A Study Of The Role And Underlying Mechanisms Of CTRP9 In Sepsis-mediate Acute Cardiopulmonary Injury

Part Ⅰ The role and mechanisms of CTRP9 in sepsis-mediate acute pulmonary injury BackgroundSepsis is a syndrome of organ dysfunction induced by dysregulated host responses to infection,and is characterized by fast deterioration,high mortality and poor prognosis.Studies have found that about 30%of patients with sepsis subsequently develop multiple organs dysfunction syndrome(MODS).It has been reported that the lung is one of the most vulnerable target organs for sepsis.Acute lung injury(ALI)is the first injury organ among the sepsis induced multiple organs dysfunction.One of the pathogenesis of ALI is the imbalance of inflammatory response.A series of inflammatory mediators released by neutrophils,macrophages,endothelial cells and mast cells lead to increased pulmonary microvascular permeability,interstitial edema,thickened alveolar walls and hyaline membrane formation and further results in acute hypoxic respiratory or respiratory failure.Therefore,regulating the production of inflammatory cells and the release of inflammatory mediators is an important treatment for ameliorating sepsis-related lung injury.As one of the main sources of various inflammatory factors,macrophages play an important role in sepsis-induced ALI.Interleukin-1β(IL-1β)is an initiating factor of inflammation and is involved in the occurrence and development of inflammation.More and more evidence has shown that interleukin-1β(IL-1β)significantly increased in acute lung injury and is closely related to the severity of ALI.The production of IL-1β relies on the activation of inflammasomes.The most widely studied and most important inflammasome is the NLRP3 inflammasome,which is involved in the occurrence and development of various inflammatory diseases.The NLRP3 inflammasome is a multiprotein complex that includes NLRP3,ASC(CARD domain-containing apoptosis-associated speck-like protein),and Pro-cysteinyl aspartate speacific proteinase-1.The NLRP3 protein,ASC and the precursor of Caspase-1(Pro-Caspase-1)are assembled to form NLRP3 inflammasome,induced by extracellular adenosine triphosphate(ATP),mitochondrial damage,the changes of intracellular ion concentration,and lysosome damage caused by cholesterol crystallization.The NLRP3 inflammasome acts as a platform to cleave Pro-Caspase-1 into active Caspase-1.On the one hand,active Caspase-1 promotes the activation of IL-1β precursors into mature IL-1β forms.On the other hand,active Caspase-1 can cut the gasdermin(GSDMD)to expose its N-terminus and transfer it to the cell membrane to make a hole,and then the cell swells and ruptures,resulting in the release of a large amount of IL-1β to the outside of the cell,amplifying the inflammatory cascade.This inflammasome-dependent programmed cell death mediated by gasdermins is called pyroptosis.Complement lq/tumor necrosis factor-related proteins(Clq/tumor necrosis factor-related proteins,CTRPs)family is a widely expressed and highly conserved adipokine family of adiponectin paralogs.CTRP9 is a member of the CTRP family.Studies have shown that CTRP9 can be detected in various tissues and organs such as skeletal muscle,brain,liver and kidney,which suggests that CTRP9 may exert its biological effects by different modes of secretion.Studies have found that CTRP9 was involved in a variety of physiological processes,such as glucose and lipid metabolism,immunity and inflammation,apoptosis and oxidative stress.Recently,the role of CTRP9 in inflammation has attracted much attention.Our previous research showed that CTRP9 promoted the polarization of macrophages to M1 type by activating the JNK pathway,resulting in increased release of inflammatory mediators and aggravating inflammatory responses.The NLRP3 inflammasome is an important intervention target for inflammatory diseases.However,it is unclear whether CTRP9 participate in the inflammatory responses of sepsis-related lung injury by affecting the activation of NLRP3 inflammasome.Therefore,in this study,WT mice and CTRP9 knockout mice were enrolled to construct septic acute lung injury model and explore whether CTRP9 participate in the inflammatory responses of acute lung injury by affecting NLRP3 inflammasome.In addition,in vitro,we extracted mouse peritoneal macrophages to further explore the effect of CTRP9 on NLRP3 inflammasome and its molecular mechanism.Objective1.To clarify the role of CTRP9 in sepsis-induced acute lung injury.2.To explore