| Background and ObjectivesChronic inflammatory pain,one of the main types of clinical pathological pain,if not timely and effectively treated,will be a costly health problem.It not only affects the patient’s quality of life,but also bring heavy economic burden and social responsibility to individuals,families and society.Due to its complex pathophysiological mechanism,chronic inflammatory pain brings a great challenge and opportunity to clinical practice（clinical diagnosis and treatment）and basic science research.Previous studies showed that neuro-inflammation and oxidative stress is one of the main mechanisms of chronic inflammatory pain.Under the condition of peripheral tissue damage or infection or tumor growth,spinal cord glial cells（including microglia and astrocytes）synthesis and release multifarious inflammatory factor and oxidative stress products to reduce the anti-oxidation capacity.All these factors build a local inflammatory minienvironment by interacting with each other,and eventually change the synaptic plasticity in the spinal cord,thus lead to the central sensitization.The main symptoms of chronic inflammatory pain are spontaneous pain,allodynia and hyperalgesia.Methane（CH4）,which has small molecular weight and simple structure,is the main ingredient of gas fuels such as natural gas,coal gas and combustible ice.Methane could be produced by anaerobic bacteria utilzing the anaerobic glycolysis of carbohydrate in mammalian intestine.Methane is inflammable gas,hence methane rich saline has been universally used to explore its physiology function.But recent studies have concluded that under hypoxia,methane can also be generated by eukaryotic cells and mitochondria in rat liver,and moreover,it has antioxidant,anti-inflammatory and anti-apoptosis capacity.To date,the above role of methane have been proved in model of ischemia reperfusion,sepsis,colitis,and so on.At the same time,there are some studies in which methane has been found to have neuroprotective effects.However,the therapeutical effect of methane on chronic inflammatory pain has not been explored.The purpose of this study is to investigate the analgesic effect of methane on chronic inflammatory pain induced by complete friend’s adjuvant and its possible mechanism.METHODWe utilize complete friend’s adjuvant induced monoarthritis rat as chronic inflammatory pain model,and use various experimental methods such as Immunohistochemistry,Flow Cytometry,Western Blot,enzyme linked immunosorbent assay etc,to observe the effect of methane rich saline on pain behavior in chronic inflammatory pain model rat,and explore the related mechanism.1.The effect of methane rich saline on the pain behavior of chronic inflammatory pain ratsSelect SD rats,weight about 200-300g,establishing monoarthritis rats（MA）inflammatory pain models.Observe the bilateral paw withdrawal threshold（PWT）changes of MA model after single or repetitive intraperitoneal injection of methane rich saline.2.Observe the effect of methane rich saline on the activation of glial cell in chronic inflammatory painEstablish the MA model,on post-operative days 10,after intraperitoneal inject the Methane rich saline or normal saline,we use western blot to detect the expression of Glial fibrillary acidic protein（mark for astrocytes）and Ionized calcium-binding adaptor molecule-1（marker for microglia）,to investigate whether the methane rich saline treat chronic inflammatory pain by adjusting the activity of glial cells.3.Obeserve the effect of methane rich saline on peripheral T lymphocyte infiltration into spinal cord in chronic inflammatory pain ratEstablish the MA model,on post-operative days 10,we use flow cytometry to detect T cell number in Lumbar enlargement segmental to detect the relationship between methane rich saline and peripheral T lymphocyte infiltration into spinal cord in chronic inflammatory pain rat.4.Observe the effect of methane rich saline on oxidative stress in spinal cord of chronic inflammatory ratEstablish the MA model,after intraperitoneal inject the Methane rich saline or normal saline,we use spectrophotometry to detect oxidative stress’s capacity（superoxide dismutase,catalase,malonaldehyde）on 0 day,3rd day,7th day,10th day in the ipsilateral spinal dorsal horn,and use