Rapamycin Ameliorates Inflammatory Pain Induced By Complete Freund’s Adjuvant(CFA) Via Activating Autophagy In The Rat Spinal Dorsal Horn

Background:Inflammatory pain is a kind of chronic pain which caused by injury or infection.Autophagy is a transcytoplasmic transport system that controls the transfer of cytoplasmic materials to lysosomes,promoting the degradation of defective organelles,misfolded or abnormally aggregated proteins and other substances to regulate the homeostasis of cells and the internal environment.Recent studies have found that rapamycin can alleviate mechanical hyperalgesia and thermal hyperalgesia in various neuropathic pain models.While the effect of autophagy on inflammatory pain remains unclear,this study was to investigate the role of autophagy in the model of inflammatory pain induced by Complete Freund’s Adjuvant and whether rapamycin could be used as a potential therapeutic agent for inflammatory pain.Methods: Male SD rats,weight 220-250 g,with left plantar subcutaneous injection of Complete Freund’s Adjuvant.The study was divided into two parts: The first part was to discuss the changes of autophagy in CFA model.24 rats were randomly divided into two groups,12 for each group.Control group(CON): 100 μl normal saline(NS)was subcutaneously injected into the left posterior foot;model group(CFA): 100 μl CFA was subcutaneously injected into the same position.Behavioral experiments were performed on 6 rats in each group: paw withdrawal threshold(PWT),paw withdrawal latency(PWL)and left posterior foot swelling were measured before modeling and 1,3,5,7 and 14 days after modeling.The rest of the rats were put to death on the third day.Lumbar segments(L4-6)and left posterior foot was taken to perfrom molecular biology experiment.Using western bolt method to detect autophagy related protein p62 and LC3 Ⅱ expression in spinal cord dorsal horn;HE staining was used to observe the infiltration of inflammatory cells in left posterior.The second part was to discusses the role of rapamycin in CFA inflammatory pain model.Forty-eight rats were randomly divided into four groups with 12 in each.Control group(CON): 100 μl NS was subcutaneously injected into the left posterior foot with no special treatment;model group(CFA): CFA inflammatory pain model was established without drug treatment.Model + normal saline group(CFA+NS): CFA inflammatory pain model was established and injected NS for 10 μl/ time by means of intrathecal tube technique.Model + rapamycin group(CFA+RAP): CFA inflammatory pain model was established,and rapamycin was injected 10 μl/ time through the intrathecal tube technique.Behavioral experiments were conducted with 6 rats in each group: before modeling,the PWT,PWL and the swelling of left posterior foot of each group was measured;after modeling,the rats in CFA+NS and CFA+RAP groups were given corresponding drug for 3 consecutive days,and the other two groups were given no drug treatment;the swelling of PWT,PWL and left posterior foot of each group was detected 1 hour after administration.The rest of the rats were subjected to molecular biology experiments: Each group was given corresponding drug treatment for 3 consecutive days after modeling.On the third day,the rats were sacrificed 2 hours after injection and the l4-6 spinal cord and the left posterior foot were collected.The expressions of autophagy-related proteins p62,LC3Ⅱand Beclin1 in the spinal dorsal horn were detected bywestern blot.Enzyme-linked Immunosorbent Assay was used to determine the inflammatory factors TNF-α,Il-1βand Il-6 in the spinal cord.The morphology and number of activated microglia in spinal dorsal horn were observed by immunofluorescence technique.HE staining was used to observe the infiltration of inflammatory cells in the ipsilateral foot.Results:(1)Autophagy proteins:Compared with CON group,p62 and LC3 Ⅱ were increased in CFA group.While p62 was decreased and LC3 Ⅱ was increased in CFA+RAP group compared with CFA and CFA+NS groups(P<0.05).(2)Inflammation:Compared with CON group,inflammatory cytokines TNF-α、IL-1β and IL-6 in spinal cord tissues were increased in CFA and CFA+NS groups.While these cytokines were decreased in CFA+RAP group compared with CFA and CFA+NS groups(P<0.05).(3)Microglia: Compared with CON group,the number of activated microglia in the spinal dorsal horn was increased in CFA and CFA+NS groups and the number was decreased in CFA+RAP group in comparison with CFA and CFA+NS groups(P<0.05).(4)Swollen feet: Compared with CON group,there was thicker left foot and more inflammatory cells in CFA and CFA+NS groups.While there was thiner left foot and less inflammatory cells in CFA+RAP group in comparison with the CFA and CFA+NS groups(P<0.05).(5)Behavior:Compared with CON group,the paw withdrawal threshold(PWT)and paw withdrawal latency(PWL)descended in CFA and CFA+NS groups.While the PWT and PWL in CFA+Rap group rose compared with CFA and CFA+NS groups(P<0.05).Conclusion: The autophagy flux was blocked in CFA model.Rapamycin could activated the autophagy in the spinal dorsal horn and attenuated the inflammatory pain.The mechanism may be related to the inhibition of microglia activation and the secretion of inflammatory factors after autophagy activation.