| T-2 toxin is a secondary metabolite produced by Fusarium,and it is also the most toxic one in type A trichothecenes.T-2 toxin is widely distributed in nature and can be detected in growing economic crops like barley,wheat,oats and corn in the field,as well as in stored grains.T-2 toxin can cause a variety of toxic effects in animals after ingestion of feeds contaminated by this toxin,and the intestine and liver are sensitive to T-2 toxin.T-2 toxins can reduce the appetite and weight gain of animals,which can cause great economic loss.T-2 toxin may also reach the dining table through the food chain,which is a potential threat to human health.Growth inhibition is one of the important toxic effects of T-2 toxin in food animals.T-2 toxin can cause chronic toxic effects,including anorexia,growth retardation and weight loss in animals after long-term intake of low dose of T-2 toxin,which can lead to a significant decrease in feed utilization rate of animals.Although there have been some studies on the growth inhibition induced by T-2 toxin,the underlying mechanism is still not quite certain.T-2 toxin has been shown to reduce the appetite of animals by changing the level of 5-hydroxytryptamine?5-HT?in the brain.T-2 toxin can also inhibit the proliferation of intestinal epithelial cells,damage intestinal microvilli,which lead to disruption of intestinal nutrition absorption.Previously,our laboratory investigated the growth inhibition effect of T-2 toxin toxin in the animals.Our results showed that the inhibition of growth hormone synthesis and secretion in pituitary cells by T-2 toxin could be an important factor in the growth inhibition of animals.It was also reported that T-2 toxin could damage the function of blood-brain barrier and increase its permeability.T-2 toxin could then enter the brain,finally causing pathological damage in pituitary of animals.Until now,although the pituitary injury induced by T-2 toxin in animals have been observed,the specific mechanism of this damage is not clear.Oxidative stress refers to the excessive ROS production by mitochondria or other sources in cells,and ROS can not be effectively eliminated,which changes the redox balance in cells?shift to oxidation?and results in oxidative damage.Increasing evidences have reported that T-2 toxin can significantly increase the ROS generation and induce oxidative stress in various cells,which is always related to the mitochondrial dysfunction in cells.Mitochondrial respiratory chain produces more than 90%of the ROS.The mitochondrial toxicity and oxidative stress can cause a variety of toxicities elicited by T-2 toxin,including immunotoxicity,reproductive toxicity,genotoxicity and neurotoxicity.etc.It is not known whether mitochondrial toxicity and oxidative stress can underlie the pituitary damage of T-2 toxin and lead to growth inhibition in animals?Based on the present evidence,we postulate that the mitochondrial dysfunction and oxidative stress may promote the pituitary injury in animals.To prove this,we select the rat anterior pituitary GH3 cells as the in vitro model?GH3 cells is a classical in vitro model to study the animal growth?in this study to investigate the mitochondrial dysfunction and oxidative stress,and the underlying mechanism by which T-2 toxin cause mitochondrial dysfunction.1.Mitochondrial dysfunction and oxidative stress induced by T-2 toxinIn this study,we firstly determined the IC50 of T-2 toxin in GH3 cells by CCK-8assay which was about 15.67 nM.To manifest a certain cytotoxicity and also assure a certain cell survival rate of GH3 cells,the doses of 10 and 40 nM of T-2 toxin and the exposure time of 24 h were chosen in this study.We treated the GH3 cells by 10 and 40nM T-2 toxin for 24 h,and then measured the parameters of oxidative stress and mitochondrial dysfunction.The results showed that T-2 toxin could significantly increase the generation of ROS and mitochondrial superoxide.The ROS significantly depleted the reduced glutathione?GSH?and caused DNA damage which was indicated by the increase of 8-hydroxydeoxyguanosine?8-OHdG?,a marker of oxidative DNA damage.Upon oxidative stress,T-2 toxin also activated the antioxidant system of GH3cells.T-2 toxin significantly increased the activity of superoxide dismutase?SOD?and gene expression of antioxidant genes GPx-1,SOD-2,CAT,UCP1,UCP2 and UCP3.Specificlly,the CAT and UCP3 were upregualted by 53 and 86 folds higher than the controls.These results indicated that T-2 toxin could greatly activate the antioxidant genes to combat the oxidative stress.T-2 toxin also induced many mitochondrial alterations in GH3 cells.T-2 toxin significantly decreased the mitochondrial membrane potential???m?and mitochondrial DNA?mt-DNA?copy numbers,and increased the content of 8-OHdG generated from mt-DNA,which indicated that T-2 toxin induced mt-DNA oxidative damage.With transmission electron