Mechanism Of Spinal Cord Neurons Autophagy In The Treatment Of Neuropathic Pain Treated With Hydrogen

Neuropathic pain is a long-term chronic pain caused by a physical sensory nervous system injury or disease.The formation of the mechanism is complex,is now difficult to treat a class of medical diseases,often delayed healing,to patients with great pain,quality of life is extremely low.A large number of studies have shown that cell autophagy dysfunction is involved in the formation and development of neuropathic pain,and appropriate induction of autophagy is a breakthrough in the treatment of neuropathic pain.In recent years,many studies have shown that low concentrations of hydrogen molecules(<4%)and hydrogen-rich liquid with selective antioxidant,anti-inflammatory,anti-apoptotic effect,can be applied to a variety of diseases research and treatment.Therefore,more and more scholars believe that hydrogen and hydrogen-rich saline is a new type of medical gas molecules.Some studies have found that intraperitoneal injection of hydrogen-rich saline has a therapeutic effect on neuropathic pain to improve behavior in neuropathic pain rats,the specific mechanism is still being explored.Hypoxia-inducible factor-1(HIF-1)is a key factor in regulating cell autophagy under hypoxia conditions and is a key regulator of cellular adaptation to hypoxic conditions.Studies have shown that it increases expression in the primary pathway of neuropathic pain.In this study,we established the neuropathic pain rat model by intraperitoneal injection of hydrogen-rich saline and herpes zoster neuralgia inhalation of hydrogen,to explore the possible mechanism of hydrogen on neuropathic pain.Experiment 1 :Effect of hydrogen-rich saline activates spinal cord neuronal autophagy in neuropathic painAutophagy is one mechanism of neuropathic pain.It is a new target for the treatment of neuropathic pain by inducing autophagy.Hydrogen-rich saline have a certain therapeutic effect on the neuropathic pain,the specific mechanism is not clear.In this study,we used the establishment of neuropathic pain rat model(CCI)to explore the effect of hydrogen-rich saline on spinal cord autophagy in rats with neuropathic pain.Method: 60 male Sprague-Dawley rats were randomly divided into 5 groups(n=12): sham operation group(S group),neuropathic pain model group(group C),neuropathic pain model group+ hydrogen-rich saline group(C+H group),neuropathic pain model group+3-methyladenine group(C+M group),neuropathic pain model+ hydrogen-rich saline+3-methyladenine group(C+M+H group),neuropathic pain model using chronic sciatic nerve ligation model(CCI).C+H group and C+H+M group began intraperitoneal injection of hydrogen-rich saline(0.6 mmol/L)5ml/kg Bid,Autophagy inhibitor 3-methyladenine was injected 1h after CCI in C+M group and C+H+M group,other groups administration the same amount of saline abdominal / intrathecal Bid.The rats were detected at 1 day(-1d),1 day(1d),3 days(3d),7 days(7d),10 days(10d)and 14 days(14d).The levels of autophagy-related genes and protein LC3?,Beclin-1 were measured by Western blot and Real-time PCR.The expression of superoxide dismutase(SOD)and malondialdehyde(MDA),IL-6,TNF-? were measured by enzyme-linked immunosorbent assay(ELISA).Autophagosomes in the spinal dorsal horn of rats were observed by electron microscopy.Result: Compared with group S,MWT decreased,TWL shorted,cold pain threshold the was lower,SOD activity decreased,MDA,IL-6 and TNF-? were up-regulated(P <0.05),the expression of Beclin-1,LC3-II gene and protein were up-regulated,p62 expression was down-regulated and the number of autophagosomes was decreased in group C after 3d(P<0.05).Compared with group C,MWT increased,TWL extend,cold pain threshold rised,the expression of Beclin-1 and LC3-II in the spinal cord of rats was up-regulated,p62 gene and protein were down-regulated,the autophagosomes increased,SOD activity increased the MDA,IL-6 and TNF-? were increased in group C+H(P<0.05).Compared with group C,the expression of Beclin-1 and LC3-II was down-regulated and the expression of p62 gene was increased in the group C+3-MA,MWT decreased,TWL shorted,cold pain threshold the was lower,the levels of MDA,IL-6 and TNF-? were up-regulated(P <0.05).Conclusion : The mechanism of hydrogen-rich saline reduced neuropathic pain is related to the induction of autophagy of spinal cord neurons,the reduction of oxidative stress and the inhibition of inflammatory response.Experiment 2: Effect of HIF-1?-mediated cell autophagy on the treatment of neuropathic pain by hydrogen-rich salinePurpose : The hypoxia-hypothermic microenvironment is an important factor in the pathogenesis of neuropathic pain,activating a series of adaptive responses under hypoxia,and the hypoxia-inducible factor HIF-1? plays a central role in it by transcription of various cytokines Adapt to the hypoxic environment,the study confirmed that it and the downstream pathway is hypoxic regulation of autophagic classical pathway.In this study,HIF-1? agonists and inhibitors were used to investigate the effect of HIF-1? on the autophagy of spinal cord cells in rats with neuropathic pain.Method : Healthy male 96 SD rats were randomly divided into 8 groups(n=12)by random number table: sham operation group(S group),sham operation + hydrogen-rich saline group(S+H group),neuropathic pain +hydrogen-rich saline group(C+H group),neuropathic pain + 2-methoxyestradiol group(C+2Me2 group),CCI + H2 group+2-methoxyestrogen group(C+H+ 2Me2 group),CCI + 3,4-dihydroxybenzoate group(C+EDHB group),CCI+H2+3,4-dihydroxybenzoate group(C+H+EDHB group).The method of hydrogen-rich saline is the same as that of experiment 1.The HIF-1? inhibitor 2ME2 was dissolved in 0.5% dimethyl-sulfoxide and administered intraperitoneally at a dose of 10mg/kg for 10 days.The HIF-1? antagonist EDHB was administered intraperitoneally at a dose of 100mg/kg for 30 minutes after CCI.Behavioral changes at The 0 day(-1d),1 day(0d),3 days(3d),7 days(7d),10 days(10d)and 14 days(14d)were used to study.The expression of autophagy-related proteins and genes Beclin-1,HIF-1? and BNIP3 were measured by Western blot and Real-time PCR in spinal cord.The levels of serum IL-6,TNF-? and SOD and MDA in the spinal cord were measured by enzyme-linked immunosorbent assay(ELISA).Result: Compared with group S,Beclin 1,HIF-1? and BNIP3 were expressed in group C,C+H,C+H+2Me2,C+EDHB and C+H+EDHB.MWT decreased,TWL shorted,SOD activity decreased,MDA,IL-6 and TNF-? were up-regulated at 14 days postoperatively(P<0.05).Compared with group C,the levels of MDA,IL-6 and TNF-? were significantly down-regulated,SOD activity and the expression of Beclin 1,HIF-1? and BNIP3 was up-regulated in the spinal cord in the C+H group and C+EDHB and C+H+EDHB group,reversing the hyperalgesia of the limb,MWT increased,TWL extend at 14 days postoperatively(P<0.05).The expression of Beclin 1,HIF-1?,and BNIP3 was down-regulated in the spinal cord,the hyperalgesia of the limb increased,the MWT decreased,the TWL was shortened,the SOD activity decreased,MDA,IL-6 and TNF-? was up-regulated in the C+2Me2 group(P<0.05).Compared with C+H group,the expression of Beclin 1,HIF-1? and BNIP3 was up-regulated in the spinal cord,MWT decreased,TWL shorted,SOD activity increased,MDA,IL 6 and TNF-? were down-regulated in the C+H+EDHB group;in the C+H+2Me2 group,the expression of Beclin 1,HIF-1? and BNIP3 in the spinal cord was down-regulated,MWT decreased,TWL was shortened,SOD activity decreased,MDA,IL-6 and TNF-? were up-regulated(P<0.05).Conclusion: Hydrogen-rich saline has a therapeutic effect on neuropathic pain rats via HIF-1? pathway-mediated cell autophagyExperiment 3: Randomized controlled clinical study of aerosolized hydrogen in patients with postherpetic neuralgiaPurpose: Posterior herpetic neuralgia(PHN)is a persistent pain more than three months after healing of the rash of herpes zoster caused by varicella zoster virus(VZV).It belongs to neuropathic pain,PHN treatment can be described as a worldwide problem,safe and effective treatment is essential to study.In this study,randomized controlled study was conducted to determine the safety and efficacy of aerosolized hydrogen in the treatment of PHN,and to explore the therapeutic mechanism of hydrogen on neuropathic pain.Method : From January 2016-2016 June at the Second Hospital of Tianjin Medical University,postherpetic neuralgia 60 patients,31 males and 29 females,aged 45 to 79 years old(N=20): control group(S group),low frequency group(H1 group)and high frequency group(H2 group)were randomly divided into three groups.S group: the basic drug + 100% oxygen at 3L/min inhalation for 30 minutes Qd,H1 group:basic drugs+67% H2-33% O2 mixed gas inhalation 30 minutes Qd,H2 group:basic drugs +67%H2-33% O2 mixed gas inhalation for 30 minutes Bid,were treated continuously for 7 days.pain was assessed and venous blood sampling were dected 0 day(0d),1 day after treatment(1d),3 days(3d),5 days(5d),7 days(7d),1 month(1M),March(3M),6 months(6M)for outpatient with PHN.Main evaluation indicators: visual analogue scale(VAS)and secondary evaluation index: 1)simplified Mc Gill pain questionnaire score 2)sleep score 3)analgesic and antiepileptic drug use.The treatment process begins daily 8 points to detect vital signs.laboratory tests contain blood routine,liver and kidney function to assess its safety.IL-6,TNF-?,LC3 and ? Beclin-1 were measured by enzyme-linked immunosorbent assay(ELISA).Result : Three group patients was normal in vital signs,blood routine,liver and kidney function(P>0.05).The VAS,SF-MPQ,SIS scores,tramadol and gabapentin in H1 group and H2 group were significantly lower than those in control group after 3 days(P<0.05),and there was significant difference between the two groups(P<0.05).Compared with the S group,the levels of VAS,SF-MPQ,SIS,tramadol and gabapentin were significantly decreased in the H1 group after 5 days,and decreased in the H2 group after 3 days.the VAS,SF-MPQ(P<0.05).Compared with H1 group,the levels of VAS,SF-MPQ,SIS,tramadol and gabapentin in H2 group were significantly lower than those in the control group,and the difference between the two groups was statistically significant(P<0.05).Compared with the S group,the levels of SOD,LC3? and Beclin-1 were decreased and MDA,IL-6 and TNF-? were increased in the H1 and H2 groups(P<0.05),compared with the H1 group,SOD,LC3? and Beclin-1 were increased,MDA,IL-6 and TNF-? were decreased in H2 group,the difference was statistically significant(P<0.05).Conclusion: Inhaled hydrogen has a therapeutic effect on patients with herpes zoster neuralgia and may be associated with reduced oxidative stress,reduced inflammatory response,and activated autophagy.