whether CTRP9 participates in sepsis-induced acute lung injury by regulating the NLRP3 inflammasome.3.To explore the molecular mechanism how CTRP9 mediates NLRP3 inflammasome activation.Method1.Establishment of animal modelsFirstly,CTRP9 gene knockout mice were constructed by Shanghai Southern Model Biotechnology Co.,Ltd.using CRISPR/Cas9 technology.12-week-old male C57BL/6J mice and CTRP9 knockout mice(CTRP9-KO mice)were enrolled to construct sepsis mouse model by intraperitoneal injection of LPS(10 mg/kg)for12 hours.Then the mice were euthanized and tissue samples such as serum,lung,heart,liver and kidney were collected.2.Histopathology of the lungsThe general morphology of lung tissue was observed by H&E staining,the infiltration of macrophages in lung tissue was observed by Immunofluorescence Staining,and the expression of IL-1β in lung tissue was detected by Immunohistochemistry and Immunofluorescence Staining.3.Enzyme linked immunosorbent assay(Elisa)The levels of IL-1β in macrophage culture medium and mouse serum were detected by Elisa detection kit.4.Extraction of mouse peritoneal macrophagesWT mice and CTRP9 knockout mice were injected intraperitoneally with 6%starch.After 3 days,macrophages were collected by repeatedly perfusing the peritoneal cavity with cold PBS.Then,peritoneal macrophages were cultivated in DMEM containing 10%FBS and 1%penicillin streptomycin.Then,the cells were seeded in plates and incubated in a humidified incubator at 37℃ with 5%CO2.5.Western blottingTotal proteins were extracted from mouse peritoneal macrophages and lung tissue to detect the expression of NLRP3,ASC,Pro-caspase-1,Pro-IL-1β,Caspase-1β10,IL-1β p17,GSDMD-FL,GSDMD-NT and NOX2.6.Real-time quantitative PCRThe RNA of peritoneal macrophages was extracted,and the RNA was reverse transcribed into stable cDNA using a reverse transcription kit.Then the mRNA levels of NLRP3 and IL-1βwere detected by fluorescence quantitative detection.7.Cellular immunofluorescenceThe formation of ASC speck and the production of intracellular ROS were detected by cellular immunofluorescence.8.PI stainingMacrophages were cultured in a 12-well plate with 5 ng/mL PI dye for half an hour in the dark.Then macrophages were observed using a fluorescence microscope to evaluate the rate of pyroptosis.9.Cell transfectionMacrophages were incubated with NOX2 small interference.After 8 hours,cell culture medium containing small interference was discarded and replaced with fresh medium.After 24 hours,other stimuli were added into culture medium and the interference efficiency was detected by western blotting.10.Flow cytometryPeritoneal macrophages were incubated with serum-free medium containing DCF-DA at 37℃ for 20 minutes in the dark.After washing with PBS,macrophages were transferred to flow tubes,and the content of intracellular ROS was detected by flow cytometry.11.Statistical analysisQuantitative data are presented as mean ± standard deviation(mean±SD).For all normally distributed data,firstly,we used Log-rank test to analyze the survival rate of septic mouse and made the survival curves.Then we used independent samples t-test to compare the data between two groups.And we used one-way ANOVA to compare the data of more than three groups.p<0.05 was considered statistically significant.Results1.The construction of CTRP9 knockout miceWe extracted the lung,heart tissues and peritoneal macrophages from the WT mice and CTRP9-KO mice,and Western blotting results demonstrated that CTRP9 protein was hardly expressed in lung,heart tissues and peritoneal macrophages of CTRP9-KO mice,indicating that the CTRP9 knockout mice were successfully constructed.2.The expression of CTRP9 is significantly up-regulated in the lung tissues of septic miceIn order to investigate whether the expression of CTRP9 changes in the sepsis disease model,we injected WT mice with PBS or LPS(10 mg/kg)intraperitoneally,and the mice were euthanized after 12 h.The expression of CTRP9 was detected by Western blotting.The results showed that the expression of CTRP9 in lung tissue of septic mice was significantly higher than that in normal mice.3.CTRP9 knockout alleviates septic lung injury and attenuates inflammatory responseFirst,WT mice and CTRP9-KO mice were enrolled,and LPS was injected intraperitoneally to construct a sepsis model.The state and survival time of the two groups of mice were observed every 8 h,and the survival curve was drawn.The results showed that the survival rate of CTRP9-KO sepsis mice was significantly higher