The Mechanism Of Neuronal Pyroptosis In Brain Of Mice Induced By Paraquat Combined With Maneb Exposure

ObjectiveParkinson’s disease(PD)is a common neurodegenerative disease,with a high prevalence and increasing year by year.The exact pathogenesis of PD is unknown.Among the environmental factors related to PD,pesticide exposure and PD have received extensive attention.Paraquat(PQ)and maneb(MB)have been shown to be associated with PD.Recent epidemiological data suggest that exposure to chronic low-dose PQ from occupational,drinking and food sources may increase the risk of chronic neurodegenerative diseases such as PD and Alzheimer’s disease(AD).PQ can enter the brain through the blood-brain barrier,produce a large number of superoxide radicals,damage endogenous antioxidant function and present neurotoxicity.MB is a fungicide commonly used in agriculture.The parent compounds other than manganese in MB and manganese have neurotoxic effects and are associated with PD.MB can cause imbalance of mitochondrial function,leading to oxidative damage to cells and inducing neurotoxicity.At present,the combined chronic exposure of PQ and MB has become a recognized method for constructing PD animal models.But the mechanism of neurotoxicity associated with PQ and MB is not fully understood and there is also a lack of specific drugs for the prevention and treatment of poisoning.Epidemiological investigations and experimental studies have found that PQ and MB can cause abnormalities in the brain.Long-term low-dose exposure to multiple pesticides including PQ and MB can increase PD prevalence.It was observed that PQ and MB caused oxidative stress in the brain,increased phosphorylation of NF-κB and activation of microglia.It is known that the activation of microglia includes M1 polarizations with pro-inflammation and M2 polarization with anti-inflammation.In particular,in recent years,a new way of programmed cell death,pyroptosis,has been discovered and plays an important role in diseases of the CNS.Compared to apoptosis,pyroptosis occurs more rapidly and is an inflammatory death dependent on cysteinyl aspartate specific proteinase(caspase-1),accompanied by a large number of proinflammatory cytokines release such as interleukin(IL-1β).Is the injury caused by the combination of PQ and MB associated with microglial M1/M2 polarization imbalance?Do pyroptosis exist?No report has yet been made.Therefore,in this experimental study,we observed microglial activation and neuronal pyroptosis in mice examined the trend of polarized molecular markers of M1 and M2 polarizations.We also observed NLRP3 and caspase-1 expressions in midbrain and pons-medulla and detected the expression of NF-κB and ROS content.The preventive and reversal effect of glibenclamide(Gli)on the chronic exposure to PQ combined with MB neurotoxicity was further observed.The neurobehavior of mice were examined.We tested whether the indicators in midbrain and pons-medulla were changed,evaluated the preventive effect of Gli on joint toxicity,explored the mechanism of NLRP3 inflammasome-mediated pyroptosis in the induction of PD-like manifestations of chronic toxicity combined with PQ and MB.Methods1.Grouping and processing of experimental animals(1)Prevention experiment:100 male C57BL/6 8-week-old SPF mice,weighing 18-20 g,after acclimation for 5 days,were randomly divided into control group,PQ+MB group,PQ+MB+Gli group and Gli group,25 mice in each group.Animals were injected intraperitoneally with Gli 10 mg/kg.bw before being poisoned.PQ and MB were intraperitoneally injected at 10 mg/kg.bw and 30 mg/kg.bw,twice a week for 8 weeks.The animals were sacrificed the next morning after being exposed to the PQ and MB.After some animals were anesthetized and the brain tissue was taken for pathological section observation.After partial animals were sacrificed by exsanguination,the midbrain was peeled off on ice.(2)Therapeutic experiment:92 male C57BL/6 8-week-old SPF mice(18-20 g)were fed adaptively for 5 days.They were randomly divided into control group and PQ+MB group according to body weight,46 mice in each group.PQ and MB were intraperitoneally injected at 10 mg/kg.bw and 30 mg/kg.bw.The control group was given an equal volume of solvent twice a week for 8 weeks to establish an animal model of PD-like manifestations.From the 10th week on,PQ+MB animals were equally divided into Gli group and solvent control group.The Gli was injected intraperitoneally at 10 mg/kg.bw twice a week for 5 weeks.Another group is given an equal volume of solvent.The control group was also divided into Gli group and blank control group.Gli and solvent were given as above.At the end of experiment the animals were sacrificed the next morning.Some animals were anesthetized and fixed in vivo.Tissues were taken for pathological section observation.Some animals were sacrificed by exsanguination and the midbrain were separated and stored at-80℃.2.Rotarod test:The rotarod experiments were performed on the 2nd,4th,6th,8th,12th and 14th week of the experiment,respectively.When the mice fell from the rotarod,the instrument automatically recorded latency.Ten mice were tested in each group.3.Fecal collection experiments:Animal feces were collected on the 2nd,4th,6th,8th,12th and 14th weekends of the experiment.The experiment lasted 60 minutes.The number of defecation pellets was recorded.The fecal water content was calculated based on the wet weight and dry weight of the feces.4.Immunohistochemical detection:Frozen sections of the brains of each group were prepared and the morphology of midbrain microglia was observed by immunohistochemistry.5.qPCR assay:The key molecules of M1/M2 polarization in mice brains were detected for expression of IL-1β,iNOS,TNF-a,Arginase-1,YM-1,CD206 mRNA and the expression of NLRP3 