Effects And Mechanism Of Paclitaxel-induced Neuropathic Painon The Function Of Learing And Memory

Along with the changes in life style and surrounding environment,The morbidity of malignant tumor has been constantly increasing.As a result,malignant tumor has become the primary deadly disease.Paclitaxel has significant curative effect on various kinds of malignant tumor.But it may exerts dosage reliant neurotoxicity effects in clinical use.Among them,neuropathic pain is the most serious.While,due to the functional mechanism hasn’t been clarified,and paclitaxel induced neuropatic pain has no sensitive effect to analgesic,there is no effective treatment to this side effect has been found in clinic.Meanwhile,with the deeper research continues,more and more researches discovered that,paclitaxel may not only occurs neuropathic pain,but also injures cognitive function.So,the deeper research on neuropathic pain and cognition impairment induced by paclitaxel,may reveal the mechanism of neurovirulence induced by chemotherapeutic drugs,provide new therapeutic target for clinic treatment.Numerous researchers considered chemotherapeutic drugs cannot access to brain and damage central nervous,due to the exist of blood brain barrier.So paclitaxel induced neuropathy was considered only appears in peripheral sensory nerves.Thanks to the application of positron emission tomography（PET）,paclitaxel with radioactive tags were detected in brain,which indicates that,paclitaxel can penetrate through blood brain barrier and work in brain.Cerebral hippocampus,cortex and corpus striatum,as the core region of limbic system,play pivotal role in spatial learning memory and cognition process.Among the known mechanism of neuropathic pain,MAPKs pathway,GABA_Breceptor andNF-κB pathway may prompt the generation of oxidative stress factor like inducible nitic oxide synthase（iNOS）,reactive oxygen species（ROS）,reactive oxygen species（ROS）and proinflammatory factor like interleukin 1β（IL-1β）,tumor necrosis factor（TNF-α）,and further,induce ache.Oxidative stress response may take place when brain is stimulated by drugs,then neure abnormal discharge frequency and neuronal excitability will accordingly increase,kinds of pain mediums and inflammatory factors will be released.As a result,neuron activation threshold and central sensitization will be consequently descended and formed.Eventually,hyoerpathia will be developed and cerebral nerve cell apoptosis will be triggered.Because of the massive expression of NF-κB in hippocampus region,we consider hippocampus neuron may be damaged by paclitaxel through NF-κB/TNF-αpathway,learning and memory function may be damaged.Thus,this research firstly took NF-κB and GABA_Breceptor as pointcut to reveal the effect of hippocampus GABA_Breceptor and NF-κB path and their regulating effect on proinflammatory factor in paclitaxel induced neuralgia.Further,we explored the impact of proinflammatory factor TNF-αand oxidative stress in paclitaxel induced brain learning and memory impairment,and clarified the specific function of NF-κB path and proinflammatory factor.The researches above have great theoretical values on illuminating the pathogenetic mechanism of paclitaxel induced neuralgia and brain learning and memory impairment and developing a new target in drug therapy.Part one Effects of the expression of NF-κB/TNF-αpathway in paclitaxel-induced apoptosisObjective:To investigate the effects of NF-κB and TNF-αin paclitaxel-induced apoptosis by cultureing the primary hippocampal neurons.Methods:The primary cultured hippocampal neurons which had been cultivated for 5 days in vitro were randomly selected,the concentration is about 1×10⁶/ml.These neurons were separately given different concentrations of paclitaxel（0.1μmol/L、1μmol/L、10μmol/L）.The changes of hippocampal neuronral inhibitory rate and apoptosis rate were detected through MTT method and flow cytometry,as a result,the optimal concentration of paclitaxel to induce apoptosis were determined.Another 5 days of primary cultured hippocampal neurons were selected,and randomly divided into two groups:control group（group C）and the optimal concentration of paclitaxel group（N group）.The NF-κB and TNF-αexpression of hippocampal neurons were detected by Western blot.Results:1.Effect of paclitaxel on the hippocampal neurons viability had duration and concentration interaction（P<0.05）.2.The early apoptosis rate induced by 1μmol/L paclitaxel for 24h was（47.83±5.34）%.This is the