Protective Effect Of Apelin-13 On Sepsis-induced Myocardial Dysfunction And Its Mechanism

Sepsis can cause multiple organ dysfunction.Some existing works have shown that sepsis-induced myocardial dysfunction(SIMD)is closely related to increased mortality of sepsis patients.However,the specific mechanism of SIMD is still currently unclear.Possible mechanisms include inhibition of myocardial inflammation by inflammatory factors,apoptosis,mitochondrial dysfunction,and calcium influx disorders,etc.,where the uncontrolled inflammatory response is one of the most significant mechanisms among them.lipopolysaccharide(LPS)secreted by Gram-negative bacilli is one of the main substances that initiate inflammation and immune imbalance in sepsis.Some preliminary works shown that there is also a mechanism of myocardial hibernation in sepsis.Heart dysfunction may be the self-protection of the body,suggesting that there is an endogenous protective mechanism in the body.Apelin is an endogenous ligand for an angiotensin-II receptor-like-G Protein-Coupled Receptor APJ,and a polypeptide consisting of 77 amino acids.The Apelin/APJ system is widely distributed in both the periphery and the center.Recent studies have demonstrated that apelin plays a role in regulating myocardial contraction and vascular tone and regulating inflammation,immunity,energy metabolism,and water balance etc.Apelin has various subtypes such as apelin-36,apelin-28,apelin-17,apelin-13 and so on.Apelin-13 can be converted to a more stable form of pyroglutamic acid-[Pyr1]apelin-13 in vivo.Studies have shown that apelin-13 and[Pyr1]apelin-13 are the most important subtypes of apelin in the cardiovascular system.Apelin-13 has strong biological activity and plays an essential role in the regulation of cardiovascular function.Although existing literature has shown that apelin can enhance myocardial contractility,reduce the load before and after the heart,and have anti-inflammatory effects,whether the apelin /APJ system has a protective effect on LPS-induced cardiac dysfunction is not clear.In our work,LPS were injected intraperitoneally into C57BL/6 mice to establish a model of SIMD.Apelin-13 and its antagonist,[Ala]-apelin-13(F13A),were given before or after LPS injection to explore the protective effect of apelin on SIMD and its mechanism.The experiment contains five parts as follows.Part one Establishment of SIMD mouse model and changes of apelin expression in myocardial tissueObjective: To successfully establish a mouse model of LPS-induced SIMD and to explore the relationship between myocardial apelin level and SIMD.Methods: SIMD model was established by intraperitoneal injection of LPS 20 mg/kg in C57BL/6 mice.Eight hours(LPS 8h group)and 24 hours(LPS 24 h group)after LPS injection,transthoracic echocardiography was performed,and tissue and serum samples were collected.The pathological changes of the myocardium were observed by HE staining,and the ultrastructural changes of the myocardium were observed by transmission electron microscope.ELISA was used to detect the changes of inflammatory factors TNF-α and IL-6 in plasma and myocardial homogenate.The m RNA and protein expression of apelin were detected by Q-RT PCR and Western blot.Results:1.Compared with the control group,the left ventricular systolic function indexes,including LVEF,LVFS and SV in both LPS 8h group and LPS 24 h group were significantly decreased.2.Compared with the control group,only slight pathological changes occurred in the myocardium after LPS injection,including telangiectasia,intermuscular erythrocyte exudation,and granulocyte edge aggregation.3.Compared with the control group,levels of TNF-α and IL-6 in the plasma and myocardial tissue of the LPS8 h group increased sharply,and showed significant regression in LPS 24 h group,but it was still higher than the control group.4.Compared with the control group,the expression of apelin m RNA and protein in the LPS8 h group was significantly decreased.Additionally,the apelin m RNA level was significantly decreased in the LPS24 h group comparing with the control group,but the apelin protein expression was not significantly changed.Conclusions: The SIMD model was successfully established by LPS.The SIMD showed a decrease of cardiac systolic function,accompanied by a marked increase in inflammatory factors,but a slight pathological change.There was a significant decrease in apelin expression in myocardium after LPS intervention.Apelin may participate in the occurrence of SIMD.Part two Effects of apelin-13 pre-treatment on LPS-induced myocardial dysfunctionObjective: To explore the role of endogenous Apelin/APJ system on SIMD.Methods: C57BL/6 mice were divided into two subgroups,the first subgroup include 4 groups: control group,LPS group,apelin+NS group,apelin+LPS group;the second subgroup include 3 groups: LPS group,apelin+LPS Group,F13A+LPS group.The LPS group was modeled as before;Apelin+LPS group,apelin+NS group and F13A+LPS group: receive apelin-13(1mg/kg)or F13A(1mg/kg)as pre-treatment 1 hour before intraperitoneal injection of LPS or NS.Transthoracic echocardiography examination and specimen collection were performed 24 hours after LPS or NS injection.The inflammatory factors TNF-α and IL-6 in plasma and myocardial homogenate were detected by ELISA.The effects of apelin-13 pre-treatment on mouse cardiac ultrasound and inflammatory factors and the changes of inflammatory factors after pre-treatment with F13 A were observed.Results:1.Compared