Evaluation Of The Protective Effect Of Naringin On Myocardial Injury Induced By LPS Using Two-dimensional Speckle Tracking Imaging And Its Molecular Mechanism

Objectives:Sepsis is a life-threatening organ dysfunction caused by the host's maladjusted response to infection.Lipopolysaccharide?LPS?is the main component of gram-negative bacterial outer membrane,which can cause sepsis in humans or animals and lead to multiple organ damage and failure.Heart is one of the most frequently involved organs,sepsis can lead to weakened myocardial contractility,slow heart rate,etc.,seriously affecting the function of the heart,which has become the main cause of morbidity and mortality in patients with sepsis.Therefore,it is very important to understand the pathogenesis between sepsis and myocardial injury and further find effective treatment methods to alleviate the myocardial injury of sepsis.Naringin?Nar?is a kind of natural flavonoids,the main source of which is the grapefruit,orange and orange of rutin.Meanwhile,Naringin is also one of the main active components of many Chinese herbs.Studies have shown that flavonoids intake is negatively correlated with the incidence and mortality of cardiovascular disease.Two-dimensional speckle tracking imaging?STI?is a relatively new ultrasonic technology to quantitatively evaluate the global and segmental myocardial function.It is independent of front and rear cardiac load,beam direction,angle and sampling position,with higher sensitivity and repeatability,and is able to detect early sub-clinical cardiac dysfunction.Compared with the global strain,the layer-specific strain is more consistent with the left ventricular myocardium of the three-layer myocardial band anatomy,so as to evaluate the cardiac function more comprehensively.The purpose of this study was to evaluate the effect of naringin on the global and segmental strain of left ventricular myocardium in septic cardiomyopathy rats by STI,and to detect whether naringin has protective effect on sepsis myocardial injury and the possible molecular mechanism of effect,so as to provide experimental basis for the treatment of sepsis myocardial injury by naringin.Methods:1.2D-STI was used to evaluate the effect of naringin on left ventricular function in rats with myocardial injury induced by LPS:?1?A model of sepsis-induced myocardial injury in rats was induced by intraperitoneal injection of LPS.75 male SD rats were randomly divided into 5 groups?15 in each group?:Control group;LPS group;LPS+Nar₅₀ group;LPS+Nar₁₀₀ group;LPS+Dexa group.Six hours after LPS injection,anesthesia with pentobarbital sodium was performed.Five groups of rats were examined by conventional echocardiography and STI using GE Vivid E9 color ultrasound diagnostic apparatus.Conventional echocardiography was used to measure left ventricular end diastolic diameter?LVEDd?,end diastolic Volume?EDV?,left ventricular end systolic diameter?LVEDs?,end-systolic volume?ESV?,left ventricular ejection fraction?LVEF?,short axis shortening rate?FS?.The global and segmental motion of left ventricular myocardium in longitudinal,circumferential and radial dimensions was measured by 2D-STI.?2?blood was collected from the rats under anesthesia after ultrasound examination,and fully automatic biochemical analyzer was used to detect serum creatine kinase?CK?and lactate dehydrogenase?LDH?levels in serum.?3?HE staining was used to observe the pathological changes of myocardium in the five groups and histopathological score was made according to the degree of injury.?4?Myeloperoxidase?MPO?activity was detected after myocardial tissue homogenization.2.The inhibitory effect of naringin on the inflammatory response in LPS-induced myocardial injury rats.The expression of TNF-?protein in serum was detected by Elisa method using rat model of sepsis myocardial injury.Real-time quantitative PCR was used to detect the expression of messenger RNA of TNF-?,IL-1?,IL-6 and iNOS in myocardial tissue.Myocardial NF-?B p65 expression was detected and scored by immunohistochemistry.The protein expression of NF-?B/iNOS signaling pathway and nuclear translocation of NF-?B in myocardial tissue of SIMD rats were detected by western blot.3.Regulation of the PI3K/AKT/NF-?B pathway affects the effect of Nar on H9c2 cardiomyocyte injury induced by LPS.?1?Cultured H9c2cardiomyocyte cell line,after adding different concentrations of naringin