Analytical Investigation Of Selenium In Improving Cardiomyocyte Injury Induced By Lipopolysaccharide

Sepsis is a disease associated with multiple organ dysfunction.The systemic inflammatory response syndrome is usually caused by the imbalance between the immune response and the inflammatory response when the host is infected with an external pathogen.Heart disease caused by sepsis inflammation is one of the main reasons for the danger to human health,and it also brings a heavy economic burden.Septic cardiomyopathy is accompanied by an increase in reactive oxygen species,inflammatory factors,and apoptosis in cardiomyocyte,which further aggravates myocardial remodeling.Selenium is an essential trace element.Many studies have found that it has the functions of anti-oxidative stress,anti-inflammatory,and anti-apoptosis.However,whether selenium can improve lipopolysaccharide(LPS)-induced cardiomyocyte damage is not yet known.Therefore,In this study,lipopolysaccharide(LPS)was used to induce myocardial cell injury in mice to construct the effect of sepsis on myocardium,to study the mechanism of selenium on myocardial injury induced by lipopolysaccharide.First,in the first part,we took in vivo culture,injected lipopolysaccharide into mice to construct a model of myocardial dysfunction caused by sepsis,and fed the mice with selenium to observe the effect of selenium on the myocardial function of lipopolysaccharide mice.In the second part,we cultured H9C2 cardiomyocytes in vitro to construct a model of lipopolysaccharide-induced H9C2 cardiomyocyte myocardial injury,and observe whether selenium can improve the myocardial injury of H9C2 cardiomyocytes induced by lipopolysaccharide in the in vitro model.In the third part,we injected mice with lipopolysaccharide to detect the expression of Stimulator of interferon genes(Sting)and Phospho Interferon regulatory factor 3(P-IRF3).Sting inhibitor C178 interferes with lipopolysaccharide-induced H9C2 cardiomyocyte myocardial injury model,observating does lipopolysaccharide-induced myocardial injury mediate apoptosis through the Sting signaling pathway?This study includes three parts:Part I: Selenium ameliorates lipopolysaccharide-induced myocardial injury in mice Objective:To observe whether selenium ameliorates lipopolysaccharide-induced myocardial injury in mice.Methods:1.Mouse model establishment and group: eight-week-old male C57BL/ 6 J mice were injected with 10mg/kg normal saline or 10mg/kg LPS for 30 days,and some mice were fed 0.2mg/kg selenium in advance for 2 weeks.The experimental groups were as follows:(1)Control group(Control),(2)Lipopolysaccharide group(LPS),(3)Selenium +lipopolysaccharide group(Se+LPS).2.Echocardiographic detection: The Left ventricular end diastolic dimension(LVEDD),the Left ventricular end diastolic dimension(LVESD),Left ventricular ejection Fraction(LVEF),and Left ventricular systolic function(LVSF)were detected by ultrasonography for each group.3.Tissue retention: The experimental mice were fed for 30 days and weighed.After ultrasonic treatment,the mice were sacrificed under abdominal anesthesia.4.Serological detection of LDH and CK-MB: After feeding the experimental mice for 30 years,blood was taken from the eyeball before death,centrifuged at 4℃ 12000 g for 5min,reagent was added to the lactate dehydrogenase(LDH)kit and creatine kinase isoenzyme(CK-MB)kit instructions.5.Heart tissue inflammatory indicators: TRIzol reagent was used to purify total RNA from mouse heart tissue,and reverse transcription system was used to synthesize c DNA.The SYBR green PCR master kit and ABI PRISM 7500 assay system were used for real-time quantitative PCR assay of IL-1β,IL-6 and TNF-A.The mouse tissues were homogenized,supernatant was collected,and the levels of IL-1β,IL-6 and TNF-a were measured by the kit.6.Oxidative stress detection: Mouse heart tissue was weighed and homogenized on ice in PBS.After centrifugation at 10000 g at 4℃ for 20 minutes,the supernatant was obtained and analyzed.Superoxide dismutase(SOD)kit,glutathione peroxidase(GSH-PX)kit and malondialdehyde(MDA)kit were used to determine the content.7.Assay of apoptosis: Homogenate mouse