Analgesic Mechanism Of Hyperbaric Oxygen On Mitophagy In CCI Rats

Objective: Neuropathy pain is a kind of chronic pain stimulated when the central or peripheral nerve damage and tissue inflammation.There are many factors involved in this complex process.It is also one of the urgent problems in the chronic pain.Hyperbaric oxygen(HBO),as a non-invasive treatment,has been widely investigated which can effectively relieve neuropathic pain.But the molecular mechanism of HBO is unclear.Hyperbaric oxygen can enhance the antioxidant activity,accelerate the scavenging of free radicals such as ROS and repair or protect the damaged nerve tissue.These effects are similar to mitophagy.In order to specify whether the alleviated effect of hyperbaric oxygen on neuropathic pain is associated with mitophagy and its possible mechanism.Our study intends to observe the effect of hyperbaric oxygen on neuropathic pain through mitophagy in the spinal cord of rats.And through discussing the mechanism of CaMKK?/AMPK signaling pathways on this duration,we could investigate the further study on the molecular mechanism of hyperbaric oxygen on neuropathic pain by mitophagy.Methods: This experiment is divided into two parts.At first,we determine the promotive effect of hyperbaric oxygen on neuropathic pain through mitophagy.Rats were randomly divided into four groups: blank control group(C)and Sham operative group(S),sciatic nerve chronic constriction treatment group(CCI)and hyperbaric oxygen treatment group(CCI+HBO).The sham operative group only exposed the sciatic nerve.The CCI+HBO group performed hyperbaric oxygen therapy at 6h after operation.The behavior test of pain were applied at 9 ~ 11 o 'clock in the morning on preoperative day and 1,3,5,7 days postoperatively followed the sciatic nerve chronic constriction(CCI),including mechanical withdrawl threshold(MWT)and the thermal withdrawl latency(TWL).On the 7th day,spinal cord was observed under the electron microscope to observe the mitochondrial morphology changes.The examinations were also done to test the level of NIX,BNIP3 and Drp1 protein by western blotting.Immunofluorescence method was also applied to observe the expressive level of mitochondrial inner and outer membrane protein(TIM23 and TOM20)in the spinal cord specimen of rats.Then inhibitor of mitophagy(CsA)was given.We observate the variation of pain and mitophagy.Secondly,we discussed the mitophagic mechanism of hyperbaric oxygen on antineuropathic pain mediated by CaMKK?/AMPK signal pathway.The rats were divided into four groups: blank control group(C),sham operative group(S),sciatic nerve chronic constriction surgery group(CCI),hyperbaric oxygen treatment group(CCI+HBO).Through a series of mitophagic detective methods,hyperbaric oxygen are comfirmed to regulate mitophagy by CaMKK? and pAMPK.On the basis of current results,we first gave AMPK inhibitor(Compound C),then CaMKK? inhibitor(STO609).The pain behavior and mitochondrial proteins NIX,BNIP3 and Drp1 were tested to explore the antineuropathic pain effect of hyperbaric oxygen mediated by CaMKK?/AMPK pathway.Results: 1.Hyperbaric oxygen can significantly induce mitophagy and relieve chronic pain after chronic constriction of the sciatic nerve.(1)pain behavior results: each observation point of the mechanical contraction of the MWT and TWL in CCI+HBO group are significantly higher than in the CCI group(P<0.05).The hyperbaric oxygen can relieve mechanical and temperatural abnormal pain caused by sciatic nerve ligation.(2)The results of electronic microscopy: The mitochondria showed the mitochondrial double-layer membrane structure in the CCI+HBO group.At the same time,a large number of lysosomes.And the mitochondrial ridge structures disappeared.The mitophagy of CCI+HBO group was obvious.(3)mitophagy related protein western blotting results: LC3 II,NIX,BNIP3,Drp1 expression level in CCI+HBO group increased significantly than that in CCI group.And P62 expression level in CCI+HBO group decreased significantly than that in CCI group.The hyperbaric oxygen can alleviate mitophagic block caused by sciatic nerve ligation.(4)mitochondrial autophagy inner and outer membrane protein immunofluorescence results: TIM23,TOM20 and NeuN can coexpress in CCI+HBO group.Hyperbaric oxygen can enhance mitophagy caused by sciatic nerve ligation.2.Hyperbaric oxygen mediated CaMKK? /AMPK signal pathway to relieve chronic pain after chronic constriction of the sciatic nerve.At first,hyperbaric oxygen can effectively improve the pAMPK and CaMKK? protein expressive levels.And after giving AMPK inhibitors,the alleviated pain effect of hyperbaric oxygen was reversed.Finally,when STO609 was given,the alleviated pain effect of hyperbaric oxygen was also reversed.(1)pain behavior results: There was no significant difference between the values of MWT and TWL in CCI and CCI+HBO group.The alleviated pain effect of hyperbaric oxygen was reversed after application of Compound C and STO609.(2)There was no significant difference between CCI and CCI+HBO group on NIX,BNIP3 and Drp1 expression level.After Compound C and STO609 application,The increasing mitophagic protein effect of hyperbaric oxygen was reversed.Conclusions: 1.Hyperbaric oxygen can significantly induce mitophagy and relieve neuropathic pain after chronic constriction of the sciatic nerve.The analgesic effect of hyperbaric oxygen on neuropathic pain may be related to mitophagy.2.The role of hyperbaric oxygen to relieve chronic pain after chronic constriction of the sciatic nerve is related to upregulation of CaMKK?,activation of AMPK and increasing phosphative AMPK.It was concluded that hyperbaric oxygen can relieve neuropathic pain mediated by CaMKK?/AMPK signal pathway through mitophagy.