| Objective This study intends to establish two models in vitro—the blood-brain barrier model(bEnd.3)and the dopaminergic neuron model(SH-SY5Y).Firstly,we investigated whether the classical protein kinase C(cPKC:PKC?/PKC?)could cause the permeability abnormalities of mouse brain microvascular endothelial cells(bEnd.3)induced by paraquat(PQ)by regulating the expression of tight junction proteins(TJPs).Secondly,we researched whether SH-SY5Y cells could release the high mobility group protein 1(HMGB1)stimulated by PQ,thereby activating RAGE/NF-?B signaling pathway and causing neuronal inflammatory damage.Methods PKC?/?participates in abnormal permeability of cerebral microvascular endothelial cells induced by paraquat by regulating tight junction protein1.To evaluate the validity of the blood-brain barrier model in vitro.The Transwell chamber was used as the carrier of the co-culture system to observe the mouse brain microvascular endothelial cells(bEnd.3)cultured for 1-7 days.The transendothelial electrical resistance(TEER)was measured by cell resistance meter.The permeability of sodium fluorescein(Na-FLU)was measured by fluorescence spectrophotometry(FS).Which were ensured that the model that we established can simulate the blood-brain barrier.2.To determine the dose/time-effect relationship of PQ exposure on brain microvascular endothelial cells.bEnd.3 cells were treated with final concentration of 0,50,100,200,300?mol/L PQ for 24 hours and 200?mol/L PQ for 0,6,12,24,48,72 hours respectively.Relative survival rates of bEnd.3 cells were measured by MTT to determine the dose-effect relationship and time-effect relationship.3.To investigate the effect of PQ exposure on the barrier function of brain microvascular endothelial cells.bEnd.3 cells were exposed to 0,50,100,200,300?mol/L PQ for 24 hours.The changes of TEER value and permeability were detected to determine whether PQ damaged to the function of blood-brain barrier model in vitro.4.To investigate the effects of PQ exposure on tight junction protein and gene expression in brain microvascular endothelial cells.bEnd.3 cells were treated with 0,100,200,300?mol/L PQ for 24 hours.The expression levels of tight junction proteins(ZO-1,Occludin,Claudin-5)and the corresponding genes were determined by immunofluorescence assay(IF)and real-time fluorescence quantitative PCR(qRT-PCR).5.To investigate the effect of PQ exposure on the expression of F-actin in brain microvascular endothelial cells.bEnd.3 cells were treated with 0,100,200,300?mol/L PQ for 24 hours.The expression of F-actin protein was determined by immunofluorescence assay(IF).6.To investigate the effect of PQ exposure on the expression of P-glycoprotein(P-gp).bEnd.3 cells were treated with 0,100,200,300?mol/L PQ for 24 hours.Immunocytochemistry(ICC)and Western Blot(WB)were used to detect the expression of P-gp protein.7.To investigate the effect of PQ exposure on the expression of PKC?/PKC?protein in brain microvascular endothelial cells.bEnd.3 cells were treated with 0,100,200,300?mol/L PQ for 24 hours.The expression levels of PKC?,PKC?,p-PKC?,p-PKC?were determined by Western Blot.8.To investigate the effect of classical PKC inhibitor(Go 6983)on TJPs expression in brain microvascular endothelial cells.The cells were divided into three groups:control group,200?mol/L PQ exposure group,intervention group(Go 6983+200?mol/L PQ exposure group).The classical PKC inhibitor(Go 6983,1?M)was pretreated for 1 hour and 200?mol/L PQ was exposed for 24 hours.Western blot was used to determine the expression levels of ZO-1,Occludin,Claudin-5 and PKC?,PKC?protein.Mechanism of RAGE/NF-?B signaling pathway mediated by HMGB1 in neuronal inflammatory injury induced by paraquat1.We observed the morphology of SH-SY5Y cell line and detected the dose-effect/time-effect relationship of PQ on cell damage.Cells were treated with PQ at the final concentration of 0,50,100,200,400,600,800?mol/L for 48 hours and 400?mol/L PQ for 0,6,12,24,48,72,96 hours respectively.Relative survival rates of cells were measured by MTT to determine the dose-effect relationship and time-effect relationship.2.To investigate the effect of PQ exposure on the release and migration of HMGB1 in SH-SY5Y cells.SH-SY5Y cells were treated with 400?mol/L PQ for 0,12,24,36,48 hours.The expression of HMGB1 protein in nucleus,cytoplasm and supernatant was detected by Western Blot and ELISA.The migration of HMGB1 induced by PQ was detected by immunofluorescence assay(IF).3.To investigate the effect of