Study On The Mechanism Of Curcumin Protecting Copper-injured HLD Model Cells Through Inhibiting Ferroptosis

Hepatolenticular degeneration（HLD）,also known as Wilson’s disease（WD）,is an autosomal recessive disorder of copper metabolism.The aetiology is ATP7B mutation in the patient’s gene,which leads to abnormal synthesis of ceruloplasmin and disorder of copper excretion in bile.Excessive copper is deposited in the body,causing damage to multiple major tissues and organs such as the liver,brain,kidney,and cornea.Ferroptosis is a form of cell death that depends on the accumulation of iron and reactive oxygen species（ROS）.The accumulation of lipid peroxides in cells is a direct factor of ferroptosis.Copper overload is also accompanied by iron overload in patients with HLD,lipid peroxidation caused by both can cause liver cell damage.Therefore,in addition to copper overload,ferroptosis induced by iron overload may play an important role in HLD liver cell injury.The previous research showed that curcumin has a protective effect on HLD liver cells from copper damage,but its protective mechanism is still unclear.Objective:To investigate whether ferroptosis is involved in the death of HLD model cells induced by CuSO₄,to study whether curcumin can play a protective role by inhibiting the ferroptosis of HLD model cells from copper damage.Methods:Detecting cell viability was used to screen the different treatment times and concentrations of CuSO₄ acting on BRL-3A cells transfected with sh ATP7B,and determining the optimal CuSO₄ modeling concentration and time to establish HLD cell models.At the same time,MTT assay was used to detect the effect of Ferrostain-1 and curcumin on the survival rate of BRL-3A cells.In order to explore the form of cell death in the HLD model,MTT and microplate reader assay were used to detect the cell survival rates and LDH leakage rates after the pre-protection of Z-VAD-FMK,Ferrostain-1,and Necrostain-1,combined with the corresponding cell death detection indicators:Annexin-APC/7-AAD assay to detect cell apoptosis,DCFH-DA probe to detect ferroptosis,Hoechst/PI assay to detect cells necroptosis.The kits are used to detect relevant oxidative stress indicators（MDA,SOD,GSH,GPX）,flow cytometry is used to detect ROS and mitochondrial membrane potential,PP-Cu probe and atomic absorption are used to detect changes in Cu²⁺content,western blot is used to detect the expressions of Nrf2,HO-1,GPX4 protein,combined with changes in cell morphology,studying the mechanism of ferroptosis in HLD and the protective mechanism of curcumin inhibiting ferroptosis on BRL-3A cells.Results:1.Preliminary exploration of the mechanism of ferroptosis in HLD model cells（1）300μM CuSO₄ treatment for 24 hours is the best modelling condition for HLD hepatocyte model;compared with the normal control group,the survival rate of BRL-3A cells transfected with sh ATP7B damaged by 300μM CuSO₄ decreased significantly,and the LDH leakage rate increased significantly;Necrostain-1.After Ferrostain-1 and Z-VAD-FMK pre-protected BRL-3A cells,the survival rate increased to varying degrees,and the LDH leakage rate of the three different cell death mode specific inhibitors administration group was compared with the model group All were significantly reduced.The ferroptosis inhibitor Ferrostain-1 can reduce the ROS fluorescence of HLD cells;the apoptotic necrosis inhibitor Necrostain-1 can reduce the staining rate of PI;the apoptosis inhibitor Z-VAD-FMK can reduce the apoptosis rate of HLD cells.（2）After pretreatment with 2.5,5,10μM Ferrostain-1,the survival rate of HLD model cells can be increased in a concentration-dependent manner.Ferrostain-1 can inhibit the occurrence of cell ferroptosis by reducing MDA content,increasing GSH level,increasing SOD activity,GPX enzyme activity,and reducing intracellular reactive oxygen species.（3）Transmission electron microscope observation revealed that the mitochondrial membrane spine of the HLD model cell group became thicker,the membrane density increased,and the mitochondria became smaller.After Ferrostain-1 was administered,the mitochondrial morphology of the cells was significantly improved.The mitochondrial membrane potential in HLD model cells decreases,and Ferrostain-1 can reverse this change.（4）300μM CuSO₄ induced a decrease in the expression of GPX4 protein in BRL-3A cells transfected with sh ATP7B,and after pre-protection with different concentrations of Ferrostain-1,Ferrostain-1 dose-dependently promoted the expression of GPX4 protein in cells.After 10μM Ferrostain-1 pre-protection,the difference between the expression of GPX4 and the HLD model group was statistically significant.2.Curcumin inhibits ferroptosis and protects copper-damaged HLD model cells（1）Curcumin is administered at low,medium and high doses of 2.5,5,and 10μM.Curcumin can reduce the damage caused by 300μM CuSO₄ on BRL-3A cells transfected with sh ATP7B in a low concentration-dependent manner.（2）Curcumin can promote the excretion of excess Cu²⁺,reduce the accumulation of Cu²⁺in a concentration-dependent manner,and reduce the Cu²⁺content in HLD liver cells.2.5,5,10μM curcumin can protect the BRL-3A cells transfected with 300μM CuSO₄and sh ATP7B,which inhibited the occurrence of cell ferroptosis by reducing MAD content,increasing GSH level,increasing SOD activity,GPX enzyme activity,and reducing the production of intracellular reactive oxygen species.（3）Transmission electron microscope observation revealed that the mitochondrial membrane spine of the cells in the HLD model cell group became thicker,the membrane density increased,and the mitochondria became smaller.The mitochondrial morphology of the cells was significantly improved after curcumin was administered.The results of flow cytometry showed that curcumin can reverse the decrease of mitochondrial membrane potential of HLD model cells.（4）300μM CuSO₄ promotes the protein expression of Nrf2 and HO-1 in BRL-3A cells transfected with sh ATP7B,and inhibits the expression of GPX4 protein,10μM curcumin can inhibit the Nrf2 and HO-1 protein of HLD model cells after pre-protection Expression and significantly increased GPX4 protein expression.Conclusion:Ferroptosis occurs in HLD model cells.Curcumin can inhibit the occurrence of ferroptosis in HLD model cells through the Nrf2/HO-1/GPX4 signaling pathway and exert a protective effect.