microscope?TEM?,we showed that T-2 toxin obviously changed the GH3 cell morphology and led to shrinkage of cell and nucleus,and appearance of many vacuoles in the cells.In addition,mitochondria underwent a variety of changes such as mitochondria condensation,decreased mitochondrial volume and increased mitochondrial matrix density.The cristae in some mitochondria have disappeared completely and formed vacuolar mitochondria.Mitochondrial swelling could also be ovserved.The mitochondrial structural changes suggested that T-2 toxin had obvious toxic effects on mitochondria and could reduce mitochondrial membrane integrity.Additionly,T-2 toxin induced the apoptosis of GH3 cells,as T-2 toxin induced the formation of apoptotic bodies.T-2 toxin inhibited the expression of anti-apoptotic gene Bcl-2,and increased the expression of anti-apoptotic gene Bax,p53 and caspases including caspase-3 and-8.These results suggested that T-2 toxin induced apoptosis by mitochondria-dependent apoptosis pathway in GH3 cells.Interestingly,the present results also showed that T-2 toxin significantly increased the ATP level and respiratory chain complex I enzyme activity in GH3 cells.We also determined mRNA levels of nuclear DNA-encoded respiratory chain iron-sulfur protein,flavo-protein and hemo-protein-containing subunits,13 mt-DNA encoded subunits,and genes involved in mitochondrial biosynthesis,such as PGC-1 family members,nuclear respiratory factors?NRFs?and mitochondrial transcription factors Tfam,Tfb1m and Tfb2m.The results showed that T-2 toxin significantly increased the expression of most of these genes.Combined with the literatures,we proposed that all these changes may due to a mitochondrial function compensation effect after mitochondrial stress response induced by T-2 toxin,in which the dose and time of incubation of T-2 toxin were two critical factors.That is,low dose and short incubation time can activate mitochondrial biogenesis and stimulate mitochondrial function,while high dose and long incubation time tend to inhibit mitochondrial biogenesis and function and induce apoptosis.2.The role of Ndufs3 in mitochondrial dysfunction induced by T-2 toxin.To explore the role of mitochondrial respiratory chain core iron-sulfur subunit Ndufs3 in mitochondrial dysfunction induced by T-2 toxin,we knocked down or over-expressed the Ndufs3 in GH3 cells,and treated the cells with 40 nM T-2 toxin for 24hour,and then measured the mitochondrial superoxide generation,complex I activity,mitochondrial??m and ATP level.The results showed that inhibition of Ndufs3 in GH3 cells?without T-2 toxin?significantly inhibited the activity of complex I and the mitochondrial??m,and slightly increased mitochondrial superoxide and decreased the ATP level.After the addition of T-2 toxin,knockdown of Ndufs3 further significantly increased mitochondrial superoxide production,decreased the activity of complex I and mitochondrial??m.These results indicated that inhibition of Ndufs3 could decrease mitochondrial complex I activity,mitochondrial??m and ATP level,and increase mitochondrial superoxide generation,and increase sensitivity of GH3 cells to T-2 toxin.Meanwhile,we over-expressed the Ndufs3 in GH3 cells,treated the cells with 40nM T-2 toxin for 24 h,and determined alterations in mitochondrial functions mentioned above.The results showed that overexpression of Nduf3?without T-2 toxin?did not significantly change the activity of complex I,superoxide generation,mitochondrial??m and ATP level.After addition of 40 nM T-2 toxin,these parameters did not change significantly in Ndufs3 overexpressing GH3 cells when compared with the normal cells.The results showed that overexpression of Nduf3 in GH3 cells did not provide some protective effects on mitochondrial function in GH3 cells against T-2 toxin.3.The role of NRF-2 in mitochondrial dysfunction induced by T-2 toxinTo explore how T-2 toxin elicit the mitochondrial dysfunction in GH3 cells,we investigated the role nuclear respiration factor 2?NRF-2?played in these changes.We firstly determined the subcellular localization and expression of NRF-2 in GH3 cells.After treatment of GH3 cells with 40 nM T-2 toxin for 0,2,4,6,8 and 12 h,we observed the subcellular localization of NRF-2 with indirect immunofluorescence and confocal microscope.The results showed that NRF-2 was located in the nucleus at all time points.which was in line with the property of NRF-2 as a transcription factor.After treatment of cells with 10 and 40 nM T-2 toxin for 24 h,NRF-2 gene and protein expression were increased significantly with a time-dependent manner,suggesting that T-2 toxin could induce increase of NRF-2 expression.Afterwards,we knocked down the NRF-2 in GH3 cells,treated these cells with 40nM T-2 toxin for 24 h,and then measured the mitochondrial ROS,ATP level,complex I activity and mt-DNA copy numbers.The results showed that