Astragalus Polysaccharide Protect Against Cadmium-induced Cytotoxicity Through The MDA5/NF-?B Pathway In Chicken Peripheral Blood Lymphocytes

Cadmium?Cd?is an environmental pollutant causing great harm,which is associated with inflammation,oxidative stress,and cell apoptosis.It may produce toxic effects in the cardiovascular system,the livers,the kidneys,the reproductive system and the immune system.MDA5 is the member of pattern-recognition receptor?PRR?family in animal organism.Previous study has found that the expression of chicken MDA5?chMDA5?in the chicken with Cd poisoning is enhanced,suggesting that chMDA5 and its signaling pathway may be involved in the Cd-induced damage process.Astragalus polysaccharide?APS?is a major component of Astragalus membranaceus,a vital qi-reinforcing herb medicine with favorable biological activities including immunregulation properties,oxidation resistance and anti-inflammatory properties.It has been discovered that APS can play an immunomodulatory role in vivo and in vitro through a pattern recognition receptor TLR4-mediated MyD88 dependent signaling pathway.There are no reports on the effects of APS on Cd poisoning animals at present.Thus this study first made the peripheral blood lymphocytes?PBLs?of chicks as the targets and based on Cd exposure in PBLs of normal chickens in vitro to study the effect and mechanism of APS on Cd-induced cytotoxicity in chickens PBLs.First,to clarify the expression of chMDA5 in PBLs,we collected PBLs at 7,14,21,28,35,42-day-old,separately.The transcription level and protein expression of chMDA5 in PBLs were detected by real-time fluorescent quantitative PCR?qRT-PCR?and western blot.It was found that chMDA5 had transcription and protein expression in PBLs from 7 to 42 days,and the expression was higher in 14²8 days.On this basis,in order to study the damage of Cd-induced PBLs through MDA5/NF-?B signaling pathway,we collected PBLs at 15-day-old and then the collected PBLs were divided into C?control?group,10^-6mol/L Cd group,40?g/mL APS group,10^-4 mol/L BAY 11-7082?a nuclear factor kappa-light chain-enhancer of activated B cells[NF-?B]inhibitor?group,5mg/L anti-chMDA5 mAb group,APS+Cd group,BAY11-7082?BAY?+Cd group and anti-chMDA5mAb+Cd group.chMDA5 protein expression and the subcellular localization were measured by western blotting and the laser scanning confocal microscope.The transcription levels of chMDA5,chicken interferon promoter-stimulating factor 1?chIPS-1?,chNF-?B,and inflammatory factors chicken tumor necrosis factor?chTNF?-?,chicken interleukin?chIL?-1,and chIL-6 were measured by qRT-PCR.The morphological change of PBLs was measured by transmission electron microscopy.3-?4,5-Dimethylthiazol-2-yl?-2,5-diphenyltetrazolium bromide?MTT?assay was used to detect the cell viability of PBLs.Levels of malondialdehyde?MDA?,superoxide dismutase?SOD?and glutathione peroxidase?GSH-Px?were measured by corresponding antioxidant kit.The results showed that?1?the MDA content of PBLs in Cd group is significantly higher than the control group?P<0.05?,and the SOD and GSH-Px activities in the Cd group were significantly lower than the control group?P<0.05?.However,and MDA content of the PBLs in the APS+Cd group decreased significantly compared with Cd group?P<0.05?,and the SOD and GSH-Px activities were significantly higher than that of Cd group?P<0.05?;?2?Ultrastructural pathological changes of PBLs in Cd group showed that the apoptotic bodies,autophagosomes,cell necrosis and necrocytosis appeared and cell viability was significantly reduced.However,APS can efficiently improve Cd-induced cell damage and decreasing cell viability;?3?The results of laser confocal microscopy and Western-blot showed that chMDA5proteins are mainly distributed in the mitochondria of PBLs.The expression of chMDA5 in Cd group was higher in PBLs mitochondria,and the expression of chMDA5 in the APS+Cd group was significantly decreased in the mitochondria of PBLs;?4?Cd significantly increased the expression of the expression of chMDA5 mRNA and chMDA5 protein?P<0.05?.However,the expression of chMDA5 in the APS+Cd group was lower than that in the Cd group?P<0.05?.The effects of cells pretreated with anti-chMDA5 mAb were similar with the APS+Cd group.The mRNA expression change regularity of related molecules of chMDA5 signaling pathway chIPS-1,chNF-?B,chTNF-?,chIL-1?and chIL-6 was consistent with the chMDA5 and the effects of cells pretreated with anti-chMDA5 mAb and BAY were similar with the APS+Cd group.In conclusion APS can enhance the antioxidant enzymes activity,can decrease the content of MDA and levels of inflammation factor?chTNF-?,chIL-1?and chIL-6?,can improve the cell vitality of chicken PBLs,and can reduce the damage in Cd-induced PBLs through inhibiting the activation MDA5/NF-?B signaling pathway and plays a protective role in the Cd-induced cytotoxicity in chicken PBLs.This study provides a scientific basis for further study on the mechanism of injury induced by Cd poisoning.