HMGB1 release on RAGE/NF-?B signaling pathway.The expression levels of RAGE,RAS,P38/p-P38,I?B/p-I?B and P65 were detected by Western blot with 500?mol/L 1-methyl-4-phenylpyridlnium cation(MPP~+)as positive control at the final concentration of 0,75,150,300?mol//L PQ for 24 hours.4.To investigate the effect of HMGB1 release on inflammatory factors.The cells exposed to the final concentration of 0,75,150,300?mol/L PQ for 24 hours.The expression of inflammatory factors TNF-?and IL-6 was detected by Western blot with 500?mol//L MPP~+as positive control.5.To explore the effect of PDTC,an inhibitor of NF-?B,on RAGE/NF-?B signaling pathway.The cells were divided into four groups:control group,400?mol//L PQ exposure group,PDTC+400?mol//L PQ exposure group,PDTC treatment group,50?M PDTC pretreatment cells 1 h and 400?mol//L PQ exposure cells 24 hours.Western blot was used to determine the expression levels of IkB/p-IkB,P65 and inflammatory factors TNF-?,IL-6protein.Results 1.The transendothelial electrical resistance(TEER)increased gradually with the cell culture time,and the peak value of TEER reached 114.3±6.9(?·cm2)on the 6th day;Whereas the permeability of cells decreased to 1.7±0.2(cm/min)on the same day,which showed that the barrier function of cells was good.MTT assay showed that compared with the control group,the survival rate of bEnd.3 cells exposed to 100,200,300?mol/L PQ decreased significantly after 24 h(P<0.05),and that of 200?mol/L PQ cells exposed to 6,12,24,48,72h was significantly decreased(P<0.05),which showed a dose-dependent and time-dependent patterns,respectively.Immunofluorescence and qRT-PCR showed that the expression of ZO-1,Occludin,Claudin-5 protein and gene significantly decreased with increasing concentrations of PQ(P<0.05).Immunofluorescence showed that F-actin was distributed around the cytoplasm in the control group,forming actin ribbons with complete and continuous lines and no obvious gap formation.After PQ exposure,the fluorescence intensity was significantly reduced and the lines were broken,especially in the high dose group(P<0.05);ICC showed that the expression of P-gp showed obvious generation in 100?mol/L PQ exposure(P<0.05).Western blot analysis showed that exposure to PQ at concentrations of 0,100,200,300?mol/L for 24 h significantly increased the expression of p-PKC?,p-PKC?(P<0.05),whereas no significant effect on the expression of PKC?,PKC?was observed.Compared with PQ group,the protein expression of ZO-1,Occludin,Claudin-5 were increased while p-PKC?,p-PKC?were decreased in Go 6983 pretreated group(P<0.05).2.Morphological observation of SH-SY5Y cells showed that the number of cells increased and the cell-to-cell arrangement was close;MTT results showed that the relative survival rate of SH-SY5Y cells in each group decreased significantly with the increase of PQ concentration for 48 hours,and the cell survival rate decreased significantly at 0,12,24,48,72,96 hours(P<0.05).Western Blot and ELISA showed that the expression of HMGB1 in nucleus,cytoplasm and supernatant increased first and then decreased,and reached the peak at 12,24 and 48 hours after PQ exposure respectively.IF showed that HMGB1 mainly accumulated in nucleus of control group,and the expression level of HMGB1 increased after PQ exposure,and migrated from nucleus to cytoplasm over time.Western blot showed that the relative expression of RAGE,RAS,P38/p-P38,I?B/p-I?B,P65 and inflammatory factors TNF-?,IL-6 increased with the increase of PQ concentration(P<0.05).Compared with the400?mol/L PQ group,the relative expression of I?B/p-I?B,P65 and IL-6 increased after PDTC pretreatment.The expression levels of inflammatory factors TNF-?and IL-6 were significantly decreased(P<0.05).Conclusion Paraquat can induce the injury of cerebral microvascular endothelial cells,and at the same time,PQ can decrease the expression of tight junction protein/gene,which lead to the permeability of mouse brain microvascular endothelial cells increasing.These two factors together lead to the abnormal permeability of the blood-brain barrier.Then PQ entering the central nervous system via the blood-brain barrier,by stimulating dopaminergic neurons to release HMGB1,binding to the cell membrane receptor RAGE,and activating the NF-?B signaling pathway.The release of inflammatory factors is out of control,which resulting in inflammatory injury of neurons.And it may be one of the mechanisms of paraquat-induced Parkinson's disease. |
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