Molecular Mechanism Of HIF-1α Mediates T-2 Toxin-Induced Human Neuroblastoma Cellular Senescence

T-2 toxin is mainly produced by Fusarium graminearum,constitutes a major agricultural and food safety problem in the world.Cell senescence is a permanent stagnation of cell growth,and is considered to be an important factor in aging-related diseases including Alzheimer’s disease（AD）.Studies have shown that T-2 toxin associated with crops is closely linked to hypoxia.However,previous studies have mainly focused on the toxic effect of T-2 toxin on animal cells or models,and there is little information about its neurotoxicity to human cells.The mechanism of cell senescence caused by T-2 toxin,and how hypoxia-inducible factor-1α（HIF-1α）is involved in this mechanism are still unclear.Therefore,in this study,human neuroblastoma cells SH-SY5Y was selected to establish an in vitro toxicity model to explore the mechanism of neuronal senescence induced by T-2 toxin and the possible regulation of HIF-1αon cell senescence and AD key proteins.The cultured SH-SY5Y cells were treated with different concentrations of T-2 toxin for 4,12,24,36 and 48 h,and the cell viability was determined by cell counting kit-8（CCK-8）method.Western blot and Real-time PCR（q PCR）were used to detect the expression of HIF-1α,senescence-related proteins,long non-coding RNA（lnc RNA）and AD key proteins.Flow cytometry was used to detect the effects of T-2 toxin and HIF-1αinhibitor（YC-1）on cell cycle at 6 and 24 h.The activity of senescence-associatedβ-galactosidase（SA-β-gal）,a biomarker of senescent cells,was detected by staining method.Finally,transmission electron microscopy was used to observe the ultrastructural changes of SH-SY5Y cells treated with T-2 toxin and YC-1.The results showed that T-2 toxin inhibited the viability of SH-SY5Y cells,and the toxic effect increased in a dependent manner with the time and concentration of T-2 toxin.In the follow-up study,the working concentration of T-2 toxin was determined to be 6 n M.By increasing the expression of cell cycle arrest related proteins p16,p21 and p53,T-2 toxin arrested the cell cycle at G₁and G₂/M phase,upregulated the expression of interleukin-6（IL-6）and lnc RNA DINO,and down regulated the expression of lnc RNA TERC and lnc RNA PINT.T-2 toxin rapidly stimulated the secretion of SA-β-gal within 2 h,and induced early senescence in SH-SY5Y cells.Characteristic features of T-2 toxin-induced cellular senescence were observed in ultrastructural observations,with flattened cellular morphology,enlarged nucleus,and disproportionate increase in the ratio of cytoplasm to nucleus.In addition,HIF-1αhas a double-edged effect on cell senescence induced by T-2 toxin.It induces p16,p21,p53,CXC chemokine ligand 8（IL-8）and monocyte chemoattractant protein1（CCL2）expression,which acts as a stressor to promote cellular senescence.HIF-1αcan also reduce T-2 toxin induced cell senescence and protect cells by inhibiting the expression of IL-6and nuclear factor kappa-B（NF-κB）.There was an additional relationship between T-2 toxin and AD,phosphorylated Tau（p-Tau）was highly expressed after T-2 toxin treatment for 6-24 h.Different from p-Tau,the expression level of amyloid precursor protein（APP）was negatively correlated with the time-dependent effect of T-2 toxin,and the expression level of BACE1-AS was down-regulated at the same time,suggesting that T-2 toxin may reduce the deposition of amyloid beta protein（Aβ）.Moreover,HIF-1α,as a target of T-2 toxin,can positively regulate the expression of p-Tau,and negatively regulate the expression of APP,suggesting that hypoxia mediates cell senescence induced by T-2 toxin and plays a key role in the pathophysiology of AD.In summary,in this study,we found that T-2 could induce hypoxia in SH-SY5Y cells,up-regulate the expression of senescence-associated secretory phenotype（SASP）factor,lnc RNA DINO and SA-β-gal,and induce early senescence of SH-SY5Y cells by increasing the expression of cyclin-dependent kinase inhibitors p21 and p16.HIF-1αis an important toxic target of T-2 toxin.It can not only be used as a stress-induced cell senescence,but also play a protective role in cell senescence induced by T-2 toxin.HIF-1αcan also regulate the key proteins of AD positively and negatively,respectively.In summary,this study provides further information for understanding the relationship between hypoxia and the mechanism of neuronal senescence,expands the understanding of the mechanism of controlling cell senescence,and also provides further evidence for the study of the neurotoxic mechanism of T-2 toxin.