| Background Rotenone(Rot),a plant insecticide,belongs to the isoflavone family.Recent studies have shown that long-term exposure of Rot results in dopaminergic(DA)neurodegeneration,leading to increase of the risk of Parkinson's disease(PD).PD is a common neurodegenerative disease.The crucial pathological features of PD are the loss of DA neurons in the substantia nigra pars compacta(SNpc)and the formation of Lewy body(LB)in the survival neurons.Studies have shown that Rot can also induce the loss of DA neurons in mice,and typical clinical symptoms similar to observed in PD patients,such as motor retardation,rigidity and tremor.However,the molecular mechanism of Rot-induced DA neurodegeneration and increase of the risk of PD still remains unclear.The blood brain barrier(BBB)is a continuous endothelial membrane in the microvessels of the brain,which can maintain the homeostasis of the brain environment.As the most basic structure of BBB,neurovascular unit(NVU)is mainly composed of cerebrovascular endothelial cells,pericytes,basement membrane,glial cells and adjacent neurons.Endothelial cells are connected by the proteins of tight junction(TJs)and adherens junctions(AJs).TJs mainly include ZO-1,Occludin,Claudin-5,etc.Studies have shown that the destruction of TJs can increase the permeability of BBB,leading to synaptic damage and neurological dysfunction,and promoting the occurrence of Alzheimer's disease(AD),PD and other diseases.Microglia are one of the components of NVU and play an important role in maintaining the structure and function of BBB.However,overactivated microglia can damage BBB through release of cytotoxic factors,such as reactive oxygen species(ROS),interleukin-1 beta(IL-1?),Tumor necrosis factor-alpha(TNF-?),matrix metalloproteinase(MMPs)and nitric oxide.Rot has been reported to cause oxidative stress in the brain of mice and promote the activation of microglia.However,whether Rot can induce BBB damage and subsequent neurodegenerative necrosis of DA through microglial activation and induce has not been reported.Objectives Therefore,we explored the effects of Rot on BBB permeability by using Rot-intoxicated mouse model.To further explore the role of microglial activation in Rot-induced BBB damage and DA neurodegeneration,colony-stimulating factor 1receptor(CSF1R)signal inhibitor PLX3397(Plx)and antibiotic Minocycline(Mino)were used to remove microglia and inhibit microglial activation,respectively.Methods1.Rot intoxication model: Adult SPF male C57BL/6J mice were randomly divided into 1 control group and 2 Rot intoxication groups(n = 15 for each group).Rot-intoxicated mice were divided into 0.75 mg/kg group and 1.5 mg/kg group according to the dosage.Rot was injected intraperitoneally to mice once a day(5m L/kg)for consecutive 21 days.Mice in Con group were intraperitoneally injected with equal saline(5 m L/kg).2.Plx and Mino treatment in Rot-intoxicated mice: Adult SPF male C57BL/6J mice were randomly divided into Con,1.5 mg/kg Rot,1.5 mg/kg Rot+Plx and 1.5mg/kg Rot+Mino groups(n = 15 for each group).Rot poisoning method as above.The Plx was given at doseage of 40 mg/kg one week before Rot poisoning,once a day,and the was administrated once every other day 30 mins prior to Rot after 7 days.Mino was given intraperitoneally at dosage of 50 mg/kg every other day 30 mins prior to Rot.Mice in Con group were intraperitoneally injected with equal saline(5 m L/kg).3.BBB permeability detection: Evans Blue(EB)rapidly binds to serum albumin to form macromolecular complexes when it enters the bloodstream.Immunoglobulin G(Ig G)is the main antibody component in the serum.Neither of them can cross through the BBB.Therefore,we used EB dye penetration test and Ig G immunohistochemical staining to detect BBB permeability in mice.The expressions and m RNA levels of TJs proteins(ZO-1,Occludin,Claudin-5)in mice were detected by Western blot,immunofluorescence staining and q RT-PCR,respectively.4.Microglial activation and production of inflammatory factors detection: The activation of microglia in the substantia nigra was detected by immunohistochemical staining with Iba-1 antibody.The expressions of i NOS and arginase-1(arg-1)in the midbrain of mice were detected by Western blot.The m RNA levels of i NOS,TNF-?,IL-1? were detected by q RT-PCR.5.Expression and transcription level of MMP-2 and MMP-9 detection : The expressions of MMP-2 pro,MMP-2 active,MMP-9 pro and MMP-9 active were detected by Western blot,and the m RNA levels of MMP-2 and MMP-9 were detected by q RT-PCR.6.Detection of DA neuron damage and gait abnormality: Immunohistochemistry with anti-TH antibody was used to detect the damage of DA neurons in substantia nigra and striatum in mice.The movement function of mice was detected by gait measurement.Results1.Effects of Rot on permeability of BBB in mice: EB experiments showed that the brains of mice in 0.75 mg/kg and 1.5 mg/kg Rot group turned to blue and the contents of EB were increased compared with controls,in which the alterations in 1.5mg/kg Rot group were significantly(P<0.01).Ig G staining showed that compared with Con