than that of WT sepsis mice.Next,we divided the mice into four groups,the control group of WT mice,the control group of CTRP9-KO mice,the sepsis group of WT mice,and the sepsis group of CTRP9-KO mice.The mice in four groups were euthanized after 12 hours,and the tissue samples were collected.H&E staining was performed to observe the general morphology of lung tissue and the infiltration of inflammatory cells in the lung tissues.The results showed that compared with the control group,septic mice showed thickened septa in the lung tissue accompanied by massive inflammatory cell infiltration.Compared with WT septic mice,there was insignificant thickening of the lung tissue septa and less inflammatory cell infiltration in CTRP9-KO septic mice.We used ELISA Kit to detect the content of IL-1β in the serum of the four groups.The results showed that the content of IL-1β in the serum of the septic mice significantly increased,and CTRP9 knockout could reduce the content of IL-1β in the serum of septic mice.In addition,we detected the expression of IL-1β in the lung tissue of the four groups of mice by immunohistochemical and immunofluorescence staining,and the results confirmed that CTRP9 knockout could significantly reduce the level of IL-1β in the lung tissue of septic mice.4.CTRP9 knockout inhibits NLRP3 inflammasome activation in lung tissues of septic miceNext,we further detected the expression of NLRP3 inflammasome-related proteins NLRP3、Caspase-1 p10、IL-1β p17 and pyroptosis-related protein GSDMD-NT in lung tissues of four groups.Western blotting results showed that the expression of NLRP3、Caspase-1 p10、IL-1β p17 and GSDMD-NT in lung tissues of septic mice was significantly increased,and CTRP9 knockout could reduce the expression of NLRP3、Caspase-1 p10、IL-1β p17 and GSDMD-NT in lung tissues of septic mice.5.CTRP9 knockout inhibits the activation of NLRP3 inflammasome in macrophagesThe peritoneal macrophages of WT mice and CTRP9-KO mice were extracted.First,we detected the expression of CTRP9 protein in macrophages by Western blotting,and found that the expression of CTRP9 in peritoneal macrophages of CTRP9-KO mice was extremely low.Then,the macrophages were stimulated with NLRP3 inflammasome inducers LPS and ATP.Western blotting results showed that once CTRP9 knockout,the amount of Caspase-1 p10 and IL-1β p17 released into the cell supernatant decreased significantly.The expression of NLRP3 and Pro-IL-1β was also significantly decreased,but the expression of Pro-Caspase-1 and ASC did not change significantly.6.CTRP9 knockout inhibits macrophage pyroptosisPI staining was performed on macrophages stimulated by LPS and ATP,and the results showed that the the pyroptosis ratio of peritoneal macrophages in CTRP9-KO mice was significantly reduced.We further detected the cleavage of the pyroptosis-related protein GSDMD by Western blotting,and found that the expression of the active GSDMD protein nitrogen terminus(GSDMD-NT)in the peritoneal macrophages of CTRP9-KO mice was significantly decreased,suggesting that CTRP9 knockout inhibited the cleavage of GSDMD protein.7.Exogenous CTRP9 promotes the activation of NLRP3 inflammasome in macrophagesIn order to further clarify the role of CTRP9 on the activation of NLRP3 inflammasome in macrophages,we pretreated macrophages with exogenous CTRP9 recombinant protein,and then administered macrophages with LPS and ATP.PCR results showed that the mRNA levels of NLRP3 and Pro-IL-1β in CTRP9+LPS+ATP group were significantly higher than those in LPS+ATP group.Western blotting showed that the release of bioactive Caspase-1 P10 and IL-1β P17 in the supernatant of CTRP9+LPS+ATP group was also increased significantly.We also found that after pretreatment with CTRP9,the expression of NLRP3 in macrophages increased significantly,but the expression of ASC and Pro-Caspase-1 did not change significantly.Furthermore,the formation of ASC speck is thought to be another hallmark of NLRP3 inflammasome activation.ASC specks were labeled by immunofluorescence staining.The results showed that ASC was evenly distributed in macrophage stimulated by LPS alone,and ATP stimulation promoted the formation of ASC specks.In addition,we found that the formation of ASC specks was significantly increased in macrophages in the CTRP9+LPS+ATP group.Taken together,the above results show that CTRP9 promotes the activation of the NLRP3 inflammasome.8.Exogenous CTRP9 promotes macrophage pyroptosisPI staining was used to label pyroptotic macrophages.We found