Research On The Role Of CYP1B1 In Manganese-induced Parkinson’s Syndrome And Its Mechanism

Object:Manganese is one of the most used metals in the industry and agriculture,and the occupational and environmental exposures to manganese have continued to increase.Recently,a causal relationship between manganese overexposure and Parkinson’s syndrome has been revealed.The cytochrome P450 1B1（CYP1B1）is abundantly expressed in the brain and participates in the metabolism of various endogenous and exogenous substances.This paper aims to study the possible role of CYP1B1 in manganese-induced Parkinson’s syndrome using in vivo and in vitro experimental models.Methods:Immunofluorescence was used to detect changes in tight junction protein 1（ZO-1）and fibrin（Fibrin）in the cortex and hippocampus of wild-type and CYP1B1knockout mice at 12 months of age.A1Si MP high speed upright two-photon microscopychange was used to detect the change of capillary length and integrity.The content of striatum arachidonic acid metabolites in wild-type and CYP1B1 knockout mice at 8 weeks old was detected by UPLC/MS-MS.Wild-type and CYP1B1knockout mice at 8 weeks old were received 2μl of Mn Cl₂（30 mg/kg）via intranasal instillation for 21 days.The open field test and rota-rod test were used to detect behavioral changes;immunofluorescence was used to detect dopaminergic neurons andα-synuclein in the substantia nigra;real-time RT-PCR was used to detect CYP1B1,the genes related with anti-oxidative stress and tight junction.Human glioma cell line U251 was treated with Mn Cl₂（200μM）,AUDA,14,15-EET.The expression of CYP1B1 and the genes related to anti-oxidation were detected by real-time RT-PCR.Human brain microvascular endothelial cells h CMEC/D3（in vitro model of blood-brain barrier）were treated with Mn Cl₂（200μM）,5-HETE,15-HETE.Real-time RT-PCR was used to detect the expression of CYP1B1 and the genes related with tight junction.Results:Compared with wild-type mice,the expression of tight junction protein 1（ZO-1）was significantly reduced in the cortex and hippocampus from 12-month-old CYP1B1 knockout mice,and fibrin exudation was observed outside the blood vessels.The total length of microvessels in the parietal cortex was reduced by 36%（P<0.05）.The amount of fluorescent substances entering the brain parenchyma through the tail vein was significantly increased.Compared with wild-type mice,the HETEs and EETs in striatum were significantly reduced in CYP1B1-/-mice.After manganese treatment in U251 and h CMEC/D3 cells,the m RNA level of CYP1B1 was significantly decreased.The level of CYP1B1 in striatum was significantly decreased after 21 days of manganese nasal drop,suggesting the inhibition of brain CYP1B1 by manganese.Compared with wild-type animals exposed to manganese,the spontaneous activity of CYP1B1 knockout mice was reduced by 32%（P<0.05）,and the time spent on the rota-rod was shortened by 45%（P<0.01）after manganese exposure.The amount of manganese in the brain was increased by 1.6-fold（P<0.01）.The co-localization images showed the loss of dopaminergic neurons in the substantia nigra from manganese-injured mice,and the accumulation ofα-synuclein,while the damage was aggravated in CYP1B1 knockout mice.Compared with wild-type animals,Nrf2 and its downstream genes including SOD,CAT and GPX1 related to mitochondrial antioxidation in striatum were significantly decreased in CYP1B1 knockout mice.In the manganese exposure model,the expression of Nrf2 in striatum from wild-type mice was decreased by 40%（P<0.01）,and the levels of SOD,CAT and GPX1 were respectively decreased by 35%（P<0.001）and 45%（P<0.001）,42%（P<0.001）;the levels of Nrf2,SOD,CAT and GPX1 were further decreased in CYP1B1 knockout animals after manganese exposure.In the manganese exposure model,the expression of PPARγin striatum from the wild-type mice was decreased by 27%（P<0.001）,and the levels of tight junction proteins including ZO-1,CLDN1,and OCLN were respectively decreased by 26%（P<0.001）,53%（P<0.001）,56%（P<0.001）.Compared with the control,a further decrease in PPARγ,ZO-1,CLDN1 and OCLN levels were found in CYP1B1 knockout animals after manganese exposure.In h CMEC/D3 cells,5-HETE up-regulated PPARγ,ZO-1,CLDN1,and OCLN m RNA levels by 1.32-fold（P<0.001）,1.55-fold（P<0.001）,and 1.42-fold（P<0.001）,1.82 times（P<0.001）compared with the manganese group;additionally,15-HETE up-regulated PPARγ,ZO-1,CLDN1,and OCLN m RNA levels by 1.84-fold（P<0.001）,1.68-fold（P<0.001）,1.3 Times（P<0.001）,1.68 times（P<0.001）.The data suggest that the propylene oxidized metabolites from arachidonic acid can attenuate the inhibitory effect of manganese on tight junction proteins.In U251 cells,AUDA and 14,15-EET co-treatment up-regulated Nrf2 m RNA levels by 1.98-fold（P<0.001）compared with manganese group;meanwhile,SOD1,CAT,and GPX1 m RNA levels were up-regulated by 1.24-fold（P<0.001）,1.91 times（P<0.001）,1.71 times（P<0.001）,respectively.The data suggest that the epoxidized metabolites from arachidonic acid can antagonize the inhibitory effect of manganese on mitochondrial antioxidant function.Conclusions:Manganese inhibited the expression of CYP1B1 and the antioxidant capacity of nerve cells,and disrupted the integrity of the blood-brain barrier.Hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids produced from arachidonic acid mediated by CYP1B1 could up-regulate the levels of Nrf2 and PPARγwhich are involved in the regulation of blood-brain barrier and mitochondrial antioxidant function.Deletion of CYP1B1 may lead to the acceleration of manganese accumulation in the brain and the exacerbation of the damage to dopaminergic neurons.CYP1B1 may be a susceptibility gene for manganese-induced Parkinson’s syndrome.