Based On The T-2 Toxin-induced Oxidative Stress Injury Of Pituitary And The Injury Of Blood-brain Barrier And The Discovery Of Small Molecule Antagonists

As a secondary metabolite,T-2 toxin isproduced by a variety of Fusarium species(mainly Fusarium cladosporium).In recent years,the neurotoxicity of T-2 toxins has received special attention from researchers.Animal studies have shown that exposure to T-2 toxin causes obvious neurological symptoms in mice or rats,including muscle weakness,ataxia,and anorexia,and even significant edema around blood vessels in the central nervous system of the brain.T-2 toxin exposure could also damage the function of the blood-brain barrier(BBB)and increase its permeability,so that mannitol and Evans blue can cross the BBB.In addition,in vitro cell model studies have found that T-2 toxin can accumulate on the side of the nerve,suggesting that T-2 toxin might cross the BBB and damage organs such as the brain and pituitary.However,it is unclear whether T-2 toxin can directly enter the brain and damage brain tissues and organs in vivo.PGC-1?,an effective activator of mitochondrial biosynthesis and respiration,could regulate the expression of a variety of ROS metabolic detoxification enzymes(SOD-1,SOD-2,GPX-1,CAT)and the expression of mitochondrial UCP-2,and then reduce intracellular ROS levels and oxidative stress damage.In addition,PGC-1? plays a protective role in BBB damage caused by various diseases.Therefore,maintaining and increasing the level of PGC-1? is an important means to improve brain damage caused by diseases or external stimuli,especially BBB damage and changes in BBB permeability.The previous genomic results of our laboratory showed that incubating GH3 cells with T-2 toxin can increase the gene expression level of PGC-1? in the cells.However,whether PGC-1?can be used as a target to reduce the toxicity of T-2 toxin remains to be further studied.Therefore,in this study,T-2 toxin induced neurotoxicity was used as an entry point to investigate whether T-2 toxin can directly cross the BBB in vivo and the role of PGC-1? in T-2 toxin induced brain injury,and PGC-1 ? was used as a target to screen small molecule compounds that antagonize T-2 toxin toxicity and validate them in vitro and in vivo.In addition,whether antagonists have a protective effect on BBB damage caused by toxic and harmful substances other than T-2 toxin was studied,which will lay the foundation for the development of compounds as drugs to alleviate BBB damage.1 T-2 toxin enters the rat brain and causes autophagy and pituitary apoptosis in the rat brainLC-MS/MS was used to detect the amount of T-2 toxin in the brain of the rats was0.012 ?g/m L.The damaging effect of T-2 toxin on the brain decreases with increasing exposure time.Significant parenchymal hemorrhage was observed in the brain after 3 days of the T-2 toxin exposure.However,in rats investigated 7 days after treatment with T-2toxin,the bleeding situation was improved seven days after treatment with T-2 toxin;transmission electron microscopy observation shows thatthe mitochondria exhibited swelling,vacuoles,loss of crests,and damaged membrane structure one day after treatment with T-2 toxin.7 days after treatment with T-2 toxin,the structure of most mitochondria had begun to return to normal.Results of gene and protein expression showed that the m RNA level of autophagy-related genes LC3,atg5 and m TOR of cerebral cortex were significantly increased T-2 toxin,with increasing protein expression of LC3,and the expression of caspase-3 and Bcl-x was significantly decreased,and the expression of caspase-9 and Bax was not significantly changed in the rat cerebral cortex.The damage effect of T-2 toxin on pituitary increased with the increase of exposure time.The hyperemia was most pronounced with deeply stained nuclei after 7 days of T-2 toxin treatment,indicating that pituitary cells may have undergone apoptosis;After 1 day of T-2 toxin exposure,some mitochondria began to swell,and after 3 days,mitochondria showed swelling,vacuolization,cristae shedding,and damaged membrane structures.After 7 days,mitochondrial cristae were arranged in a disordered manner,and the mitochondrial area changed into an electron lucent zone.q RT-PCR results in pituitary tissues showed that the gene expression of LC3 and atg5 increased expression only at a specific time,and immunohistochemistry results showed that LC3 protein did not increase expression.A significant increase in Bax,Bcl-x,and caspase-3 expression levels was detected in pituitary tissue after exposure to the T-2 toxin,and these results shown that T-2 toxin can inhibit the apoptosis of rat brain cortex and cause the apoptosis of rat pituitary.The relationship between apoptosis and autophagy indicates that the damage of T-2 toxin to the pituitary gland is harmful than the damage to the cerebral cortex,and the damage of T-2 toxin to the brain is caused by its direct entry into the brain.2 PGC-1? is an important factor for GH3 cells to resist oxidative stress caused by T-2toxinIn this study,LC-MS/MS was first used to detect the T-2 toxin inside and outside GH3 cells.The results showed that the results showed that the proportion of 10 n M(4.67 ?g/kg)and 40 n M(18.66 ?g/kg)of T-2 toxin into GH3 cells reached 7.37% and 11.82%,which is0.34±0.09 ?g/kg and 2.09±0.13 ?g/kg.This indicated that T-2 toxin can directly enter GH3 cells,and as the concentration of T-2 toxin increases,the concentration and the proportion of the concentration of T-2 toxin entering the cell continues to increase.This shows that as the concentration of T-2 toxin increases,it may bring