Involvement Of Cyclooxygenase-2 In Lipopolysaccharide (LPS)-induced Dopaminergic Neurons Degeneration

Parkinson's disease(PD) is a common chronic neurodegenerative disorder in elderly people.The mean age of onset usually is over 55 years of age,and the incidence and prevalence of PD increase with age.The clinical manifestations include resting tremor,rigidity,slowness of movements,and postural instability.Degeneration of dopaminergic neurons in the substantia nigra par compacta (SNpc) and the conseguent loss of their projecting nerve fibers in the striatum is its pathological hallmark.The exact cause of neuronal loss in PD remains unknown.Consequently,current therapeutic interventions with dopamine replacement are aimed to controlling the symptoms of the disorder and fail to halt the underlyingprogressive degeneration of dopaminergic neurons process.Moreover,this therapy graduatly loses efficacy following its long-time taking.Therefore,it is important to deeply understand the pathophysiological mechanism of PD for developing new therapeutic strategies that will halt or even reverse the progress of the disease.Recently,increasing evidence from clinical and animal studied has suggested that the neuroinflammation,characterized by the activation of microglia,is involved in the pathogenesis of PD,Inflammatory process is a determinant factor for the degeneration of substantia nigra dopaminergic neurons.Lipopolysaccharide(LPS) is a active immunostimulant and potent microglial activator.It is different from 6-OHDA and MPTP killing neurons directly,LPS acts indirectly,via stimulation of microglial activation and release of neurotoxic products,such as TNF-α,IL-1β,NO,ROS and PG,consequently induced degeneration of dopaminergic neurons.Therefore,the models in vitro and vivo of LPS-induced dopaminergic degeneration are powerful tools for neuroinflammatory mechanistic studies of PD.However,there are many controversial reports about the roles of these neurotoxic factors in neurodegenerative process of PD.Indeed,inflammation-mediated degeneration of dopaminergic neurons in PD is a complex matter,these factors released by activated microglia interact and regulate one other.So it is important to identify the key factor and molecular pathway for the development of therapeutic strategies of PD.COX is a rate-limiting enzyme which catalyze the conversion of arachidonic acid to prostaglandins.COX-2,one of its isoforms,is a important inflammatory mediator.Inflammatory processes associated with an increased expression of COX-2 and elevated concentrations of PGE2 have been implicated in the cascade of deleterious events leading to neurodegeneration in several pathological settings.and many studies strongly suggest COX-2 may play a detrimental role in degeneration of dopaminergic neurons.However,the role of COX-2 in the loss of dopaminergic neurons induced by LPS ,ideal inflammatory model of PD, is still not fully understood. Clarifying the mechanisms of action,cellular and molecular pathway of COX-2 will help to develop new agents for the treatment of PD.The present study first investigated the change of COX-2 expression,the cellular origin of COX-2 and the neuroprotective effects of celecoxib,a specific COX-2 inhibitor,on dopaminergic neurons in rat primary midbrain cell cultures treated by LPS.Furthermore,we have built a inflammation-mediated model of PD by the intranigral injection of LPS,and observed the change of COX-2 protein expression in rat SNs at the different survial time.Finally,we examined the effects of celecoxib on the dopaminergic neurons in this rat model of PD for further exploring the role of COX-2 involved in inflammation-mediated neurodegeneration of PD .1,COX-2 mediated LPS-induced degeneration of dopaminergic neurons in rat primary midbrain cell culturesObjective: LPS-induced dopaminergic degeneration in primary midbrain cell cultures is a powerful model in vitro for neuroinflammatory mechanistic studies and screening potential therapeutic agents for PD.To establish an in vitro model of inflammation-mediated DA neuronal degeneration and investigate the neuroprotective effects and possible mechanism of celecoxib,a selective COX-2 