The Intervention Effect And Mechanism Of Quercetin On Mitochondrial Oxidative Damage Under ZEN Exposure

Mycotoxin pollution is a crucial risk factor with the most significant concern for agricultural products and food safety,which seriously threatens human health.Zearalenone(ZEN),as the main mycotoxin polluting agricultural products and grain deep-processing food,has various toxic effects such as reproductive toxicity,immunotoxicity,genotoxicity,which is harmful to animal production performance and human health.Prevention and control of ZEN contamination is a pivotal problem to be solved urgently to achieve grain and feed safety.At present,it is a part of the research trends to use the biological activity of food-borne natural ingredients to control the toxicity of hazard factors.Quercetin(Que)is a dietary flavonoid compound widely existing in vegetables and fruit,extensively studied in nutritional intervention and the treatment of various disease models.This paper used Caco-2cells and Caenorhabditis elegans(C.elegans)as research objects.In vivo and in vitro experiments were conducted to study the effects of Que on oxidative damage and mitochondrial function induced by ZEN,as well as explore the intervention mechanism of Que in regulating mitochondrial through silencing information regulator Sirt3.The main research results and conclusions are as follows:(1)Caco-2 cells were used as models to construct two modes of Que intervention before ZEN exposure(Intervention mode A)and Que intervention after ZEN exposure(Intervention mode B),respectively.The oxidative damage induced by ZEN and the intervention effect of Que were studied by cell viability,nuclear damage,ROS content,and total antioxidant capacity.During the construction of ZEN induced oxidative damage model,ZEN concentration was negatively correlated with cell viability,and the nucleus was ill-damaged showing a high aggregation or fracture state.In addition,ZEN treatment under semi-inhibitory concentration(20 and 40μmol/L)increased the intracellular ROS content by 21.43% and 45.24%,and decreased the total antioxidant capacity significantly to 86.11% and 66.67% of normal physiological state cells(P<0.05),indicating that the balance between intracellular oxidation and antioxidant was broken.In intervention mode A,the intervention ratio of 2:1(Que:ZEN)could significantly improve the cell viability,reduce ROS content by 35.72%,increase the total antioxidant capacity by 33.33%,and make the nuclear morphology complete and clear under ZEN exposure,which suggested that Que intervention can effectively protect cells from oxidative damage induced by ZEN.In intervention mode B,the intervention ratio of 2:1(Que:ZEN)had no significant effect on the cell viability,reduced the ROS content by 16.67%,increased the total antioxidant capacity by 15.32%,and the nucleus distributed in green fragments under ZEN exposure,which suggested that cells showed a certain degree of oxidative damage.As a result,the two modes showed intervention effects in oxidative damage induced by ZEN,although they both had significant differences.The oxidative stress level of mode A is relatively lower than that of mode B,indicating that Que intervention before ZEN exposure has a more apparent alleviating effect on oxidative damage induced by ZEN.(2)In the study of the intervention mode A,the influence of ZEN on mitochondrial function and the intervention mechanism of Que on toxicity through regulating mitochondrion were analyzed by studying the changes in cell morphology,mitochondrial function indicators,mRNA and protein expression related mitochondrial function under ZEN induction and Que intervention.The results showed that ZEN induced oxidative damage of Caco-2 accompanied by mitochondrial dysfunction.20 μmol/L ZEN reduced the mitochondrial membrane potential by42.23%,ATP content by 72%,SOD and CAT enzyme activities by 51.43% and38.97%.The protein expression of Sirt3 and Nrf2 was reduced by 57.83% and63.76%,respectively,using Western blotting.Subsequently,the changes in mRNA expression related mitochondrial function using RT-q PCR showed that ZEN significantly affected the ability of mitochondrial biosynthesis by extremely down-regulating PGC-1α mRNA expression,which was only 9% of the normal physiological state cells.While,the mode A intervention of Que(Que:ZEN=1:2,1:1,2:1)can ameliorate mitochondrial dysfunction induced by ZEN,and Que:ZEN=2:1has the best effect on damage.Under the condition of this intervention proportion,Que could stabilize the mitochondrial membrane potential,provide 2.49 times of ATP synthesis,as well as increase the activities of SOD and CAT by 51.04% and 40.38%,which help Caco-2 cells maintain the normal physiological function of mitochondria under oxidative stress.RT-q PCR and Western blot results showed that Que promoted the transcription expression of Sirt3 