Abstract: Objective To find potential effect of crocin (Cro) to alleviate retinal ischemia-reperfusion injury (RI/RI) in rat′s model. Methods Rats were divided into 6 groups (n=12): sham operation group (sham), RI/RI group (rats with RI/RI model established by intraocular perfusion), 25Cro group, 50Cro group (intraperitoneal injection of 25 and 50 mg/kg Cro, respectively), 50Cro+OSS (OSS_128167) group (intraperitoneal injection of 50 mg/kg Cro and 20 mg/kg Sirt6 inhibitor OSS), and 50Cro+Erastin group (intraperitoneal injection of 50 mg/kg Cro and 15 mg/kg ferroptosis inducer Erastin). The administration time was 30 min before RI/RI modeling. Rats were sacrificed 24 h after RI/RI treatment, retinal injury and cell apoptosis were evaluated by hematoxylin-eosin (HE) staining microscopy and terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) staining. The level of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), reduced glutathione (GSH) and Fe²⁺ in the retina was detected. The mRNA level of Sirt6, glutathione peroxides 4(Gpx4), solute carrier family 7 member 11(Slc7a11), ferritin heavy chain 1(Fth1), ferroportin 1(Fpn1), transferrin receptor (Tfrc), interleukin-6(Il-6), tumor necrosis factor-α(Tnf-α) and cyclooxygenase-2 (Cox-2) in the retina was detected by RT-qPCR. The protein level of Sirt6, GPX4, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax) and cleaved caspase-3 in the retina was detected by Western blot. The positive expression of Sirt6 and GPX4 in the retina was detected by immunohistochemical staining. Results Compared with sham group, the retina of rats in RI/RI group showed significant damage. The proportion of TUNEL⁺ in the retina, the level of ROS, MDA and Fe²⁺, mRNA of IL-6, TNF-α, COX-2 and TFRC, and the protein level of Bax and cleaved caspase-3 were all increased(P＜0.05). The level of GSH and SOD activity, the mRNA level of Fth1, Fpn1, Gpx4, Slc7a11 and Sirt6, and the protein level of Bcl-2, GPX4 and Sirt6 were all decreased (P＜0.05). Compared with RI/RI group, the retinal damage in 25Cro group and 50Cro group was significantly alleviated, and the other indicators were significantly relieved (P＜0.05). Compared with 50Cro group, the retinal injury of rats in 50Cro+OSS group and 50Cro+Erastin group were exacerbated. The proportion of retinal TUNEL⁺, the level of ROS, MDA and Fe²⁺, the mRNA levels of Il-6, Tnf-α, Cox-2 and Tfrc, and the protein levels of Bax and cleaved caspase-3 all increased (P＜0.05). The levels of GSH and SOD activity, the mRNA levels of Fth1, Fpn1, Gpx4, Slc7a11 and Sirt6, and the protein level of Bcl-2, GPX4 and Sirt6 was all decreased (P＜0.05). Conclusions Crocin alleviates RI/RI in rats by inhibition of Sirt6-mediated ferroptosis.

Key words: crocin, ischemia-reperfusion, sirtuin6(Sirt6), ferroptosis, iron metabolism