The Study On The Neuroprotective Effects Of The Non-steroidal Anti-inflammatory Drugs Combining With Neurotrophic Factor Against Degeneration Of Dopaminergic Neurons

Objective:Parkinson's disease (PD) is one of the most common chronic neurodegenerative diseases.Although the exact mechanisms responsible for this cell loss are unclear,emerging evidence suggests the involvement of inflammatory events played important roles in etiopathogenisis and pathogenesis of PD. Increased data has shown that the microglias might initiate the inducement of inflammatory reaction that resulted in the injury of dopaminergic neurons.And then the activation of microglias released a great deal of proinflammatory cytokines, superoxide(SOD), free radicals and so on, which resulted in the cascade reaction of the down-regulation of neurotrophic factors,the dysfunction of mitochondria and oxidative stress,etc.Such pathologic process above could contribute to the progression of PD slowly.Therefore, the suppression of inflammation processes and nerve protective therapy have become a hot spot of research to postpone and prevent the development of neurodegeneration. Since celecoxib can block enzymatic reaction of cyclooxygenase-2(COX-2),and inhibit the occurrence a series of inflammation reactions by activation of microglias.It could protect dopaminergic neuron.Ciliary neurotrophic factor(CNTF) is known to be crucial for the growth,differentiation,maturity of neurons.Moreover,it can maintain survivals of the neuron and promote the regeneration and recovery of impaired one.Finally,it has the function to make the astrocyte to secrete various neurotrophic factors to make up for the deficiency of such material. We used lipopolysaccharide (LPS)-induced inflammator reaction model of PD in vitro,and used celecoxib combining with CNTF against degeneration of dopaminergic neurons. Therefore, the purpose is to explore the injury of inflammatory reaction to dopaminergic neuron and the effect of non-steroidal anti-inflammatory drugs combining with neurotrophic factor(NTF) against degeneration of dopaminergic neurons induced by LPS in vitro. Then it may supply an ideal theoretical base for radical curing Parkinson's.Little research of non-steroidal anti-inflammatory drugs combining with NTF against degeneration of dopaminergic neurons in vitro has been reported.Methods: We dislodged ventral mesencephalon from SD newborn rat in 24h respectively, and single cell suspensions were achieved by trypsin digestion, centrifugalization and mechanical dissociation in sterile condition. These cells were planted into DMEMP/F12 culture medium with B27(2%) ,bFCF(20ng/ml) in 2×106/ml. After several days of incubation at 37℃, in 5%CO2,the medium was exchanged a half with flesh culture medium on the third culture day, followed by medium replacements at 2-3 d intervals for 7th days. When the cells covered with bottom and synapse of the cells inosculated each other.We used LPS-induced inflammatory lesion model of PD in vitro.The experiment was divided into control group and experiment groups.The control was cultured without LPS any agents. The experiment groups were divided into four subgroups according to media which contained the agents after being added in LPS as fowllows,respectively:(1)LPS (0.01mmol/l);(2) CNTF (10ng/ml); (3) CB(2.5umol/ml);(4) CNTF(10ng/ml) + CB(2.5umol/ml).After challenged by LPS for 12h and 24h,did cell molecular biology, respectively to observe the change of tumor necrosis factor-α(TNF-α) at translation levels in the culture medium using ELISA method,and did cell immunohistochmisry respectively to observe the change the quantity of the surface markers of TH-positive neuron in induction media for 12h and 24h.Results: The primary mesencephalic neural cell deriving from SD newborn rat can be cultured easily,and can maintain several days by action of neurotropic factors(NTFs) in vitro.Parts of cells were adherence after culture 24h of culture. Parts of cells stretched out 1-2 synapses and the cells distributed single layer after 48h. 72h later almost all cells stretched out synapses. Many cells exhibited augmented cell bodies, and stretched out long neurodendrite and neuraxon ,most of which were bipolar and multipole 7d later.The result of ELISA discovered that the concentration of TNF-αwas very low in control group in same time point.It had a remarkable statistical significance (P<0.01) compared with the experiment groups.In the same timepoint, the the concentration of TNF-αof CB+CNTF group( which was 12h,218.49±15.78, 24h,310.91±25.69 pg/ml ) was significantly lower than other subgroups.There is a remarkable statistically significant (P<0.01) compared between each subgroup.The result of cell immunocytochemical method showed that the amount of TH-positive cells were significantly statistical difference (P<0.01) among the control group and each subgroups in same timepoint. Additionally, in the experimental ,the amount of TH-positive cells of CB+CNTF group (12h,50.65±1.76, 24h,46.67±1.62) at an average view was significantly more than other subgroups (P<0.01). Although the amount of TH-positive cells of CB+CNTF group and CB group induced less and it had a statistical significance (P<0.05) compared between 12h and 24h,and moreover, it had a remarkable significance in LPS groups and CNTF groups(P<0.01).Conclusion: The primary mesencephalic neural cell culture from SD newborn rat can be successfully cultured for a long time in vitro. The inducer of LPS-induced inflammatory lesion maked the model of PD in vitro.Celecoxib and CNTF have neuroprotective effect on the protection of doparminergic neuron from LPS,and Celecoxib is better than CNTF.Celecoxib combining with CNTF can markedly lessen the degeneration of dopaminergic neurons induced by LPS in vitro.