and caspase-1 mRNA in NLRP3 inflammasome.6.Western blot detection:We detected the relative protein expression of iNOS,Arginase-1,Iba-1,NLRP3,IL-1β,caspase-1,NF-κB and NF-κB phosphorylation in the midbrain and pons-medulla of the mice.7.ROS detection:Fluorescence dye was used to detect the content of ROS in mice brain.Results1.The combined effects of PQ and MB on body weight of mice:Compared with the control group,there was no significant difference in body weight between the PQ+MB group and the Gli prevention and treatment group(P>0.05).2.The combined effects of PQ and MB on neurobehavioral effects in mice:(1)Prevention experiment:In rotarod test,compared with the control group,the rotarod latency of the mice in the PQ+MB group was significantly reduced(P<0.01)and Gli prevention can significantly improve the coordination ability of the exposed mice.The latency of Gli prevention group was significantly higher than that of PQ+MB group(P<0.05).In the fecal collection experiment,the fecal water content and frequency of defecation were significantly lower in PQ+MB group than in control group(P<0.05).Gli was effective as a preventive drug to increase the number of defecations(P<0.05)and the feces contained more water.It is suggested that Gli can prevent constipation caused by PQ and MB.(2)Therapeutic experiment:In rotarod test,the rotarod latency of mice in the PQ and MB exposure group decreased from the 4th week and was significantly lower than that in control group at the 8th week(P<0.01).From the 8th week of Gli treatment to the 14th weekend,the rod latency of PQ+MB-treated animals was significantly different from that of control animals(P<0.01).In the fecal collection experiment,since the 2nd week,the feces water content and the number of feces pellets of mice in the PQ+MB group were significantly lower than those in control group(P<0.01).Gli treatment can improve exposed animals of constipation.At 12th week,the feces water content and the number of feces pellets in the Gli treatment group were higher than those in PQ+MB group(P<0.01).3.The effects of PQ combined with MB on the morphology of midbrain microglia:In PQ+MB group,the soma of small glial cells in the midbrain became large and round and the pseudopods were obvious.The amoeba-like changes suggest that microglialcells are activated and Gli prevention and treatment significantly reduced activation.4.The effects of PQ combined with MB on the polarization of microglia:Animals were given PQ and MB in combination and Iba-1 and iNOS in the midbrain of mice were significantly higher than that in the control group(P<0.05).At the same time,the expression of TNF-α mRNA in midbrain and pons-medulla was increased(P<0.01).It suggests that PQ and MB can lead to M1 polarization of microglia.The expression of Arginase-1 mRNA in the midbrain of the PQ+MB group was also significantly higher than that in control group(P<0.05,P<0.01).The expression of CD206 mRNA in the pons-medulla was higher than that in control group(P<0.01).Exposed to PQ+MB can cause partial microglia M2 polarization.Gli prevention can significantly inhibit the activation of microglial cells and the expression level of Iba-1 in the pons-medulla is lower than that in the exposure group(P<0.01).The expression of TNF-a mRNA and iNOS protein in midbrain and pons-medulla in Gli prevention group was significantly lower than that in PQ+MB group(P<0.01)but the expression of Arginase-1,YM-1,and CD206 mRNA was not affected significantly.5.The effects of PQ combined with MB on activation of NLRP3 inflammasome and neuronal pyroptosis:Compared with the control group,the expression of NLRP3,IL-1β and caspase-1 mRNA of PQ+MB group had a tendency to increase and the expression levels of IL-1β and caspase-1 mRNA were statistically different(P<0.01,P<0.05).In PQ + MB group,NLRP3,mature IL-1β,caspase-1 p10 protein levels were higher than that in control group(P<0.05).Compared with the PQ+MB group,the expressions of NLRP3,IL-1β and caspase-1 mRNA of Gli prevention group decreased and the protein content of the three groups was significantly lower than that of the PQ+MB group(P<0.01,P<0.05).After treatment with Gli,the expression of NLRP3,IL-1β,caspase-1 mRNA and the protein expressions was lower than that of PQ+MB group(P<0.05).6.The effects of PQ combined with MB on the activation of NF-κB of mice:The expressions of NF-κB and NF-κB phosphorylation were increased in the PQ+MB group compared with the control group(P<0.05,P<0.01).Compared with the PQ+MB group,the NF-κB and phosphorylated NF-κB in the midbrain of Gli prevention group had a tendency to decrease compared with the control group.In pons-medulla,Gli reduced the phosphorylation of NF-κB(P<0.01)but had no significant effect on NF-κB.Gli treatment can effectively reduce the level of NF-κB and phosphorylated NF-κB protein,suggesting that PQ+MB can activate NF-κB and increase the production of phosphorylated NF-κB.7.The effects of PQ combined with MB on ROS content of mice:The content of ROS in the midbrain of mice in the PQ+MB group was higher than that in the control group(P<0.05),suggesting that PQ and MB may lead to oxidative stress and inflammation occurred within the brain.ROS of Gli-preventive mice was significantly less than that in PQ+MB mice(P<0.01,P<0.05).After Gli treatment,the PQ+MB+Gli group was significantly lower than the exposed mice(P<0.05).Conclusion1.PQ combined with MB exposure can cause PD-like symptoms and neuronal pyroptosis in experimental animals.2.The combined chronic toxicity of PQ and MB can lead to M1 polarization of microglia,imbalance of M1/M2 polarization and cause the neuroinflammation.3.The K+ channel inhibitor glibenclamide can effectively prevent inflammation and neuronal pyroptosis induced by PQ and MB in the brain of animals.