Atorvastatin Inhibits Ferroptosis of H9C2 Cells by regulatingSMAD7/Hepcidin Expression to Improve Ischemia-Reperfusion Injury

Abstract

Background. Ferroptosis plays a key role in cardiomyopathy. Atorvastatin (ATV) has a protective effect on ischemia-reperfusion (I/R) cardiomyopathy. The purpose of this study is to elucidate the mechanism of ATV in I/R injury. Methods. H9C2 cells and cardiomyopathy rats were induced by hypoxia/reoxygenation (H/R) and I/R to construct in vitro and in vivo models. Cell viability was determined by CCK8. Cardiac histopathology was observed by HE staining. Transmission electron microscope (TEM) was used to observe the mitochondrial morphology. The reactive oxygen species (ROS) content in cells was analyzed by the biochemical method. ELISA was conducted to calculate the concentrations of total iron/Fe2+ and hepcidin. The expression of ferroptosis and SMAD pathway-related genes were detected by qPCR. Western blot was performed to detect the expression levels of ferroptosis and SMAD pathway-related proteins. Results. In H9C2 cells, ATV reversed the decline in cell viability, mitochondrial shrinkage, and ROS elevation induced by erastin or H/R. The concentration of total iron and Fe2+ in H/R-induced H9C2 cells increased, and the protein expression of FPN1 decreased. After ATV treatment, the concentration of total iron and Fe2+ decreased, and the protein expression of FPN1 increased. The expression of the SMAD7 gene in H/R-induced H9C2 cells decreased, and the expression of the hepcidin gene increased, which were reversed by ATV. When SMAD7 was knocked down, ATV treatment failed to produce the above effect. ATV also improved ferroptosis in I/R rat myocardium through the SMAD7/hepcidin pathway. Conclusions. ATV reversed the decline in H9C2 cell viability, mitochondrial shrinkage, and ROS elevation, and improved the myocardium ferroptosis through the SMAD7/hepcidin pathway in I/R rat.

Ferroptosis Regulated by Hypoxia in Cells

“…From understanding the mechanism involved in the inhibition of ferroptosis caused by hypoxia, avoiding the ferroptosis inhibition caused by hypoxia could be a new strategy for treating tumors with ferroptosis inducers [ 180 , 181 ]. Considering that ferroptosis is the major reason for IRI, many drugs have been developed to inhibit hypoxia-induced ferroptosis based on its mechanism [ 129 , 140 , 141 , 150 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 ]. Intriguingly, hypoxia is a symptom of the Coronavirus Disease-2019 (COVID-19); HIF-1α plays an important role in the early phase of SAR-CoV-2 infection and is also associated with secondary organ damage [ 182 , 183 ].…”

“…H/R also induces PTGS through HIF-1α to induce ferroptosis in H9C2 cardiomyocytes [ 139 ] and a rat model of coronary microembolization (CME)-induced myocardial injury [ 140 ]. H/R treatment decreased SMAD7 expression and increased Hamp1 expression to promote erastin-induced ferroptosis in H9C2 cardiomyocytes [ 141 ]. Atorvastatin blocked the HIF-1α/COX-2 axis and the SMAD7/hepcidin pathway to inhibit CME- or erastin-induced ferroptosis of cardiomyocytes [ 140 , 141 ].…”

“…H/R treatment decreased SMAD7 expression and increased Hamp1 expression to promote erastin-induced ferroptosis in H9C2 cardiomyocytes [ 141 ]. Atorvastatin blocked the HIF-1α/COX-2 axis and the SMAD7/hepcidin pathway to inhibit CME- or erastin-induced ferroptosis of cardiomyocytes [ 140 , 141 ]. It is reported that H/R treatment induces USP7 to activate p53, leading to the increased TFRC levels to promote ferroptosis of H9C2 cardiomyocytes, as well as in a rat model of myocardial IRI [ 142 ].…”

“…However, studies that employed both hypoxia and H/R treatment found that hypoxia alone could promote ferroptosis, which could not be further elevated by reoxygenation in some cases [ 146 , 151 ]. Another study focused on the reoxygenation process showed that reoxygenation did not alter Nrf2 or HIF-1α activity [ 156 ], whereas these two pathways have been reported to be involved in the H/R treatment in several studies [ 138 , 139 , 140 , 141 , 149 , 150 ]. These studies suggested that hypoxia/ischemia at least partially contributes to promoting ferroptosis.…”

Targeting ferroptosis as a promising therapeutic strategy to treat cardiomyopathy

“…13 functions as a new type of ferroptosis inhibitor through inhibiting Fe 2+ accumulation and restoring mitochondrial functions in H9c2 cells and reduced Fe 2+ deposition and lipid peroxidation in a myocardial I/R injury mouse model . Atorvastatin ( 14) is a potent, orally available inhibitor of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the major rate-limiting enzyme in cholesterol synthesis.14 reversed erastin or H/R-induced cell injury in H9C2 cells through inhibiting ferroptosis by decreasing Fe 2+ via upregulating expression of FPN1 (Peng et al, 2022). 14 increased the expression of the SMAD7 and decreased the expression of the hepcidin in H/R-induced H9C2 cells (Peng et al, 2022).14 protects myocardium against ischemia-reperfusion injury through various mechanisms (Zuo et al, 2016;Chen et al, 2017;Yang et al, 2018;Cao et al, 2019).…”