| Hydrogen sulfide?H2S?,as a gas molecule,participates in many physiological and pathological processes of organisms.Hydrogen sulfide is involved in regulating the pathophysiology of systems,such as nervous system,endocrine system,cardiovascular system,etc.Endogenous and exogenous hydrogen sulfide play an important role in pain.N-methyl-D-aspartic acid?NMDA?receptor is a ligand gated ionic glutamate receptor,whose subtype B plays an important role in the pain conduction in the spinal cord.Studies have shown that activation of NMDA receptors mediates formalin induced hyperalgesia,which is associated with peripheral tissue and nerve injury.In central nervous system,hydrogen sulfide induces the long-term potentiation?LTP?in the hippocampus,which is mainly produced by increasing the phosphorylation mediated current of the NMDA receptor.Diabetic mellitus?DM?is a metabolic disease characterized by hyperglycemia.Hyperglycemia could cause chronic damage and dysfunction in the cardiovascular system,the urinary system,and the nervous system.Diabetic peripheral neuropathy?DPN?is one of the most common chronic complications of DM.The affected areas often occur acupuncture,cauterization,and electric shock pain,which seriously affect the quality of patients'life.It has been reported that the incidence of peripheral neuropathy and degenerative pain in diabetic patients is about 7.5%-24%.Cognitive dysfunction is mainly manifested by memory impairment and intellectual decline,accompanied by a decline in learning and executive ability,as well as personality and behavior changes.Studies have shown that hyperglycemia can cause neuronal damage in the brain.Cognitive dysfunction has been reported to be one of the late complications of DM.Hydrogen sulfide has a variety of neuroprotective effects,which have been confirmed by a large number of experiments at home and abroad,mainly including anti-neuroinflammatory reaction,anti-oxidative stress injury and anti-hypoxic ischemic nerve injury.Studies have shown that the level of hydrogen sulfide in the brain of Alzheimer's patients is significantly lower,and the level of homocysteine is increased,and the degree is positively correlated with the severity of the disease.Hydrogen sulfide can increase the plasticity of neural cells and reduce the apoptosis of hippocampal neurons.Clinically,the risk of cognitive dysfunction in diabetic patients is significantly higher than that of non diabetic patients.The relationship between cognitive dysfunction and endogenous hydrogen sulfide is unclear.Dexmedetomidine is a highly selective alpha 2adrenergic receptor agonist,which can reduce the formation of inflammatory cytokines in the immune inflammatory response through the central and peripheral mechanisms.This study investigates the mechanism of hydrogen sulfide participated in pain and the role and mechanism of hydrogen sulfide in cognitive function of diabetic rats with neuropathic pain,and evaluates the effect of dexmedetomide on postoperative cognitive function and the effect of hydrogen sulfide in plasma to provide a reference for clinical treatment.Part one Hydrogen sulfide is involved in the formation of hyperalgesia in ratsObjective:Hydrogen sulfide?H2S?is an endogenous neurotransmitter that importantly regulates various physiological and pathological events including pain signal transduction.In this study,we observed the effects of hydrogen sulfide on the behavior of rats and the effects of mRNA and protein of N-methyl-D-aspartic acid?NMDA?receptor 2B in the spinal dorsal horn and dorsal root ganglion?DRG?.Methods:After subcutaneous injection of Na HS and formalin in 5 days,we observed the bilateral PWT as well as NMDAR-2BmRNA and protein levels in spinal dorsal horn and dorsal root ganglion?DRG?.Before subcutaneous injection of NaHS,hydroxylamine or methemoglobin was intrathecally injected.We observed the changes of contralateral PWT after intrathecally injected two hours later.Before NaHS intrathecal injection,memantine was intrathecally injected.We observed the contralateral PWT in15min,30min,45min,60min and 120min.Results:Subcutaneous injection of NaHS significantly decreased the PWT in the contralateral paw of rats,while formalin did not decrease the PWT in the contralateral paw of rats,and NMDAR-2BmRNA and protein levels in spinal dorsal horn and dorsal root ganglion were significantly increased in hyperalgesia induced by formalin.MNDAR-2BmRNA and protein levels in spinal dorsal horn were significantly increased in hyperalgesia induced by NaHS,while MNDANR-2BmRNA and protein levels in dorsal root ganglia had no obvious change.Intrathecal injection hydroxylamine or methemoglobin could alleviate the hyperalgesia in the contralateral paw induced by NaHS.Intrathecal injection memantine could also alleviate the hyperalgesia in