greater cytotoxicity.In this study,GH3 cells were incubated with 10 n M and 40 n M T-2 toxins for 24 h to detect cellular ROS levels,the activities of antioxidant enzyme such as SOD and GSH-Px,and the expression of mitochondrial-related antioxidant stress genes PGC-1? and Tfam.The results showed that T-2 toxin dose-dependently increased ROS levels,antioxidant enzyme activities and the expression of PGC-1? and Tfam in GH3 cells.Subsequently,PGC-1? and Tfam were silenced in GH3 cells,the results shown that silencing the expression of PGC-1? in GH3 cells can significantly reduce the activity of antioxidant enzymes and inhibit the expression of Tfam.However,after T-2 toxin is added,the activity of oxidase will increase compensatory.Silencing the expression of Tfam in GH3 cells could significantly reduce the activity of antioxidant enzymes and increase the level of ROS.However,after the addition of T-2 toxin,the activity of oxidase will increase compensatory,and the level of ROS will be higher.It shows that PGC-1? plays an anti-oxidative stress role by positively regulating the expression of Tfam in GH3 cells,suggesting that PGC-1?is an important factor for GH3 cells to resist oxidative stress.3 PGC-1? is an important gene for h BMEC cells to resist T-2 toxin cell tight damageCCK-8 assay showed that with the increase concentration of T-2 toxin,the viability of h BMEC cells reduced by a dose-dependent manner.The results of in vitro BBB model showed that T-2 toxin significantly reduced the value of TEER in h BMEC cells and increased the Na F permeability coefficient,indicating that T-2 toxin destroyed the tightness of the BBB.In addition,the gene expression showed that the gene expression of PGC-1?,SOD,GSH-Px,IL1?,TNF?,IL6,CCL2,CLCX1 and CCL3 was increased by T-2 toxin in a dose-dependent matter.However,T-2 toxin dose-dependently down-regulated the gene and protein expression of ZO-1,CLDN5 and occuldin.These results suggest that T-2 toxin caused h BMEC cell toxicity and BBB damage by reducing the expression of tight junction proteins.This study applied overexpression or inhibition of PGC-1? expression combined with indirect immunofluorescence assay to further investigate the effect of PGC-1? on tight junction proteins.Overexpression of PGC-1? in h BMEC cells significantly induced the fluorescence intensity of ZO-1,CLDN5 and Occludin in the cell membrane,and improve T-2 toxin induced-the blurring of the cell membrane.However,SR-18292,an inhibitor of PGC-1?,further aggravated the decrease in tight junction protein expression caused by T-2 toxin,and the tight connections between cells became more blurred or even lost.The results indicate that PGC-1? is an important protective factor for HBMEC cells against BBB damage caused by T-2 toxin.4 Small molecule compounds of traditional Chinese medicine activate PGC-1? and resist the BBB damage caused by T-2 toxin and LPS exposure3-131 was screened from 166 traditional Chinese medicine by dual luciferase reporter assay binding molecular docking technique.Compound 3-131 directly binds to the promoter of human PGC-1? and activates the transcription of PGC-1?.The point mutation test verified the binding sites and found 71-73 sites mutation caused a 70% reduction in dual luciferase activity compared with P7.These results suggested that 3-131 could directly activate the transcriptional activity of the promoter of PGC-1?,and the upstream of the transcription starting site-1100 bp?-1000 bp was the core binding region.Meanwhile,binding of 3-131 to bases 71-73 in the-1100 bp?-1000 bp fragment in the form of hydrogen bonds activates the transcriptional activity of PGC-1 ?.In this study,in vivo and in vitro BBB damage models were constructed by T-2 toxin and LPS,and the permeation of Na F at the cellular and animal levels and the expression of tight junction proteins in vitro and in vivo were examined to comprehensively analyze the protective effects of small molecule compounds 3-131 on the BBB.In vitro BBB model results showed that T-2 toxin or LPS can significantly decrease TEER,increase Na F permeability,decrease the protein expression of ZO-1,OCLN and CLDN5 and the addition of 3-131 significantly increase intercellular TJs,make the cell membrane structure clear and relieve BBB damage caused by T-2 toxin and LPS.In vivo animal model results showed that T-2 toxin and LPS significantly increased Na F in the mouse brain,makes the structure of brain tissue abnormal,loose and disordered in the arrangement of neurons in the hippocampus,massive neuron degeneration,pyknotic and deeply stained of the cells,and fibrous tangles,decrease the protein expression of ZO-1,OCLN and CLDN5 and increase the expression of IL1? and TNF?.In mice,3-131 could increase the tightness of BBB,reduce the pathological damage of the brain,and alleviate the inflammatory response of the brain,thereby alleviating the brain damage caused by T-2 toxin or LPS.In summary,this study investigated that the injury effect of T-2 toxin directly across the BBB on the cerebral cortex and pituitary gland,and the role of PGC-1? in the oxidative stress in GH3 cells caused by T-2 toxin,and role of PGC-1? in BBB injury induced by T-2 toxin,the screening of antagonists targeting PGC-1?,and protective effect of antagonists on BBB injury caused by T-2 toxin and LPS.From the perspective of neurotoxicity,this study takes PGC-1? as the target and BBB injury as the research object,which provides a new basis for pituitary and brain injury caused by T-2 toxin,and provides a new research direction for the study of T-2 toxin antagonists,and a basic for the study of BBB protective agents.