| Background: Sepsis is a systemic inflammatory response syndrome caused by a pathogen invading human blood.Severe trauma,infection,acute pancreatitis,and severe pneumonia are common predisposing factors.Patients with severe disease will develop into septic shock or multiple organ dysfunction syndrome.It has high mortality and already has become the third leading cause of death in patients.Severe sepsis and septic shock can cause myocardial damage and ultimately lead to cardiac insufficiency,mainly due to heart enlargement,myocardial contractile dysfunction,and decreased left ventricular ejection fraction.Clinical studies have found that endotoxin and inflammatory factors play an important role in the pathogenesis of cardiovascular damage caused by sepsis.In addition,hypoxia,acidosis,hypotension and hypovolemia,metabolic disorders,abnormal coagulation,and increased peroxide production after oxidative stress injury are thought to be involved in heart and macrovascular damage during sepsis,but its exact pathophysiological mechanism is still unclear.The statin is a lipid-lowering drug that primarily inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase,resulting in the reduction of cholesterol synthesis.Recent studies have shown that statins not only have lipid-lowering effects,but also have a wide range of anti-inflammatory and cellular immune functions.Given that sepsis is an inflammatory disease and statins have a broad anti-inflammatory effect,we speculate that statins may have protective effects against cardiovascular damage caused by sepsis.Multiple clinical studies have confirmed that phosphoinositide-3 kinase(PI-3K)/serine/threonine kinase(Akt)-endothelial nitric oxide synthase(eNOS)signaling pathway is one of the important signaling pathways for protecting myocardium.After activation of PI3K-Akt pathway,activated Akt promotes the production of endogenous nitric oxide(NO)through the phosphorylation of eNOS,and then NO is generated as a signal.Therefore,NO plays an important role in protecting the heart muscle and blood vessels.Therefore,whether statins can promote NO production,reduce inflammation and myocardial damage by regulating the expression of PI3K-Akt-eNOS signaling pathway,and thus may have a good therapeutic effect on patients with sepsis---there is no clinical and basic research in this area currently.Aim: This study was to evaluate the effect of rosuvastatin on cardiovascular damage in rats with sepsis induced by lipopolysaccharide(LPS),and meanwhile to evaluate the effect of oxidative stress of rosuvastatin on LPS-induced rat aortic endothelial cells,andto explore the mechanism of cardiovascular protection,hence we can provide new ideas and strategies for the treatment of clinical sepsis.Methods: The first part evaluates the protective effect of rosuvastatin on cardiovascular damage induced by lipopolysaccharide in septic rats.SD rats were randomly divided into control group,LPS sepsis group,LPS sepsis + rosuvastatin low dose group(10mg/kg/d)and LPS sepsis + rosuvastatin high dose group(20mg/kg/d).SD rats in sepsis group were injected intraperitoneally with LPS 20 mg/kg to establish sepsis model.In the control group,SD rats were given a single intraperitoneal injection of the same volume of normal saline,and immediately given the same volume of autoclaved double distilled water.Rosuvastatin treatment group: rosuvastatin(10 mg/kg or 20 mg/kg)was orally administered once immediately after intraperitoneal injection of LPS.(1)The growth status and survival rate of sepsis rats were observed from 6 hours to 12 hours after modeling.(2)SD rats were injected 3 hours,6 hours and 9 hours after intraperitoneal injection of LPS,the rat heart was isolated and the blood of the apex was extracted to detect the levels of plasma tumor necrosis factor-?(TNF-?),interleukin-6(IL-6),and troponin I(cTNI),N-terminal pronatriuretic peptide(NT-proBNP)and high-mobility group box 1 protein(HMGB1).(3)The RNA and protein were extracted from myocardial tissue of four groups of rats,and the expression levels of ANP,BNP and inflammatory factors TNF-?,IL-6 and IL-2 were measured.(4)Echocardiographic evaluated the cardiac structure and function changes in the four groups of rats.(5)Masson staining was used to observe the morphological effects of rosuvastatin on myocardial tissue.