Vitamin C Alleviates LPS-induced Myocardial Injury By Inhibiting Pyroptosis Via The ROS-Akt/mTOR Signalling Pathway

Sepsis is a clinical syndrome associated with fatal organ dysfunction caused by a systemic inflammatory response to infection and is an important cause of death in severe clinical patients.The heart,which is a vital organ in humans,is easily and rapidly affected during the development of sepsis.Overall,myocardial injury occurs in more than 50% of cases of sepsis,and the mortality can exceed 50%.Thus,a better understanding of sepsis-induced myocardial injury is urgently needed.However,the pathophysiologic understanding of sepsis-induced myocardial injury is not fully exposed.Multiple mechanisms have been proposed to be involved in the pathophysiologic processes of sepsis-induced myocardial injury,including persistent inflammatory responses,energetic starvation,mitochondrial dysfunction and oxidative stress.However,recent evidence indicates that pyroptosis may be a main cause of heart failure and death.Pyroptosis is a newly discovered mode of cell death that is different from necrosis and apoptosis.It is a suicidal behavior after cells are seriously injured,a pathological suicide,and release of cell contents and inflammatory factors,including IL-1 β and IL-18 to induce inflammatory reaction.Lipopolysaccharide(LPS),can induce ROS production and pyroptosis.However,to the best of our knowledge,no studies have explored which signalling pathway participates in the process that ROS initiates pyroptosis.The Akt/ m TOR pathway is a critical cellular cascade in the cellular response to extracellular stimuli.It has been demonstrated that the Akt/m TOR signalling pathway is driven by ROS in many diseases.However,whether the Akt/m TOR pathway participates in pyroptosis is unclear.Vitamin C,is a common antioxidant that can reduce the inflammatory response and damage of cell and tissue.The level of vitamin C in patients with sepsis is significantly decreased,and most patients exhibit vitamin C deficiency.It is also found that the intravenous administration of vitamin C at the early stage of sepsis could reduce the mortality of septic patients.However,whether vitamin C can alleviate LPS-induced myocardial injury by inhibiting pyroptosis has not been studied.We hypothesize that vitamin C attenuates LPS-induced myocardial injury by inhibiting ROS-dependent pyroptosis.We also explored whether the Akt/m TOR signalling pathway is involved in the process of ROS initiating pyroptosis.Our study aimed to obtain a new perspective on the pathophysiological process of sepsis-induced myocardial injury and to provide a novel prevention strategy.This study is divided into four parts.Part One LPS induces ROS production and pyroptosis in H9C2 cells in vitroObjective: To explore the level of oxidative stress and pyroptosis caused by LPS-induced myocardial injury.Methods:1.The rat cardiomyocyte cell line H9C2 was culture in a suitable environment,H9C2 cells were divided into a control group and LPS treatment groups,which were exposed to different final LPS concentrations(0.1,0.5 or1.0 μg/m L).2.Cell viability was assessed by a CCK-8 assay.3.The concentrations of LDH,CK-MB,IL-1β and IL-18 were measured using ELISA kits according to the manufacturer’s instructions.4.The levels of intracellular ROS were measured with the fluorescent probe dichlorodihydrofluorescein diacetate according to the manufacturer’s instructions.5.Western blotting was performed to detect the expression of NLRP3,Caspase-1 and GSDMD-N to study the level of pyroptosis in H9C2 cells,and the expression of NOX4 was detected to study the level of intracellular oxidative stress.Results:1.A significant dose-dependent decrease in cell viability was observed in H9C2 cells.LDH and CK-MB release increased in a dose-dependent manner.2.The intracellular ROS level increased in a dose-dependent manner,similar to the expression of NOX4.3.The analysis of pyroptosis revealed that the expression of associated proteins(NLRP3,Caspase-1 and GSDMD-N)was upregulated in H9C2 cells by LPS,and the levels of the pyroptosis-associated inflammatory cytokines IL-1β and IL-18 were also positively correlated with the LPS concentration.Conclusions:1.LPS induces death and ROS production in H9C2 cells.2.LPS induces pyroptosis in H9C2 cells.Part two LPS induces pyroptosis through the Akt/m TOR pathway in H9C2 cellsObjective: To explore whether the Akt/m TOR pathway involved in the process of H9C2 pyroptosis in LPS.Methods:1.The rat cardiomyocyte cell line H9C2 was culture in a suitable environment,H9C2 cells were divided into a control group and LPS treatment groups,which were exposed to different final LPS concentrations(0.1,0.5 or 1.0 μg/m L).2.Western blotting was performed to detect the expression of p-Akt and p-m TOR.3.H9C2 cells were then divided into a control group,LPS group and MK-2206 + LPS group.The H9C2 cells in the MK-2206 + LPS group were pretreated with 0.5 μM MK-2206 for 1 h,whereas those in the control and LPS groups were pretreated with equal volumes of DMSO for 1 h.The H9C2 cells in the LPS and MK-2206 + LPS groups were then treated with 0.5 μg/ml LPS for 3 h.4.Cell viability was assessed by a CCK-8 assay in the three groups.5.The concentrations of LDH,CK-MB,IL-1β and IL-18 were measured using ELISA kits according to the manufacturer’s instructions.6.The levels of intracellular ROS were measured with the fluorescent probe dichlorodihydrofluorescein diacetate according to the manufacturer’s instructions.7.Western blotting was performed to detect the expression of NLRP3,Caspase-1,GSDMD-N,p-Akt and p-m TOR to study the level of pyroptosis and Akt/m TOR pathway in H9C2 cells,and the expression of NOX4 was detected to study the level of intracellular oxidative stress.Results:1.The expression of p-Akt and p-m TOR was upregulated in H9C2 cells by LPS.2.Similar to p-Akt and p-m TOR,decreased expression of the pyroptosis-associated proteins NLRP3,caspase-1 and GSDMD was detected in the MK-2206 + LPS group.Furthermore,the release of IL-1β and IL-18 was also inhibited compared with the LPS group.3.The release of LDH and CK-MB found for the LPS group was higher than that obtained after MK-2206 pretreatment,and increase in cell viability in MK-2206 + LPS group compared with the LPS group.4.There was no significant difference between the two groups in the expression of ROS and NOX4.Conclusions:1.LPS induces pyroptosis through the AKT/m TOR pathway in H9C2 cells.2.ROS is located at the upstream of Akt-m TOR signal pathway,which can activate Akt-m TOR signal pathway to up-regulate its expression.Part Three Vitamin C protects H9C2 cells against LPS-induced cell death through the ROS-Akt/m TOR-pyroptosis pathwayObjective: To explore the protective effect of vitamin C on LPS-induced injury of H9C2 myocardial cells and its mechanism.Methods:1.H9C2 cells were divided into a control group,LPS group,LPS + NAC group and LPS + vitamin C(LPS + Vc)group.The H9C2 cells in the LPS+NAC and LPS+Vc groups were pretreated with 2 μM NAC or 2 μM vitamin C for 1 h.All the groups except for the control group,cells were then treated with 0.5 μg/ml LPS for 3 h.2.Cell viability was assessed by a CCK-8 assay.3.The concentrations of LDH,CK-MB,IL-1β and IL-18 were measured using ELISA kits according to the manufacturer’s instructions.4.The levels of intracellular ROS were measured with the fluorescent probe dichlorodihydrofluorescein diacetate according to the manufacturer’s instructions.5.Western blotting was performed to detect the expression of NLRP3,Caspase-1,GSDMD-N,p-Akt and p-m TOR to study the level of pyroptosis and Akt/m TOR pathway in H9C2 cells,and the expression of NOX4 was detected to study the level of intracellular oxidative stress.Results:1.The LPS-induced ROS levels and NOX4 expression were decreased after pretreatment with NAC or vitamin C.2.The results show a decrease in pyroptosis-associated proteins and reductions in IL-1β and IL-18.3.The expression of p-Akt and p-m TOR was decreased in LPS+ Vc group and LPS+NAC group.4.Compared with LPS group,the cell viability of LPS + NAC and LPS+Vc groups were higher.5.Compared with LPS group,the release of LDH and CK-MB decreased in both LPS + NAC and LPS+Vc groups.Conclusions:1.Vitamin C can protect myocardial cell injury caused in sepsis by inhibiting pyroptosis.2.Vitamin C protects H9C2 cells against LPS-induced cell death through the ROS-Akt/m TOR-pyroptosis pathway.Part Four Cardiac function and plasma inflammatory factor in sepsis patientsObjective: To explore the cardiac function and plasma inflammatory factor in sepsis patients.Methods:1.21 patients with sepsis admitted in EICU of the Third Hospital of Hebei Medical University from April 2022 to July 2022 were selected as the study subjects,and 12 trauma patients without sepsis admitted in the same period were selected as the control group.21 trauma patients with sepsis were divided into sepsis cardiac dysfunction group(9 cases)and sepsis non-cardiac dysfunction group(12 cases)according to LVEF and FS values detected by bedside cardiac color Doppler ultrasound.2.The data of patients selected for the study were analyzed retrospectively,including basic information,infectivity index,biochemical index,myocardial enzymology index,cardiac function index and serum cytokines.3.Statistically analyze whether there are differences in various indicators in different groups.Results:1.Among 21 patients with sepsis,9 patients had cardiac dysfunction,and the incidence rate of cardiac dysfunction was 42.86%.2.Compared with the control group,the expression of PCT,CK-MB,c Tn I,IL-6 and IL-10 in patients with sepsis were significantly higher.3.Compared with sepsis without cardiac dysfunction group,the expression level of IL-1β and IL-6 in sepsis patients with cardiac dysfunction group was significantly higher,and the expression level of IL-10 was significantly lower.Conclusions:1.There is a difference in expression of IL-1β between the control group and the sepsis group.2.The expression level of IL-1β in sepsis cardiac dysfunction group was significantly higher than in the sepsis non-cardiac dysfunction group,and IL-1β may be a predictor of myocardial injury in sepsis.