Role Of MALT1 In Promoting Ferroptosis In Rat Heart Following Ischemia/reperfusion And The Intervention Of Micafungin

Background:Ferroptosis is a newly discovered form of regulated cell death dependent on iron and LPO,which is one of the main causes of myocardial ischemia/reperfusion injury.It can be observed that mitochondrial membrane density increases and mitochondrial cristae disappear when ferroptosis occurs.SLC7A11 is an essential component of the cystine-glutamate antiporter,which can mediate the transport of cystine into the cell to promote glutathione synthesis and inhibit ferroptosis.Nrf2 could regulate the transcription of SLC7A11 through binding to the antioxidant response element（ARE）domain of the SLC7A11 promoter.MALT1 is a paracaspase that hydrolyzes the arginine domain of its substrate.Studies have reported that inhibiting MALT1 can promote the formation of glutathione and participate in the antioxidant process mediated by SLC7A11 and Nrf2.Therefore,we speculate that MALT1 can participate in cardiomyocyte ferroptosis by inhibiting the Nrf2/SLC7A11 signaling pathway.Micafungin is an echinocandin antifungal drug commonly used in the treatment of fungemia,respiratory mycosis,and gastrointestinal mycosis caused by invasive aspergillus flavus or candida infection.Molecular docking results indicate that there is a potential binding site between micafungin and MALT1.Therefore,we speculate that micafungin may exert a protective effect against myocardial ischemia/reperfusion injury by targeting MALT1-induced ferroptosis.Purposes:1.To explore the role and mechanism of MALT1 in cardiomyocytes ferroptosis after ischemia/reperfusion.2.To evaluate the effect and mechanism of micafungin on myocardial ischemia/reperfusion injury.Methods:1.Animal experiments:a rat MI/RI model was established by ischemia for 1 h and reperfusion for 3 h.Male SD rats were randomly divided into 7 groups:control group,sham group,I/R group,+Micafungin-L group（I/R-treated rats were treated with 8 mg/kg micafungin by intraperitoneal injection on 30 min after ischemia）,+Micafungin-H group（I/R-treated rats were treated with 16 mg/kg micafungin by intraperitoneal injection on 30 min after ischemia）,+MI-2group（I/R-treated rats were treated with 15 mg/kg MI-2 by intraperitoneal injection on 30 min after ischemia）and+vehicle group（I/R-treated rats were treated with equal volume of MI-2 vehicle by intraperitoneal injection on 30 min after ischemia）.Myocardial infarct size was detected by TTC staining;myocardial damage was assessed by CK activity and HE staining;ferroptosis was assessed by LPO,total iron content and ferrous content;Western Blot was used to detect the protein levels of ACSL4,GPX4,MALT1,SLC7A11and Nrf2;The m RNA level of SLC7A11 was detected by real-time PCR.2.Cell experiments:The hypoxia/reoxygenation induced cardiomyocyte injury model was established by hypoxia for 8 h and reoxygenation for 12 h.The experiment was divided into 4 groups:control group,H/R group,+MI-2 group（0.2μM of MI-2 was incubated with H/R-treated H9c2）,and+Vehicle group（0.002%DMSO was incubated with H/R-treated H9c2）.LDH was used to detect drug cytotoxicity,and MTS was used to detect cell viability.Fe³⁺content was detected by Prussian blue staining;LPO and GSH content were detected by kit;ACSL4,MALT1,SLC7A11 and Nrf2 protein levels were detected by Western Blot;SLC7A11 m RNA level was detected by real-time PCR.Results:1.Animal experiments:1.1 Compared to the Sham group,the myocardial infarction area and CK activity in the I/R group were significantly increased,accompanied by myocardial cell arrangement disorder,increased gap,and myocardial fiber tear;the protein levels of MALT1 and ACSL4 in the heart tissue of the I/R group were significantly increased;the protein level of Nrf2 and SLC7A11 were significantly decreased,along with the obvious decrease in GPX4 and GSH content,and the increase in LPO,total iron ion and Fe²⁺content.The above results suggested that ischemia/reperfusion can induce ferroptosis in cardiomyocytes.1.2 Compared with the I/R group,intervention of MI-2 reduced myocardial infarct size,significantly decreased CK activity,and significantly improved myocardial fiber arrangement and morphological structure.In addition,the protein level of MALT1 in cardiac tissue was significantly decreased after MI-2 treatment,accompanied by obvious increase of Nrf2 protein level,SLC7A11 m RNA and protein level.Meanwhile,MI-2 could significantly restore GPX4 protein level and reduce ACSL4,total iron ion,Fe²⁺and LPO content,suggesting that MI-2could inhibit rat myocardial cell ferroptosis,thereby alleviating myocardial ischemia/reperfusion injury.1.3 Compared to the I/R group,treatment of micafungin could significantly reduce myocardial infarction size and CK activity,and significantly improved myocardial fiber arrangement and morphological structure,suggesting that micafungin has an anti-myocardial ischemia/reperfusion injury effect.In addition,micafungin treatment can inhibit the up-regulation of MALT1 protein expression induced by myocardial ischemia/reperfusion injury;significantly restore the expression of Nrf2,SLC7A11 and GPX4;significantly reduce the contents of ACSL4,total iron ion,Fe²⁺and LPO in cardiac tissue.2.Cell experiments:2.1 Compared to the Control group,LDH release,LPO and Fe³⁺contents in H/R group were significantly increased;MALT1 and ACSL4protein levels were also significantly increased;along with decreases in Nrf2,SLC7A11 and GSH content,suggesting that hypoxia/reoxygenation can induce ferroptosis of myocardial cell.2.2 MI-2 could protect H9c2 cell from suffering with H/R in dose-dependent manner.Compared to the H/R group,MI-2 intervention significantly decreased MALT1 protein expression,restored the contents of Nrf2,SLC7A11 and GSH,and significantly decreased Fe³⁺and LPO content.The above results indicated that MI-2 could inhibit H/R-induced cardiomyocyte ferroptosis.2.3 Micafungin could protect H9c2 cell from suffering with H/R in a dose-dependent manner.And 1μM and 5μM of micafungin can be used for subsequent experiments.Conclusions:1.MALT1 in myocardial tissue is up-regulated following ischemia/reperfusion,lead to inhibition of Nrf2/SLC7A11/GPX4 pathway,thereby promoting ferroptosis of rat heart and aggravating myocardial ischemia/reperfusion injury.2.Micafungin inhibits MALT1,enhances the activity of the Nrf2/SLC7A11 pathway,inhibits ferroptosis in cardiomyocytes,thereby alleviates myocardial ischemia/reperfusion injury.