Astragalus Polysaccharide Protect Against Cadmium-induced Autophagy Injury Through The ROS Pathway In Chicken Embryo Fibroblast

Cadmium?Cd?is a heavy metal pollutant that is extremely harmful to the natural environment.It can cause oxidative stress and induce cell damage.Reactive oxygen species?ROS?is a general term for substances that are oxygen-containing and active in the body.Excessive ROS can cause damage to the body.Studies have found that cadmium poisoning can induce autophagy with producing a large amount of reactive ox ygen species in the cells,suggesting that ROS may be associated with cadmium-induced autophagy.Astragalus polysaccharide?APS?is a kind of polysaccharide with anti-oxidation and anti-stress biological functions in the extract of Astragalus membranaceus.It has high research value,but there are few studies on the effects of APS on cadmium poisoning in animals.Therefore,in this study,chicken embryo fibroblasts?CEF?were used as the research object.Based on the establishment of cadmium-induced CEF autophagy and injury model,the relationship between APS and ROS as well as the effect and mechanism of APS on cadmium-induced autophagy injury were studied.Firstly,in order to establish a Cd-induced CEF autophagy and injury model,CdCl₂ was incubated with different doses and different times to detect the expression of autophagy protein LC3 and cell viability in CEF.The results show that LC3 in different concentrations of CdCl₂ was expressed in different degrees.At the concentration of 10 ?mol/L of CdCl₂ at 36 h,the expression of LC3 was the highest in CEF and the cells were damaged.Besides,CEF was divided into control group,10 ?mol/L Cd group,NAC+Cd group,NAC group,3-MA+Cd group,3-MA group,RAPA group,40 ?g/m L APS group,APS+Cd group.DCFH-DA staining was used to detect changes in intracellular ROS content,biochemical methods were used to detect changes in peroxide malondialdehyde?MDA?content and activity of superoxide dismutase?SOD?and glutathione peroxidase?GSH-Px?,the autophagosome detection kit was used to detect changes in the number of autophagosome s,western-blot method was used to detect the expression changes of autophagy proteins LC3,Beclin-1 and m TOR,transmission electron microscopy was used to observe t he pathological changes of CEF,the CCK-8 method was used to detect changes in CEF activity.The results showed that:?1?ROS content in CEF of Cd group were significantly higher than that of C group?P<0.05?,while NAC+Cd group,APS+Cd group ROS content in the CEF group were significantly lower than that in the Cd group?P<0.05?.?2?The MDA content in the CEF of the Cd group was significantly higher than that in the control group,and the activities of SOD and GSH-Px were significantly lower than those in the C group.?P<0.05?,while MDA content in APS+Cd CEF was significantly lower than that in Cd group?P<0.05?,superoxide dismutase and glutathione peroxidase activities were significantly higher in Cd group?P<0.05?.?3?The number of autophagosomes in the RAP A group and Cd group were significantly increased than that in the Cd group?P<0.05?,the number of autophagosomes in the 3-MA+Cd group was significantly lower than that in the Cd group?P<0.05?.?4?The expression of LC3 and Beclin-1 in CEF of Cd group was significantly higher than that of control group?P<0.05?,the expression of LC3 and Beclin-1 in APS+Cd group was significantly lower than that in Cd group?P<0.05?;the expression of m TOR protein was significantly lower in the Cd grou p than in the C group?P<0.05?,the expression of m TOR in APS+Cd group was significantly higher than that in Cd group?P<0.05?;the expression of LC3 in the 3-MA+Cd group was significantly lower than that in the Cd group?P<0.05?.?5?In the Cd group,The phenomenon of increased autophagosome,endoplasmic reticulum expansion and mitochondrial ridge blur in CEF were observed by transmission electron microscopy,the CCK-8 test showed that the cell viability was significantly decreased,while the APS+Cd group both in the cell morphology damage and cell viability were significantly improved compared with the Cd group.In conclusion,cadmium can induce cell damage by ROS-induced CEF autophagy,while APS can significantly reduce cadmium-induced ROS production in CEF and reduce the expr ession of LC3 and Beclin-1 protein in CEF induced by cadmium,APS increased the expression of m TOR and antioxidant levels and improved the cell viatality and morphological damage of CEF caused by cadmium,These results indicate that APS can attenuate cadm ium-induced CEF autophagy-induced cell damage by reducing ROS production.This study provides a scientific experimental basis for further exploring the role and mechanism of APS intervention in cadmium poisoning.