Effects Of Different Frequency Electroacupuncture On Type Ⅱ Diabetic Neuropathic Pain Mediated By P2X3 Receptors And CGRP In Rat Dorsal Root Ganglion

Objective To observe the effects of electroacupuncture(EA)with high and low frequencies on type Ⅱ diabetic neuropathic pain(DNP)animal model,and there effects on P2X3 receptors and calcitonin gene-related peptide(CGRP)in dorsal root ganglion(DRG),it was designed to investigate the mechanism and choose the dominant EA frequency on type Ⅱ DNP.Methods SD rats were randomly divided into two groups:normal group and model group.After fed with normal diet and high fat-glucose diet respectively,both 2 groups were determined body weight(BW),fasting plasma glucose(FPG),fasting plasma insulin(FINs),paw withdrawal latency(PWL),and calculated insulin sensitivity index(ISI).Rats with insulin resistant were further injected intraperitoneally with streptozotocin(STZ,35 mg/kg)for once.2 weeks after STZ injection,FPG and PWL were determined again.Rats with DNP were then divided into DNP group,DNP+2 Hz EA group and DNP+100 Hz EA group.2 Hz EA and 100 Hz EA were applied to bilateral Zusanli(ST36)and Kunlun(BL60)from 2 weeks after STZ injection.EA stimulation of both EA groups:continuous wave,2 Hz and 100 Hz,intensity from 1 mA,15 min to 2 mA,15 min,30 min altogether;once daily for consecutive 7 days.Sciatic nerve was observed and L4-L6 DRG P2X3 receptor and CGRP expression were determined by immunofluorescence method.Results(1)After high fat-glucose diet for 5 weeks,BW of Model group was increased(P<0.05).After high fat-glucose diet for 5 weeks and 2 weeks after STZ injection,FINs of Model group was increased(P<0.05),ISI of Model group was decreased(P<0.05).2 weeks after STZ injection,FPG of Model group was significantly increased(P<0.01).(2)Light microscope result:sciatic nerve of DNP rats shown decreased fiber density,abnormal axon structure and swell,unclear nerve fiber boundary.Electron microscope result:sciatic nerve of DNP rats shown:fiber of DNP rats with myelin disruption and dissolute axoplasm.There was a visible demyelination and separation of myelin sheath.(3)2 weeks after STZ injection,PWLs of DNP group,DNP+2 Hz EA group,DNP+100 Hz EA group were significantly decreased compared with Normal group(P<0.01).EA 3 D,5 D and 7 D,PWLs of DNP+2 Hz EA group,DNP+100 Hz EA group were significantly increased compared with DNP group(P<0.01),PWL of DNP+2 Hz EA group was significantly higher than DNP+100 Hz EA group(P<0.01).④Immunofluorescence result:Compared with Normal group,the expression of L4-L6 DRG P2X3 receptor of DNP group was increased(P<0.01).Compared with DNP group,the expression of L4-L6 DRG P2X3 receptor of DNP+2 Hz EA group was significantly decreased(P<0.01),and the expression of L5-L6 DRG P2X3 receptor of DNP+100 Hz EA group was decreased(P<0.05).Compared with DNP+100 Hz EA group,the expression of L4 DRG P2X3 receptor of DNP+2 Hz EA group was decreased(P<0.05).⑤Immunofluorescence result:Compared with Normal group,the expression of L4-L6 DRG CGRP of DNP group was increased(P<0.01).Compared with DNP group,the expression of L4-L6 DRG CGRP of DNP+2 Hz EA group and DNP+100 Hz EA group were decreased(P<0.01,P<0.01,P<0.05,respectively).Conclusion EA with different frequencies has an interventional effect on type Ⅱ DNP,and low frequency EA has a better analgesic effect on type Ⅱ DNP.The analgesic mechnisam of EA may be related to its inhibition on DRG P2X3 receptors and CGRP expression,and low frequency EA has a better inhibitory effect on L4 DRG P2X3 receptors than high frequency.