inhibitor,on this model.Methods: Rat primary midbrain cell cultures were obtained from timed pregnant Sprague-Dawley rats on embryonic day 14.Seven days after seeding,the cultures were pretreated for 30 min with vehicle or 20μM celecoxib before treatment with 20ng/ml of LPS .Three days later,the cells were fixed and followed by immunofluorescence .PGE2 and TNF-αin supernatants were measured by radioimmunoassay.Results: LPS significently decreased TH-ir neurons. Morphologically,the TH-ir neurons had a significantly fewer dendrites. LPS treatment increased the number of microglia to 348% of that of the control cultures(P<0.05) and many microglias became irregular shape with enlarged cell body.Moreover,the levels of PGE2 and TNF-αin the culture media exposed to LPS markedly increases (531.65±69.59pg/ml and 13.24±0.62pmol/L respectively,P<0.05) compared with the control cultures(65.27±33.13pg/ml and 10.75±0.09pmol/L respectively).In contrast,pretreatment with celecoxib obviously attenuated the LPS-induced reduction in the number of TH-ir neurons, the survival rate of TH-ir neurons was 69% of the control cultures(P<0.05 compared with LPS-treated cultures).and the loss of neuronal process were reversed by celecoxib pretreatment.Celecoxib alone did not show significant effect on the number of TH-ir neurons.The number of microglias reduced to 200% of control ,and the LPS-induced morphological change of microglia were partially blocked with treatment with celecoxib. Celecoxib also decrease in levels of PGE2,TNF-α((190.52±73.10 pg/ml and 11.19±0.52pmol/L respectively,P<0.05).Interestingly, the increase of cellular density which expressing COX-2 was partially attenuated by pretreatment with celecoxib.Moreover, double immunoflurescence confirm that COX-2 only expressed in microglia.Conclusion: LPS stimulated activation of microglia, and increased COX-2 expression as well as the production of PGE2 ,TNF-αin microglia,and leaded to DA neuronal degeneration in primary midbrain cell cultures.Celecoxib protect dopaminergic neurons against LPS-induced neurodegeneration in primary midbrain cell cultures ,and its protective effects may be associated with inhibiting microglial activation and suppression of COX-2 expression in activated microglia, subsequently lessed the production of neurotoxic factors.Our study indicated that COX-2 involve in LPS-induced degeneration of dopaminergic neurons in primary midbrain cell cultures. 2,Neurotoxicity effect of intranigral injection of LPS on dopaminergic neuronsObjective: to observe intranigral injection of LPS-induced neurodegeneration of dopaminergic neurons and explore the inflammation mechanism of PD.Methods: Thirty-five female Sprague-Dawley rats were randomly divided into five groups;PBS treated group and 6h,24h,14d,30d groups after intranigral injection of 15μg LPS.The number of tyrosine hydroxylase(TH)-ir neurons and the morphological changes of OX-42 positive microglias in the SN of rats at different groups were observed by immunohistochemistry.Results: To compared with the control,the significant loss of TH-ir neurons in substantia nigra of the sides injected with LPS were observed on 24h,14d and 30d after LPS treatment(the survival rates were 57.4±5.72%,16.0±5.84%,14.9±5.74%,respectively,P<0.05),but no apparent loss at 6h postinjection of LPS(the survival rate was 90.1±7.05%,P>0.05) compared to the control.There are significant progression of TH-ir neurons loss from 6h to 24h and from 24h to 14d(P<0.05),but not from 14d to 30d(P>0.05). The microglial activation was observed as early as 6h,nearly all of the OX-42-positive microglial cells became full activated at 24h and 14d post-LPS injection,by 30 days the morphology of microglial cells returned to a resting state.Conclusion: Intranigral injection of LPS induced the microglial activation and leaded to time-dependent loss of DA neurons.It may be a ideal in vivo model for studying the mechanisms of inflammation in the pathogenesis of PD.3,COX-2 protein expression in the rat substantia nigra in dopaminergic neurons degeneration induced by intranigal injection of LPS Objective: To investigated the changes of COX-2 protein expression and PGE2 content in the