Activation Of The ROS/HO-1/NQO1 Signaling Pathway Contributes To The Copper-induced Oxidative Stress And Autophagy In Duck Renal Tubular Epithelial Cells

This article aims to study the mechanism of copper（Cu）-induced cytotoxicity of renal tubular epithelial cells,and select 11-15-day-old ducklings to establish an model of duck renal tubular epithelial cells.Anhydrous Cu sulfate（Cu SO₄）was used as the Cu source,BHA was used as the ROS scavenger,and ZnPP was used as the HO-1 inhibitor.The experiment is divided into three parts,grouped as follows,control group,100μmol/L Cu²⁺group(100μmol/L Cu²⁺),200μmol/L Cu group(100μmol/L Cu²⁺);control group,200μmol/L Cu²⁺group,100μmol/L BHA+200μmol/L Cu²⁺group（Cu+BHA group）,100μmol/L BHA group（BHA group）;control group,200μmol/L Cu²⁺group,10μmol/L ZnPP+200μmol/L Cu²⁺group（Cu+ZnPP group）,10μmol/L ZnPP group（ZnPP group）.After 12 hours of exposure,we observed the morphology of autophagosomes,acidic autophagy vesicles,LC3 aggregation points,and determined cell survival,reactive oxygen species（ROS）levels,MDC fluorescence levels,and antioxidant indicators（SOD,CAT,H₂O₂,MDA,GSH and GSH-Px）,determined the antioxidant and autophagy-related genes（HO-1,SOD-1,CAT,NQO1,GCLM,m TOR,Beclin1,ATG7,ATG5,ATG3,LC3I and LC3II）m RNA expression and protein expression levels（HO-1,SOD-1,Beclin1 and LC3II/LC3I）.In the experiment of co-processing BHA and Cu,compared with the 0μM Cu²⁺group,in the two Cu treatment groups,ROS accumulated significantly,SOD activity,H₂O₂and MDA content increased significantly（P<0.01）,and GSH-Px activity decreased significantly（P<0.01）.Result showed that the number of autophagosomes increased,the number of AVOs and fluorescence intensity increased,and the concentration of LC3increased.Antioxidant-related and autophagy-related genes m RNA expression levels（HO-1,SOD-1,NQO1,m TOR,Beclin1,ATG7,ATG5,ATG3,LC3B）and protein expression levels（HO-1,SOD-1,Beclin1 and LC3BII/LC3BI）were significantly increased（P<0.05）,and the m RNA expression of CAT and LC3I was extremely significantly decreased（P<0.01）.Compared with the Cu group,in the Cu+BHA group,the accumulation of ROS was significantly reduced,and the number of AVOs and fluorescence intensity were reduced.Antioxidant index CAT activity and GSH content increased significantly（P<0.05 or P<0.001）,H₂O₂and MDA content decreased significantly（P<0.05or P<0.01）.The m RNA expression of HO-1,m TOR and Beclin1 was significantly reduced（P<0.05）,and the expression of protein LC3BII/LC3BI was significantly reduced（P<0.05）.The other indicators have the same changing trend but no significant difference（P>0.05）.In the experiment of co-processing ZnPP and Cu,compared with the Cu group,in the Cu+ZnPP group,the accumulation of ROS was significantly reduced,and the number of AVOs and fluorescence intensity were reduced.The antioxidant index SOD activity,MDA and GSH content decreased significantly（P<0.05 or P<0.001）,and CAT activity and GSH-Px activity increased significantly（P<0.05）.HO-1,SOD-1,CAT,GCLM,Beclin1and LC3II m RNA expressions were significantly reduced（P<0.05 or P<0.01）,and protein SOD-1 and LC3BII/LC3BI expressions were significantly reduced（P<0.05 or P<0.01）.The other indicators have the same changing trend but no significant difference（P>0.05）.In conclusion,excess copper can induce oxidative stress and autophagy by activating the ROS/HO-1/NQO1 pathway,while inhibiting HO-1 may attenuate the oxidative stress and autophagy of copper in duck renal tubular epithelial cells.