The Mechanism Of Inflammasome Activation Of NLRP3 In Paclitaxel Induced Neuropathic Pain And Electroacupuncture Analgesia

Part IThe mechanism of the activation of NLRP3 inflammasome in peripheral nerve involved in paclitaxel-induced neuropathic painObjective: Paclitaxel is commonly used as a cancer chemotherapy drug that frequently causes peripheral neuropathic pain.Inflammasome is a multiprotein complex consisting of Nod-like receptor proteins(NLRPs),apoptosis-associated specklike protein,and Caspase-1,which functions to switch on the inflammatory process and the release of interleukin-1β.Growing evidences have supported that peripheral interleukin-1β is critical in enhancing paclitaxel-induced neuropathic pain.However,whether activation of NLRP3 inflammasome in peripheral nerve contributes to paclitaxel-induced neuropathic pain is still unclear.In this study,we aim to study whether paclitaxel can activate NLRP3 inflammasome and release proinflammatory cytokines,thus inducing peripheral hyperalgesia by neuroimmune mechanism.In-depth study,we probe the cellular and molecular mechanism that paclitaxel activate NLRP3 inflammasome by inducing mitochondria reactive oxygen species(ROS)from macrophages.Our study will provide a new idea and intervention target for the clinical treatment of paclitaxelinduced peripheral neuralgia after chemotherapy.Method:(1)Prepare the SD rat model of paclitaxel-induced neuropathic pain.Detect the variation of mechanical hyperalgesia threshold in each group using von Frey filaments.(2)Using Western blot and real-time PCR quantitation,detect the development of the paclitaxel-induced neuropathic pain at different time points(1 week,2 weeks,3 weeks).Detect the expression of NLRP3,Caspase-1 and IL-1β m RNA in the peripheral nerve tissue such as tibial nerve,sciatic nerve and L4-L6 dorsal root ganglion(DRG)and lumbar spinal cord.Detect the expression levels of NLRP3,Caspase-1 p20 and mature IL-1β p17 protein,thus evaluating the activation of NLRP3 inflammasome.(3)Detect the expression of NLRP3 of macrophages(CD68)in sciatic nerve and DRG by immunofluorescence double labeling histochemical.(4)Observe the ultrastructure and mitochondrial morphology in DRG andsciatic nerve by scanning electron microscopy.(5)In vitro culture macrophage NR8383 cell line.Detect the number of damaged mitochondria and ROS-producing mitochondria by Flow cytometric analyses.(6)The paclitaxel-induced neuropathic pain model rats were treated by non-specific ROS scavenger phenyl-N-tert-butylnitrone(PBN).Determine the effect of PBN on paclitaxel-induced neuropathic pain and activation of NLRP3 inflammasome in L4-6 dorsal root ganglia and sciatic nerve.(7)The paclitaxel-induced neuropathic pain model rats were treated by electroacupunture(EA).Results:(1)On the third day after injection of paclitaxel,the rats presented mechanical allodynia,reached the lowest on day 9 and worsen gradually until 3 weeks after injection.(2)In contrast with the vehicle control group,the expression of NLRP3,Caspase-1 and IL-1β m RNA in L4-6 DRG,sciatic nerve and tibial nerves of paclitaxel-induced neuropathic pain rats were significantly up-regulated from the first week to the third week.At the same time,the expression level of NLRP3 and activated fragments of Caspase-1 and IL-1β was significantly increased,which implied the activation of peripheral NLRP3 inflammasome.However,during 3 weeks after paclitaxel modeling,the expression of NLRP3,Caspase-1 and IL-1β m RNA and NLRP3 and activated fragments of Caspase-1 and IL-1β in the lumbar spinal cord,was not significantly different from that of the vehicle control group.(3)After 3 weeks of paclitaxel injection,the expression of NLRP3 was located in L4-6 DRG and CD68-labeled macrophages of the sciatic nerve by double-labelling immunofluorescence,which showed the expression of NLRP3 was up-regulated in macrophages.(4)Infiltration of the macrophages and damage of the mitochondria(increased and swollen)in the sciatic nerve were observed after 3 weeks of paclitaxel injection by electron microscope.(5)In vitro experiments,paclitaxel resulted in a significant increase in the number of damaged mitochondria and ROS-producing mitochondria in rat macrophage line NR8383 cells,compared to the vehicle control group.(6)PBN significantly improved the mechanical allodynia of the paclitaxel-induced neuropathic pain model and down-regulated the the expression of component of NLRP3 inflamasome,Caspase-1 and IL-1β in the L4-6 dorsal root ganglion and sciatic nerve,which showed PBN significantly inhibited the activation of NLRP3 inflammatory in the sciatic nerve.Paclitaxel induced expression of NLRP3 in DRG and sciatic nerve were negatively correlated with mechanical pain threshold.