| BackgroundPain is the subjective sensation that people experience the most in their life.It is usually caused by actual or potential tissue damage.It can be divided into pathological pain and nociceptive pain according to the nature,location,and course of the disease.Inflammatory pain belongs to pathological pain,which is caused by exudate oppression and inflammatory mediators acting on sensory nerve fiber endings.Pain and other symptoms can persist for several weeks to years after the lesion is repaired or the noxious stimulus is removed.Persistent non-adaptive pain is the leading cause of disability worldwide,affecting one-fifth of the adult population globally,resulting in high socioeconomic burdens and costs.Pain treatment-related consumption in the United States is about $600 billion per year.With the aging of the social population,cancer,HIV infection,diabetes,and the prolonged survival of patients with other diseases that can cause pathological pain,the prevalence of pathological pain is increasing year by year.The regulation of pain is a very complicated process,and its etiology is different,and the treatment is a medical problem that needs to be solved urgently.Pain control is the main purpose of clinical drug treatment of pain,among which the most commonly used antipyretic analgesic anti-inflammatory drugs are nonsteroidal anti-inflammatory drugs(NSAIDs),by inhibiting the body cyclooxygenase(COX)activity,reduce prostaglandin(PG)synthesis,achieve the purpose of reducing inflammation and pain relief,side effects include cardiovascular system adverse reactions and the tendency to induce gastrointestinal bleeding.Other commonly used analgesics include opioids and cannabinoids,etc.,and their analgesic effects are related to stimulating opioid receptors and cannabinoid subtype receptors,but both have drug addiction and mental dependence,which can easily lead to drug abuse and drug withdrawal syndrome,so even if there are indications for medication,the frequency and dose of medication should be minimized.Therefore,it is imperative to seek analgesic drugs that are safer,more effective,and have less side effects.Traditional Chinese medicine classifies pain into "pain syndrome" and "arthritis syndrome",takes "blockage leads to pain" as the etiology,and "passes to no pain" as the treatment principle,and acupuncture and moxibustion are used to dredge the meridians and eliminate the cause of the disease.Acupuncture,as an important part of traditional Chinese medicine,has irreplaceable advantages in the treatment of various pains.Acupuncture promotes the release of endogenous opioid peptides in the human body,producing anti-inflammatory and analgesic effects.Studies have shown that the serum level of transthyretin(TTR)decreases in the state of inflammatory pain in the body.Hot plate assay showed that the pain threshold of TTR knockout(KO)mice was different from that of wild-type(WT),suggesting that TTR is closely related to pain.Although many pain syndromes belong to the indications of acupuncture and moxibustion,and can be improved or cured under acupuncture treatment,traditional acupuncture does not give specific explanations and definite answers to the mechanism of acupuncture analgesia.Both basic and clinical studies have proved that acupuncture can play a good analgesic effect on inflammatory pain,but the exact mechanism is still unclear.Therefore,discovering and determining the key molecules involved in acupuncture analgesia is crucial to elucidating the mechanism of acupuncture analgesia,and at the same time helps to improve the clinical efficacy of acupuncture analgesia.ObjectivesTo identify novel biomarkers related to acute and chronic pain,analyze the ion channel mechanism of TTR-mediated acupuncture inhibition of inflammatory pain,and provide new scientific basis for elucidating the mechanism of acupuncture analgesia.Materials and MethodsIn this study,three different strains of mice,including 129/SvJ,C57BL/6J and BALB/c mice,weighing(23.00±2.00)g,using different stimulation methods and different EA frequencies were selected to verify the universality and extensiveness of the analgesic effect of acupuncture.Then 129/SvJ mice were used as the research subject,weighing(23.00±2.00)grams.Firstly,the pain threshold of 129/SvJ TTR WT and KO mice was compared and it was determined that TTR was closely related to pain.Subsequently,the 129/SvJ TTR WT mice were divided into normal group(Control),model group(Model),electroacupuncture group(EA),and TTR group(TTR)according to the random number table method.Mice in the Model group,EA group and TTR group were injected with 100 μL of 3%carrageenan-based solution under the skin of both hind plantar feet to establish a model of chronic inflammatory pain caused by carrageenan.The Control group did not intervene at all.Comprehensive use of behavioral(hot plate method),electrophysiology(patch clamp),imaging(functional magnetic resonance imaging),morphology(HE,Masson,immunofluorescence staining),molecular biology(ELISA,WB)and many other technical methods to explore and analyze the mechanism of TTR-mediated acupuncture inhibition of inflammatory pain.Results1 Dynamic changes in mouse behavior after modeling:after subcutaneous injection of 100 μL of 3%carrageenan solution on the soles of the bilateral hind paws of the mice,the hind paws were significantly swollen and the pain threshold was significantly reduced(P<0.01),suggesting that model successfully.Set different time points and use the hot plate method to measure the pain threshold at 3h,6h,12h,1d,3d,7d,14d and other time points after injection,and dynamically observe the changes in the pain threshold of mice after modeling.The pain threshold of the mice was lower than that of the normal group.2 Changes in pain threshold of TTR gene knockout mice:select 129/SvJ TTR WT and KO female and