The Effect Of Regulating Ferroptosis On Rat Lower Limb Ischemia-reperfusion Injury

Objective(s): To verify the effect of iron death pathway on lower limb ischemiareperfusion injury in rats,and try to alleviate the injury caused by lower limb ischemiareperfusion injury by inhibiting iron death-related pathway.Methods:(1)We established a rat model of hind limb ischemia-reperfusion injury to investigate the correlation between ferroptosis and the injury.24 female SD rats were randomly assigned into three groups: the control group(CG),the group with femoral artery ligation(CFA),and the group with hind limb ligation(LE).The rats in the CG group underwent sham surgery,while the rats in the CFA and LE groups underwent femoral artery and hind limb ligation,respectively.After maintaining the rats in the model groups for 6 hours of ischemia and 6 hours of reperfusion,they were anesthetized and euthanized.Blood and gastrocnemius muscle specimens from the right hind limb were collected to measure MDA,ROS,GSH-SH,LDH,and serum iron levels using ELISA.The skeletal muscle total iron level was determined using the cellular total iron assay.The gastrocnemius muscle and femoral artery from the right hind limb were fixed and embedded,followed by HE and EVG staining to observe the pathological differences among the groups.(2)To investigate the effect of regulating the process of iron death on the rat’s lower limb ischemia-reperfusion injury,48 SD rats were randomly divided into 6 groups: control group(CG),ischemia-reperfusion group(IR group),long-term injection of deferoxamine group(LDFO group),short-term injection of deferoxamine group(SDFO group),long-term injection of ferric carboxymaltose group(LID group),and long-term injection of rapamycin group(RAPA group).The CG group underwent sham surgery,while the IR group underwent direct ligation of the lower limb to induce ischemia-reperfusion injury.The LDFO group received intraperitoneal injections of deferoxamine mesylate at a dose of 100mg/kg/12 h for one week prior to surgery.The SDFO group received intraperitoneal injections of deferoxamine mesylate at a dose of 100mg/kg one hour before reperfusion.The LID group received intraperitoneal injections of ferric carboxymaltose at a dose of60mg/kg/12 h for one week prior to surgery.The RAPA group received intraperitoneal injections of rapamycin at a dose of 1.0mg/kg/12 h for one week prior to surgery.After maintaining a 6-hour ischemic period and a 6-hour reperfusion period,the rats were anesthetized and euthanized.Blood samples and gastrocnemius muscle samples from the right lower limb were collected to detect levels of MDA,ROS,GSH-SH,LDH,and serum iron content using ELISA.The total iron level in skeletal muscle was determined using a cell total iron colorimetric method.The right gastrocnemius muscle and femoral artery were fixed and embedded for HE staining and EVG staining to observe pathological differences among groups.Results:(1)Compared with the CG group,the levels of GSH,MDA,ROS,LDH,and serum iron in the CFA and LE groups were significantly increased(p<0.05).There were no statistically significant differences in the levels of GSH,MDA,ROS,LDH,and serum iron between the CFA and LE groups.Histopathological sections showed that the vascular wall morphology and structure of the arteries’ intima,media,and adventitia were normal in the CG group.The skeletal muscle cells were also normal in shape,with intact structures and clear transverse striations,without obvious pathological changes.In the CFA and LE groups,the internal and external elastic fiber plates of the arterial vascular wall were continuous,while the structure of the medial elastic fiber plates was locally blurred or discontinuous.The skeletal muscle sections showed significant structural damage to the muscle cells,with a large number of cells undergoing degeneration and necrosis.The tissues showed a large number of infiltrating inflammatory cells and obvious edema.(2)The text describes the results of a study comparing various groups of samples(CFA,LE,CG,IR,LDFO,SDFO,LID,and RAPA)based on several parameters including GSH,MDA,ROS,LDH,serum iron levels,and muscle iron levels.The results indicate that the levels of GSH were significantly decreased and the levels of MDA,ROS,LDH,serum iron,and muscle iron were significantly increased in all groups except the CG group.Additionally,the LDFO,SDFO,and LID groups showed no significant differences in GSH levels compared to the IR group,while the RAPA group showed a significant increase.The LDFO and SDFO groups showed no significant differences in MDA,ROS,and LDH levels compared to the IR group,while the LID group showed a significant increase and the RAPA group showed a significant decrease.In terms of serum iron levels,the LDFO group showed a significant decrease,while the SDFO and LID groups showed significant increases compared to the IR group.The LID group showed a significant increase in muscle iron levels compared to the IR group,while the LDFO,SDFO,and RAPA groups showed slight decreases but no significant differences.The tissue pathology sections showed that the CG group had no significant pathological changes,while the other groups showed varying degrees of damage to the blood vessel and muscle tissue.In the IR group,LDFO group,SDFO group and LID group,there were obvious structural damage of skeletal muscle cells,large area of muscle cell necrosis and lysis,muscle edema,accompanied by a large number of inflammatory cell infiltration.The RAPA group showed slight structural damage of skeletal muscle,occasional muscle cell necrosis,and a small amount of inflammatory cell infiltration.Conclusion(s): Based on the provided information,it appears that when the body is exposed to factors that promote iron death and increase iron ion levels,it may exacerbate the damage caused by lower limb ischemia-reperfusion injury.Chelating agents like deferoxamine,which are used to reduce iron ion levels,do not appear to have a significant effect on treating this type of injury.However,using rapamycin to reduce the production of ROS and other products caused by iron death may help alleviate the damage caused by lower limb ischemia-reperfusion injury.