| Objective:Realgar is a traditional mineral Chinese medicine in China.Its main component is arsenic disulfide（As2S2）,which has the effects of detoxification,insecticidal,dryness and expectorant,and is widely used in clinic.As the main organ of drug metabolism,a large number of studies have confirmed that long-term,excessive and irregular use of realgar or its preparations can cause hepatotoxicity.However,the mechanism of realgar hepatotoxicity has not been fully clarified.Ferroptosis is a new type of programmed cell death mode triggered by excessive accumulation of iron-dependent lipid peroxide.Iron overload is the key factor leading to ferroptosis.The study found that ferritin autophagy process mediates the autophagic degradation of ferritin into lysozyme,making the iron ions stored in ferritin release into free iron,increasing the level of intracellular iron ions and promoting ferroptosis.Many studies have shown that ferroptosis is closely related to liver disease or liver injury.However,it is not clear whether ferroptosis is involved in the mechanism of realgar induced hepatotoxicity and whether ferritinophgy plays a role in it.The purpose of this experiment is to study the effect of subchronic realgar exposure on ferroptosis in mice liver and the role of ferritinophgy in it,so as to provide new laboratory data for clarifying the mechanism of realgar induced hepatotoxicity and possible intervention targets.Methods:In this study,a mouse model of exposure to realgar at different doses,intervention of iron chelating agent Deferprone（DFP）and intervention of autophagy inhibitor 3-methyladenine（3-MA）was established.Three batches of 6-week-old male healthy Kunming mice（weight 18-22 g）,the first batch of mice were randomly assigned to the control group,0.15 g/kg realgar exposure group,0.45 g/kg realgar exposure group,1.35 g/kg realgar exposure group,with 9 mice in each group.The control group was given 0.5%sodium carboxymethyl cellulose-Na（CMC-Na）by gavage;the three groups of realgar exposure groups were given 0.15 g/kg,0.45 g/kg and 1.35 g/kg realgar suspension（suspension medium is 0.5%CMC-Na）by gavage,once a day,for eight weeks.The second batch of mice were randomly divided into control group,realgar group,DFP group and DFP+realgar group.The control group and DFP group were given 0.5%CMC-Na solution by gavage,while the realgar group and DFP+realgar group were given realgar suspension（1.35 mg/kg,suspension medium was 0.5%CMC-Na）by gavage,once a day,for eight weeks;The control group and realgar group were intraperitoneally injected with 0.5%DMSO,and the DFP group and DFP+realgar group were intraperitoneally injected with DFP solution（80 mg/kg,solvent 0.5%DMSO）;In the seventh week,intraperitoneal injection was started once a day for two weeks.The third batch of mice were randomly divided into control group,realgar group,3-MA group and3-MA+realgar group.The control group and 3-MA group were given 0.5%CMC-Na solution by gavage,while the realgar group and 3-MA+realgar group were given realgar suspension（1.35 mg/kg,suspension medium 0.5%CMC-Na）by gavage,once a day,for eight consecutive weeks;The control group and realgar group were intraperitoneally injected with 0.5%DMSO,and the 3-MA group and 3-MA+realgar group were intraperitoneally injected with 3-MA solution（15 mg/kg,0.5%DMSO solvent）;In the sixth week,intraperitoneal injection was started,once every two days,for a total of three weeks.24 hours after the last exposure,3 mice in each group were fixed by cardiac perfusion;The rest of the mice took blood from eyeballs,separated plasma,extracted liver,and frozen for the following experiments:1.The activities of alanine aminotransferase（ALT）and aspartate aminotransferase（AST）in mouse plasma were determined by spectrophotometry;2.The pathological morphology of mouse liver was observed by H&E staining;3.The iron content in plasma and liver tissue of mice was detected by colorimetry;4.Prussian blue staining was used to observe the iron deposition in the liver of mice;5.The content of reactive oxygen species（ROS）in mouse liver was detected by chemical fluorescence method,the content of malondialdehyde（MDA）in mouse liver was detected by TBA method,and the content of GSH in mouse liver was detected by microplate method;6.The morphology of mouse liver mitochondria was observed by transmission electron microscope;7.Reverse transcription-polymerase chain reaction（RT-PCR）and Western blot（WB）were used to detect the m RNA and