substantia nigra(SN) following intranigral injection of LPS.Methods: Sixty female Sprague-Dawley rats were randomly divided into five groups;PBS treated group and 6h,24h,14d,30d groups after intranigral injection of 15μg LPS.At the different survial time after injection of LPS in the SN of rats,the number of TH-ir neurons were observed by immunohistochemistry,COX-2 protein expression by immunoblotting,and PGE2 production was measured with radioimmunoassay.Results: The number of TH-ir neurons is not changed on the 6st hour,but the significant loss were observed on the 24h and the maximum of loss on the 14d compared with the contral group;COX-2 protein significantly increased from 6h after LPS injection achieving maximum on the 14d(194.34±9.68A for 6h, 211.23±7.05A for 24h and 256.07±16.32A for 14d,P<0.05 compared to the PBS group),however,it persisted and elevated until the 30d(162.9±75.03A,P<0.05 compared to the PBS group).PGE2 productions in the SN and serum of rat also increased ,but did not followed the time pattern of COX-2 protein expression,reach the maximum at 6h and 24h after LPS injection,respectively.Conclusion: COX-2 protein expressions are increased in the SN of rat after intranigral injection of LPS.4,Neuroprotective effects of COX-2 inhibitor celecoxib against the degeneration of dopaminergic neurons induced by intranigral injection of LPSObjective: To explore the neuroprotective effect and possible mechanism of celecoxib,a selective COX-2 inhibitor,on degeneration of dopaminergic neurons induced by intranigral injection of LPS. Methods: Thirty female Sprague-Dawley rats were randomly divided into three groups:PBS group,saline solution +LPS group and celecoxib +LPS group. At the 14d after injection of 15μg LPS or PBS.Saline or celecoxib(20mg.kg-1.d-1) was administered by a gastric intubation,from 3 hour before intranigral injection of LPS up to 14days post LPS injection the number of tyrosine hydroxylase(TH) positive neurons and the morphological changes of OX-42 positive microglias in the SNs of rats were assayed by immunohistochemistry;COX-2 protein expression by immunoblotting,the production of PGE2 and TNF-αwere measured with radioimmunoassay.Results: The TH-ird neurons in the SN on the LPS-injected side reduced to 15.99±5.84% of the non LPS-injected side in saline treated group, compared with PBS control group(98.14±9.48%,P<0.05);COX-2 protein expression and productions of PGE2,and TNF-αwere upregulated in saline solution +LPS group(255.92±16.34A,1.77±0.41pg/mg and 4.76±0.49 pmol/L respectively);Compared with saline treated group,43% TH-ird neurons survived in the animals treated with celecoxib (P<0.05) and celecoxib significantly inhibited the protein expression of COX-2 as well as the levels of PGE2,TNF-α(95.85±8.77A,0.95±0.33pg/mg and 12.61±0.39 pmol/L respectively)and the microglial activation.Conclusion: Celecoxib exerted neuroprotective effects on DA neurons through inhibited COX-2 activity and the microglial activation as well as the neurotoxic secretions.COX-2 may be involved in degeneration of dopaminergic neurons after intranigral injection of LPS.In words,there is a vicious cycle consisting of COX-2,microglia,and dying or damaged neurons in the processes of LPS-mediated dopaminergic neuronal degeneration.LPS triggered activation of microglia, Activated microglia strongly expressed the COX-2 protein, and secreted a variety of toxic factors,including PGE2,TNF-α,and consequently damaged to dopaminergic neurons;The increase of COX-2 protein expression in turn aggravated microglial activation and exacerbated dopaminergic neuronal cell death through the generation of PGE2 and reactive oxygen species. Damaged or dying neurons also can activated microglia.The vicious cycle leaded to progressive dopaminergic neuronal loss.The blockade of COX-2 activity by COX-2 inhibitors can attenute the vicious circle and produce the neuroprotection by inhibiting microglial activation as well as reducing the production of PGE2 and TNF-α.Our study suggests that COX-2 paly a key role in LPS-induced degeneration of dopaminergic neurons.COX-2 may serve as a potential target for the development of therapeutic strategies to treat PD.