The Neurotoxicity Effect And Molecular Mechanism In Response To Coal-fired Air Pollutants(SO₂ And PM_2.5)

Benefiting from the continuous optimization of China's energy structure and the enforcement policies of industrial waste gas treatment,the sulfur dioxide?SO₂?pollution has basically been controlled in large and medium-sized cities.However,with increasing consumption of coal,a large amount of SO₂ and soot are continuously discharged into the environment,and a series of complicated chemical reactions have taken place between SO₂,soot and other pollutants in the atmosphere to aggravate fine particulate matter(PM_2.5)pollution.In addition,due to the poor and backward economic conditions,scattered small boilers and high-sulfur inferior coal are widely used for cooking and heating in rural areas of China.This coal-fired method of high-consumption and low-efficiency leads to a large amount of SO₂ and dust,causing serious indoor and outdoor air pollution and posing a serious threat to people's daily life and health.It has been found that SO₂ inhalation form the derivatives of bisulfite and sulfite after entering the organism,causing neuroinflammation and increasing the risk of cerebral ischemic stroke.However,few medications are available for preventing the inflammatory responses and relieving the subsequent harmful insults from SO₂ inhalation.PM_2.5 can directly or indirectly stimulate the brain via the lung-blood system,nasal-entorhinal cortex,eye and other exposure pathways,affecting neurological behaviors and increasing the risk of neurodegeneration such as Alzheimer's disease?AD?and Parkinson's disease?PD?.However,these studies focus on epidemiological investigations and the changes of gene and protein levels,ignoring the alterations of metabolites that directly affect brain activity.In view of the above two questions,we proposed this topic:1.The previous studies demonstrate that SO₂ exposure causes a series of ischemic brain damage,such as inflammatory responses,the alterations of synaptic structure and function and neuronal apoptosis,and cyclooxygenase 2?COX-2?-catalyzed prostaglandin E₂?PGE₂?-mediated glutamate neuronal excitotoxicity plays an important role in this process.The Non-steroidal anti-inflammatory drug?NSAID?NS398,a COX-2 inhibitor,significantly inhibited SO₂ inhalation-induced nerve injury,However,the serious gastrointestinal and cardiovascular complications in patients receiving NS398 limit the translational potential for neuroinflammatory syndromes.More and more evidences show that endocannabinoid?eCB?2-arachidonoyl glycbrol?2-AG?not only participates in neuron signal transduction,but also suppresses the COX-2 to exert anti-inflammatory and analgesic effects.To determine whether 2-AG protects against neuronal inflammation from SO₂ inhalation,the levels of COX-2 protein were first determined in primary cultured neurons exposed to SO₂ derivatives with or without 2-AG,and the results showed that exogenous 2-AG and JZL184?the inhibitor of 2-AG degrading enzyme?significantly attenuated the SO₂ derivative-induced COX-2 overexpression.Subsequently,with chemical inhibitors?SR1/2,the antagonists of cannabinoid 1/2?CB1/2?receptors?and various technologies,such as electrophysiology recordings,western blotting and fluorescent staining,we found that 2-AG significantly inhibited SO₂-induced COX-2/PGE₂ overexpression and subsequently enhanced excitatory postsynaptic potential?fEPSP?and neuronal apoptosis via the CB1 receptor.To find the signaling pathway underlying the effect of endogenous 2-AG on COX-2-mediated neuronal insults in response to SO₂ exposure,peroxisome proliferator activated receptor gamma?PPAR??,mitogen-activated protein kinase?MAPK?and nuclear factor kappaB?NF-?B?were further investigated under different treatment conditions.The results revealed that SO₂ derivatives significantly increased the phosphorylation of p38MAPK and NF-?B and inhibited the expression of PPAR?,while 2-AG restored the expression of these proteins through CB1 receptors.After pharmacological inhibition of PPAR?,the protective effect of 2-AG on NF-?B and COX-2 was inhibited.To verify the above results,we established an animal model of SO₂ inhalation exposure by intraperitoneal injecting the JZL184,SR1,SR2,or GW9662?a PPAR?inhibitor?,then measured the expression of COX-2,PGE₂,p-NF-?B,PPAR?and neurons apoptosis.The results suggestted that 2-AG restored PPAR?expression and inhibited the expression of p-NF-?B through the CB1,which