tissues,collect supernatant,and use caspase-3 kit,Caspase-8 kit and caspase-9 kit were used to detect the expression of apoptosis indicators caspase-3,Caspase-8,and caspase-9.Real-time quantitative PCR assay of caspase-3、caspase-8 and caspase-9.The expression of Bax,Bcl-2,and Cleaved caspase-3 proteins were detected by western blot.Results:1.Organ weight of experimental mice in each group:Compared with the control group(Control),the body weight(BW)and left ventricular weight(LVW)of lipopolysaccharide group(LPS)mice were significantly increased(P < 0.05,vs Control group).The heart weight/body weight(Total HW/BW)and left ventricular weight/body weight(LVW/BW)were significantly decreased(heart weight/body weight,P < 0.05;Left ventricular weight/body weight,P < 0.05;Vs LPS),and there was no significant difference between the other groups.There was no statistical difference in heart weight(HW),Lung W and Lung W/BW among all groups.2.Cardiac function test : Compared with the Control group(Control),the left ventricular end-diastolic diameter(LVEDD)and left ventricular end-systolic diameter(LVESD)in LPS group were significantly increased(left ventricular end-diastolic diameter,P < 0.05;Left ventricular end-systolic diameter,P < 0.01;Vs Control group),selenium treatment improved lipopolysaccharide-induced in left ventricular end-diastolic diameter(LVEDD)and left ventricular end-systolic diameter(LVESD)(left ventricular end-diastolic diameter,P<0.05;Left ventricular end-systolic diameter,P < 0.05;Vs LPS).Compared with the Control group(Control),the left ventricular ejection fraction(LVEF)and left ventricular systolic function(LVSF)in LPS group were significantly decreased(LEFT ventricular ejection fraction,P < 0.01;Left ventricular systolic function,P < 0.05;Vs control group),the left ventricular ejection fraction(LVEF)and left ventricular systolic function(LVSF)were significantly increased in Se+LPS group(left ventricular ejection fraction,P<0.05;Left ventricular systolic function,P<0.05;Vs LPS group).3.LDH and CK-MB were detected by serology : Lipopolysaccharide feeding increased the expression of lactate dehydrogenase(LDH)and creatine isoenzyme(CK-MB),markers of myocardial injury in serum of mice(lactate dehydrogenase,P <0.01;Creatine isozyme,P < 0.01;Vs Control group),selenium inhibited lipopolysaccharide-induced elevation of markers of myocardial injury in serum(lactate dehydrogenase,P < 0.05;Creatine isoenzyme,P < 0.05,vs LPS group).4.Detection of inflammatory indexes of heart tissue:Detection of inflammatory indexes of heart tissue:The expression of IL-1β,IL-6 and TNF-α in lipopolysaccharide group(LPS)was significantly increased compared with Control group,(IL-1β,P < 0.05;IL-6,P < 0.01;TNF-α,P < 0.01;Vs control group),selenium inhibited the increase of inflammatory cytokines induced by lippolysaccharide(IL-1β,P < 0.05;IL-6,P < 0.01;TNF-α,P < 0.01;Vs LPS).Similarly,m RNA IL-1β,m RNA IL-6 and m RNA TNF-αshowed the same trend by QRT-PCR.The expression of IL-1β,IL-6 and TNF-α in LPS group was increased(IL-1β,P < 0.01;Il-6,P < 0.01;TNF-α,P < 0.05;Vs control group),the expression of IL-1β,IL-6 and TNF-α in Se+LPS group decreased(IL-1β,P < 0.05;Il-6,P < 0.01;TNF-α,P < 0.05;Vs LPS group).5.Detection of oxidative stress in heart tissue : Superoxidase dismutase(SOD)activity and glutathione peroxidase(GSH)content were significantly decreased in lipopolysaccharide group(LPS),while malondialdehyde content was increased(super oxidase dismutase,P < 0.05;Glutathione peroxidase,P < 0.05;Malondialdehyde,P <0.01;Vs Control group),the superoxidase dismutase(SOD)activity and glutathione peroxidase(GSH)content were significantly increased,while the malondialdehyde content was decreased(superoxidase dismutase,P < 0.05;Glutathione peroxidase,P <0.05;Malondialdehyde,P < 0.01;Vs LPS group).6.Determination of cardiac tissue apoptosis : Compared with the Control group(Control),the expressions of caspase-3,caspase-8 and caspase-9 in lipopolysaccharide group(LPS)were significantly increased(caspase-3,P < 0.01;Caspase-8,P < 0.01;Caspase-9,P < 0.01;Vs Control group),selenium inhibited the expression of caspase-3,caspase-8 and caspase-9 in LPS