| Objective In this study,paraquat(PQ)was intraperitoneally injected into mice to establish a PD-like model.Firstly,we observed the neurobehavioral and DA neurons injury of PQ in mice.Secondly,we investigate the role of α-synuclein in the regulation of NOX2 via CD11BPI3 K pathway in PQ induced microglia activation.Lastly,the protective effect of taurine(Tau)on PQ induced neurotoxicity by inhibiting microglia activation was discussed.It provides a theoretical basis for the study of molecular mechanism and therapy of PD induced by PQ.Methods1.To establish the model of PD-like mice: 48 SPF male C57BL/6 mice were randomly divided into control group(n = 12)and PQ Group(n = 36).Mice in the exposed group were intraperitoneally injected with 15 mg /kg PQ twice a week,while those in the control group were injected with 0.9% Saline of the same volume for 8 weeks.2.To establish Tau protective model: 48 SPF C57BL/6 male mice were randomly divided into 4 groups: control group(Con): 0.9% Saline,Tau control group(Tau): 150 mg/kg Tau,PQ:15mg/kg PQ;Tau + PQ group: 150 mg/kg Tau was injected intraperitoneally 1 hour before PQ injection.Each group of 12 mice was injected twice a week for 8 weeks.3.Neurobehavioral observation: The body weight of mice was recorded once a week,and the general behavioral changes such as mental state,reactivity,fur and feeding condition were observed.On the second day after the last administration,the mice in each group were tested on neurobehavior.Firstly,the locomotor coordination ability and gait changes of each group were tested by the bar climbing test and gait measurement test.Then,the cognitive function and learning and memory ability of each group were evaluated by open field test and new object recognition test.Finally,the depression-like behavior and anxiety-like behavior of mice were evaluated by tail-suspension test and elevated maze test.4.Nissl staining: The number and morphology of Nissl bodies in neurons of substantia nigra were measured by Nissl staining.5.Immunohistochemical staining(IHC): IHC was used to observe the morphological and quantitative changes of Neu N and DA neurons,and to detect the activation level of Iba-1 in substantia nigra and striatum and the expression level of α-synuclein in microglia.6.Double-label staining(IF): The co-expression of α-synuclein and TH in the substantia nigra and the co-expression of Iba-1 and TH in the substantia nigra were detected by IF.7.Western blot(WB): The expression of PI3 K,PDK1,AKT,P-,AKT and P47 PHOX in the midbrain neurons Neu N,DA neurons TH,Iba-1,α-syn,DA transporter DAT,inflammatory factors(tnf-α,IL-1β,IL-6,HMGB1,i NOS),P47 PHOX in the cytoplasm and cell membrane were determined by WB.8.Superoxide anion fluorescent probe(DHE)analysis: to detect the expression level of superoxide in the substantia nigra.ResultsPart1.The mechanism of paraquat regulating NOX2 in neuroinflammation through α-synuclein mediated CD11b/PI3 K pathway.1.The changes of body weight and general behavior of mice in each group: The Mice in the control group were in good condition during the experiment.No obvious behavioral changes were observed immediately after intraperitoneal injection of PQ in mice exposed to PQ.However,with the increase of exposure time,hair appears dry and disorderly,slow reaction to escape.Individual mice appear to tilt the head,back arch,static tremor and other symptoms.The body weight of each group showed a steady rising trend,and there was no significant difference in the change of body weight.2.The results of gait analysis showed that there was no significant change in the step length(P > 0.05),but the step length decreased gradually,especially in the group of 8 weeks(P <0.05).The results of pole climbing test showed that the time of pole climbing in mice exposed to the drug at 6 and 8 weeks was significantly increased compared with the control group(P <0.05).3.The effect of PQ on cognition and memory ability of mice: compared with the control group,the moving distance and the residence time in the central region of PQ group were decreased(P < 0.05).In the new object recognition experiment,with the increase of time and dose,the time and times of touching the new object decreased in a time-dependent manner,and the difference was statistically significant compared with the control group(P < 0.05).4.The effect of PQ on the emotion of Mice: The tail suspension test showed that the tail suspension time of mice increased with the increase of PQ exposure time(P < 0.05).In the elevated maze test,the time and frequency of entering the open arm in the PQ group decreased gradually compared with the control group(P < 0.05).5.The damage effect of PQ on DA neurons in substantia nigra and striatum of mice:compared with the control group,the number of DA neurons in substantia nigra of PQ group decreased significantly(P < 0.05).IHC showed that the volume of neurons in substantia nigra of PQ group decreased,the axon fibers were scattered and the axon terminals were broken,the number of neurons in PQ group decreased significantly(P < 0.05).The positive expression of axon fibers of DA neurons in the striatum also decreased significantly(P < 0.05).Western blot results showed that the relative expression of protein of Neu N,DA neurons TH and DAT of striatum decreased with the increase of time and dose of PQ(P < 0.05).6.The result of IHC showed that the expression level of α-synuclein in the substantia nigra of mice exposed to PQ increased gradually,the positive staining was brown and yellow,and the expression level of α-synuclein in the substantia nigra of mice exposed to PQ was higher than that in the control group,especially at the 8th week after exposure.Only a few sparse TH neurons(red)were observed in the substantia nigra of the PQ group.The staining intensity was decreased and accompanied with α-syn deposition(green),and the fluorescence intensity of α-syn was enhanced obviously.Western blot showed that the relative expression of dimer and oligomer α-syn protein in the midbrain tissue of PQ exposed mice increased in a time-dependent manner(P < 0.05).The relative expression of S129-p-α-syn protein was also increased,which was significantly higher than that of control group(P < 0.05).7.The effect of PQ on microglia activation and M1 polarization: compared with the control group,the results of IHC showed that the substantia nigra and striatum microglia Iba-1 of the PQ group increased with the increase of exposure time,and the synapses became shorter and thicker,the expression level increased significantly(P < 0.05).IF observed,compared with the control group,the neurons in PQ group were obviously lost(green),the distribution was sparse and disorderly,and the fluorescence signal was obviously weaker than the control group.At the same time,the fluorescence intensity of Microglia Iba-1 increased gradually(red).Western blot showed that the relative expression of Iba-1,TNF-α,IL-1β,IL-6,HMGB1 and i NOS in PQ group were significantly higher than those in control group(P < 0.05).8.The expression of PI3 K,PDK1 and p-AKT in the CD11B/PI3K/NOX2 pathway was significantly increased in PQ group after 8 weeks of PQ exposure,the expression of P47 phox in the cytosol of PQ Group was higher than that of control group,but the expression of P47 phox in cytosol was lower than that of control group(P < 0.05).The expression of superoxide in substantia nigra of PQ group was significantly higher than that of control group(P < 0.05).Part2.Taurine protects against paraquat-mediated neuroinflammation by inhibiting microglia activation1.The body weight and general behavior of mice in each group: the hair of the PQ group was dry and disorderly,the reaction ability was slow,the action was reduced,the reaction was slow to the outside stimulation,the escape reaction was slow,the individual mice presented the symptoms of crooked head,erect tail and so on.There was no significant behavior in control group and Tau Intervention Group.There was no significant difference in the average body weight of each group(P > 0.05).2.The effect of Tau on the motor function of PQ-induced PD-like mice: gait analysis showed that compared with control group,the step length of PQ-exposed mice decreased(P <0.05),and that of Tau-exposed mice increased significantly(P < 0.05).The results of poleclimbing test showed that the time of pole-climbing in PQ group was longer than that in control group(P < 0.05),and the time of pole-climbing in Tau intervention group was shorter than that in PQ group(P < 0.05).3.Effect of Tau on cognitive function of PQ-induced PD-like mice: in open field experiment,compared with the control group,the moving distance and the residence time of the central region decreased in PQ-exposed group(P < 0.05),and compared with PQ-exposed group,the distance of movement and the residence time in the central region of the mice in the Tau intervention group were significantly increased(P < 0.05).The new object recognition experiment showed that compared with the control group,the new object recognition time of the mice in the exposed group decreased(P < 0.05),while that of the mice in the Tau intervention group significantly increased(P < 0.05).4.Effect of Tau on PQ-induced emotion in PD-like mice: compared with the control group,the tail