The Regulation Of DRG P2X3 Receptor In Rats With Type Ⅱ Diabetic Neuropathic Pain By Electrocupuncture With Low Frequency

Objective By observing the effects of electroacupuncture(EA)with low frequency on Type Ⅱdiabetic Neuropathic Pain(DNP),the expression of L4-L6 DRG P2X3 receptors and p-PKC of rats,to explore the effects of electroacupuncture with low frequency on Type Ⅱ diabetic Neuropathic Pain and its possible mechanism from behavioral observation and protein expression.Methods Part 1:Thirty five normal SD rats were randomly divided into Normal group(8 rats)and Model group(27 rats).The Model group was fed with high fat and high sugar diet compositing a small dose of streptozotocin(STZ,35 mg/kg)intraperitoneal injection.While the Normal group were fed with normal food compositing a same dose of citric acid buffer intraperitoneal injection.Rats in Model group were then divided into DNP group and DNP+EA group.EA(continuous wave,2 Hz,1mA for 15 min,2mA for 15 min)was applied bilaterally to Zusanli(ST 36)and Kunlun(BL 60)for 30min,given once every day,totally 7 times.The body weight(BW),fasting blood glucose(FPG),fasting insulin(FINS)and insulin resistance(IR)were calculated on day 0(base),5 week and 7 week after high fat and high sugar diet to evaluate the establishment of type 2 diabetes model.Sciatic nerve morphology was assessed with Transmission electron microscopy.Paw withdrawal threshold to a von Frey-like filament was measured to assess mechanical allodynia using a Dynamic Plantar Aesthesiometer on day 0(base),5 week,7 week,and 30 min after EA treatment on day 3,5,and 7.Western blotting analyses was used to determin both total and membrane protein levels of P2X3Rs in L4-L6 DRGs.we performed immunofluorescence study to detect p-PKC expressions in L4-L6 DRGs.Part 2:DNP rats were divided into EA+PBS group,EA+αβ-meATP group and EA+PMA group.Rats in the three groups received the same EA treatment as Part 1.In the first 10 minutes every time before EA treatment,rats in EA+αβ-meATP group were injected with P2X3 receptor agonist αβ-meATP(0.6μmol/L,100μL)and rats in EA+PMA group were injected with PMA solution(aprotein kinase C activator,100 μL)into the ventral surface of each hind paw,while rats in EA+PBS group received the same dose of vehicle(PBS buffer)as a control.Pain threshold of rats were measured.Western blotting analyses was used as Part 1.Results Part 1:①Compared with Normal group,the BW、FINs、IR levels of the rats in DNP group was significantly increased after 5 weeks of the high-fat,high-sugar diet(P<0.01).In addition,two weeks after STZ injection,the FPG levels in the rats receiving STZ were significantly elevated(P<0.01),all above shows the type 2 diabetes model was established;②Sciatic nerve morphology assessed with transmission electron microscopy:Normal group showing large myelinated fibers;DNP group showing a fiber with dissolute axoplasm,myelin disruption.③PWTs:The PWTs of rats in DNP group were reduced compared with rats in Normal group(P<0.01),which indicates that the type 2 DNP model was successfully established;2 Hz EA significantly increased bilateral PWTs of rats subjected to DNP from day 3 after treatment,compared to the PWTs of DNP-controlled rats(P<0.01).④Western Blotting results shows:Either STZ injection or EA treatment did not alter the total P2X3Rs protein levels in L4-L6 DRGs.Membrane protein levels of P2X3Rs in L4-L6 DRGs is upregulated in DNP rats compared to Normal group(P<0.01).Moreover,2Hz EA treatment Inhibited the upregulation of membrane protein levels of P2X3Rs in L4-L6 DRGs significantly compared to DNP group(P<0.01).⑤Immunofluorescence study shows:p-PKC expressions in bilateral L4-L6 DRGs in DNP group was significantly increased as compared to that in Normal group(P<0.01).The increases were inhibited by 2 Hz EA in L4-L6 DRGs compared to DNP group(P<0.01).Part 2:①PWTs:Injection of P2X3 receptor agonist αβ-meATP and PKC agonist PMA both greatly reduced bilateral PWTs of EA-treated DNP rats compared to EA+PBS group(P<0.01).②Western Blotting results shows:The injection of PKC activator PMA improved the membrane protein levels of P2X3Rs in L4-L6 DRGs in EA+PMA group significantly compared to EA+PBS group(P<0.01).Conclusion In conclusion,our experiments indicate that the compensatory P2X3 and p-PKC up-regulation in L4-L6 DRGs,may contribute to DNP.EA with low frequency can relieve type 2 DNP,which may be through inhibiting the activation of PKC,and then decrease the expression of P2X3 receptors in L4-L6 DRGs of rats with DNP.