in the damaged mitochondrion and increased its mRNA and protein expression levels by 152.63% and 91.51%,respectively.On the one hand,the up-regulation of Sirt3 expression mediates AMPK to regulate transcription factors related to energy metabolism,then down-regulate ACC mRNA expression by 34.82% and activate PGC-1α to increase mitochondrial biosynthesis and energy metabolism;On the other hand,it can increase the phosphorylation level of Foxo3 and activate Sirt3-Foxo3-SOD,CAT antioxidant defense pathways;In addition,the intervention of Que also increased the protein expression of Nrf2 and Bcl-2 by 136.24% and 41.89%,and decreased the expression of Bax protein by28.56%.The above results suggest that mitochondrial dysfunction induced by ZEN is related to the abnormal decrease of Sirt3 expression.What’s more,Que regulated mitochondrial energy metabolism and antioxidant defense to mediate mitochondrial homeostasis via Sirt3-AMPK-PGC-1α,ACC and Sirt3-Foxo3-SOD,CAT;which improved the ability of antioxidant and anti-apoptosis to resist the toxic effects induced by ZEN in Caco-2 cells.(3)Taking C.elegans as in vivo model,the intervention effects and its mechanism of Que on toxicity induced by ZEN were studied through physiological,biochemical,and molecular changes during nematode growth under ZEN induction and Que intervention.The results showed that the ZEN induction model severely affected the growth and development of C.elegans.With the increase of ZEN exposure concentration,the body length and spawning quantity gradually decreased.Especially under the treatment of 40 μmol/L ZEN,the body length and spawning quantity of C.elegans were 88.94% and 65.02% of that of the blank group.Likewise,the levels of ROS and MDA increased by 17.78% and 34.34%,the activities of SOD and CAT decreased by 10.23% and 60.14%,as well as ATP content and mitochondrial membrane potential decreased by 37.33% and 62.23%,which caused obvious oxidative damage and mitochondrial dysfunction in C.elegans.RT-q PCR experiment showed that ZEN(40 μmol/L)induced 55.83% down-regulation of the vital longevity factor daf-16,then 41% and 51% down-regulation of antioxidant defense factors sod-3 and clt-1,respectively.Indicating that ZEN significantly inhibited nuclear daf-16 translocation by down-regulating the expression of daf-16,and then reduced the mRNA expression of downstream target genes sod-3 and clt-1,ZEN accelerated aging and reduced longevity of C.elegans by inducing oxidative free radical damage mechanism.By analyzing the changes of physiological and biochemical,it was found that under the intervention ratio of Que: ZEN = 2:1,the levels of ROS and MDA in C.elegans decreased by 34.43% and 23.76% respectively,the activities of antioxidant enzymes SOD and CAT increased by 25.56% and 47.46%,indicating that the antioxidant capacity of C.elegans was enhanced;ATP synthesis and mitochondrial membrane potential increased by 1.82 and 2.05 times respectively,which means that the damage of mitochondrial function is reduced;the body length and spawning quantity remained at a normal level.Studies on the transcription of related genes found that Que intervention dose was significantly correlated with the up-regulation of Sir-2.1 mRNA expression.When Que:ZEN=2:1,the mRNA expression of Sir-2.1increased 1.38 times before and after the intervention.After Que promoted the over-expression of Sir-2.1,the mRNA expression levels of nhr-49 and pod-2 in ZEN-induced damage C.elegans returned to a normal level.Activation of the daf-16 pathway increased the antioxidant levels of sod-3,clt-1 and skn-1 by 240%,293%and 100%,respectively.In addition,Que intervention increased the anti-apoptotic protein ced-9 by 1.36 times and inhibited the over-expression of pro-apoptotic protein egl-1 induced by ZEN.In vivo experiments further proved that sir-2.1/sirt3 might be a significant regulatory site of Que.Que in mitochondrial regulatory,anti-oxidation ability,and inhibition of apoptotic protein over-expression play an important role in improving the oxidative stress state of C.elegans.In conclusion,Que alleviates oxidative damage and mitochondrial dysfunction by decreasing ROS level and MDA content,increasing the activities of SOD and CAT,as well as maintaining mitochondrial membrane potential and ATP content at normal levels under ZEN induction;so as to effectively reduce the toxic damage to Caco-2cells and C.elegans caused by ZEN.The transcription and expression of genes-related mitochondrion suggest that sir-2.1/Sirt3 played an essential role in the intervention of Que on the toxic injury of ZEN.Que regulated mitochondrial energy metabolism and antioxidant defense to mediate mitochondrial homeostasis via Sirt3-AMPK-PGC-1α,ACC and Sirt3-Foxo3-SOD,CAT;which improved the ability of antioxidant and anti-apoptosis to resist the toxic effects induced by ZEN in vivo and in vitro.