| Parkinson’s disease(PD)is a central neurodegenerative disease with the basic pathological changes of the loss of dopaminergic neurons in the substantia nigra and the aggregation ofα-synaptic nucleoprotein in neurons.At present,the prevalence rate of Parkinson’s disease in our country is in the stage of sharp rise,which will give the whole society serious challenge.The pathogenesis of PD is complex and involves a variety of pathways and mechanisms,such as abnormal protein dynamics,mitochondrial dysfunction and neuroinflammation,which promote the development of PD.It has been found that PD inflammatory injury is closely related to over-activated microglia cells.Overactivated microglia cells produce inflammatory factors that trigger neuroinflammatory responses,which lead to the degeneration and death of dopaminergic neurons.The study found that NOD like receptor protein-3(nod like receptor family pyrin domain-containing3,NLRP3)mediated nerve inflammation can cause death of dopaminergic neurons.NLRP3 inflammatory corpuscle mainly by the effect of Apoptosis related proteins Cysteine aspartic acid protease 1 joint protein Apoptosis related dot protein and NLRP3.The NLRP3 inflammasome is an inflammatory complex in microglia.Its activation promotes the secretion of inflammatory cytokines Interleukin-1β(IL-1β)and Interleukin-18(interleukin-18),which ruptures microglia and further releases IL-1β,damaging dopaminergic neurons.Therefore,intervention of microglia-mediated neuroinflammatory response and inhibition of NLRP3 inflammasome may be potential measures for prevention and treatment of PD.Black seed grass is xin,warm,sweet,with tonifying kidney and brain,menstruation,detoxification diuresis effect.Thymoquinone is the active ingredient in the volatile oil of black seed,which has antioxidant,anti-inflammatory and anticonvulsant effects.Our previous study found that the Nrf2 pathway activated by TQ up-regulates the expression of anti-heme oxygenase-1 protein,alleviates neuronal cell apoptosis and delays the occurrence of PD.In order to further verify the therapeutic effect of TQ on PD neuroinflammation and the possible related mechanisms,this study established in vitro and in vivo PD models to explore the mechanism of TQ improvement on PD neuroinflammatory injury.PartⅠThe effect Neuroprotective effects of thymoquinone on MPTP-induced PD mouse modelsObjective:To observe the effect of thymoquinone on motor function injury of PD mouse model and verify the protective effect of thymoquinone on PD dopaminergic neurons.Methods:The motor function of each group of mice was evaluated by open field test and rotarod test.The expression levels of TH andα-Synuclein in the substantia nigra were measured by immunohistochemistry.Western Blotting method was used to analyze the expression of TH andα-Synuclein in each group.The neuronal degeneration of mice in each group was observed by FJB staining.Western Blotting was used to detect the expression of NLRP3 inflammasome related protein in PD model.Results:1.Thymoquinone improved motor function injury of PD mice:Open field experiment results showed that,compared with the control group,the central distance and central time decreased and the side distance and side time increased in the model group(P<0.01);Compared with model group,the central distance and central time in model+TQ group were significantly higher than those in model group,while the side distance and edge time were lower than those in model group(P<0.05).The results of rotarod test showed that:compared with the control group,the time on rod in model group was significantly reduced,and the time on rod in model+TQ group was significantly increased compared with model group(P<0.01).2.Thymoquinone alleviated dopaminergic neuron loss in MPTP-induced PD mice:Immunohistochemical results showed that TH-positive neurons and fibers were sparse and TH-positive neurons were reduced in model group compared with control group(P<0.01).Compared with model group,TH positive neurons and fibers in TQ group were increased(P<0.01).Western Blot showed the same results:compared with control group,TH protein expression in model group was significantly decreased(P<0.01).Compared with model group,TH protein expression in TQ group was significantly increased(P<0.01).3.Thymoquinone decreased the level ofα-Synuclein in MPTP treated PD mice:immunohistochemical results showed that the level ofα-Syn in the model group was significantly increased compared with the control group(P<0.01).Compared with the model group of mice injected with MPTP,model+TQ significantly decreased the level ofα-Syn(P<0.01).Western Blot showed that compared with the control group,the level ofα-Syn protein in the model group was significantly increased(P<0.01).Compared with model group,model+TQ significantly decreasedα-Syn protein level(P<0.01).4.Ameliorating effect of thymoquinone on neuron damage in PD mouse model:FJB staining results showed that the expression of degenerated neurons in the model group was significantly increased compared with the control group(P<0.01).Compared with model group,the expression of denatured neurons in model+TQ group was significantly reduced(P<0.01).Conclusion:Thymoquinone can alleviate motor dysfunction and improve motor function injury and loss of DA neurons in PD mice.PartⅡAmeliorating effect of thymoquinone on neuroinflammatory injury in PD cell modelObjective:To observe the neuroprotective effect of TQ on PD in vitro.Methods:MTT was used to detect the effect of MPP~+on proliferation of BV-2 and SH-SY5Y cells.The effect of TQ on the proliferation of SH-SY5Y cells under BV-2condition was detected by MTT assay.TUNEL method was used to detect the apoptosis rate of conditioned SH-SY5Y