group(Caspase-3,P < 0.01;Caspase-8,P < 0.01;Caspase-9,P < 0.05;Vs LPS group).The apoptosis-related genes m RNA caspase-3,m RNA caspase-8 and m RNA caspase-9 showed the same trend.Cleaved caspase-3 and Bax expression in LPS group was significantly increased compared with Control group(Cleaved caspase3,P < 0.05;Bax,P < 0.01;Vs Control group),selenium inhibited the increase of caspase-3 and Bax protein expression in LPS group(Cleaved caspase3,P <0.01;Bax,P < 0.05;Vs lipopolysaccharide).Bcl-2 protein falled in lipopolysaccharide(LPS)group,although the trend was not obvious,but selenium improved the fall of bcl-2protein induced by LPS(Bcl-2,P < 0.05;Vs LPS group).Conclusion:Lipopolysaccharide induced myocardial remodeling in mice after myocardial injury,and selenium could improve the myocardial injury induced by lipopolysaccharide,which may be beneficial to the treatment of myocardial injury in lipopolysaccharide mice。Part II: Selenium ameliorates lipopolysaccharide-induced H9C2 cardiomyocyte damageObjective:To observe whether selenium ameliorates lipopolysaccharide-induced injury of H9C2 cardiomyocytes.Methods:1.MTT assay: MTT assay was used to detect the effects of different concentrations of Se on H9C2 injury.The groups were as follows: Control group(Control),100 n M Se(100n M Se),250 n M Se(250n M Se),500 n M Se(500n M Se),1000 n M Se(1000n M Se),2000 n M Se(2000n M Se).2.Determination of LDH in cells: Cells were lysed and LDH in cells was detected with lactate dehydrogenase(LDH)kit.The groups of LDH were as follows: Control group(Control),lipopolysaccharide group(LPS),selenium + lipopolysaccharide group(Se+LPS).3.Oxidative stress detection: Homogenating H9C2 cardiomyocytes in PBS on ice.After centrifugation at 10000 g at 4℃ for 20 minutes,the supernatant was obtained and analyzed.Superoxide dismutase(SOD)kit,glutathione peroxidase(GSH-PX)kit and malondialdehyde(MDA)kit were used to determine the content.Groups(1)Control group(Control),lipopolysaccharide group(LPS),Selenium + lipopolysaccharide group(Se+LPS);(2)Control group,hydrogen peroxide Group(H2O2),Selenium + hydrogen peroxide group((H2O2+Se).4.Determination of intracellular inflammatory factors: H9C2 cardiomyocyte lysis,IL-1β kit,IL-6 kit and TNF-a kit were used to determine the contents of IL-1β,IL-6 and TNF-a.5.Apoptotic protein detection: Cell groups(1)Control group(Control),lipopolysaccharide group(LPS),selenium + lipopolysaccharide group(Se+LPS),western blot detection of apoptosis protein caspase3,Bax and Bcl-2 protein expression;(2)The expression of Caspase 3 in the Control group(Control),lipopolysaccharide group(LPS),BAY11-7082 + lipopolysaccharide group(BAY11-7082 +LPS)was detected by Caspase 3 kit.Results:1.MTT test:The effects of selenium on H9c2 cardiomyocytes at 0 nm(control),100 n M,250 n M,500 n M,1000 n M and 2000 n M were detected by MTT method.The results showed that selenium had no effect on the survival rate of H9C2 cardiomyocytes at each concentration gradient.Therefore,we selected 100 n M selenium as the intervention concentration for cell modeling.2.Determination of LDH in cells : Compared with the control group,lipopolysaccharide induced the expression of lactate dehydrogenase(LDH)in H9c2cardiomyocytes(LDH,P < 0.01;Selenium alleviated the increase of lactate dehydrogenase(LDH)expression induced by lipopolysaccharide in H9c2cardiomyocytes(LDH,P < 0.01;Vs lipopolysaccharide group).3.Detection of cellular oxidative stress: compared with the Control group,lipopolysaccharide intervention significantly increased the content of malondialdehyde(MDA),but significantly decreased the activity of superoxide dismutase(SOD)and the level of glutathione peroxidase(GSH-Px)(superoxide dismutase,P < 0.01;Glutathione peroxidase,P < 0.001;Malondialdehyde,P < 0.001;Vs Control group).However,selenium treatment significantly reversed these changes and inhibited lipopolysaccharide induced cardiac oxidative damage,although selenium did not significantly improve superoxide dismutase(superoxide dismutase,P > 0.05;Glutathione peroxidase,P < 0.01;Malondialdehyde,P < 0.001;Vs LPS group).In order to prove that selenium can inhibit oxidative stress,we intervene