The Mechanism Of AMPK Activation Rescuing Chronic Pain

Chronic pain is a rising health problem that is predicted to affect up to 30%of adults worldwide.Due to its complex causes,the pathogenesis is not clear,the treatment is very difficult.And long-term suffering from the disease prone to anxiety,depression,insomnia,etc.,consume a lot of medical resources,seriously affecting people's quality of life.Therefore,the treatment of effective chronic pain has become a challenging problem to be solved urgently.Chronic pain comprises several different types of pain,including inflammatory pain following tissue injury(for example,arthritis),cancer pain,and neuropathic pain following nerve injury,spinal cord injury and brain injury(for example,stroke and trauma).Chronic pain is typically characterized by hyperalgesia,which is an increased response to noxious thermal and mechanical stimuli as well as allodynia,in which nociceptive responses occur to normally innocuous stimuli such as light touch(known as mechanical allodynia).Neuropathic pain is also characterized by burning pain,paraesthesia(a sensation of tingling,tickling,prickling and pricking)and dysaesthesia(an unpleasant,abnormal sense of touch).Traditional drugs such as non-steroidal anti-inflammatory drugs and opioids that relieve chronic pain are not well controlled.Currently,ozone treatment is a new type of treatment that is widely used clinically and many clinical reports confirm their effects significantly.Ozone has a hundred years,history in the application of pain.In 1988,the Italian doctor Verga injected ozone into the psoas muscle and paravertebral space for the treatment of low back pain.In 1998,Muto injected ozone into the intervertebral disc and paravertebral space for the treatment of lumbar disc herniation,the effective rate was 78%.From 1994 to 2000,Albertini reported 6665 cases of multi-center treatment of lumbar disc herniation results,the effective rate was 80.9%.Some regions of Asia and Eastern Europe,such as Italy,Rome and Russia,are widely used clinically.In China,ozone therapy as a very important alternative treatment of neuropathic pain,which is widely used in intervertebral disc herniation,joint disease,frozen shoulder,migraine,peripheral vascular ischemic disease and herpes zoster pain.Although ozone is now widely used in painful diseases,few scholars have studied the mechanism of ozone in the treatment of pain,and the experimental evidence that ozone has the exact effect on pain is blank.This normative use of ozone lacks a lot of guidance evidence.The preliminary results of this laboratory and the forefront of neuroscience research have suggested that adenosine monophosphate-activated protein kinase(AMPK)plays an important role in pain modulation.AMPK is a heterotrimeric Ser/Thr protein kinase activated by alterations in cellular AMP:ATP ratio and servers as an energy sensor that regulates energy homeostasis and metabolic stress.Once activated,AMPK inhibits ATP consuming anabolic processes such as protein translation.The evidences for a role of AMPK in pain modulation came from a variety of studies in animals.Resveratrol and metformin are classic agonists of AMPK.Resveratrol is a highly bioactive polyphenolic substance,also known as Qiditriol,is a natural antioxidant,can reduce blood viscosity,and inhibit platelet coagulation and vasodilatation.It can prevent the occurrence and development of cancer,with anti-atherosclerosis and coronary heart disease,ischemic heart disease,high cholesterol prevention and treatment.Recent studies have shown that AMPK is not only an energy responsive enzyme,it also detects redox signals.Ozone has strong oxidizing properties,so the molecular mechanism of ozone onset may be closely related to AMPK.However,whether ozone is through the activation of AMPK and thus by regulating the relevant nerve inflammation to treat pain,has not been reported.Therefore,we use ozone and resveratrol through different chronic pain model to study the role of AMPK in chronic pain.Part One:Study on the mechanism of ozone-activated AMPK in the treatment of neuropathic pain Aim:To test the mechanism of ozone-activated AMPK in the treatment of neuropathic pain.Methods:The level of phosphorylation of AMPK was analyzed by western blot;Mechanical allodynia was determined by measuring the incidence of foot withdrawal in response to mechanical indentation of Von Frey.The level of phosphorylation of NR1,NR2B,PKC and ERK were analyzed by western blot.The effect of ozone on the activation of microglia in spinal cord was detected by western blot and immunofluorescence.The effects of ozone on AMPK and NF-kB in macrophages cultured in vitro were examined by western blot and immunofluorescence.Result:In this study,we found that a single peri-sciatic nerve injection of ozone decreased mechanical allodynia and thermal hyperalgesia,normalized the phosphorylated levels of protein kinase C ?(PKC?),NMDAR and extracellular regulating kinase(ERK)in the model of chronic constriction injury(CCI)of sciatic nerve.Meanwhile,ozone could significantly suppress CCI-induced activation of spinal microglia.More importantly,we also found that the anti-nociceptive effect of ozone depends on the activation of AMP-activated protein kinase(AMPK),which proved by the fact that the level of phospho-AMPK was increased during ozone therapy and the effect of ozone was abolished by specific antagonist of AMPK in vivo and in vitro.In addition,direct injection of AMPK agonist could replicate the effect of ozone in CCI rats.Ozone could inhibit the translocation of NF-kB p65 from the cytoplasm to the nucleus in RAW264.7 cells.Part Two:Study on the mechanism of resveratrol-activated AMPK in the treatment of bone cancer painAim:To investigate the effect of resveratrol on the behavior of bone cancer pain and its mechanismMethods:Mechanical allodynia was determined by measuring the incidence of foot withdrawal in response to mechanical indentation of Von Frey.The phosphorylation ofAMPK and the effect of resveratrol on bone cancer pain were detected by western blot.The effects of resveratrol on AMPK and MAPK family in microglia and astrocytes were detected by immunofluorescence.The effect of resveratrol on the phosphorylation of AKT and mTOR was detected by western blot.The effects of resveratrol on AMPK and MAPK in vitro were detected by western blot.Results:Resveratrol can decrease TCI-induced mechanical allodynia.Resveratrol significantly increased the phosphorylation of AMPK on neurons,astrocytes and microglia,and decreased the levels of phosphorylation of MAPK family,AKT and mTOR caused by TCI.Moreover,resveratrol can inhibit the phosphorylation of MAPK family on astrocytes and microglia in vitro,and the phosphorylation of AKT and mTOR on neurons.Conclusion:Resveratrol can inhibit the MAPK signaling pathway by activating AMPK,inhibit the activation of astrocytes and microglia,and the phosphorylation of AKT and mTOR,relieve bone cancer pain.