group,the optical density of Ig G was significantly increased in the substantia nigra of mice treated with 1.5 mg/kg Rot(P<0.01),indicating increased BBB permeability.2.Effects of Rot on TJ proteins in the midbrain of mice: Western blot showed that the expressions of ZO-1,Occludin and Claudin-5 in the midbrain of mice in 0.75mg/kg and 1.5 mg/kg Rot groups were decreased compared with those in the Con group,in which the decrease of TJ proteins was significant in 1.5 mg/kg Rot group(P<0.05).Consistent with that of Western blot,immunofluorescence staining revealed thatcompared with Con group,1.5 mg/kg Rot significantly reduced the expression levels of ZO-1,Occludin,Claudin-5 in the midbrain of mice.3.Effects of Rot on microglial activation in the midbrain of mice:Immunohistochemical staining showed that compared with Con group,the activation level of microglia in the nigra region of mice in 0.75 mg/kg and 1.5 mg/kg Rot groups was increased and the cell body was large with high optical density of Iba-1 staining.The alteraions of these indexes in 1.5 mg/kg Rot group was significant(P<0.05).4.Effects of Plx and Mino on the number and activation of microglia in the midbrain of mice,respectively: Immunohistochemical staining showed that,compared with Con group,the activation level of microglia cells in the nigra region of mice in the 1.5 mg/kg Rot group was increased and the optical density of Iba-1 staining was elevated.Mino treatment significantly reduced the microglial activation and increase of optical density of Iba-1 staining(P<0.05).Quantificationresults of Iba-1+ microglia showed that the number of microglia in the substantia nigra of the Plx group decreased significantly,which was 89.3% less than that in the Con group(P<0.01).5.Effects of Plx and Mino on Rot-induced BBB injury in mice: BBB permeability test showed that compared with Con,1.5 mg/kg Rot intoxication resulted in increase of blue color,EB contents and Ig G optical density in the brain of mice.Plx and Mino treatment significantly reduced the increase of EB content and the optical density of Ig G staining induced by 1.5mg/kg Rot,and the difference was statistically significant(P<0.05).Western blot and q RT-PCR showed that the expressions and m RNA levels of three TJs proteins including ZO-1,Occludin and Claudin-5,in the midbrain of mice in the 1.5 mg/kg Rot group were significantly lower than those in the Con group(P<0.05).Plx and Mino treatment significantly attenuated the decrease of TJs protein expression and m RNA level induced by 1.5 mg/kg Rot,and the difference was statistically significant(P<0.05)compared with the 1.5 mg/kg Rot group.6.Effects of Plx and Mino on expression and m RNA levels of toxic factors in the midbrain of Rot-induced mice: Western blot showed that,compared with Con group,i NOS expression in mice in the 1.5 mg/kg Rot group was significantly increased and Arg-1 content was significantly decreased(P<0.05).Plx and Mino treatment significantly(P<0.05)reduced the expression of i NOS and decrease of Arg-1 content induced by 1.5 mg/kg Rot in mice.Compared with the Con group,the contents of MMP-9 pro,MMP-9 active,MMP-2 pro and MMP-2 active were significantly increased in the 1.5 mg/kg Rot group(P<0.05).Plx and Mino treatment significantly decreased the content and activity of MMP-9 and MMP-2 induced by 1.5 mg/kg Rot,and the difference was statistically significant(P<0.05).q RT-PCR showed that compared with the Con group,the m RNA levels of TNF-?,i NOS,IL-1?,MMP-2 and MMP-9 were significantly increased in the 1.5 mg/kg Rot group(P<0.05).Plx and Mino treatment significantly reduced 1.5 mg/kg Rot-induced gene expressions of inflammatory factors,MMP-2 and MMP-9,and the difference was statistically significant compared with that of 1.5 mg/kg Rot alone group(P<0.05).7.Effects of Plx and Mino on the decrease of DA neurons and gait abnormality in Rot-intoxicated mice: Immunohistochemistry showed that compared with Con group,the cell number and axon fibers of DA neurons in the substantia nigra in the 1.5 mg/kg Rot group were decreased significantly(P<0.01).Plx and Mino treatment significantly alleviated 1.5 mg/kg Rot-induced DA neuron damage,and the difference was statistically significant compared with 1.5 mg/kg Rot alone group(P<0.01).Gait detection showed that compared with Con group,the stride length of mice in the 1.5mg/kg Rot group was significantly reduced,and the stride distance was significantly increased(P<0.05).Plx and Mino treatment significantly reduced gait abnormality induced by 1.5 mg/kg Rot in mice,and the difference was statistically significant compared with that in the 1.5mg/kg Rot alone group(P<0.05).Conclusions1.Rot exposure increased BBB permeability and simutenuously decreased the expressions of TJs protein in the midbrain of mice.2.The activation of microglia and the release of inflammatory cytokines were important factors for Rot-induced BBB injury in mice.3.BBB injury mediated by microglial activation was involved in Rot-induced DA neurodegeneration and behavioral abnormalities in mice. |
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