and/or LPS,the cell viability was measured by CCK-8 colorimetry,and the optimal concentration of naringin to protect cardiomyocytes was screened.?2?H9c2 cardiomyocytes were divided into 4 groups according to different treatments:control group;LPS group;naringin low-dose treatment(LPS+Nar₄₀)group;naringin high-dose treatment(LPS+Nar₈₀)group.Messenger RNA expression levels of TNF-?,IL-1?,IL-6 and iNOS in cardiomyocytes were detected by Real-time PCR.The protein expression levels of NF-?B,p-NF-?B,I?B?,p-I?B?and iNOS were detected by Western blot.?3?Add PI3K inhibitor LY294002 to detect downstream pathway protein expression.The H9c2 cardiomyocytes were divided into five groups:control group;LPS group;LPS+Nar₈₀ group;LPS+Nar₈₀+LY294002;LY294002.The expression of NF-?B p65 protein and nuclear translocation were detected by immunofluorescence.The expression levels of p-AKT,AKT,and NF-?B protein levels in nuclear and cytoplasm were detected by Western blot.Results:1.2D-STI was used to evaluate the effect of naringin on left ventricular function in rats with myocardial injury induced by LPS.?1?Six hours after LPS injection,there were no significant abnormalities in the control group and positive control group,and the other three groups were slow to move.The rats in the LPS group showed rapid increase in breathing,listlessness,lethargy,vertical hair,and loose stool watery etc.The general condition of rats in LPS+Nar₅₀ group and LPS+Nar₁₀₀ group was less than that in the model group,the rats in LPS+Nar₅₀ group had slight apathy,vertical hair,a small amount of watery stools,the spirit of LPS+Nar₁₀₀ group rats is still good,with a little dilute water samples.?2?Compared with the control group,the left ventricular diameter increased and the LVEF decreased in the LPS group.The LVEDd,EDV,and ESV in the LPS+Nar₅₀ group were improved compared with the LPS group,while LVEDs,LVEF and FS showed no statistical difference.As expected,the LPS+Nar₁₀₀ and LPS+Dexa groups improved significantly.?3?The global strain and strain rate in the LPS group were lower than those in the control group.The global longitudinal strain and strain rate and circumferential strain in the LPS+Nar₅₀ group were higher than those in the LPS group,while the global circumferential strain rate,radial strain and strain rate were not statistically different between the two groups.The global strain and strain rate in the LPS+Nar₁₀₀ group and the LPS+Dexa group were all increased.?4?The system automatically traced the inner,middle,and outer layers of the myocardium,and the circumferential and longitudinal strain gradually decreased from the subendocardial myocardium to the subepicardial myocardium.The parameters in the LPS group were lower than those in the control group,and subendocardial myocardial injury was the most severe.In the LPS+Nar₅₀ group,except for the circumferential strain of the myocardial layer in the epicardium,the other indicators increased.Both the LPS+Nar₁₀₀ group and the LPS+Dexa group alleviated myocardial injury in the context of SIMD.?5?Cardiac enzymes CK and LDH in the LPS group were significantly higher than those in the control group.The LPS+Nar₅₀,LPS+Nar₁₀₀ group,and LPS+Dexa group were all reduced,and the differences were statistically significant.?6?HE staining showed that the myocardial fiber myofilaments of rats in the control group were neatly arranged,with normal cell structure and clear transverse stripes.In the LPS group,myocardial cells became edema,the intercellular space widened,some myocardial fibers were broken,inflammatory cells increased,and histopathological scores increased.However,the above pathological changes in the naringin treatment groups and the positive control group were obviously relieved and the scores were decreased.MPO activity is an indicator of neutrophil aggregation at the site of inflammation.The myocardial tissue MPO activity of the LPS group was higher than that of the control group,while the myocardial tissue MPO activity of the LPS+Nar?50/100 mg/kg?group and the LPS+Dexa group were significantly lower than that of the control group.2.The inhibitory effect of naringin on the inflammatory response in LPS-induced myocardial injury rats.?1?The protein level of TNF-?in serum in the LPS group was significantly higher than that in the control group,and naringin low-and high-dose treatment groups and the positive control group were lower than