inhibition of NRF-2?without T-2 toxin?slightly increased ROS generation,decreased ATP level,mt-DNA copy numbers and activity of complex I.After addition of T-2 toxin,inhibition of NRF-2 further significantly increased ROS generation and decreased mt-DNA copy numbers.As expected,inhibition of NRF-2 significantly reversed T-2 toxin-induced increase in ATP level and complex I activity.These results suggested that NRF-2 was involved in regulation of mitochondrial function,and NRF-2 could protect mitochondrial function in GH3 cells against T-2 toxin.Furthermore,we determined the target genes of NRF-2 in GH3 cells by chromatin immunoprecipitation?ChIP?combined with PCR and high throughput sequencing?ChIP-Seq?.The results showed that NRF-2 could bind to the promoters of genes including mitochondrial transcription factor Tfam,Tfb1m and Tfb2m,respiratory chain complex I core iron-sulfur subunits Ndufs3 and Ndufs7,complex I assembly factor Ndufaf1,Ndufaf2 and Ndufaf4,complex IV assembly factor 5?COA5?.These genes encoded the structural subunits of mitochondrial respiratory chain and promoted the assembly of complex.In addition,NRF-2 also could bind to promoters of many other genes such as DNM1L,AR,DIAPH2,DDX55,FGF14,FAT3,NSFL1C,ZSCAN23,MYO3A and PEX5.Bioinformatics analysis showed that the promoter region of these target genes contained NRF-2's characteristic tandem repeats of DNA core binding motif GGAA or TTCC.We designed primers to amplify the promoter regions of these genes containing the tandem repeats of GGAA or TTCC motif,and performed PCR and qRT-PCR with the DNA template obtained from ChIP.PCR products were then subject to agarose gel electrophoresis.The reslults showed that the expected size of products were amplified by PCR for all the selected target genes,and the size of products in NRF-2 group were in accord with that of Input group.The sequencing results showed that these amplified products belonged to respective promoters of target genes,indicating that NRF-2 could indeed bind to the promoter regions of these target genes.The results of qRT-PCR also showed that the enrichment of promoter regions of target genes by NRF-2 antibody were all significantly increased compared with that by IgG antibody,which further confirmed the reliability of ChIP-Seq results.We knocked down the NRF-2 in GH3 cells,and then treated these cells with 40 nM T-2 toxin for 24h.The mRNA levels of these target genes were detected,which showed that knock down of NRF-2?without T-2 toxin?slightly decreased the mRNA levels of these target genes.T-2 toxin treatment of normal GH3 cells significantly increased the mRNA levels of these genes,while knock down of NRF-2 significantly reduced T-2 toxin-induced increase of these genes,which confirmed the accuracy of ChIP-Seq results.The above results suggested that NRF-2,as an important transcription factor in mitochondrial biogenesis,could regulate expression of many mitochondrial respiratory chain genes and mitochondrial function-related genes to control mitochondrial function.4.Induction of autophagy and mitophagy by T-2 toxin in GH3 cellsTo investigate whether T-2 toxin could activate autophagy and mitophagy in GH3cells that provide protection to the cells against the toxin,we firstly determined the T-2toxin-induced autophagy and mitophagy in GH3 cells with double fluorescent protein mRFP-eGFP labeled autophagy marker LC3?mRFP-eGFP-LC3?and fluorescent protein mKeima labeled mitochondria?Mito7-mKeima?.The mRFP-eGFP-LC3protein was stably expressed in GH3 cells by adeno-associated virus.The mRFP-eGFP-LC3 stably expressing GH3 cells were treated with 10 and 40 nM T-2 toxin for 24 h,and autophagy was observed by confocal microscopy.The results showed that the mRFP-GFP-LC3 puncta were obviously observed in GH3 cells treated with T-2 toxin,while the mRFP-GFP-LC3 protein in the control group was evenly distributed in the cytoplasm,indicating that T-2 toxin could induce autophagy in GH3 cells.In addition,when GH3 cells were treated with 40 nM T-2 toxin,the Merge revealed some mRFP puncta since the GFP protein was quenched in these puncta in the environment of pH<4,indicating that the autophagosomes have developed to the next stage of autolysosomes.We stably expressed the Mito7-mKeima fusion protein in the GH3 cells to lable the mitochondria,and then treated these cells with 10 and 40 nM T-2 toxin for 24 h.The mitophagy was observed by fluorescence microscope.The results showed that the red fluorescence intensity of mKeima was increased significantly in GH3 cells treated with 10 and 40 nM T-2 toxin,indicating that the mKeima labeled mitochondria have been gulfed by lysosomes in which the environment was pH<4 and the mKeima could emit red fluorescence in acidic environment.This indicated that T-2 toxin could also induce the mitophagy in GH3 cells.The results of TEM showed that the damaged mitochondria