that the number of PI-positive macrophages significantly increased in the LPS and ATP-stimulated group compared with the LPS-stimulated group,suggesting that LPS and ATP co-stimulation could successfully induce macrophage pyroptosis model.The proportion of PI-positive macrophages in the CTRP9+LPS+ATP group significantly increased.At the same time,Western blotting results showed that CTRP9 increased the cleavage of the pyroptosis-related protein GSDMD.The above results suggest that CTRP9 could promote macrophage pyroptosis.9.CTRP9 promotes the activation of NLRP3 inflammasome and macrophages pyroptosis through NOX2/ROS signaling pathwayThe content of ROS in macrophages was detected by flow cytometry and immunofluorescence staining.All results confirmed that exogenous CTRP9 could promote ROS generation in macrophages.After adding ROS inhibitor N-acetyl-L-Cysteine,flow cytometry showed that ROS generation was reduced.Western blotting showed that the expression of NLRP3 inflammasome and pyroptosis-related proteins was significantly inhibited.It has been reported that NOX2 is one of important sources of intracellular ROS.Western blotting showed that CTRP9 significantly increased the expression of NOX2.Next,we transfected macrophages with small interference of NOX2,and found that after NOX2 was silenced,the generation of ROS was reduced,and the promotion effect of CTRP9 on NLRP3 inflammasome activation and macrophages pyroptosis was also significantly inhibited,indicating that NOX2/ROS signaling pathway is involved in the process of CTRP9 on NLRP3 inflammasome activation.Conclusions1.CTRP9 knockout attenuated inflammatory response in septic lung injury.2.CTRP9 knockout inhibited the activation of NLRP3 inflammasome.3.CTRP9 promotes the activation of NLRP3 inflammasome through NOX2/ROS signaling pathway and aggravates the inflammatory response.Part Ⅱ The role and mechanisms of CTRP9 in sepsis-mediate acute myocardial injuryBackgroundSepsis is a systemic inflammatory syndrome triggered by infection which is usually accompanied by multiple organs dysfunction.Myocardial injury is a common complication of sepsis,with the incidence of as high as 40%.Studies have found that the pathogenesis of septic cardiomyopathy can be related to mitochondrial dysfunction,metabolic abnormalities,oxidative stress and autonomic nervous dysfunction.Among them,excessive oxidative stress has always been regarded as the key pathological process of septic cardiomyopathy.Redox imbalance is mainly reflected in increased oxygen free radical generation and decreased antioxidant capacity.When sepsis occurs,macrophages and neutrophils recruited into the myocardial tissue can promote the production of reactive oxygen species(ROS)through NADPH oxidase,and excessive ROS can damage the structure of biological membrane,especially mitochondrial and lysosomes,resulting in myocardial dysfunction;the decreased antioxidant capacity is reflected in the decrease of antioxidant enzymes,like superoxide dismutase(SOD),Glutathione peroxidase(GSH-PX),catalase(CAT)and low molecular weight antioxidants like vitamin E、vitamin A、vitamin C and coenzyme Q10,which results in the disability of clearance of oxygen free and myocardial systolic dysfunction.Ferroptosis is a novel cell death mode caused by imbalance of oxidative stress,which is manifested by the decreased expression of glutathione and glutathione peroxidase 4(GPX4)in the antioxidant system and increased iron-dependent lipid peroxidation.However,the mechanism of ferroptosis has not fully understood,and previous studies have shown that there are lots of mechanisms involved in ferroptosis,including transport of iron ions,depletion of L-Glutathione(GSH),inactivation of GPX4 and excessive accumulation of ROS.Studies have shown that ferroptosis is associated with Parkinson’s disease,cancer,diabetes and cerebrovascular disease.What’s more,ferroptosis is also involved in myocardial hypertrophy,diabetic cardiomyopathy and doxorubicin-induced cardiotoxicity.Ning Li’s team found that ferroptosis plays an important role in sepsis induced myocardical injury.Nuclear factor-E2-related factor 2(nuclear factor erythroid 2-related factor 2,Nrf2),as an important transcription factor in anti-oxidative stress,can promote the expression of downstream antioxidant enzymes(such as GPX4 and HO-1,etc.).In addition,studies have confirmed that almost genes related to ferroptosis are transcriptionally regulated by Nrf2.Therefore,Nrf2 is considered to be a key regulator of