The Mechanism Of Microglial MHCⅡ Expression-induced Dopaminergic Neurodegeneration And Protective Effects Of Doxycycline

PartⅠEffects of nigral lipopolysaccharide administration on dopaminergic dysfunction and microglial cell activationObjective To investigate the effects of nigral lipopolysaccharide (LPS) administration on dopaminergic dysfunction and microglial cell activation.Methods 60 rats were divided randomly into five groups:Control, 1day,7d, 14d and 60d groups. LPS was stereostatically injected into unilateral substantia nigra of rats. At different survival times,the circling behavior was observed by intraperitoneal injection of Apo morphine; the damage to the substantia nigra DA neurons was observed by using tyrosine-hydroxylase (TH) immunohistochemical staining; specific antibody OX-42 was used to detect the changes in morphology and the numbers of microglia; the contents of monoamine in the nigrostriatal system were measured by high performance liquid chromatography (HPLC);the degenerative neurons in the substantia nigra were detected by Fluoro-JadeB staining.Results The circling behavior ipsilateral to LPS injected-side was induced in some rats 14d to 60d following injection. Compared with the control rats, the 12% to 71.5% reduction of TH-positive cells in the lesion of substantia nigra was found at Id, 7d,14d and 60d time groups after LPS treatment. Compared with the control rats, the 28.2%-65.7% reduction of dopamine and it s metabolite (DOPAC) in the lesion of striatum and substantia nigra was found at 7d,14d and 60d time groups. The activation of microglia was also observed from Id to 60d time groups after LPS treatment. There were a number of the positive degenerative neurons in the substantia nigra.Conclusions LPS intranigral injection could induce the activation of microglia and the degeneration of dopaminergic neurons in the substantia nigra.PartⅡThe expression of microglia MHCⅡis affected by LPS injected in substantia nigraObjective To investigate the effect of Lipopolysaccharide(LPS)on the expression of Major histocompatibility complex classⅡ(MHCⅡ) in microglia on LPS-induced rat mode of Parkinson's disease(PD).Methods LPS was stereostatically injected into unilateral substantia nigra (SN) of rats. Immunohistochemistry was used to detect midbrain MHCⅡ-positive microglia. Western blot were used respectly to detect the express of MHCⅡprotein. In order to determine the possible colocalization of inducible nitric oxide synthase (iNOS) and p47phox with microglia, double immunofluorescence was performed on nigral sections.Results MHCⅡ-positive microglia appearnced at 1 d following nigral LPS administration. The number of MHCⅡ-positive microglia reached the peak at 7 day and significantly decreased at 60days following nigral LPS administration. The levels of MHCⅡdetected by Western blot have a similar tendency. Double immunofluorescence for iNOS and p47phox with MHCⅡ-ir microglial marker OX6 showed a very close match between iNOS-ir, p47phox-ir and OX6-ir cells.Conclusions LPS intranigral injection could induce the expression of MHCⅡon microglia in SN. MHCⅡ-ir microglia could also induced by LPS to express iNOS and p47phox in a rat mode of PD.PartⅢProtective effects of Doxycycline on dopaminergic neuron in LPS-induced rat model of Parkinson's diseaseObjective To explore the protective effect of Doxycycline (DC) on dopaminergic neuron in lipopolysaccharide (LPS)-induced rat model of Parkinson's disease (PD).Methods 60 SD rats were randomly divided into three groups:the control, LPS group and Doxycycline treatment group. LPS was stereostatically injected into unilateral substantia nigra (SNc) of rats to form the PD models. The damage to the substantia nigra DA neurons was observed by using tyrosine-hydroxylase (TH) immunohistochemical staining. Specific antibody OX6 was used to detect the changes in morphology and the numbers of microglia. The contents of dopamine and DOPAC in the striatum were measured by high performance liquid chromatography (HPLC). Western blot were used to detect the express of MHC II proteinum.Results After Doxycycline treatment, the number of TH-positive cells remained in the SNc increased from 37.86±5.43% to 78.57±4.28% (P<0.01). the contents of dopamine and DOPAC in the striatum increased respectly from 4.89±0.27 and 0.70±0.07 to 7.00±0.34 and 1.10±0.10 (P<0.01); There was a significant decrease in rotational asymmetry in the Doxycycline treatment group (79.86±11.76 turns/30min) when compared with the LPS group(207.71±13.61 turns/30min) (P<0.01). However, the number of MHCⅡ-ir microglia decreased significantly(the LPS group: 834.80±82.41; the Doxycycline treatment group:354.20±59.365,P<0.01) after Doxycycline treatment.Conclusions Doxycycline might inhibit dopaminergic neuron degeneration by down-regulating MHCⅡexpression in microglia.PartⅣDoxycycline down-regulates MHCⅡexpression in BV-2 microgliaObjective To explore the inhibitory effects of Doxycycline (DC) on expression of MHCII in BV-2 microglia.Method 1. BV-2 was cultured as a substitute of microglias, which were divided into 3 groups:the control group (no any special handling), lipopolysaccharide (LPS) group (LPS+BV-2), and Doxycycline pre-treatment group(BV-2 was stimulated by LPS after co-incubation with Doxycycline at 3 concentration gradients for 30min).2. BV-2 microlia were detected with immunofluorescence confocal microscopy.3. The levels of MHCⅡ, IRF-1,phospho-STAT1 and phospho-PKCa were evaluated by Westen Blot,and the levels of CIITAmRNA were evaluated by RT-PCR.Results 1. Doxycycline pre-treatment could inhibite MHCⅡexpression in BV-2 by immunofluorescence, with quantity-effect relation.2.Weastern blot displaied that Doxycycline pre-treatment blockaded MHCⅡand phospho-PKCa expression(P<0.01),but unaffected IRF-1,phospho-STAT1(P>0.05).3. G66976 down-regulated obviously MHCⅡexpression induced by LPS (P<0.01).4. RT-PCR displaied that Doxycycline pre-treatment inhibited CIITAmRNA expression in BV-2(P<0.01).Conclution Doxycycline inhibited MHCⅡexpression in BV-2 microlia through inhibition of Protein Kinase Ca.