| BACKGROUNDPain is a nociceptive feeling inside of human body, and plays the critical role in defensive protection system. However, long-term severe pain will induce physical and psychological torture and a variety of clinical diseases. Pain has become one of the most common clinical disorders. According to the clinical survey, patients taking rapamycin are suffering from the side effect of pain. However, there is no ideal treatment for it. In clinical, opium alkaloids are often used as an analgesic drug to relieve the pain, but it causes toxicity and addiction in the long-term use. Currently, analgesic effects of nicotine have been revealed on the molecular and cellular level. The mechanisms underlying the analgesic effect of nicotine are not well known. Elucidation of analgesic mechanism of nicotine will provide a reliable basis for finding targets for the treatment of pain.AIMSRapamycin-induced spontaneous pain model was used to detect the mechanism of nicotine in the process of spontaneous pain, as well as the interaction between nicotine and m TORC1. It provides a new target and theoretical basis for the development of analgesia drugs.METHODS1. Mice were injected with rapamycin to induce the hyperalgesia. Behavioral experiments including von frey test, heat stimulation, and tail flick tests were performed to detect hyperalgesia. Western blot and immunofluorescence were used to detect the m TORC1 signaling pathway in the brain region of anterior cingulate cortex(ACC) which is closely related to pain processing. 2. Changing of pain threshold was measured in the mice treated with nicotine. Western blot and immunofluorescence were used to detect the effect of nicotine on m TORC1 signaling pathway. DH?E(?4?2n ACh R antagonist) and MLA(?7n ACh R antagonist) were used to observe the function of nicotine receptor subtypes. 3. Whole-cell patch clamp recording was used to detect the effects of rapamycin on cell excitability as shown by the number of action potentials, latency to first action potential, and resting membrane potentials.RESULTS1. Hyperalgesia behaviors were induced by intraperitoneal treatment of rapamycin(10 mg/kg) in mice as shown by the significant decrease in withdrawal threshold, hot-plate latency, and tail-flick latency. Western blot and immunofluorescence showed a down-regulation of m TORC1 signaling pathway in the ACC. 2. Treatment with nicotine(1.5 mg/kg) could relieve the hyperalgesia behaviors in the mice injected with rapamycin as shown by the significant increase in withdrawal threshold, hot-plate latency, and tail-flick latency. Western blot and immunofluorescence showed the inhibition of S6K1 feedback pathway by nicotine and subsequent up-regulation of m TORC1 signaling pathway. Na?ve mice treated with nicotine showed a significantly increase the threshold of mechanical pain and thermal stimulate pain as compared with the control group. The western blot results were consistent with the data in the model mice.3. Whole-cell patch clamp results indicated that neuronal excitability was increased in rapamycin mice as shown by increased number of action potentials, decreased the latency to first action potential, and decreased resting membrane potentials. Pretreatment of nicotine in the artificial cerebrospinal fluid inhibited the effects of rapamycin on the the numbers of action potentials, latency to first action potential, and the resting membrane potentials. 4. Pretreated mice with DH?E(?4?2n ACh R antagonist) and MLA(?7n ACh R antagonist) abolished the analgesic effects of nicotine. It indicated that rapamycin-induced hyperalgesia was inhibited by nicotine through ?4?2n ACh R and ?7n ACh R subunits.CONCLUSIONSThe present study provided evidences that nicotine could inhibit the hyperalgesia behaviors induced by rapamycin. Western blot showed that nicotine could inhibit the feed back pathway of S6K1 through up-regulation of m TORC1. Whole-cell patch clamp recordings indicated that nicotine could reduce the neuronal excitability induced by rapamycin. |
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