| Background Cancer-induced bone pain is one of the common and serious complications in patients with intermediate to advanced malignancies.Autophagy is a physiological function of the body,responsible for the removal of denatured proteins and damaged organelles to maintain pathophysiological homeostasis and prevent apoptotic programs,and alterations in autophagic activity are involved in the development of pain,and the NLRP3 inflammatory cascade mediated by inflammatory vesicles plays an important role in pain.And the activation of NLRP3 inflammatory vesicles is closely related to the regulation of autophagy.As one of the common means of pain treatment,electroacupuncture has been increasingly studied in the field of bone cancer pain in recent years due to its rare side effects and favorable analgesic effect,while the specific mechanism of action still needs to be studied in depth.Objective To investigate the analgesic effect of electroacupuncture and its effect on spinal autophagy,and to explore the possible mechanism of action of electroacupuncture in relieving bone cancer pain based on the regulation of spinal autophagy-NLRP3 in rats with bone cancer pain,and to provide a theoretical basis for the treatment of cancer pain by acupuncture.Methods PartⅠ:(1)Female SD rats with pain thresholds meeting the conditions were randomly divided into sham and model group.Rats in the model grou Pwere injected with Walker-256 breast cancer cells into the bone marrow cavity of the left hind limb to construct a rat bone cancer pain model,while rats in sham grou Pwere injected equal amounts of inactivated Walker-256 breast cancer cells.(2)Changes in thermal and mechanical pain thresholds were examined in each grou Pat days 0,3,6,9,12,15 and 18 of modeling,and the rats were radiographed at days 6,9,12 and 18 after modeling to observe the broken ring in the tibia.(3)Western blot and immunofluorescence dual labeling were used to detect the expression of rat spinal cord autophagy proteins LC3 B,Beclin1 and P62.PartⅡ:(1)Female SD rats with pain thresholds meeting the conditions were randomly divided into sham group,model group,electroacupuncture group,sham electroacupuncture group,and Ra P(autophagy agonist)group.In the electroacupuncture group,the rats were given electroacupuncture intervention starting on day 6 after modeling,and the bilateral Zusanli and Kunlun points were selected and given sparse and dense waves of 2/100 Hz at an intensity of 0.5-1.5 m A for30 min per point for 7 times.Sham electroacupuncture group: the same acupuncture point as the electroacupuncture grou Pwas selected and subcutaneous acupuncture was performed at 5mm next to this point without electricity.Ra P(autophagic agonist)group: Ra P5 mg/kg was injected intraperitoneally for 5times starting on the 6th day after modeling.Equal amounts of saline were injected intraperitoneally in the sham and model grou P(2)Each grou Pof rats was tested for thermal pain thresholds at days 0,2,5,8,11,14,and 17 of modeling,and for changes in mechanical pain thresholds on the following day.(3)Western blot and immunohistochemistry were performed to detect the expression of ratspinal cord autophagyproteins LC3 B,Beclin1 and P62,respectively.PartⅢ:(1)Female SD rats with pain thresholds meeting the conditions were randomly divided into sham group,model group,electroacupuncture group,3-MA(autophagy inhibitor)group,and electroacupuncture + 3-MA group.3-MA group: 15 mg/kg was injected intraperitoneally on the 6th day after modelling,every other day.Electroacupuncture + 3-MA: electroacupuncture intervention in the same way and frequency as before,3-MA,15 mg/kg,was administered intraperitoneally 30 min before each acupuncture intervention until day 18.The sham grou Pand the model grou Pwere given equal amounts of saline treatment.(2)Pain behavior was measured as in Part II.(3)Western blot and immunohistochemistry were performed to detect the expression of LC3 B,Beclin1,P62,NLRP3,ASC,caspase1 protein in rat spinal cord.(4)Transmission electron microscopy was performed to observe the structural and quantitative changes of autophagosomes in each group.