The Effect Of The AMP-Activated Protein Kinase Agonists On Neuropathic Pain In Mice

Neuropathic pain(NP) is a chronic state of nervous system damage. Which is difficult to treat for its complex mechanism, and there was no significantly effective conventional analgesics. NP is characterized as hyperalgesia, allodynia and spontaneous pain, and the spinal cord is the primary neuron of pain. In this paper, we study the change of proteins associated with neuropathic pain in spinal cord, thus proposing a new method for the treatment of neuropathic pain.AMPK(Adenosine monophosphate-Activated Protein Kinase) is an important endogenous kinases in regulating energy metabolism. Until now this is unique kinase actived by agonists in the field of pain in pharmacologically. It has studied that the AMPK agonists can cure peripheral nerve damage and regulate pain plasticity caused by incisions, and block the development of chronic pain after surgery.Objectives⑴ Construct the neuropathic pain model of mice, and provide a good foundation for studying the neuropathic pain.⑵ Observe the therapeutic effect of metformin and AICAR on mice with neuropathic pain, and providing a scientific basis for the use of metformin in clinical neuropathic pain patients.⑶ Observe the mRNA expression of AMPKα1 and AMPKα2 before and after curing neuropathic pain with AMPK agonists in spinal cord and sciatic nerve. And compared the expression of AMPKα2 with AMPKα1, inferring which AMPK subtype is more importent in treatment of neuropathic pain.⑷ Further observe the change of P-AMPKα after AMPK agonists treatment in spinal cord and sciatic nerve of mice, for investigating the effects of AMPK on activation treatment of neuropathic pain in mice.Methods⑴ SNI model was established by using adult male BALB/C mice, then ligated and cut the left sciatic nerve branches the tibial nerve and the common peroneal nerve, and sural nerve was reserved. In the case of sham-operated animals, an identical exposure was performed, but the nerve was not ligated. Then measured the left foot mechanical threshold(50%MWT) before and after surgery with von Frey cilia, if after 7 days 50% MWT≥1.0g, which is recognized the model failed and excluded from the experiment.⑵ Intraperitoneal injection metformin or intrathecal injection AICAR treatment of neuropathic pain in mice.⑶ After the mice were transcardially perfused with NS, we collected L4-L6 spinal cord and ipsilateral sciatic nerve, extracted RNA in the sample, using RT-q PCR testing the expression of AMPKα1 and AMPKα2 in different groups mice.⑷ After the mice were transcardially perfused with NS, we collected L4-L6 spinal cord and ipsilateral sciatic nerve, extracted protein in the sample, and detected the expression of pAMPKα and AMPKα by western-blot in different groups mice.Results⑴ The paw on the operative side presented moderate eversion and the toes were held together. Abnormal posture and spontaneuous pain also were found in nerve ligated mice. Three days after surgey, the mechanical pain threshold of the operated side foot in SNI model significantly decreased(P<0.05), which proved the neuropathic pain model successed, and the decline of mechanical pain threshold reached peak after 12 days, lasted 21 days after surgey.⑵ Treatment with metformin and AICAR significantly increased the mechanical threshold(p <0.05), Which proved metformin and AICAR have a therapeutic effect on hyperalgesia induced by SNI mice.⑶ RT-q PCR detected the expression of different groups mice, and the change of AMPKα1 and AMPKα2 was not statistically significant(p> 0.05), but the expression of AMPKα2 is more than AMPKα1.⑷ Compared with normal mice, the western-blot proved that P-AMPKα in spinal cord and sciatic nerve of SNI mice model is unchanged; the P-AMPKα expression was significantly increased after treatment with metformin and AICAR, and presented time dependence, But there is no change about AMPKα in each group, which means the AMPK agonists therapeutic effect on neuropathic pain in mice by activating AMPK.