRP were mainly expressed in the superficial layer of SDH of ratExperiment 2 Study on the analgesic effect of EA at different frequencies in DNP model rats.At 1 week,compared with vehicle group,the body weight of DNP group,2Hz EA group and 100 Hz EA group decreased significantly(P <0.01),but the fasting blood glucose increased significantly(P <0.01);At 2 weeks,compared with vehicle group,the PWL and PWT of DNP group rats,2Hz EA group and 100 Hz EA group decreased significantly(P <0.01);At 3 weeks,compared with DNP group,the PWL and PWT of2 Hz EA group and 100 Hz EA group increased significantly(P <0.01),and the PWL and PWT of 2Hz EA group significantly increased compared with 100 Hz EA group(P<0.01).Experiment 3 Study on the mechanism of electroacupuncture alleviates DNP by inhibitng activation of microglia B1 R at SDH.1.Observation of the BW,FBG,PWL and PWT of rats: 1week after STZ injection,compared with the normal group,the body weight of DNP group and 2Hz EA group decreased significantly(P <0.01),and the fasting blood glucose increased significantly(P <0.01);At 2 weeks,compared with the normal group,the PWL and PWT of DNP group and 2Hz EA group decreased significantly(P <0.01);At 3 weeks,compared with DNP group,the PWL and PWT of 2Hz EA group increased significantly(P <0.01);2.Observation of the p-PKC、NF-κB p65、CD11b、B1R、IL-1β protein expression in SDH of rats:Compared with the normal group,the expression of p-PKC,NF-κB p65,CD11 b,B1R,TNF-α and IL-1β in SDH of DNP group increased(P <0.01,P <0.01,P<0.01,P <0.05,P <0.05,P <0.05);Compared with DNP group,the expression of p-PKC,NF-κB p65,CD11 b,B1R,TNF-α and IL-1β in SDH of DNP+EA group decreased(P <0.05,P <0.01,P <0.05,P <0.05,P <0.05,P <0.01).3.Observation of B1 R,TNF-α,IL-1β m RNA levels in SDH of rats: Compared with the normal group,there were no significant differences in the level of TNF-α m RNA in SDH of DNP group(P >0.05),the levels of B1 R and IL-1β m RNA in SDH of DNP group increased(P <0.01);Compared with the DNP group,there were no significant differences in the levels of TNF-α m RNA in SDH of DNP group(P >0.05),the levels of B1 R and IL-1β m RNA in SDH of DNP group decreased(P <0.01).4.Immunofluorescence results: Compared with the normal group,the positive expression of CD11 b,B1R,SP and CGRP in SDH of DNP group was increased(P<0.01);Compared with DNP group,the positive expression of CD11 b,B1R,SP and CGRP in the spinal Dorsal Horn of DNP+EA group were decreased(P <0.05,P <0.01,P <0.01).Conclusions:1.Diabetic rats develop neuralgia at 2 weeks after STZ injection,the occurrence and maintenance of DNP may be related to PKC/NF-κB pathway and activation of B1 R in microglia,as well as up-regulation of inflammatory cytokines and pain promoted neuropeptides at spinal dorsal horn.2.Both 2Hz electroacupuncture and 100 Hz electroacupuncture have analgesic effect on DNP model rats,but the analgesic effect of 2Hz electroacupuncture is better.3.The mechanism of 2Hz electroacupuncture alleviate DNP may be related to regulating PKC/NF-κB pathway,inhibiting activation of B1 R in microglia and downregulating the expression of inflammatory cytokines and pain promoted neuropeptides at spinal cord horn. |
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