cells.Western Blotting was used to determine apoptosis-related proteins in cultured SH-SY5Y cells.Results:1.The optimal conditions for the intervention of MPP~+in BV-2 cells:The results of MTT showed that 100μM MPP~+treated BV-2 cells for 8h had little effect on cell viability and the activation state of BV-2 cells was observed under the microscope.SH-SY5Y cells were further treated with 100μM MPP~+directly or indirectly for 24h,and the survival rate of SH-SY5Y cells was significantly decreased after BV-2 cell intervention(P<0.01).2.Thymoquinone can alleviate the damage of SH-SY5Y cells induced by MPP~+conditioned culture:Compared with the control group,the survival rate of SH-SY5Y cells in the model group was significantly reduced(P<0.01).Compared with model group,the survival rate of SH-SY5Y cells in TQ group was significantly increased(P<0.01).3.Thymoquinone can reduce the apoptosis rate of SH-SY5Y cells cultured under MPP~+condition:TUNEL staining results indicated that the number of apoptotic SH-SY5Y cells in the model group was significantly increased compared with the control group(P<0.01),and the number of apoptotic SH-SY5Y cells in TQ group was significantly decreased compared with the model group(P<0.01).4.Effects of thymoquinone on apoptosis-related proteins of SH-SY5Y cells cultured under MPP~+conditions:Compared with the control group,the value of Bcl-2/Bax decreased and the expression of Caspase-3 increased in the model group(all P<0.01);compared with model group,Bcl-2/Bax value increased and Caspase-3 expression decreased in TQ group(all P<0.01).Conclusions:Thymoquinone can alleviate the apoptosis of SH-SY5Y cells induced by BV-2 cells and improve the damage of PD cell inflammatory model neurons.Part III Thymoquinone inhibited NLRP3 inflammasome activation and microglial cell activation in PD modelObjective:To observe the effect of NLRP3 inflammatory bodies on nerve injury in PD model,and to explore the possible mechanism of thymoquinone’s neuroprotective effect.Methods:The m RNA expression of TNF-α,IL-6 and IL-18 were analyzed by RT-q PCR.The expression of NLRP3 inflammasome related genes was analyzed by RT-q PCR.The expression of inflammatory factor IL-1βwas detected by ELISA.Western Blotting was used to detect the expression of NLRP3 inflammasome related protein in PD model.Results:1.Effects of thymoquinone on the relative m RNA expressions of inflammatory factors TNF-α,IL-6 and IL-18in the PD mouse model:Compared with the control group,the relative m RNA expressions of inflammatory factor-related genes TNF-α,IL-6and IL-18were significantly up-regulated in the model group(all P<0.01);Compared with model group,the m RNA relative expressions of inflammatory factor-related genes TNF-α,IL-6 and IL-18 in TQ treatment group were down-regulated(all P<0.01).2.Thymoquinone inhibits the activation of NLRP3 inflammasome in PD mouse model:Western Blotting showed that compared with the control group,NLRP3,ASC,Caspase-1 and IL-1βprotein expression levels were significantly increased in the model group(all P<0.01),and NLRP3 inflammasome-related protein expression levels were significantly decreased after TQ intervention(all P<0.01).The PCR results showed that compared with the control group,the m RNA relative expressions of NLRP3,ASC,Caspase-1,IL-1βin the model group were significantly up-regulated(all P<0.01).Compared with model group,the m RNA relative expressions of NLRP3,ASC,Caspase-1 and IL-1βin TQ treatment group were significantly down-regulated(all P<0.01).3.Thymoquinone decreased IL-1βexpression in PD cell model:Compared with control group,IL-1βexpression in model group was significantly increased(P<0.01);Compared with model group,TQ treatment group significantly decreased the expression of inflammatory factor IL-1β(P<0.01).4.Thymoquinone inhibited the activation of NLRP3 inflammasome in PD cell model:Compared with the control group,the protein expression levels of NLRP3,ASC,Caspase-1,IL-1βin the model group were significantly increased(all P<0.01),and the protein expression levels of NLRP3,ASC,Caspase-1,IL-1βin the TQ group were significantly decreased(all P<0.01).Conclusions:Thymoquinone protects dopaminergic neurons by inhibiting the activation of NLRP3 inflammatory bodies,reducing the expression of inflammatory factors,and inhibiting the activation of microglia cells.Part IV Thymoquinone promotes autophagy clearance of damaged mitochondria in PD cell models through a Parkin-dependent pathwayObjective:To observe the protective effect of TQ on mitochondrial damage in PD model.Methods:Western Blotting was used to detect the autophagy related proteins the expression levels of P62,LC3-Ⅱ,LC3-Ⅰ,Parkin and PINK1.Results:Thymoquinone can promote mitochondrial autophagy in PD cell model:Compared with the control group,the protein expression level of P62 in model group was significantly increased,the ratio of LC3-Ⅱ/LC3-Ⅰwas significantly decreased,and the protein expression levels of Parkin and PINK1 were significantly decreased(all P<0.01).Compared with model group,the expression level of P62 protein in TQ group was significantly decreased,the ratio of LC3-Ⅱ/LC3-Ⅰwas significantly increased,and the expression levels of Parkin and PINK1 protein were significantly increased(all P<0.01).Compared with TQ group,the protein expression level of P62 in TQ+inhibitor group(CQ)was significantly increased,the ratio of LC3-Ⅱ/LC3-Ⅰwas significantly decreased,and the protein expression levels of Parkin and PINK1 were significantly decreased(all P<0.01).Conclusions:Thymoquinone may have protective effect on dopaminergic neuron injury by promoting mitochondrial autophagy clearance and reducing neuroinflammation. |
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