the contralateral paw induced by NaHS.Summary:Repeated administration of H2S in peripheral tissue leads to a contralateral hyperalgesia through upregulating MNDAR-2B subunit in the spinal cord.Part two Hydrogen sulfide in hippocampus is involved in the formation of cognitive dysfunction in diabetic neuropathic ratsObjective:To observe the behavioral changes of diabetic neuropathic pain rats and determine the content of hydrogen sulfide and CBS m RNA and protein levels in the hippocampus of diabetic neuropathic cognitive dysfunction rats.Methods:Twenty-four clean male SD rats were divided into the control group and the diabetic neuropathic group?n=12?,and the diabetic neuropathic rat model was made by intraperitoneal injection of streptomycin 60 mg/kg.The adaptive training of Morris water maze was carried out before the experiment.Behavioral analysis was performed on the day of modeling and7d,14d,21d and 28d after modeling and the escape latency and time of crossing platform of each rat were recorded.HE staining was used to observe the pathological changes of hippocampal CA1 area and the cell apoptosis was detected by Tunel.The content of hydrogen sulfide and the levels of CBSmRNA and protein in hippocampus were measured.Results:The escape latency was significantly prolonged after injection of NaHS for 14d,21d and 28d,the time and numbers of crossing the platform were decreased after injection of NaHS for 21d and 28d in diabetic neuropathic rats.The cells in the hippocampal CA1 area of diabetic neuropathic pain rats were arranged in disorder and the pyramidal cells were loosely arranged and irregular.There was a lack of nerve cells in diabetic neuropathic rats.The apoptosis of hippocampus were significantly increased in the diabetic neuropathic rats.The hydrogen sulfide content,CBSmRNA and protein expression were decreased.Summary:Diabetic neuropathic rats have cognitive dysfunction and the cognitive dysfunction is aggravated over time.Cognitive dysfunction in diabetic neuropathic rats is related to the apoptosis of nerve cells.The cognitive dysfunction of diabetic neuropathic rats is related to reduction of hippocampal hydrogen sulfide and reduction of CBSmRNA and protein leveds in hippocampus.Part three Effects of exogenous hydrogen sulfide on central cognitive dysfunction in diabetic neuropathic ratsObjective:To observe the behavioral changes and mechanisms of exogenous hydrogen sulfide on diabetic neuropathic cognitive dysfunction rats.Methods:Sixty clean male SD rats were divided into 5 groups:control group?group C?,diabetic neuropathic group?group DNP?,low concentration hydrogen sulfide protection group?group DSL?,high concentration hydrogen sulfide protection group?group DSH?and pure hydrogen sulfide control group?group S??n=12?.The Morris water maze was detected at 0d,7d,14d,21d and28d respectively and the escape latency of each rat was recorded.HE staining was used to observe the pathological changes of the hippocampal CA1 area and the cell apoptosis was detected by Tunel.We detected the BDNF/TrkB,NMDAR-2A/2BmRNA and protein levels in hippocampus of five groups rats.Immunohistochemical determination was used to detect A beta-40 and APP levels at rats'hippocampus.We used RT-PCR method to determine the hippocampal APPmRNA and BACE1mRNA levels in hipoocampus of five groups of rats.Inflammatory factors such as IL-1betamRNA,TNF-alphamRNA,NF-kappaB65mRNA were detected by RT-PCR method.Results:The results of Morris water maze:the escape latency of group DSL was shorter than those in group DNP at 21d and 28d,the escape latency of group DSH were shorter than those in group DNP at 14d,21d and 28d.The arrangement and morphology of hippocampal cells in group DSL and group DSH were better than those in group DNP.The IHS score analysis showed that the IHS score in group DNP was significantly higher than that in group C,and the IHS score of the group DSH was significantly lower than that in group DNP.There was no significant change in IHS score in group DSL compared with that in group DNP.Compared with group C,the expression of BDNF/TrkBmRNA and protein in hippocampus of group DNP was significantly decreased.Compared with group DNP,the expression of BDNF/TrkBmRNA and protein in hippocampus of group DSL and group DSH increased significantly in a dose-dependent manner.The expression of NMDAR-2A/2BmRNA and protein in hippocampus of rats in group DNP was significantly higher than those in group C,and compared with group DNP,the levels of NMDAR-2A/2BmRNA and protein in hippocampus of group DSL and group DSH decreased significantly and were dose-dependent.The immunofluorescence showed that the positive cells of A beta-40 and APP in the group DNP were significantly increased and the cytoplasm coloring was obviously deepened.The expression of APP mRNA and BACE1 