gulfed by autophagy vacuoles were ovserved in GH3 cells treated with T-2 toxin,which also confirmed that T-2 toxin could induce mitophagy in GH3 cells.We knocked down the autophagy-associated gene Atg5 in GH3 cells and treated these cells with 40 nM T-2 toxin for 24 h,and then determined the autophagy and mitophagy by the mRFP-GFP-LC3 and Mito7-mKeima methods mentioned above.The results showed that normal GH3 treated with 40 nM T-2 toxin revealed many strong mRFP-GFP-LC3 puncta,while the number and fluorescence intensity of mRFP-GFP-LC3 puncta in Atg5-knock-down cells were decreased significantly after T-2 toxin treatment.Similarly,the normal GH3 cells?expressing Mito7-mKeima?treated with 40nM T-2 toxin for 24 h revealed stronger red fluorescence intensity compared with that in T-2 toxin-untreated normal cells.T-2 toxin treatment for 24 h in Atg5-knock-down cells also increased the red fluorescence intensity compared with that in T-2 toxin-untreated cells.However,the red fluorescence intensity in Atg5-knock-down cells was significantly weaker when compared with the normal cells after T-2 toxin treatment.These results suggested that Atg5 knock down could inhibit the autophagy and mitophagy induced by T-2 toxin in GH3 cells.We also determined mRNA levels of the autophagy-associated proteins LC3 and Atg3 and mitophagy genes Pink1,Parkin and NIX by qRT-PCR.The results showed that 10 nM T-2 toxin did not obviously change the mRNA levels of Pink1 and Parkin,yet significantly increased the mRNA levels of NIX.40 nM T-2 toxin significantly increased the mRNA levels of these three genes.10 nM and 40 nM T-2 toxin treatment in GH3 cells for 24 h significantly increased the protein levels of Pink1 and Parkin and LC3-II/LC3-I ratio.10 nM T-2 toxin significantly increased protein expression of Atg3.We measured the activation of cAMP-PKA-CREB signaling pathway in GH3 cells after incubation with T-2 toxin for 24 h.The results showed that 10 nM and 40 nM T-2toxin significantly increased cAMP level and activity of PKA kinase.However,pre-treatment of cells with H89?PKA kinase inhibitor?significantly inhibited T-2 toxin-induced increase of PKA activity.40 nM T-2 toxin significantly increased the CREB protein level,and 10 nM and 40 nM T-2 toxin also significantly increased phosphorylated CREB?p-CREB?level.However,H89 pre-treatment significantly inhibited the protein levels of CREB and p-CREB.These results suggested that in GH3cells,T-2 toxin increased the cAMP level that could activate PKA activity.PKA then phosphorylated the CREB to form p-CREB,and thus the cAMP-PKA-CREB signaling pathway was activated by T-2 toxin.We knocked down Nrf2 in GH3 cells,and treated these cells with 40 nM T-2 toxin for 24 h,and then measured the mRNA level of Pink1.The results showed that Nrf2 inhibition significantly decreased T-2 toxin-induced Pink1expression.Also,H89 significantly decreased T-2 toxin-induced expression of Nrf2 and Pink1.These results indicated that PKA kinase could regulate the expression of Nrf2and Nrf2 was a upstream regulator of Pink1.Taken together,in this study,we showed that the cAMP-PKA-CREB-Nrf2-Pink1 signaling pathway was a critical pathway that could regulate the mitophagy in GH3 cells.To explore whether mitophagy could protect mitochondrial function of GH3 cells and reduce the cell death induced by T-2 toxin,we knocked down two critical genes involved in mitophagy?Pink1 and NIX?,and measured the mRNA levles of genes?Drp-1,Fis-1,Mfn-1?related to mitochondrial fusion and fission,the mitochondrial ATP level and apoptosis.The results showed that knock down of Pink1 or NIX,or knock down of both genes Pink1 and NIX in GH3 cells decreased expression of Drp-1,Fis-1and Mfn-1 to some extent.10 and 40 nM T-2 toxin significantly increased the expression of these genes in GH3 cells with a dose-dependent manner.However,knock down of Pink1 or NIX,or simultaneous knockdown of Pink1 and NIX decreased T-2toxin-induced increase in mRNA level of Drp-1,Fis-1,Mfn-1 and mitochondrial ATP level,and also promoted the apoptosis caused by T-2 toxin in GH3 cells.This indicated that inhibition of mitophagy could inhibit the activity of mitochondrial fusion and fission and lead to mitochondrial dysfunction and cell death of GH3 cells.In conclusion,we investigated the following contents,including T-2 toxin-induced oxidative stress,mitochondrial changes in structure and function,the role of respiratory chain core iron-sulfur subunit Ndufs3 and mitochondrial biogenesis regulator NRF-2in mitochondrial dysfunction.We also studied T-2 toxin-induced autophagy and mitophagy and the protective effects of mitophagy in mitochondrial function and cell survival in GH3 cells.From the perspective of mitochondria,this study provided new evidences for the mechanism of animal growth inhibition induced by T-2 toxin. |
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