those in the LPS group.?2?Compared with the control group,the mRNA expression levels of TNF-?,IL-1?,IL-6and iNOS in the myocardial tissue of the LPS group were significantly increased.The mRNA expression levels of TNF-?,IL-1?and IL-6 were all decreased in the LPS+Nar₅₀group compared with the LPS group,and the m RNA expression level of iNOS was not statistically different between the two groups..All indexes in the LPS+Nar₁₀₀ group and the LPS+Dexa group were decreased,and the differences were statistically significant.?3?IHC staining was used to detect the expression of NF-?B p65 in rat heart tissues after 6hours of LPS treatment.In the control group,NF-B p65 protein was stained yellow,mostly in cytoplasm.In the LPS group,the expression of NF-?B was enhanced in the nucleus of myocardial cells,with yellow nuclei and shallow cytoplasm staining.The nuclear expression of NF-?B was decreased in both LPS+Nar group and LPS+Dexa group.After LPS treatment,IHC score was significantly higher than that of the control group,and the IHC score was significantly lower after Nar and Dexa treatment.?4?The ratio of nuclear to cytoplasmic expression of NF-?B p65 in the LPS group was higher than that in the control group.The ratio in LPS+Nar groups and LPS+Dexa group were reduced,which effectively inhibited the nuclear transposition of NF-?B.?5?The protein expression level of iNOS in LPS group was significantly higher than that in the control group,while the expression level of iNOS in LPS+Nar₁₀₀ treatment group and LPS+Dexa group decreased.3.Regulation of the PI3K/AKT/NF-?B pathway affects the effect of Nar on H9c2 cardiomyocyte injury induced by LPS.?1?CCK-8colorimetric method was used to detect the effect of Nar on the viability of H9c2 cardiomyocytes.Adding 10?M,20?M,40?M,80?M,160?M,and 320?M Nar did not affect the viability of cardiomyocytes.After 24 h treatment with LPS?10 ug/ml?,the cell survival rate droped to 62.33%±0.02%,and the difference between the two groups was statistically significant?P<0.05?.In the LPS+Nar groups,the cell survival rates of the 10?M and 20?M Nar treatment groups were not statistically different from that of the LPS group.The cell survival rates of the Nar treatment groups of 40?M,80?M,160?M,and 320?M were significantly increased,with statistical differences.?2?The messenger RNA expression levels of TNF-?,IL-1?,IL-6 and iNOS in H9c2cardiomyocytes in LPS group were significantly higher than those in the control group,and the LPS+Nar groups?40/80?M?were decreased,with statistically significant differences.?3?The protein expression levels of NF-?B p65and I?B phosphorylation and iNOS in the LPS group were higher than those in the control group.The level of NF-?B p65phosphorylation and iNOS protein expression in the LPS+Nar₄₀ group were reduced,which were statistically significant compared with the LPS group,while I?B phosphorylation level was not statistically significant.Those levels in LPS+Nar₈₀ group were all significantly reduced.?4?The expression level of AKT phosphorylated protein in the LPS group was lower than that in the control group,and the NF-?B p65 expression level in nucleus and cytoplasm ratio?N/C value?increased.Immunofluorescence test showed that NF-?B p65 fluorescent was expressed in the nucleus.The phosphorylated protein level of AKT in the LPS+Nar₈₀ groups were higher than that in the LPS group,the N/C value of NF-?B p65was lower,and nuclear fluorescence expression of NF-?B p65 was significantly reduced.The phosphorylation level of AKT in LPS+Nar₈₀+LY294002 group was lower than that of the LPS+Nar₈₀ group,the N/C value of NF-?B p65 was increased,and the fluorescence expression of NF-?B p65 in nucleus was increased.Conclusions:1.The STI-based layered strain technique can be used to evaluate the effect of naringin pretreatment on early left ventricular function in rats with septic myocardial injury.Moreover,for septic myocardial injury rats,the subendocardial myocardial injury is the most serious,which gradually decreases from the subendocardial myocardial layer to the subepicardial myocardial layer.2.Naringin inhibits the inflammatory response of septic myocardial injury in rats and plays a protective role in myocardial injury.3.Naringin inhibits LPS-induced H9c2 cardiomyocyte injury by regulating the PI3K/AKT/NF-?B pathway,providing experimental evidence for naringin in the treatment of sepsis myocardial injury.