ferroptosis.Clq/tumor necrosis factor-related protein 9(Clq/TNF-relative protein9)is a novel adipocytokine concerned by more and more people rencently.The expression level of CTRP9 in cardiac tissue is highest.CTRP9 is cleaved into active globular domain form(gCTRP9)and secreted into local heart tissue and circulatory system and plays an important role in biological function.Studies have shown that CTRP9 has protect fuction for cardiaovescular disease,however,some studies shown CTRP9 also has negative effect.On the one hand,it has been reported that CTRP9 can alleviate myocardial injury induced by ischemia-reperfusion.CTRP9 can improve atherosclerosis by inhibiting inflammation,regulating endothelial function,and inhibiting the transformation of VSMCs into macrophage-like cells.On the other hand,it has been reported that compared with normal people,the expression of CTRP9 in hypertension patients is significantly increased,and is related to the develop of hypertension with atherosclerosis.Upregulation of CTRP9 in hypertrophic cardiomyopathy can promote myocardial remodeling and even lead to left heart failure.In summary,the different effects of CTRP9 may be related to different disease models.it may be because of binding with different receptors to produce different effects.Therefore,the specific roles and mechanisms of CTRP9 in cardiovascular diseases deserve further study.Our previous studies show that CTRP9 is upregulated in myocardium of sepsis mice.It indicated that CTRP9 may have relationship with myocardial injury.However,the effect of CTRP9 on ferroptosis has not been reported,and this study aims to investigate whether CTRP9 is involved in sepsis-induced myocardial injury through inducing ferroptosis.Objective1.To clarify the role of CTRP9 in sepsis-induced myocardial injury.2.To explore whether CTRP9 aggravates sepsis-induced myocardial injury by inducing cardiomyocyte ferroptosis.3.To explore the molecular mechanism how CTRP9 mediates cardiomyocyte ferroptosis.Method1.Establishment of animal modelsFirstly,CTRP9 gene knockout mice were constructed by Shanghai Southern Model Biotechnology Co.,Ltd..12-week-old male C57BL/6J mice(WT mice)and CTRP9 knockout mice(CTRP9-KO mice)were enrolled to construct sepsis mouse model by intraperitoneal injection of LPS(10 mg/kg)for 12 hours.Then the mice were euthanized and tissue samples such as serum,lung,heart,liver and kidney were collected.2.Mouse genotype identificationThe mouse tail was clipped and DNA in mouse tail was extracted using a tissue DNA extraction kit.After RCR amplification,agarose gel electrophoresis was performed to identify the DNA.At the same time,the protein of mouse heart tissue was extracted.Then the expression of CTRP9 was detected by western blotting to evaluate the gene knockout efficiency of CTRP9.3.Cardiac function assessmentMice were anesthetized by inhalation of isoflurane.Depilatory cream was used to remove the hair of chest.Then mice were fixed on the platform in a supine position,electrode pads were attached to the limbs,and coupling agent was applied to the chest.Parasternal M-mode echocardiography was used.After analyzing the acquired data,we could get left ventricular ejection fraction(LVEF)and fractional shortening(LVFS).4.H&E staining of myocardial tissueThe general morphology of myocardial tissue was observed.5.Western blottingThe expression of Nrf2,HO-1,GPX4,cystine/glutamate antiporter xCT,transferrin receptor(TFR)and transferrin(TRF)in primary rat cardiomyocytes(NRCMs)was detected.6.Statistical analysisQuantitative data are presented as mean±standard deviation(mean±SD).For all normally distributed data,we used independent samples t-test to compare the data between two groups and we used one-way ANOVA to compare the data of more than three groups.p<0.05 was considered statistically significant.Results1.CTRP9 knockout improves cardiac systolic function in septic miceWe analyzed the results of cardiac ultrasound examination of four groups of mice.The data showed that the LVEF and LVFS of the septic mice group were significantly reduced compared with the control group.Compared with the WT septic mice,the LVEF and LVFS of CTRP9-KO septic mice were significantly increased.These data suggests that knockouting the CTRP9 gene could improve the cardiac systolic function of the septic mice.2.CTRP9 knockout attenuates sepsis-induced acute myocardial injuryH&E staining showed that the myocardium was neatly arranged in the normal control group,while the myocardium was significantly swollen