with the LPS group,LVEF and SV were significantly increased in the apelin pre-treatment group;2.Apelin pre-treatment significantly inhibited the increase of serum IL-6induced by LPS,and decreased the serum TNFα and inflammatory factors in myocardial tissue;3.Apelin pre-treatment caused a decrease in LVEF in normal mice,but had no significant effect on CO;4.There is no further increase of inflammatory factor levels in the F13 A pre-treatment group.Conclusions: Apelin pre-treatment improves LPS-induced cardiac dysfunction and has a protective effect on SIMD;Pre-treatment with apelin-13 may have adverse effects on cardiac function in normal mice;Under the conditions of this study,F13 A has no obvious antagonistic effect on endogenous apelin.Part three Protection of exogenous apelin-13 on LPS-induced cardiac dysfunctionObjective: To investigate the effect of exogenous administration of apelin-13 on LPS-induced SIMD.Methods: C57BL/6 mice were divided into 4 groups,LPS group,LPS+apelin-L group,LPS+apelin-H group,LPS+apelin+F13A group.LPS group: same as before;LPS+apelin-L group,LPS+apelin-H group and LPS+apelin+F13A group: receive apelin-13 lower dose(100ug/kg),or apelin-13 higher dose(500ug/kg),or apelin-13 high-dose combined with F13A(500ug/kg)1 hour after LPS injection,then once every 5 hours,4 times total.The effects of apelin-13 and F13 A treatment on inflammatory factors,myocardial pathology and ultrastructure,and the effect of a high dose apelin-13 on cardiac ultrasound in mice were observed 24 hours after LPS.Results:1.Compared with the LPS group,high-dose apelin-13 treatment significantly increased LVEF and SV.2.Both low-dose and high-dose apelin-13 treatment significantly inhibited IL-6 elevation induced by LPS,more significant in the high-dose group;3.Compared with the apelin-13 treatment group,treatment combined with F13 A increased inflammatory factors levels.Conclusions: Apelin-13 significantly improved LPS-induced cardiac dysfunction,significantly antagonized the elevation of inflammatory factors induced by LPS.F13 A partially antagonized the effect of apelin-13 on inflammatory factors.Part four The mechanism of the protective effect of apelin-13 on LPS-induced myocardial dysfunctionObjective: To investigate the protective mechanism of apelin-13 on LPS-induced SIMD.Methods: The heart tissue of control group,LPS(24h)group,apelin+LPS group,LPS+apelin-Hgroup,and LPS+apelin+F13A group in the part 2 and the part 3 experiment were used for detecting.Apoptosis and autophagy indicators were detected: the protein expression of caspase-3,caspase-8,LC3 II and beclin1 were detected by Western blot,caspase-3 m RNA levels was detected by Q-RT PCR m RNA,TUNEL for apoptotic cells.The protein expression of TLR4,ERK,NF-κB were measured by Western blot.at the same time.Results:1.Apoptosis of cardiomyocytes increased after LPS intervention.Compared with the LPS group,apelin-13 treatment significantly reduced apoptotic cell ratio,cleaved-caspase-3 and cleaved-caspase-8 protein expression,caspase-3 m RNA level,and this effect is clearly antagonized by F13 A.2.Compared with the control group,the expression of LC3 II and beclin1 protein in LPS group increased;the expression of both was further increased after apelin-13 treatment,while F13 A significantly antagonized the effect of apelin-13.3.Apelin intervention significantly inhibited the increase of TLR4 protein expression and the phosphorylation of ERK and NF-kappa Bp65 induced by LPS injection,and F13 A significantly antagonized this effect.Conclusion: Apelin-13 may inhibit the inflammatory response and apoptosis through TLR4/ERK/NF-κB signaling pathway and protect LPSinduced cardiac dysfunction.Part five Protective effects of apelin-13 on LPS-induced lung injuryObjective: To investigate the effect of apelin-13 on lung injury induced by LPS.Methods: C57BL/6 mice were injected with LPS 20mg/kg to make a sepsis model as before.There were six groups in this part,control group,LPS8 h group,LPS 24 h group,apelin+LPS group,LPS+apelin-H group,and LPS+apelin+F13A group.The pathological changes of lung tissue were observed by HE staining,and the ultrastructural changes were observed by transmission electron microscope.ELISA was used to detect the inflammatory factors TNFα and IL-6 in lung tissue homogenate,and TUNEL for cell apoptosis.Results:1.Compared with the control group,the LPS group showed obvious interstitial edema and inflammatory cell infiltration;the LPS 24 h group changed significantly compared with the LPS 8h group;apelin-13 treatment significantly reduced the pathological changes caused by LPS;the lesions were aggravated in the LPS+apelin+F13A group.2.Compared with the control group,levels of TNF-α and IL-6 in plasma and myocardial tissue of LPS 8h group were significantly increased,and the LPS 24 h group showed significant regression,but still higher than the control group;compared with the LPS 24 h group,levels of inflammatory factors in apelin+LPS group and LPS+apelin-13-H group showed a downward trend,and F13 A intervention partially antagonized this effect of apelin-13.3.Compared with the normal control group,the apoptotic cells in the LPS group increased significantly,and the apelin-13 treatment significantly reduced the apoptotic cell ratio.Conclusions: Apelin-13 attenuated lung pathological changes induced by LPS,down-regulated the levels of inflammatory factors,reduced the cell apoptosis of lung tissue,and protect against LPS-induced lung injury.