male mice,detect their pain threshold by hot plate method,compare the difference in pain threshold between WT and KO 129/SvJ mice of different genders.The results showed that after knocking out the TTR gene,the pain threshold of 129/SvJ mice of different sexes was significantly reduced(P<0.01).3 The effect of intraperitoneal injection of TTR versus morphine on the pain threshold of mice:select the mice that have successfully established the model,and when the pain threshold drops to the lowest value,that is,about 14d,inject TTR or morphine intraperitoneally,and use the hot plate method to observe the pain threshold of the mice respectively.The pain threshold of 10min,0.5h,1h,3h,6h,12h,1d,3d,7d and other time points after drug administration,long-term,multi-time point,dynamic observation of the dynamic changes of pain threshold in mice after drug administration,found that the pain threshold of model mice was significantly increased,which was higher than the control value after modeling,and the maintenance time was longer.The pain threshold of mice injected intraperitoneally with morphine was significantly increased,but the maintenance time was shorter.4 Analgesic effect of acupuncture:In normal mice,different acupuncture methods and different EA frequencies can significantly increase the pain threshold of mice of different strains,suggesting the universality of acupuncture analgesic effect.In model mice,2/15 Hz EA significantly increased animal pain threshold within 0.5 h,indicating that acupuncture has a definite analgesic effect.5 Pathological changes in the liver and choroid plexus of inflammatory pain model mice:HE and Masson staining were used to observe the pathological changes in the liver tissue of the mice in the model group,with destruction of cell structure,disappearance of nucleus,fatty degeneration of liver tissue,significant fibrosis,and the winding arrangement is disordered.IF staining was used to detect the expression of TTR in the liver and choroid plexus of the mouse model group and the EA group.The results showed that the expression of TTR was significantly reduced after modeling,and the expression of TTR in the liver and choroid plexus was significantly increased after EA treatment.6 The effect of TTR on cell membrane ion channels:Patch clamp technique was used to detect the effect of TTR on capsaicin-activated dorsal root ganglion(DRG)neuron cell membrane TRPV1 ion channel,and to analyze the ion channel of TTR analgesic effect mechanism.The results showed that TTR had a 24.32%inhibitory effect on capsaicin-activated DRG neuron cell membrane TRPV1 ion channel,and TTR had no significant effect on resting state DRG neuron cell membrane potential,suggesting that TTR had no obvious toxicity to normal cells.7 fMRI analysis of the central nuclei involved in the analgesic effect of TTR:detection of the BOLD signal response amplitude in the brain at rest.The neural activity of the nuclei in the brain area was inhibited.In addition,there was increased functional connectivity between the periaqueductal gray matter and the hippocampus,and between the hippocampus and the prefrontal cortex.It is suggested that multiple central nuclei are involved in the pain-inhibiting effect of TTR.Conclusions1 Carrageenan can prepare long-term and stable pathological models of chronic inflammatory pain.Mice were subcutaneously injected with 100μL of 3%carrageenan solution in the soles of bilateral hind paws,and the pain threshold of the mice was significantly reduced and maintained The low pain threshold can reach two weeks,indicating that the mouse model of inflammatory pain prepared by carrageenan has good reproducibility and high success rate,and is suitable for related experimental research on pathological pain.2 After TTR gene knockout,the pain threshold of mice was significantly reduced,suggesting that TTR is a potential pain suppressing molecule.3 Intraperitoneal injection of TTR can significantly improve the pain threshold of inflammatory pain model mice for more than two weeks,and no significant weakening of the drug effect was found after continuous administration,suggesting that TTR can produce stable analgesic effects and does not produce tolerance effects,while the same amount of intraperitoneal injection of the positive control drug morphine sulfate significantly increased the pain threshold of mice,but the duration of the effect was short,and repeated administration tended to quickly develop tolerance.4 Acupuncture has an analgesic effect.Different acupuncture methods and different EA frequencies can significantly increase the pain threshold of mice of different strains,suggesting the universality of acupuncture analgesic effects;2/15 Hz EA treats inflammatory pain models In mice,the pain threshold of the mice was higher than that of the model group,which produced obvious acupuncture analgesic effect,and acupuncture had a definite analgesic effect.5 TTR has a 24.32%inhibitory rate on capsaicin-activated DRG neuron cell membrane TRPV1 ion channel,suggesting that TTR has a good analgesic effect.TTR has no effect on the cell membrane channels of DRG neurons in the resting state,suggesting that it does not produce obvious toxicity to normal cells.6 TTR-mediated acupuncture reduces peripheral inflammatory injury in inflammatory pain model mice,especially the inflammatory edema of liver tissue.TTR content in serum of inflammatory pain model mice is significantly reduced,suggesting that TTR has a certain anti-inflammatory effect.7 Nuclei in multiple central brain regions,including the thalamus,hippocampus,gray matter of the central aqueduct,amygdala,and prefrontal cortex,participate in the central pain modulation of TTR.Changes in functional connectivity between nuclei may also be involved in the analgesic effect of TTR. |
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