Homocysteine regulates ADAMTS8 to mediate ferroptosis in atherosclerosis

AimTo investigate the role and mechanism of homocysteine(Hcy)in regulating ADAMTS8-mediated ferroptosis in atherosclerosis (AS).MethodsApoE-/- mice(n=12)were randomly divided into two groups to establish a normal diet group(ND, n=6) and a high-methionine diet group (HMD, n=6). RAW264. 7 macrophages were cultured and divided into the control group(0 μmol·L-1 Hcy)and Hcy intervention group(100 μmol·L-1 Hcy), NC adenovirustransfected with Hcy intervention group(Hcy+Ad-NC) and ADAMTS8 adenovirus-transfected with Hcy intervention group (Hcy+Ad-ADAMTS8), NC siRNAtransfected with Hcy intervention group(Hcy+si-NC) and ADAMTS8 siRNA-transfected with Hcy intervention group (Hcy+si-ADAMTS8), ADAMTS8 adenovirus-transfected with Hcy intervention group (Hcy+Ad-ADAMTS8) and ADAMTS8 adenovirustransfected with Hcy intervention+ferroptosis inhibitor group (Fer-1+Hcy+Ad-ADAMTS8). Immunofluorescence staining was used to observe the expression differences of ADAMTS8, xCT and ACSL4. Pearson correlation coefficient was used to analyze the correlation between ADAMTS8 and ferroptosis related indexes xCT and ACSL4. Western blot was used to detect the expression of xCT and ACSL4. The activity of malondialdehyde and superoxide dismutase was used to assess the oxidation level and antioxidant capacity of cells.ResultsCompared with ND, the expression of ADAMTS8 in the HMD group significantly increased, the expression level of ferroptosiia-related protein xCT decreased, while the expression level of ACSL4 significantly increased. Compared with the Control group, in the Hcy group, the expression of ADAMTS8 protein increased, the expression of xCT protein decreased, while the expression of ACSL4 protein increased, the level of MDA increased, and the activity of SOD significantly decreased. Pearson correlation analysis showed that ADAMTS8 expression level was negatively correlated with xCT expression level and positively correlated with ACSL4 expression level. Compared with the Hcy+si-NC group, the expression level of xCT protein in the Hcy+si-ADAMTS8 group significantly increased, the expression level of ACSL4 decreased, the MDA level was reduced, and the SOD activity was enhanced. On the contrary, compared with the Hcy+AdNC group, the expression level of xCT protein decreased, the expression level of ACSL4 increased, the MDA level was enhanced, and the SOD activity decreased in the Hcy+Ad-ADAMTS8 group. Compared with the Hcy+Ad-ADAMTS8 group, the expression level of xCT protein increased, the expression level of ACSL4 decreased, the level of MDA decreased, and the activity of SOD was enhanced in the Fer-1+Hcy+ Ad-ADAMTS8 group.ConclusionADAMTS8 regulates ferroptosis and affects the balance of oxidation and antioxidant in macrophages, thereby promoting the occurrence and development of Hcy-induced AS.