experimental conditions for the determination of NF-κB and TNF-αproteins.3.Compared with group C,the expression of NF-κB and TNF-αprotein was significantly up-regulated in group N（P<0.05）.Conclusion:Paclitaxel can induce cell apoptosis by up-regulating the expression of NF-κB and TNF-αprotein.Part two Effects of NF-κB/TNF-αpathway and GABA_B receptor in central hippocampus on paclitaxel-induced neuropathic painObjective:To evaluate the effects of paclitaxel on the expression of NF-κB pathway、proinflammatory factor and GABA_B receptors in central hippocampus of rats by using nuclear factor-kappa B（NF-κB）inhibitor（SN50）and gamma-aminobutyric acid type B（GABA_B）receptor agonist（baclofen）.Methods:Pathogen-free male Sprague-Dawley rats,weighing 160-180g,were used in the study.All the Fifty rats were randomly divided into two groups:normal control group（C group,10）and Paclitaxol-induced neuropathic pain model group（40）,which rats were intraperitonealy injected saline or paclitaxel respectively.The forty rats were made the model rats of Paclitaxol-induced neuropathic pain in two weeks after received paclitaxol（i.p）2mg/kg in four alternate day injections reaching a cumulative dose of 8mg/kg per rat.Mechanical paw withdrawal threshold（MWT）to confirm the successful model was made at 2nd weeks after the first administration of Paclitaxol,time points were T1.Forty Paclitaxol-induced neuropathic pain model rats were randomly divided into 4 groups（n=10,each group）according to the injection medicines:saline 10μl were injected intrathecally in N group,baclofen 0.5μg/10μl in B+N group,SN50 200ng/10μl in SN+N group,SN50200ng+baclofen 0.5μg in SN+B+N group;saline 10μl were injected intrathecally in ten normal rats（C group）.There was an interval of 15minutes between twice intrathecal injections in five groups.The MWTwere measured at before and 120 minutes after intrathecally injection,time points were T2 and T3 respectively.After the end of behavior test,the hippocampus of rats was removed for detection of GABA_B1 receptor,NF-κB and inflammatory factor（IL-1β,TNF-α）expression using Western blotting and the number of positive cells with Immunohistochemical staining.Results:1.Compared with N group,MWT was significantly higher in groups B+N、SN+N and SN+B+N at T3（P<0.05）.2.Compared with C group,the expression of GABA_B1 receptor was down-regulated but which of NF-κB p65 and inflammatory factor（IL-1β,TNF-α）was up-regulated in group N（P<0.05）.Compared with N group,the expression of GABA_B1 receptor was significantly up-regulated,while the expression of NF-κB p65,inflammatory factor（IL-1β,TNF-α）were significantly down-regulated in groups B+N,SN+N and SN+B+N（P<0.05）.3.There were the same founding in immunohistochemistry assay analysiscompared with the results of western blot.Conclusion:Paclitaxel can induce neuropathic pain bydown-regulating of NF-κB,IL-1β,TNF-α,and up-regulating the expression of GABA_B receptors.Activing GABA_B receptors can relieve thepaclitaxol-induced neuropathic pain by increasingly blocking NF-κB pathway in coordination with NF-κB pathway antagonist.Part three Effects of TNF-αon the function of learing and memory of the rats suffering paclitaxel-induced neuropathic painObjective:To evaluate the effects of proinflammatory factor TNF-αon the function of learing and memory of rats with paclitaxol-induced neuropathic pain by using TNF-αreceptor inhibitor（Thalidomide）.Methods:Pathogen-free male Sprague-Dawley rats were used in the study.All the Forty-eight rats were randomly divided into four groups:normal control group（Vehicle group,12）,paclitaxol-treated group（PTX group,12）,thalidomide treated group（THD group,12）and paclitaxol+thalidomide（PTX+THD group,12）.Paclitaxel or vehicle was injected administered to these 2 groups of rats on 4 consequent days（days 1,3,5,and 7）with paclitaxel at a dose of 2mg/kg to reach a final cumulativepaclitaxel dose of8mg/kg.TNF-αinhibitor thalidomide was administered orally（100mg/kg/day）for 4 days during paclitaxel treatment.The MWTand Morris Water Maze（MWM）tests were performed before and every 4 days after paclitaxel and vehicle treatment（an average of values of 4 tests during the testing day）.The escape latency and the number of crossings over the platform location were analyzed and plotted.After the end of behavior test,the hippocampus of rats was removed for detection ofproinflammatory factor（IL-1β,TNF-α）expression using Western blot and the content of TNF-αwith ELISA.Results:1.Compared with