in turn inhibited the overexpression of COX-2 and the release of PGE₂,which in turn improved SO₂-induced neuronal damage.It was found that 2-AG?CB1-dependent?PPAR??the inhibited of MAPK/NF-?B?the inhibited of COX-2 and PGE₂?the inhibited of excitatory synaptic transmission and improvement of neuronal apoptosis.These findings provide evidence for a possible therapeutic effect of endogenous 2-AG regulation for protecting against neurological dysfunction from SO₂ inhalation in polluted areas.2.Epidemiological investigations demonstrate that air pollutant impairs the brain's neurobehavioral in people of different ages various mechanisms.However,whether PM_2.5 induces susceptibility of neurotoxicity,and whether metabolite levels directly reflecting the brain's activities are changed in the process have not yet been studied.In the present study,the different ages?4 weeks,4 months,10 months?of C57BL/6 mice received oropharyngeal aspiration of PM_2.5?3 mg/kg?every other day for 4 weeks.The Morris water maze showed that PM_2.5 exposure caused deterioration of spatial learning and memory in young?4-weeks?mice.Gas chromatogram mass spectrometry?GC-MS?was then used to analyze metabolite changes in hippocampus of 4 weeks mouse,and 20metabolites belonging to different metabolite classes were significantly changed by PM_2.5exposure.Based on metabolic pathway analysis,we speculated that the decline in spatial learning and memory ability from PM_2.5 exposure may be directly or indirectly associated with hippocampal region-specific metabolic alterations involving energy metabolism?citric acid,succinic acid,malic acid,maltose and creatinine?;cholesterol metabolism?desmosterol,lanosterol and campesterol?;arachidonic acid metabolism?methyl arachidonic acid,nonanoic acid and linoleic acid?;inositol phosphate metabolism?myo-inositol,myo-inositol-1-phosphate and methyl-phosphate?and aspartic acid metabolism?aspartic acid,asparagine and homoserine?.3.We previously examined the effect of PM_2.5 on the hippocampus by GC-MS metabolomics and found that AA metabolism was affected by PM_2.5.The half-life period of free AA in cells is very short,that is either quickly recombined into the cell membrane phospholipids or metabolized by COXs to PGs.These PGs are involved in brain inflammation,allergic reactions and vascular diseases,especially,PGE₂ stimulates the release of glutamate and enhances excitatory synaptic transmission and involves in neuroinflammation and synaptic plasticity.It is reported in the literature that COX-2 is involved in PM_2.5-induced nerve injury,but the mechanism is still not clear.Therefore,we hypothesized that COX-2 may catalyze the AA to PGE₂ to participate in PM_2.5-induced neuroinflammation and synaptic dysfunction.Our results revealed that PM_2.5 dose-dependently elevated COX-2 expression in primary cultured hippocampal neurons.And the significant enhancement of PGE₂ and excitatory synaptic transmission were observed at 20 min after PM_2.5 treatment.These results suggested that COX-2/PGE₂associated neuroinflammation and synaptic transmission played an important role in PM_2.5-induced neurological damages.PM_2.5 also induced intracellular reactive oxygen species?ROS?generation accompanied with glutathione?GSH?depletion and the loss of mitochondrial membrane potential?MMP?,and the ROS inhibitor,N-acetyl-L-cystein?NAC?suppressed the COX-2 overexpression and the increased of excitatory synaptic transmission.This result suggested that ROS is involved in PM_2.5-induced COX-2overexpression and excitatory synaptic transmission.In addition,a binding activity of NF-?B p65 was detected in the promoter positions?-419-428 bp?of COX-2,and this interaction was enhanced by PM_2.5 and inhibited by SC-514?NF-?B inhibitor?.These findings indicated that PM_2.5 activated COX-2 expression and enhanced the synaptic transmission through ROS-NF-?B pathway.In this study,on the one hand,we investigated the protective effect of 2-AG on neuronal injury from SO₂ inhalation in‘vivo'and‘vitro';on the other hand,we screened the possible metabolic pathway of PM_2.5-induced neuronal injury via GC-MS,and we also confirmed that COX-2-AA-PGE₂ metabolism is involved in PM_2.5-induced neuronal injury through electrophysiology,ChIP and other techniques.Our study not only could generate some novel mechanistic hypotheses of neurological impairment caused by PM_2.5and SO₂,but also provide experimental evidence for regional air pollution control and health protection.