and disordered,and the infiltration of inflammatory cells increased in septic mice.In addition,compared with WT septic mice,the myocardial tissue of CTRP9-KO septic mice was slightly swollen,and the number of inflammatory cell infiltration was significantly reduced.In addition,cardiac troponin I(cTnI)and creatine kinase isoenzyme(CK-MB)were significantly elevated in serum of septic mice.Compared with WT septic mice,cTnI and CK-MB in serum of CTRP9 knockout septic mice were significantly decreased.3.CTRP9 knockout inhibits ferroptosis in myocardial tissue of septic miceWe detected the expression of ferroptosis-related proteins in myocardial tissue of four groups of mice by Western blotting.The results showed that the expression of iron metabolism-related protein transferrin receptor,transferrin in myocardial tissue of LPS-induced septic mice significantly increased,but knockout of CTRP9 inhibited the expression of transferrin receptor and transferrin.At the same time,the expression of GPX4 and xCT decreased in the myocardial tissue of LPS-induced septic mice,while knockout of CTRP9 reversed this phenomenon.4.Exogenous CTRP9 promotes LPS-induced ferroptosis in NRCMsTo further verify the effect of CTRP9 on cardiomyocyte ferroptosis in vitro,we stimulated NRCMs with LPS to simulate a septic cardiomyocyte injury mode.The expression of ferroptosis-related proteins transferrin receptor,transferrin,GPX4 and xCT in NRCMs was detected by Western blotting.The results showed that compared with LPS stimulation group,the expression of transferrin receptor and transferrin in the LPS+CTRP9 group significantly increased and the expression of GPX4 and xCT decreased,suggesting that CTRP9 promotes LPS-induced ferroptosis in NRCMs.5.CTRP9 knockout attenuates oxidative stress in myocardial tissue of septic miceTo further explore the mechanism of CTRP9-mediated myocardial injury in sepsis,we next detected the levels of oxidative stress in the myocardial tissue of four groups of mice.Immunofluorescence staining found that the generation of ROS in the myocardial tissue of the septic mice significantly increased compared with the control group.Compared with WT septic mice,the level of ROS in the myocardial tissue of CTRP9-KO septic mice significantly reduced.Meanwhile,knockdown of CTRP9 increased the production of antioxidant superoxide dismutase(SOD)and inhibited the generation of the malondialdehyde(MDA)in sepsis-induced myocardial tissue.6.Exogenous CTRP9 promotes LPS-induced ROS generation in NRCMsIn order to verify the effect of CTRP9 on ROS generation in cardiomyocyte,we used immunofluorescence staining to observe the level of ROS in NRCMs.The results showed that compared with the LPS group,the generation of ROS in the LPS+CTRP9 group significantly increased.7.CTRP9 knockout promotes the expression of Nrf2 and HO-1 in myocardial tissue of septic miceTo further explore whether CTRP9 regulates septic myocardial injury through the Nrf2/HO-1 signaling pathway,we performed immunohistochemical staining and found that the expression of the antioxidant protein Nrf2 was significantly decreased in sepsis-induced myocardial tissue,and CTRP9 knockout could reverse this phenomenon.Western blotting results showed that CTRP9 knockout increased the expression of Nrf2 and HO-1 in sepsis-induced myocardial tissue.8.Exogenous CTRP9 inhibits the expression of Nrf2 and HO-1 in LPS-induced NRCMsIn order to further clarify the role of CTRP9 on Nrf2/HO-1 signaling pathway,we detected the expression of Nrf2 and HO-1 proteins in LPS-induced NRCMs by Western blotting.The results showed that the expression of Nrf2 and HO-1 proteins in LPS group was inhibited compared with the control group.Meanwhile,compared with the LPS group,the expression of Nrf2 and HO-1 in CTRP9+LPS group was significantly decreased.9.SFN reverses the upregulation of CTRP9 on LPS-induced ferroptosis in NRCMsTo further verify that CTRP9 promotes LPS-induced ferroptosis in NRCMs through the Nrf2/HO-1 signaling pathway,we pretreated NRCMs with the Nrf2 agonist SFN.Western blotting results showed that compared with the LPS+CTRP9 group,the expression of transferrin receptor in the LPS+CTRP9+SFN group was significantly decreased,and the expression of xCT and GPX4 proteins was significantly increased.Conclusions1.CTRP9 knockout alleviates sepsis-induced acute myocardial injury.2.CTRP9 knockout inhibits sepsis-induced myocardial ferroptosis.3.CTRP9 promotes LPS-induced ferroptosis in NRCMs through the Nrf2/HO-1 signaling pathway.