MWT on the 0 day of PTX and PTX+THD group,MWT on 14 day was significantly higher（P<0.05）.There was no significance difference in Vehicle and THD group.2.Paclitaxel treatment significantly increased the duration of escape latencies and decreased the number of crossings over the platform compared with vehicle-treated rats（P<0.05）,while it had no effect on escape latencies and the number of crossings in thalidomide treated rats.However,thalidomide treatment can restored the increased escape latencies and the number of crossings in paclitaxel-treated rats（P<0.05）.3.Compared with vehicle group,the expression of IL-1βand TNF-αwere significantly upregulated in PTX group（P<0.05）.The expression of IL-1βand TNF-αcan be downregulated when the paclitaxel rats treated with thalidomide（P<0.05）.There was no significance difference between Vehicle and THD group.4.Compared with vehicle group,the content of TNF-αin plasma and Cerebro-Spinal Fluid（CSF）were significantly increased in PTX group（P<0.05）.The content of TNF-αin plasma and CSF can be decreased when the paclitaxel rats treated with thalidomide（P<0.05）.Conclusions:1.The function of learning and memory were impaired in rats with paclitaxel-induced neuropathic pain.2.The learning and memory impairment induced by paclitaxel is associated with an increase in TNF-αand IL-1βexpression level in the hippocampus.3.Inhibition of TNF-αsynthesis recovered the impairment of learning and memory in rats with paclitaxel-induced neuropathic pain.Part four Effects of cell apoptosis on learing and memory ofthe rats suffering paclitaxel-induced neuropathic painObjective:To evaluate the effects of cell apoptosis onthe function of learing and memory of rats with paclitaxol-induced neuropathic pain by using tumor necrosis factorα（TNF-α）receptor inhibitor（Thalidomide）.Methods:Pathogen-free male Sprague-Dawley rats were used in the study.All the Thirty-six rats were randomly divided into three groups:normal control group（Vehicle group）,paclitaxol-treated group（PTX group）and paclitaxol+thalidomide（PTX+THD group）.Paclitaxel or vehicle was injected administered to these 2 groups of rats on 4 consequent days（days 1,3,5,and 7）with paclitaxel at a dose of 2mg/kg to reach a final cumulative paclitaxel dose of 8mg/kg.TNF-αinhibitor thalidomide was administered orally（100mg/kg/day）for 4 days during paclitaxel treatment.The MWTand Morris Water Maze（MWM）tests were performed before and every 4 days after paclitaxel and vehicle treatment（an average of values of 4 tests during the testing day）.The escape latency and the number of crossings over the platform location were analyzed and plotted.After the end of behavior test,the cerebral cortex and hippocampus of rats was removed for detection of apoptosis with TUNEL Apoptosis Detection Kit,cleaved caspase-3 and TNF-αexpression using Western blot and the number of positive cells with Immunohistochemical staining（caspase-3,TNF-α）and the content of MDA.Results:1.Compared with MWT on the 0 day of PTX and PTX+THD group,MWT on 14 day was significantly higher（P<0.05）.There was no significance difference in Vehicle and THD group.2.Paclitaxel treatment significantly increased the duration of escape latencies and decreased the number of crossings over the platform compared with vehicle-treated rats（P<0.05）,while it had no effect on escape latencies and the number of crossings in thalidomide treated rats.However,thalidomide treatment can restored the increased escape latencies and the number of crossings in paclitaxel-treated rats（P<0.05）.3.Compared with vehicle group,the expression of TNF-αand cleaved caspase-3were significantly upregulated in PTX group（P<0.05）.The expression of TNF-αand cleaved caspase-3 can be downregulated when the paclitaxel rats treated with thalidomide（P<0.05）.4.There were the same founding in immunohistochemistry assay analysis and TUNEL Assay compared with the results of western blot.5.Compared with vehicle group,the content of MDAin plasma and CSF were significantly increased in PTX group（P<0.05）.The content of MDA in plasma and CSF can be decreased when the paclitaxel rats treated with thalidomide（P<0.05）.Conclusions:The behavior impairment induced by paclitaxol-induced neuropathic pain is associated with an increase in MDA,TNF-αand cleaved caspase-3expression level and apoptotic cells in the cerebral cortex and hippocampus.Inhibition of TNF-αsynthesis recovered the impairment of learning and memory in rats with paclitaxol-induced neuropathic pain.