mRNA in the hippocampus of DNP rats was significantly higher than thoes in group C.Compared with group DNP,the hippocampus APPmRNA and BACE1mRNA were decreased in group DSL and group DSH.Compared with group C,the hippocampus proinflammatory factors such as IL-1betamRNA,TNF-alphamRNA and NF-kappaB65mRNA were increased in group DNP and the expression of IL-1betamRNA,TNF-alphamRNA,and NF-kappaB65 mRNA in the hippocampus of both group DSL and group DSH decreased significantly compared with group DNP.Summary:Diabetic neuropathic rats can produce cognitive dysfunction.Intraperitoneal injection of NaHS can improve the cognitive function in a dose-dependent manner,and its mechanism is related to the reduction of Abeta,the BDNF/TrkB pathway,the decrease of inflammatory factors and the downregulation of NMDAR-2A and 2B receptors.Part four Effects of hydrogen sulfide on postoperative cognitive dysfu-nction in diabetic patients during perioperative periodObjective:To observe the effect of dexmedetomidine on postoperative cognitive dysfunction indiabetes patientsundergoing spinal surgery.To observe the role of hydrogen sulfide in cognitive function of diabetic patients during perioperative period.Methods:Sixty diabetes patients?ASA?-??undergoing lumbar spinal with general anesthesia from December 2016 to May 2017 in our hospital were randomly divided into two groups?n=30?,control group?group C?and dexmedetomidine group?group D?.The blood samples were obtained from the patients at 30 min before anesthesia induction?T0?,the end of the operation?T1?and the second day of postoperative?T2?for the expression of TNF-?,IL-6,IL-10 and CRP as well as blood lactate and glucose.The blood samples were also obtained from the patients at T0 and T2 for the levels of S100bata and hydrogen sulfide in serum.Mini Mental State Examination?MMSE?score was performed the day before operation and the second day and 1 month after operation.The amount of total general anesthetic drugs during the surgery,the postoperative cognitive dysfunction and hospital stay were compared between the two groups.Results:TNF-?,IL-6 and CRP levels of the two groups significantly increased at T2 than those at T0?P<0.05?.IL-10 level significantly increased at both T1 and T2,while the TNF-?and IL-6 in group D were significantly lower than those in group C and IL-10 was higher in group D than that in group C?P<0.05?.The blood lactate and glucose in T1 were higher than those in T0.The level of S100bata at T2 was higher than that at T0 in both groups.While the levels of serum hydrogen sulfide before and after operation didn't have statistical significance in two groups?P>0.05?.There was no significant difference in MMSE score between the two groups at the day before operation?P>0.05?,and the MMSE score at 2d after operation was significantly lower than that one day before operation?P<0.05?.Compared with group C,MMSE scores in group D at 2d after operation were significantly higher?P<0.05?.Compared with group D,group C had more postoperative cognitive dysfunction?P<0.05?at 2d after operation.The remifentanil and propofol dosages in group D were significantly less than those in group C?P<0.05?.Summary:The use of dexmedetomidine in diabetes patients undergoing lumbar spine surgery could reduce postoperative cognitive dysfunction,which may be related with the reduction of dosage of combined drugs and the reduction of TNF-??IL-6 and the increase of IL-10.The role of hydrogen sulfide in perioperative cognitive function of diabetic patients needs further discuss.Conclusion:1.Repeated administration of H2S in peripheral tissue leads to a contralateral hyperalgesia through upregulating MNDAR-2B subunit in the spinal cord.2.Diabetic neuropathic rats have cognitive dysfunction and the cognitive dysfunction is aggravated over time.Cognitive dysfunction in diabetic neuropathic rats is related to the apoptosis of nerve cells.The cognitive dysfunction of diabetic neuropathic rats is related to reduction of hippocampal hydrogen sulfide and reduction of CBSmRNA and protein leveds in hippocampus.3.Diabetic neuropathic rats can produce cognitive dysfunction.Intraperitoneal injection of NaHS can improve the cognitive function in a dose-dependent manner,and its mechanism is related to the reduction of Abeta,the BDNF/TrkB pathway,the decrease of inflammatory factors and the downregulation of NMDAR-2A and 2B receptors.4.The use of dexmedetomidine in diabetes patients undergoing lumbar spine surgery could reduce postoperative cognitive dysfunction,which may be related with the reduction of dosage of combined drugs and the reduction of TNF-??IL-6 and the increase of IL-10.The role of hydrogen sulfide in perioperative cognitive function of diabetic patients needs further discuss. |
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