Study In The Effect Of Astragalin On Neuropathic Pain Mediated By P2X4 Receptor In The Spinal Dorsal Horn And Dorsal Root Ganglion

Research background: Neuropathic pain is a chronic pain caused by nervous system damage.Its pathogenesis is complicated,and there is still no effective therapeutic drug.Traditional pain treatments are mainly aimed at neurons,but studies have shown that glial cells play a vital role in chronic pain.Therefore,a more suitable target for pain treatment may be glial cells.The P2X4 receptor is one of the members of the purinergic receptor family,which is expressed in the microglia of the spinal dorsal horn(SDH)and the satellite glial cells(SGCs)of the dorsal root ganglion(DRG).P2X4 receptor on the microglial cells of SDH and the SGCs of DRG was upregulated when neuropathic pain,while promoting the release of ATP from primary sensory neurons to activate P2X4 receptor,and allowing SGCs and microglia to release biologically active molecules to increase the excitability of DRG or spinal dorsal horn neurons and enhance nerve Signaling of pathological pain.Astragalin(AST)has various pharmacological effects such as anti-inflammatory and anti-oxidation.Combined with our pre-experiment,it was observed that the pain behaviors in rat neuropathic pain models were reduced after application of astragalin.Astragalin may have anti-injury and analgesic effects.Objective: In this study,chronic constriction injury(CCI)was used as rat neuropathic pain model.We explored whether astragalin can reduce the expression and function of P2X4 receptor in the dorsal horn and dorsal root ganglion to relieve pain behaviors in neuropathic pain model rats.We will observe the effect of AST on CCI rats and explore its possible mechanism.It is helpful to broaden and deepen the understanding of the role AST and its mechanism,which will explore new methods for the prevention and treatment of neuropathic pain.Methods: In this experiment,the CCI rat model was first established,and the SD rats were randomly divided into six groups: control group(Ctrl group),sham operation group(Sham group),model group(CCI group),control + Astragalin group(Ctrl + AST group),Model + Astragalin group(CCI + AST group),Model +Pharmaceutical solvent group(CCI + DMSO group).(1)Behavioral measurement:The changes of pain behaviors in rats were detected by mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL).(2)Quantitative real-time PCR(qPCR)was used to detect the changes of P2X4 mRNA in dorsal root ganglion(DRG)and spinal dorsal horn(SDH)of rats in each group.(3)Detection of P2X4,tumor necrosis factor –receptor1(TNF-R1),GFAP,OX-42,extracellular regulated protein kinase(ERK)1/2 and phosphorylated(p)-ERK1/2 protein expression by Western blot(WB).(4)Co-expression of P2X4 receptor and GFAP in DRG and co-expression of P2X4 receptor and OX-42 in SDH by immunofluorescence double-label method.(5)The whole cell patch clamp technique was used to observe the effect of astragalin on ATP-activated current of HEK293 cells transfected with P2X4 recombinant plasmid.Results: CCI rat neuropathic pain model was successfully established.(1)Astragalin reduced the pain behaviors of CCI rats: Compared with the Ctrl group,the mechanical withdrawal threshold(MWT)and thermal withdrawal latency(TWL)of CCI rats were significantly reduced(p <0.001);MWT and TWL in the CCI + AST group was significantly higher than those in the CCI group(p <0.001);there was no significant difference between the CCI + DMSO group and the CCI group(p> 0.05).(2)Astragalin reduced the expression of P2X4 receptor in DRG and SDH of CCI rats.qPCR results showed that the expression levels of P2X4 mRNA in DRG and SDH of CCI group were significantly higher than that in Ctrl group(p <0.01);the expression level of P2X4 mRNA in CCI + AST group was significantly lower than that in CCI group(p <0.01);There was no significant difference between the CCI + DMSO group and the CCI group(p> 0.05).WB results showed that the expression levels of P2X4 protein in DGR and SDH in CCI group were significantly higher than those in Ctrl group;the expression levels of P2X4 proteins in CCI + AST group were significantly lower than those in CCI group(p <0.01);There was no significant difference between CCI + DMSO group and CCI group(p> 0.05).(3)Astragalin inhibits CCI-induced glial cell activation.Immunofluorescence double labeling results showed that: P2X4 receptorwas co-expressed with satellite glial cell marker glial fibrillary acidic protein(GFAP)in DRG,P2X4 receptor was co-expressed with microglia marker OX-42 inSDH,the fluorescence intensity in CCI group was significantly higher than that in Ctrl group(p <0.001);the fluorescence intensity of CCI + AST group was significantly lower than that in CCI group(p <0.001);there was no significant difference between CCI + DMSO group and CCI group(p> 0.05).At the same time,the expression levels of GFAP protein in DRG and OX-42 protein in the dorsal horn of the spinal cord increased in the CCI group were increased,and after application of Astragalin,the expression levels of GFAP protein and OX-42 protein in the CCI rats twere significantly decreased(p <0.001).(4)Astragalin treatment reduced the expression of TNF-R1 in CCI rats: the expression level of TNF-R1 protein in CCI rats was significantly higher than that in Ctrl group;the expression level of protein in CCI + AST group was significantly lower than that in CCI group(p <0.01).There was no significant difference between CCI + DMSO group and CCI group(p> 0.05).(5)P2X4 receptor activation involves the activation of ERK signaling pathway.The expression level of p-ERK1/2 protein in CCI rats was significantly higher than that in Ctrl group;the expression level of p-ERK1/2 protein in CCI + AST group was significantly lower than that in CCI group(p <0.01);There was no significant difference between CCI + DMSO group and CCI group(p> 0.05).(6)Astragalin can inhibit the ATP-activated current of HEK293 cells transfected with the pEGFP-hP2X4 plasmid.Conclusion: Astragalin treatment may reduce the expression of P2X4 receptor in DRG and SDH,inhibit glial cell activation,and down-regulate p-ERK1/2 levels,reduce the expression level of TNF-R1,reduce the communication between the neuron and the glia cells,then relieve the pain behaviors of CCI rats.