Astaxanthin Alleviates Aflatoxin B1-induced Damage In IPEC-J2 Cells

A powerful antioxidant,astaxanthin(AST)is a member of the lutein family of carotenoids.The fungal toxin aflatoxin B1(AFB1),which is frequently present in the feed,is extremely poisonous.Oxidative stress is one of the main ways in which it exerts its toxicity,and finding a suitable antioxidant is an effective measure to mitigate its toxicity.Pigs are particularly sensitive to AFB1,serious damage to the intestinal tract can be caused by AFB1.Therefore,this study was conducted in IPEC-J2 cells to investigate whether AST can alleviate AFB1-induced damage in IPEC-J2 cells and its specific mechanism.This study is a two-part study,study 1 is the effect of AST on IPEC-J2 cells,and study 2 is the effect of AST to alleviate the damage of AFB1 on IPEC-J2 cells.CCK-8 was used to assess cell viability 24 h following exposure to various concentrations of AST(0,5,10,20,40,80,and 100 μM).The results showed that AST(5～100 μM)had no significant effect on IPEC-J2 cell viability,and cell viability was not decreased.The impact of AST on IPEC-J2 cells’ antioxidant capacity was assessed by assessing SOD activity using a SOD kit,T-SOD and CAT gene expression using q-PCR.The findings demonstrated that SOD activity increased considerably as AST concentration increased,T-SOD and CAT gene expression also increased significantly in comparison to the CON(AST of 0 μM).This implies that AST improves antioxidant capability in IPEC-J2 cells(P<0.05).TNF-α,My D88,and IL-1βgene expression substantially decreased following AST(10 μM)in the AST group compared to the CON(P<0.05).After AST(20 μM),the gene expressions of Caspase-9 and the Bax/Bcl-2 ratio substantially decreased compared to the CON.Bax protein expression and the ratio of Bax/Bcl-2 protein expression were significantly reduced by AST(80 μM)(P<0.05).AST is an activator of Nrf2,an important transcription factor that regulates antioxidant function.Increasing AST concentration led to a gradual increase in Nrf2 gene expression,with the highest Nrf2 gene expression at AST(100 μM).HO-1,NQO-1,HSP70,and SOD2 are Nrf2 downstream genes,these genes expression was substantially augmented,Nrf2 and HO-1 protein levels were significantly increased by AST(80 μM)(P<0.05).In conclusion,AST(5～100 μM)is non-toxic to IPEC-J2 cells and can activate the Nrf2 signaling pathway in IPEC-J2 cells,among which AST(80 μM)has a more stable and better effect on enhancing cellular antioxidant,anti-inflammatory and anti-apoptotic capacity.IPEC-J2 cells were exposed to AFB1 at various concentrations for 24 h(1,5,10,20,40,and 80 μM),a significant decrease in cell viability was measured by CCK-8starting from AFB1(5 μM)(P<0.01),and AFB1(10 μM)induced a decrease in cell viability to about 80%,so 10 μM AFB1 was chosen for subsequent experimentations.According to the results of previous studies,AST(5～100 μM)did not affect IPEC-J2 cell viability,then,the cells were exposed to AFB1(10 μM)in combination with varying concentrations of AST(5,10,20,40,80,and 100 μM).The addition of AST(10～100 μM)greatly increased the viability of IPEC-J2 cells as compared to the AFB1 group,and AST(80 μM)greatly rescued the decrease in cell viability of IPEC-J2 cells caused by AFB1(10 μM)(P<0.01).Therefore,in subsequent experiments,IPEC-J2 cells were treated with AFB1(10 μM)and AST(80 μM)together.Divided into 3 treatment groups,the control group(CON),the AFB1(10 μM)group,and the AFB1(10 μM)+ AST(80 μM)group.Compared to the CON,the AFB1 group augmented LDH,ROS,and MDA levels substantially and reduced the activity of GSH and SOD.However,compared to the AFB1 group,the addition of AST caused a significant decline in LDH and ROS levels(P<0.05);MDA levels also tended to decline but did not differ statistically.GSH and SOD activities were considerably raised in the AFB1+AST group compared to the AFB1 group.The apoptosis rate was detected using flow cytometry,and the consequences revealed that AFB1 resulted from the rise in the apoptosis rate greatly compared to the CON,while the supplementation of AST significantly alleviated the increase in the apoptosis rate(P<0.01).The AFB1 group expression of pro-apoptotic proteins,such as cytochrome C,Bax,the Bax/Bcl-2 ratio,and Caspase-3 protein,was significantly higher than that of the CON.Additionally,the anti-apoptotic protein Bcl-2 was downregulated significantly.And compared with the AFB1 group,the AFB1+AST group greatly reversed the expression of the above-mentioned apoptosis-related proteins(P<0.05).Compared to the CON,AFB1 induced a meaningful decline in HO-1 and Nrf2 protein expression in IPEC-J2 cells,and Nrf2 downstream genes,including NQO-1,SOD2,and HSP70,whose expression was substantially diminished by AFB1.The complementation of AST successfully activated the Nrf2 signaling pathway,which significantly increased the expression of Nrf2,HO-1,SOD2,and HSP70 genes,HO-1and Nrf2 proteins were expressed much more(P<0.05),while NQO1 also showed an upward trend.In conclusion,the negative effects of AFB1(10 μM)on oxidative stress and apoptosis in IPEC-J2 cells,AST(80 μM)could mitigate these hazards by triggering the Nrf2 signaling pathway.In summary,AST reduces AFB1-induced IPEC-J2 cell damage,providing a theoretical basis for AST to promote intestinal health in pigs and its application in pig breeding.