(5)ELISA assay was performed to detect the content of spinal cord inflammatory factors(IL-1β,IL-18).Results PartⅠ: Pain behavioral measures showed that thermal andmechanicalpain thresholds were significantly lower in the model grou Pcompared to the sham grou Pat day 9(P< 0.01).The X-ray images showed that the bone of the upper tibia of the rats started to show damage on the 12 th day after molding,and the degree of damage gradually increased with the duration of molding.Western blot results showed thatcompared with the sham-operated group,the spinal cord LC3 B,Beclin1 and P62 proteins in the model grou Pshowed a gradual increase with the modeling time,with LC3 B and Beclin1 increasing significantly on the 12 th day after modeling and P62 on the 9th day(P<0.01).Immunofluorescence double-labeling results showed that most of the autophagy proteins LC3 B,Beclin1,and P62 were co-locally expressed with neurons in the spinal cord of the model group.PartⅡ:Pain behavioral measures showed that andthermal mechanical pain thresholds were significantly lower in the model grou Pcompared to the sham grou P(P< 0.01 or P< 0.05),compared with the model group,the thermal and mechanical pain thresholds were significantly higher in the bone cancer pain rats in the electroacupuncture and autophagy agonist groups.(P< 0.01 or P< 0.05).But no significant differences were found between the model and sham electroacupuncture groups(P>0.05).Western blot and immunohistochemicalresults showed that the expression of LC3 B,Beclin1,and P62 proteins were increased in the spinal cord of rats in the model grou Pcompared with the sham grou P(P<0.01);compared with the model group,the expression of LC3 B and Beclin1 protein increased(P< 0.01 or P< 0.05)and P62 protein expression decreased significantly(P< 0.01 or P< 0.05)in the electroacupuncture grou Pand autophagy agonist group,and no significant differences were found between the model and sham electroacupuncture groups(P(29)0.05).PartⅢ: Pain behavioral measures showed that: compared with the model group,thethermal andmechanical pain thresholds of rats in the electroacupuncture grou Pwere significantly increased(P< 0.01 or P< 0.05),and the 3-MA grou Pwere significantly decreased(P< 0.01);compared with the3-MA,mechanical pain and thermal pain thresholds increased in the electroacupuncture +3-MA grou P(P< 0.01).Western blot and immunohistochemicalresults showed that compared with the sham group,the expression of LC3 B,Beclin1,P62,NLRP3,ASC,and caspase1 proteins were significantly increased in the model grou P(P< 0.01 or P< 0.05);compared with the model group,the expression of LC3 B and Beclin1 proteins were increased in the electroacupuncture grou P(P< 0.01 or P< 0.05),and the expression of P62,NLRP3,ASC,and caspase1 proteins were significantly decreased(P< 0.01 or P< 0.05)in the electroacupuncture group,and the expression of LC3 B and Beclin1 proteins were decreased(P< 0.01 or P< 0.05)and the expression of P62,NLRP3,ASC,and caspase1 proteins were significantly increased in the 3-MA grou P(P< 0.01 or P< 0.05);compared with the 3-MA group,LC3 B and Beclin1 protein expression increased in the electroacupuncture + 3-MA grou P(P< 0.01 or P< 0.05),and P62,NLRP3,ASC,and caspase1 protein expression decreased(P< 0.01 or P<0.05).ELISA assay results showed that: compared with the sham group,the expression of IL-1β and IL-18 increased significantly in the model grou P(P< 0.01 or P< 0.05);compared with the model group,the expression of IL-1β and IL-18 was significantly decreased in the electroacupuncture grou P(P< 0.01 or P< 0.05)and increased in the 3-MA grou P(P< 0.01 or P< 0.05);compared with the 3-MA group,the expression of IL-1β and IL-18 was decreased in the electroacupuncture + 3-MA grou P(P< 0.01 or P< 0.05).Transmission electron microscopy results showed that the neuronal structure was approximately normal in the sham-operated group,with few autophagosomes,and neuronal damage and increased number of autophagosomes were seen in the remaining groups;neuronal damage was better in the electroacupuncture group,with increased number of autophagosomes,and neuronal damage was worse in the 3-MA group,with decreased number of autophagosomes;neuronal damage was less in the electroacupuncture + 3-MA grou Pthan in the 3-MA group,with increased number of autophagosomes.Conclusion Activation of spinal cord autophagy inhibits NLRP3 inflammasome activation-mediated inflammatory cascade response and reduces the release of inflammatory factors,which may be one of the mechanisms by which electroacupuncture relieves bone cancer pain. |
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