protein expression of ferritinophagy and ferroptosis related molecules;8.Immunofluorescence staining was used to observe the co-localization of nuclear receptor coactivator 4（NCOA4）and microtubule-associated protein 1 light chain 3 beta（LC3B）.Results:1.Subchronic realgar exposure induced liver damage in mice.Compared with the control group,the plasma ALT and AST activities in the 1.35 g/kg realgar group were significantly increased（P<0.05）.The H&E staining results showed that compared with the control group,the liver of mice exposed to realgar showed cytoplasmic vacuoles,nuclear shrinkage,necrosis,and inflammatory infiltration.2.Subchronic realgar exposure induced liver ferroptosis in mice.As the dosage of realgar exposure increases,the iron content in the plasma and liver tissue of mice gradually increases,and iron deposition in liver tissue increases;Compared with the control group,the liver MDA level of the realgar group mice significantly increased,while the GSH level significantly decreased;The mitochondrial cristae in liver of mice in realgar group was significantly reduced and the Outer mitochondrial membrane was damaged.The results showed that the protein expression of Acyl Co A synthase long chain family member 4（ACSL4）in 0.45 g/kg and 1.35 g/kg realgar was higher than that in the control group（P<0.05）;The expression of GPX4 protein in the 1.35 g/kg realgar group was lower than that in the control group（P<0.05）;Compared with the control group,the expression of Gpx4 m RNA in the 0.45 g/kg and 1.35 g/kg realgar groups decreased,the expression of Prostaglandin endoperoxide synthase 2（Ptgs2）and Acls4 m RNA in the 1.35 g/kg realgar group increased,and the expression of Cha C glutamate specific gamma glutamylcyclotransferase 1（Chac1）m RNA in the 0.45 g/kg realgar group increased,the differences were statistically significant（P<0.05）;The m RNA expression of member 11 of the solute carrier family 7（Slc7a11）in the realgar group showed no significant difference compared to the control group.3.DFP intervenes to reduce iron content and inhibit realgar induced liver ferroptosis in mice.Compared with the realgar group,the DFP+realgar group showed a decrease in plasma and liver tissue iron levels（P<0.05）,and a significant decrease in liver iron deposition;MDA levels significantly decreased（P<0.05）,while GSH levels significantly increased（P<0.05）.The ROS levels in the liver tissue of mice in the realgar group were higher than those in the control group（P<0.05）,while the ROS levels in the DFP+realgar group were lower than those in the realgar group（P<0.05）.Compared with the realgar group,the expression of ACSL4 protein in the liver of DFP+realgar group mice decreased（P<0.05）,while the expression of GPX4 protein increased（P<0.05）.4.Realgar induces ferroptosis in mouse liver by affecting ferritinophagy.Compared with the realgar group,the 3-MA+realgar group showed a decrease in plasma and liver tissue iron levels（P<0.05）,and a significant decrease in liver iron deposition;The MDA level significantly decreased（P<0.05）,while the GSH level significantly increased（P<0.05）;The expression of ferroptosis related protein ACSL4 decreased（P<0.05）,and the expression of GPX4 increased（P<0.05）.The results of detection of ferritinophagy related protein and gene expression levels in mouse liver showed that the protein expressions of NCOA4,LC3II/I,SQSTM1/p62,and Ferritin heavy chain 1（FTH1）in Realgar group were higher than those in the control group,the protein expressions of p62 and FTH1 in 3-MA+Realgar group were higher than those in Realgar group,and the protein expressions of NCOA4 and LC3II/I were lower than those in Realgar group,with statistical significance（P<0.05）.Compared with the control group,the m RNA levels of autophagy related gene 5（Atg5）,autophagy related gene 7（Atg7）,Ncoa4,and Fth1 in the realgar group increased（P<0.05）;Compared with the realgar group,the m RNA levels of Atg5,Atg7,and Ncoa4 decreased after 3-MA intervention（P<0.05）,while the Fth1 m RNA levels did not show significant changes.The immunofluorescence results showed an increase in co localization of NCOA4 and LC3B in the liver tissue of mice treated with realgar,after3-MA intervention,the co localization of NCOA4 and LC3B decreased compared to the realgar group.Conclusion:Subchronic realgar exposure affects ferritinophagy in mouse liver,which releases iron ions,leads to iron overload,induces ferroptosis,and ultimately leads to liver injury in mice. |