Immunohistochemistry to detect the expression of 8 hydroxy deoxyguanosine,which is the biomarker of the intracellular oxidative stress,on 10th day in ipsilateral spinal cord dorsal horn.We will use the results to preliminary analysis that methane rich saline whether apply anti-oxidative stress to affect the chronic inflammatory pain.5.Observe the effect of the regulation effect of methane rich saline on the expression of inflammatory mediators in the spinal cord dorsal horn of chronic inflammatory pain ratEstablish the MA model,on post-operative days 10,after intraperitoneal inject the Methane rich saline or normal saline,we use enzyme linked immunosorbent assay to detection the expression of inflammatory cytokines（interleukin 1 beta,tumor necrosis factor alpha,interleukin 10）in spinal cord dorsal.horn.To investigate weather methane can inhibit the expression of inflammatory mediators in the spinal cotd dorsal horn.Results1.Methane rich saline can relieve mechanical allodynia in chronic inflammatory pain rats.Compared with NS treatment group,Single intraperitoneal injection methane rich saline at the dose of 10ml/kg,can partially reverse the PWT of MA rat,which difference is statistically significant（p<0.05）.And the peak effect appear 1 h after the treatment,and then began to reduce,and the total effective time is about 4 h.However,repeated intraperitoneal injection（1 h before opretaion and postoperative,once a day,a total of 11days）of methane rich saline,can improve the PWT of MA rat on postoperative day 3,and at least until the 10th day（p<0.05）.2.Methane rich saline can impact the PWT of chronic pain rat by inhibiting the activation of glial cellsAfter the left ankle inflammation,the astrocyte and microglia were significantly activated in ipsilateral spinal dorsal horn（p<0.05）.Compared with NS group,the activation intensity of microglial and astorcyte notly reduced in the opration side spinal cord dorsal horn of MS group rats（p<0.05）.3.Methane rich saline can impact the PWT of chronic pain rat by inhibiting the infiltration into spinal cord of peripheral T lymphocytesCompared with sham group,the ankle inflammation can induce peripheral T lymphocytes to infiltrate into spinal cord dorsal horn,which difference is statistically significant（p<0.001）.Compared with NS group,the proportion of CD3 positive T cells/mononuclear cells significantly decreased in the opration side spinal dorsal horn of MS group rat（p<0.01）.4.Methane rich saline can significantly decrease the oxidative stress in spinal cord dorsal horn of chronic inflammatory model ratsCompared with sham group,the activity of SOD and CAT decreased,and the expressin of oxidative stress product（MDA and 8-OHDG）increased in spinal cord of MA rat group（p<0.01）.Methane rich saline can partial recover the activity of SOD and CAT,and reduce the expression of oxidation product（MDA and 8-OHDG）in ipsilateral spinal cord dorsal horn of chronic inflammatory rats（p<0.05）.5.Methane rich saline can reduce the expression level of inflammatory mediators in spinal cord dorsal horn of chronic inflammatory pain ratCompared with sham group,the expression of proinflammatory cytokines（IL-1 beta,TNF alpha）increase and the expression of anti-inflammatory cytokines IL-10decrease in the opration side spinal dorsal horn of MA group（p<0.05）.However,continuous dosing of methane rich saline can inhibit the expression of IL-1 beta and TNF alpha,at the same time promote the expression of IL-10 in the opration side spinal dorsal horn of MA rat（p<0.05）.CONCLUSIONIn the chronic pain model rat induced by CFA,the intraperitoneal injection of methane rich saline can inhibit the activation of glial cells and the infiltration of T cell in spinal cord,and decrease the expression of proinflamatory mediators and oxidative stress products,as well as promote the expression of anti-inflammatory factors and protect the anti-oxidative stress capability in spinal dorsal horn,eventually relieve chronic inflammatory pain.This study suggests that methane rich saline play an analgesic role,and anti-oxidative stress effect and anti-inflammation effect maybe involved in this process.Methane rich saline maybe be new therapeutic choice of chronic inflammatory pain. |
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