(7)EA significantly improved the mechanical allodynia of the paclitaxel-induced neuropathic pain model.Conclusion:Paclitaxel-induced mechanical allodynia is parallel to the activation of NLRP3 inflammasome in L4-6DRG,sciatic nerve and tibial nerve.The activation of NLRP3 inflammasome continues from week 1 to week 3.The mechanism of paclitaxel-induced neuropathic pain may be: Paclitaxel causes macrophage infiltrating into peripheral nerve tissue.The damage of mitochondrial and ROS production in macrophage lead to the activation of NLRP3 inflammasome and the production of IL-1β activated fragments,which excites nociceptive nerve endings and causes paclitaxel-induced neuropathic pain.Part IIThe mechanism of CB2 receptors involved in inhibiting the activation of NLRP3 inflammasome by electroacupuncture to relieve paclitaxel-induced neuropathic painObjective:Our previous studies have shown that electroacupuncture(EA)inhibited the expression of inflammatory cytokines by activating endogenous cannabinoid type II receptor(CB2R)and exerted analgesic effects.Previous studies have shown that paclitaxel-induced neuropathic pain is due to the sensitization of peripheral nerve sensitiztion caused by the activation of peripheral NLRP3 and the increased expression of IL-1β.Then,whether EA can activate the peripheral CB2 receptors to inhibit the aggregation and activation of NLRP3 inflammasome to reduce neuropathic pain remains unknown.In this study,we aim to clarify the new mechanism of peripheral neuro-immuninteraction by EA treating paclitaxel-induced neuropathic pain,and provide new ideas for the clinical treatment of neuropathic pain caused by chemotherapy drugs.Methods:(1)Prepare the animal model of neuropathic pain induced by intraperitoneal injection of paclitaxel.The mechanical threshold was measured by the ‘‘up-down’’ method.EA was administered at 2 Hz on double “Huantiao”(GB30).(2)Remove sciatic nerve of the mouse,using western blot to research the expression of NLRP3,Caspase-1 p20,IL-1β p17 and the expression of CB2 receptor in peripheral nerve tissue.(3)Construct the model of paclitaxel-induced neuropathic pain.CB2 receptor-specific agonist AM1241 was intraperitoneally injected into the mice.Before the treatment of EA,CB2 receptor antagonist AM630 was intraperitoneally injected into another group.Detect the mechanical threshold by behavioral test.Detect the activation of NLRP3 inflammasome,the mature fragment protein expression levels of caspase-1 and IL-1β in sciatic nerve by western blot.We aim to verify whether the endocannabinoid CB2 receptors were involved in analgesic effect of EA and inhibition effect of EA on NLRP3 inflammasome activation process.(4)Using CB2 receptor knockout mice and their wild-type littermates,the model of paclitaxel-induced neuropathic pain was established.Use the mechanical threshold and western blot to study the involvement of endocannabinoid CB2 receptor in analgesic effect of EA and inhibition effect of EA on NLRP3 inflammasome activation process.Results:(1)Our study demonstrated that EA significantly improved mechanical hyperalgesia in the models of paclitaxel-induced neuropathic pain.(2)EA significantly down-regulated the expression of NLRP3,Caspase-1 and IL-1β active fragments in paclitaxel-induced mouse sciatic nerve,thus inhibiting the activation of NLRP3 inflammasome and up-regulating the expression of CB2 receptor in sciatic nerve.(3)In the model of paclitaxel-induced neuropathic pain,endocannabinoid type II(CB2)receptor agonist AM1241 significantly relieves the mechanical allodynia of the model of paclitaxel-induced neuropathic pain;the CB2 receptor antagonist AM630 can significantly the analgesic effect of EA.(4)In the model of paclitaxel-induced neuropathic pain,the CB2 receptor agonist AM1241 could significantly down-regulate the expression of NLRP3,Caspase-1 and IL-1β active fragment of sciatic nerve,thus inhibiting the activation of NLRP3 inflammasome.CB2 receptor antagonist AM630 could reverse the inhibitory effect on the activation of NLRP3 inflammasome in sciatic nerve by EA.(5)In the CB2 knockout mice,EA had no significant effect on paclitaxel-induced mechanical hyperalgesia.In the control experiment with wild-type mice,the EA could significantly reduce paclitaxel-induced mechanical hyperalgesia.(6)In the CB2 knockout mice,EA had no significant effect on the expression level of NLRP3,Caspase-1 and IL-1β active fragments in sciatic nerve of paclitaxel-induced neuropathic pain model,which did not inhibit paclitaxel-induced activation of the NLRP3 inflammasome.In the control experiment with wild-type mice,EA can significantly inhibit the activation of NLRP3 inflammasome induced by paclitaxel in sciatic nerve.Conclusions:(1)In this study,the EA can significantly inhibit the activation of NLRP3 inflammasome in the sciatic nerve,and play an analgesic effect in the model of paclitaxel-induced neuropathic pain.(2)The analgesic effect of EA on paclitaxel-induced neuropathic pain in mice model was mediated by activation of endocannabinoid receptor 2.(3)The analgesic effect of EA on paclitaxel-induced neuropathic pain may be mediated by activating the peripheral endocannabinoid CB2 receptor and then inhibiting the expression of NLRP3 inflammasome.