(6)The rat thoracic aorta was isolated and subjected to aortic ring-induced vascular endothelial cell sprouting experiments to evaluate the level of angiogenesis.The second part evaluates the protective effect of rosuvastatin on oxidative stress injury induced by lipopolysaccharide in rat aortic endothelial cells and explores its mechanism.(1)Effect of LPS on the activity of Rat Aortic Endothelial Cells(RAOEC): Different concentrations(10,100,500,1000,10000 ng/ml)of LPS were administered to rat aortic endothelial cells for 24 hours.Cell viability and inhibition rate were determined by MTT colorimetric assay.The damage of LPS on RAOEC was studied,and then selected the appropriate concentration of LPS in the next experiment.(2)Effect of rosuvastatin(RSV)on LPS-induced RAOEC activity:Rat aortic endothelial cells were pretreated with different concentrations of rosuvastatin for 2 hours,and then treated with 1000 ng/ml(1 ?g/ml)of LPS for 24 hours,divided into5 groups: negative control group,LPS group,LPS+2?M RSV group,LPS+4?M RSV group and LPS + 8?M RSV group.Cell viability and inhibition rate were determined by MTT colorimetric assay.The protective effect of RSV on LPS-induced cell injury was evaluated,and then selected the appropriate concentration of RSV in the next experiment.(3)Effects of RSV on LPS-induced reactive oxygen species(ROS)and Malonicdialdehyde(MDA)in rat aortic endothelial cells: They were divided into 4 groups,negative control group,LPS(1?g/ml)group,RSV(4 ?M)group,and LPS(1?g/ml)+RSV(4?M)group.LPS+RSV group: 4?M rosuvastatin was used to pretreat rat aortic endothelial cells for 2 hours,and/or(1?g/ml)LPS was administered for 24 hours;The intracellular ROS production was measured by H2DCHF-DA probe,and the lipid oxidation level MDA content was measured by thiobarbituric acid method.(4)Effects of LPS,RSV and Perifosine(Akt inhibitor)on NO production in rat aortic endothelial cells.Grouping and processing:1)negative control group: only an equal volume of medium was given;2)LPS intervention group: 1?g/ml LPS treatment for 24 hours;3)LPS+RSV intervention group: 4?M RSV treatment for 2 hours,and then treated with 1?g/ml LPS for24 hours;4)LPS+RSV+Perifosine intervention group: The cells were pretreated with 5?M Perifosine(Akt inhibitor)for 1 hour,then treated with 4?M RSV for 2 hours and treated with 1 ?g/ml LPS for 24 hours.(5)Research on the mechanism of protection:Grouping and processing are the same as group(4)NO generation.The effect of rosuvastatin on LPS-induced phosphorylation of PI3K-Akt-eNOS signaling pathway in RAOEC was studied by immunoblotting.Results: The first part is about the protective effect of rosuvastatin on cardiovascular damage induced by lipopolysaccharide in rats with sepsis.1.SD rats which were injected LPS intraperitoneally began to die after 6 hours.After 12 hours of continuous observation,LPS-induced sepsis SD rats were given low-dose and high-dose rosuvastatin,which could significantly reduce the mortality rate of sepsis rats(12-hour survival rate: 20% in LPS group,50% in LPS + low-dose rosuvastatin group,70% in LPS + high-dose rosuvastatin group,P=0.0165 in Mantel-Cox test).2.The levels of cTNI and NT-proBNP in plasma were measured at 3 hours,6 hours and 9 hours after intraperitoneal injection of LPS in SD rats.It was found that the levels of plasma cTNI and NT-proBNP gradually increased after LPS induction,and peaked at 6 hours(P< 0.05).3.The levels of plasma cTNI and NT-proBNP of LPS-induced septic rats after 6 hours were compared between the three groups.Both low-dose and high-dose rosuvastatin significantly reduced cTNI and NT-proBNP levels(P<0.05).4.The levels of TNF-?,IL-6 and HMGB1 in plasma of septic rats after 6 hours of LPS induction were compared.Both low-dose and high-dose rosuvastatin significantly reduced the levels of plasma TNF-?,IL-6,and HMGB1(P<0.05).5.In sepsis-induced myocardial tissues,we found that low-dose and high-dose rosuvastatin could significantly reduce the expression of ANP and BNP,and the levels of TNF-a,IL-6 and IL-2 were also significantly lower than those in LPS group(P<0.05).6.Masson staining of rat myocardial specimens showed that myocardial fibrosis was significantly aggravated,myocardial hypertrophy,gap relaxation and vacuolization in myocardial tissue after LPS induction,while after giving low-dose and high-dose rosuvastatin,myocardial fibers chemotherapy,cardiac hypertrophy and vacuolizationwere significantly alleviated,and it showed that rosuvastatin was effective in reducing