suspension time of PQ-exposed mice increased,while that of Tau-exposed mice decreased.The residence time and times of open arms in the PQ group were lower than those in the control group(P < 0.05).The residence time and times of open arms in the Tau intervention group were increased compared with those in the exposed group(P < 0.05).5.The effect of Tau on PQ-induced neurons in substantia nigra and striatum of PD-like mice: compared with control group,most of the neurons in substantia nigra of PQ-exposed mice were not clear and axons disappeared,the number of Neu N and TH positive cells and the expression of axon fibers were significantly decreased(P < 0.05),compared with PQ group,the morphology of neurons in substantia nigra was more intact in Tau Intervention Group,the number of Neu N and TH positive cells and the expression of axon fibers were significantly increased(P < 0.05).Western blot showed that the protein expression of TH,Neu N and DAT in mesencephalon of mice exposed to Tau was significantly lower than that in control group(P< 0.05),and the protein expression of Th,Neu N and DAT in Tau intervention group was significantly higher than that in PQ group(P < 0.05).6.The expression level of α-synuclein in the substantia nigra of PQ group was significantly higher than that of PQ group(P < 0.05),and the expression level of α-synuclein in the substantia nigra was significantly higher than that of PQ group(P < 0.05),the positive staining of α-syn in substantia nigra of mice in Tau intervention group was significantly decreased(P <0.05).Western blot showed that protein expression of dimer,oligomer and s129-p-α-syn in PQ group was significantly higher than that in control group(P < 0.05).The protein expression of dimer,oligomer and S129-p-α-syn in Tau intervention group was significantly lower than that in control group(P < 0.05).7.Effect of Tau on PQ induced microglia in PQ-like mice: IHC results showed that compared with the control group,in PQ Group,the microglia of Iba-1 positive staining in substantia nigra were enlarged,round or oval,thick synapses,dark brown and more.The number of Iba-1 positive cells in the substantia nigra of the Tau intervention group was small,the positive staining was light,the distribution was sparse and the positive cells were few.The results of IF and IHC showed that the number of Neu N positive neurons in substantia nigra of PQ group was decreased,the fluorescence signal of IBA1 was decreased(green),and the fluorescence signal of Iba-1 was enhanced(red).Compared with PQ group,Neu N fluorescence signal of neurons in substantia nigra of Tau intervention group was stronger than that of PQ group,but the microglia fluorescence signal was weaker.The results of Western blot showed that the expression of IBA-1 protein in mesencephalon of the exposed group was higher than that of the control group,but the expression of Iba-1 protein in the PQ group was lower than that in the Tau group(P < 0.05).8.The expression of IL-6,IL-1β,TNF-α,HMGB1 and i NOS in PQ group was significantly higher than that in control group(P < 0.05).The relative expression of IL-6,IL-1β,TNF-α,HMGB1 and Inos in Tau intervention group was significantly lower than that in PQ group(P< 0.05).9.Effect of Tau on activation of CD11b/PI3K/NOX2 signaling pathway induced by PQ:Western blot showed that the relative expression levels of CD11 b,PI3K,PDK1 and p-AKT in the midbrain of PQ treated mice were significantly increased.The expression of P47 phox decreased in cytoplasm and increased in membrane(P < 0.05).The expression levels of CD11 b,PI3K,PDK-1 and p-AKT protein in the midbrain of the Tau intervention group were significantly lower than those in the PQ group.Meanwhile,the expression of P47 phox in the cytoplasm and the expression of P47 phox in the cell membrane were significantly lower than those in the PQ group(P < 0.05).The level of ROS in PQ group was higher than that in control group,while that in Tau group was lower than that in PQ group(P < 0.05).Conclusion 1.PQ activates CD11b/PI3K/ NOX2 pathway by inducing pathological a-syn aggregation,and then mediates abnormal activation of microglia,causing the uncontrolled release of oxygen free radicals and inflammatory factors,damaging DA-ergic neurons,and inducing depression,anxiety,cognitive function and motor dysfunction in mice.2.Tau can inhibit the activation of CD11B/PI3K/NOX2 pathway,down regulation of microglia M1 marker protein,reduce the injury of DA neurons and abnormal aggregation of α-syn induced by PQ,and improve the motor,emotional and cognitive function of PD-like mice. |
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