H9c2 cardiomyocytes with exogenous reactive oxygen species H2O2.The results show that selenium can improve oxidative damage by inhibiting oxidative stress.4.Determination of intracellular inflammatory factors: lipopolysaccharide increased expression of IL-1β、IL-6 and TNF-α(IL-1 β,P<0.001;IL-6,P<0.001;TNF-α,P<0.01;Vs Control group),selenium treatment alleviated expression of IL-1β、IL-6 and TNF-α in H9c2 cell(IL-1 β,P<0.01;IL-6,P<0.001;TNF-α,P<0.05;Vs LPS group).5.Detection of apoptotic proteins: the expression of apoptotic proteins Cleaved caspase 3,Bax and anti apoptotic protein Bcl-2 were detected by Western blot.Compared with the Control group,Cleaved caspase-3 in lipopolysaccharide group(LPS)increased,although there was no significant difference,Bax expression increased significantly(Bax,P < 0.01;Selenium inhibited the expression of Cleaved caspase-3 and Bax protein in lipopolysaccharide group(LPS)(Cleaved caspase-3,P < 0.001;Vs LPS group).Bcl-2protein falled in lipopolysaccharide group(LPS),although the trend was not obvious,selenium improved the fall of Bcl-2 protein induced by lipopolysaccharide(Bcl-2,P <0.01;Vs LPS group).In order to determine whether inflammation causes apoptosis,we intervened H9c2 cells with inflammatory inhibitor BAY11-7082.The results showed that inflammatory inhibitor BAY11-7082 reversed the increase of apoptotic protein Caspase 3induced by lipopolysaccharide(Caspase-3,P < 0.001;Vs LPS group).Conclusion:Lipopolysaccharide induces oxidative stress,inflammatory response and apoptosis of H9c2 cell.Selenium inhibits this phenomenon and inhibits the production of oxidative stress,inflammatory response and apoptosis,which will provide a new idea for the treatment of lipopolysaccharide induced H9c2 cardiomyocyte injury.Part III: Selenium improves lipopolysaccharide induced myocardial injury through sting pathwayObjective:Sting activation eventually leads to the occurrence of inflammatory response.To observe whether selenium improves the injury of cardiomyocytes induced by lipopolysaccharide through sting pathway.Methods:1.Expression of sting and P-IRF3 protein in heart tissue: divided into control group(control),lipopolysaccharide group(LPS)and selenium + lipopolysaccharide group(Se+ LPS),QRT-PCR was used to detect the expression of sting and P-IRF3 protein in the heart tissue of each experimental group,and Western blot was used to detect the expression of sting and P-IRF3 protein in the heart tissue of each experimental group.2.Effect of sting pathway on H9c2 cardiomyocyte inflammation: divided into control group(control),lipopolysaccharide group(LPS)and C178 + lipopolysaccharide group(C178 + LPS).The inflammatory factor IL-1β 、 IL-6 and TNF-α Protein expression of H9c2 cardiomyocytes in each experimental group was detected by Western blot.Result:1.Expression of Sting and P-IRF3 protein in heart tissue: QRT-PCR results showed that compared with the Control group,lipopolysaccharide significantly increased the m RNA level of sting in heart tissue(Sting m RNA,P < 0.01,vs control group),and selenium inhibited the increase of m RNA-Sting level in heart tissue induced by lipopolysaccharide(sting m RNA,P < 0.01,Vs LPS group).The expression of sting protein in each experimental group showed the same trend by Western blot(sting,P <0.05,vs control group;Sting,P < 0.05,vs lipopolysaccharide group).The expression of P-IRF3 protein was significantly increased in lipopolysaccharide group(LPS),and selenium improved this phenomenon(P-IRF3,P < 0.05,vs Control group;P-IRF3,P <0.05,vs LPS group).2.Effect of Sting pathway on inflammation of H9c2 cell: compared with the Control group,lipopolysaccharide significantly increased IL-1β 、 IL-6 and TNF-α Protein expression in H9c2 cell(IL-1 β,P<0.01;IL-6,P<0.01;TNF-α,P<0.01;Vs Control group),Sting inhibitor C178 improved to increased protein expression of IL-1β、IL-6 and TNF-α induced by lipopolysaccharide in H9c2 cell(IL-1 β,P<0.01;IL-6,P<0.05;TNF-α,P<0.05;Vs LPS group).Conclusion:Lipopolysaccharide induces inflammatory response by activating Sting pathway and downstream P-IRF3.Selenium improves lipopolysaccharide induced myocarditis by inhibiting this pathway.