myocardial pathological remodeling caused by sepsis caused by cardiomyopathy.7.By echocardiography,we found that LPS-induced sepsis cardiomyopathy significant increased the left ventricular end systolic dimension(LVEDs)and left ventricular end diastolic dimension(LVEDd)compared with the control group(P<0.05),but the left ventricular ejection fraction(LVEF)decreased significantly(P<0.05).Compared with the LPS group,low-dose and high-dose rosuvastatin significantly increased the LVEDs caused by sepsis,and decreased the LVEF(P <0.05).8.After LPS intervention,we found that the number of neovascularization in aortic rings of SD rats was significantly reduced compared with the control group,while low-dose and high-dose rosuvastatin reversed the decrease of LPS-induced neovascularization(18.82± 3.64 in the control group,8.64±1.88 in the LPS group,12.52±3.76 in the LPS+low-dose rosuvastatin group,and 14.93±3.21 in the LPS+high-dose rosuvastatin group,P<0.05).The second part is to find the protective effect of rosuvastatin on oxidative stress injury induced by lipopolysaccharide in rat aortic endothelial cells and explored its mechanism.1.Compared with the control group,LPS could inhibit cell viability in a dose-dependent way,and the difference was statistically significant(P<0.05).When LPS was at 1?g/ml,the activity of RAOEC was significantly decreased,and the degree of cell inhibition was moderate(P<0.01),which may cause moderate cell damage,and it is suitable for the experimental concentration in the next study.2.Compared with the LPS-treated group,RSV attenuated the injury of LPS-induced RAOEC in a dose-dependent way,and RAOEC activity was significantly increased,the difference was statistically significant(P<0.05).The concentration of rosuvastatin(4?M)had the least effect on RAOEC activity and the least inhibition on cells,so the subsequent experiment of rosuvastatin selected 4?M concentration.3.Compared with the control group,ROS(4?M)production in aortic endothelial cells of rosuvastatin alone was not significantly different(P>0.05).1?g/ml LPS significantly increased the ROS production of RAOEC-induced(P<0.01);Compared with the LPS-treated group,4?M RSV significantly inhibited the ROS production of LPS-induced(P<0.01).4.Compared with the control group,there was no significant change in the MDA content of aortic endothelial cells in the rosuvastatin(4 ?M)treated group,it was not significantly different(P>0.05),but 1?g/ml LPS significantly increased the MDA content of rat aortic endothelial cells(P<0.01);Compared with the LPS-treated group,4?M rosuvastatin significantly decreased the MDA content of LPS-induced in rat aortic endothelial cells(P<0.01).5.Compared with the control group,1?g/ml LPS significantly inhibited NO production in rat aortic endothelial cells(P<0.01).Compared with the LPS-treated group,4?M rosuvastatin significantly increased the NO production of LPS-induced in rat aortic endothelial cells(P<0.01).Compared with the LPS+RSV group,the NO production of the Akt inhibitor Perifosine treatment group was significantly lower(P<0.01).6.In terms of molecular mechanism,compared with the control group,1?g/ml LPS treatment on rat aortic endothelial cells had no significant effect on the total protein levels of Akt and eNOS,and there was no statistical significance(P>0.05).However,the phosphorylation levels of Akt and eNOS were significantly decreased(P<0.05);Compared with the LPS-treated group,4?M rosuvastatin significantly increased the phosphorylation levels of Akt and eNOS inhibited by LPS(P<0.05).Phosphorylation levels of Akt and eNOS were significantly lower in the Akt inhibitor Perifosine treated group compared with the LPS+RSV group(P<0.05).Conclusion: 1.Rosuvastatin can inhibit myocardial injury and cardiac insufficiency induced by LPS in rats with sepsis,reduce inflammation,promote aortic ring angiogenesis in rats,improve myocardial remodeling and vascular remodeling,and reduce sepsis rat mortality.2.Rosuvastatin has an obvious protective effect on LPS-induced injury of rat aortic endothelial cells,which can significantly inhibit LPS-induced oxidative stress,reduce the production of ROS and MDA in endothelial cells,and promote the production of NO.Its possible mechanism is to play a role in protecting endothelial cells by promoting the production of NO by activating the PI3K-Akt-eNOS signaling pathway. |
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