Puerarin Protects Against Sepsis-Induced Myocardial Injury Through AMPK-Mediated Ferroptosis Signaling

Background and Purpose:Puerarin,as a natural active product extracted from wild Puerarin,has been widely used in the treatment of various cardiovascular diseases,such as atherosclerosis,ischemic cardiomyopathy,myocardial hypertrophy and arrhythmia,due to its multi-target effect in clinical application.Studies have shown that puerarin can effectively improve myocardial damage caused by sepsis,but the mechanism of its myocardial protection is not clear,and it is mainly believed to be related to the inhibition of inflammation and oxidative stress.Ferroptosis is a newly discovered cell death mode regulated by lipid oxidation.Different from the traditional cell death mode,its mechanism is mainly related to the inhibition of cystine entry into cells by ferroptosis promoters（Erastin）,resulting in glutathione depletion and GPX4inactivation,and then a large amount of Fe²⁺enters mitochondria.A large number of reactive oxygen species（ROS）are produced,eventually leading to cell death.Ferroptosis has been shown to play a key role in myocardial damage from multiple causes,such as diabetic cardiomyopathy,ischemic cardiomyopathy,and septic myocardial damage.Studies have shown that after sepsis,lipopolysaccharides（LPS）interact with toll-like receptors（TLRs）located on immune cells and other cells to promote the production of a large number of inflammatory cytokines,affecting the oxidative respiratory chain coupling process in mitochondria.The increase of ROS further leads to the occurrence of oxidative stress,which causes damage to cardiomyocytes.Meanwhile,the accumulation of ROS can also cause iron death,which leads to cell death.Therefore,inhibiting the occurrence and development of ferroptosis can ameliorate myocardial damage in sepsis.At present,it is believed that a variety of signaling pathways can regulate the occurrence of ferroptosis,among which AMPK signaling pathway is one of its regulatory pathways,and activation of this pathway can inhibit the occurrence and development of iron death.Studies have shown that puerarin can ameliorate a variety of myocardial diseases by activating AMPK signaling pathway,including pathological myocardial hypertrophy and myocardial ischemic injury.Therefore,we hypothesized that puerarin could reduce LPS-induced myocardial injury by activating AMPK signaling pathway,thereby inhibiting inflammatory response,oxidative stress and ferroptosis.In summary,the purpose of this study was to first clarify the protective effect of puerarin on myocardial damage in sepsis by using isolated cells,and further verify whether puerarin can ameliorate myocardial damage in sepsis by inhibiting ferroptosis through activating AMPK signaling pathway through animal experiments,so as to provide a theoretical basis for clarifying its mechanism of action.Research methods:1.Chapter 1:Protective effect of puerarin on H9c2 cytotoxic injury induced by LPS.According to different treatment methods,H9c2 cells were divided into 5groups:Control group,sepsis group（LPS group）,sepsis+Puerarin group（LPS+Pue group）,sepsis+puerarin group+ferroptosis enhancer（LPS+Pue+Era group）,and sepsis+puerarin group+AMPK inhibitor Compound C（LPS+Pue+CC group）.The levels of lactate dehydrogenase（LDH）,tumor necrosis factorα（TNF-α）and iron content in the supernatant were determined.Cell viability and apoptosis were observed.The expression levels of glutathione peroxidase 4（GPX4）and phosphorylated AMPK（P-AMPK）in myocardial cells were determined.2.Chapter 2:Puerarin inhibits ferroptosis-mediated myocardial damage in sepsis.36 healthy male Sprague-Dawley rats were randomly divided into four groups:Control group,LPS group),Sepsis+LPS+Pue group and LPS+Pue+Era group.Puerarin and puerarin Combined with erastin were used to treat sepsis rat.The echocardiography was used to evaluate Cardiac function.The levels of tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）,interleukin-10（IL-10）,creatine kinase isoenzyme（CK-MB）,troponin I（c Tn I）and lactate dehydrogenase（LDH）in serum and superoxide dismutase（SOD）,reduced glutathione（GSH）,and lipid peroxidase（MDA）in myocardial homogenate were determined.Pathological changes of myocardial tissue were observed.The expression levels of glutathione peroxidase4（GPX4）,Acyl-Co A synthetase long chain family member 4（ACSL4）,Ferritin,transferrin receptor（TFR）,phosphorylated AMPK（P-AMPK）and Iron ion content in myocardial tissue were determined.The expression levels of apoptotic protein（Cleaved-caspase3）,Bcl-2-associated X protein（Bax）and anti-apoptotic protein（Bcl-2）were determined.Apoptosis of rat cardiomyocytes was observed by TUNEL staining.3.Chapter 3:Puerarin inhibits iron death by activating AMPK signaling pathway.12 healthy male Sprague-Dawley rats were randomly divided into four groups:Control group,LPS group,LPS+Pue group and LPS+Pue+CC group.Puerarin（Pue）and puerarin Combined with Compound C（CC）were used to treat sepsis rat.Western Blot was used to determine the expression levels of Ferritin,acyl-Co A synthetase long chain family member 4（ACSL4）,glutathione peroxidase 4（GPX4）,transferrin receptor（TFR）,phosphorylated AMPK（P-AMPK）and Iron ion content in rat myocardial tissue.Research Results:1.Chapter 1:1.1 Puerarine can improve the LPS-induced H9c2 cell damage,reduce the levels of LDH and TNF-α,reduce apoptosis,up-regulate GPX4 expression and reduce iron ion content。1.2 The myocardial protective effect of puerarin in sepsis can be abolished by the Era and AMPK inhibitor CC.2.Chapter 2:2.1 Puerarin can down-regulate the concentration of myocardial injury markers CK-MB,LDH and c Tn I in mouse serum.And it was found that puerarin can reduce the myocardial tissue damage induced by LPS via observing HE slices.2.2 Puerarin can improve the ejection fraction and fraction shortening of the heart of LPS model rat.Puerarin can inhibit the inflammatory response induced by LPS and down-regulate the expression of TNF-α,IL-6 and IL-10.Puerarin can inhibit the oxidative stress response induced by LPS,up-regulate the expression of GSH and SOD and down-regulate the expression of MDA.2.3 Apoptosis-related proteins were detected and TUNEL staining results were observed.Puerarin can promote the expression of Bcl-2,and inhibit the expression of Cleaved-caspase3 and Bax,thereby reducing cardiomyocyte apoptosis.2.4 Puerarin can promote the expression of GPX4 and Ferritin in the myocardial tissue of LPS mouse model,and inhibit the expression of ACSL4 and TFR.In addition,Puerarin can also promote the phosphorylation of AMPK in myocardial tissue,and erastin has no effect on the phosphorylation of AMPK.Erastin can inhibit the anti-inflammatory,anti-oxidative stress and anti-apoptotic effects of puerarin.3.Chapter 3:3.1 Puerarin increased AMPK phosphorylation in myocardial tissue of sepsis rats.3.2 AMPK inhibitor CC promoted the occurrence of iron death in myocardial tissue of sepsis,and AMPK inhibitor Compound C abrogates ferroptosis inhibiting effects of puerarin.Research conclusion:1.Both in vitro and in vivo experiments confirmed that puerarin can protect myocardial injury from sepsis and improve cardiac function by inhibiting the ferroptosis in myocardial cells induced by LPS.2.Puerarin can up-regulate AMPK phosphorylation in myocardial cytotoxic injury models with sepsis.3.Both erastin and Compound C can effectively abrogated the protective effect of puerarin on myocardial injury in sepsis.4.Puerarin protects against sepsis-induced myocardial injury via the AMPK-mediated ferroptosis signaling.