Protective Effect Of Shenfu Injection On Myocardial Mitochondrial Damage Induced By LPS In Mice

Aim:Sepsis was induced in mice by intraperitoneal injection of lipopolysaccharide(LPS)at a dose of 8 mg/kg according to the previous studies(Hu et al.,2018).Different doses of Shenfu Injection(SFI)were given before the establishment of the sepsis model and the changes of myocardial ultrastructure in septic mice were investigated.This is the first time to explore the specific protective effects of SFI on myocardial ultrastructure in septic mice,the effect of dosage,and the related mechanism of myocardial cell apoptosis.The study offers a theoretical foundation for the clinical diagnosis and treatment of sepsis and also provides the research basis for the clinical application of SFI.Methods:1.Forty C57/B6J mice were randomly divided into three groups:control(NC),sham sepsis(Sham),sepsis(Lipopolysaccharide-LPS).The sepsis group includes sepsis induced by injection at a dose of 3mg/kg,8mg/kg and 12mg/kg.The general condition and survival situation of mice were compared to select the optimized model and the appropriate time of sampling.2.In addition,sixty-one C57/B6J mice were randomly divided into five groups:control group(NC),sham group(Sham),LPS group(LPS),LPS plus low dose SFI treatment("LPS+SFI-Low",3.0 ml/kg,i.p.),LPS plus high dose SFI treatment("LPS+SFI-High",10.0 ml/kg,i.p.).The following indicators were tested and compared:(1)The performance of septic mice.(2)The indexes of cardiac injury of mice in each group were examined with the method of ELISA(enzyme-linked immunoabsorbent assay),such as CK-MB,NT-pro-BNP and CTnI.(3)The pathological changes in the myocardial tissues of mice in each group:the cardiac histopathological changes and inflammatory cell infiltration were observed using H&E(Hematoxylin-eosin)staining and the apoptosis in myocardial cells were detected by TUNEL(TdT-medicated dUTP nick end labeling)assay.(4)The damage to myocardial ultrastructure of mice in each group:the TEM(transmission electron microscopy)was used to observe the mitochondrial morphology(mitochondrial area and matrix density),judge the mitochondrial swelling and analyse the correlation between the degree of mitochondrial swelling and myocardial injury indicators.(5)The protein expression of B-cell lymphoma 2(Bcl-2),BH3 interacting-domain death agonist(Bid),truncated-Bid(t-Bid)and Caspase-9,which were related to the myocardial apoptosis of mice in each group,were determined by Western blot analysis.The expression of Bcl-2 and Bid were calculated.Result:1.The mice in the LPS-1,LPS-2,and LPS-3 groups began to die after modeling at 12 hours,11 hours,and 8 hours respectively.The 48h survival rate of the LPS-2 group was higher than that of the LPS-3 group,and the mice of the LPS-2 group showed obvious symptoms of sepsis.Thus,LPS-2 was selected as the experimental model(injected by LPS at a dose of 8 mg/kg i.p.)for this study.6 hours,12hours,and 24hours after modeling were selected as time points for taking samples.2.The 48h survival rate of mice in the“LPS+SFI-High" group was higher than that of the LPS group and the "LPS+SFI-Low" group.3.The symptoms of sepsis showed in the LPS group gradually worsened at 6 hours after establishing models and the most severe symptom of sepsis occurred at 12 hours after modeling.The symptoms of sepsis developed by mice in the "LPS+SFI-High" group were obviously alleviated,but there was no significant difference between the "LPS+SFI-Low"group and the LPS group.4.On the basis of HE staining,pathological changes such as abnormal morphology,myofilament disorder,interstitial edema and higher levels of CK-MB,NT-proBNP and CTnl were observed in the myocardial cells of the LPS group.Almost intact myocardial structure were found in the "LPS+SFI-High" group;myocardial fiber rupture was reduced,and interstitial edema and inflammatory cell infiltration were improved.These results indicated that pathological damage was significantly reduced in the "LPS+SFI-High" group.Meanwhile,the levels of CK-MB,NT-proBNP and CTnl decreased.However,there was no obvious difference between the "LPS+SFI-Low" group and the LPS group.With TUNEL method,it revealed that the variation of apoptotic cells in the Sham group was not significant compared with that in the NC group.The mean optical density of myocardial cells in the LPS and SFI groups was higher than that in the Sham group,and the apoptosis rate of the"LPS+SFI-High" group was significantly lower than that of the LPS group and the"LPS+SFI-Low" group.5.With the help of TEM,these results could be showed in the NC group:Both thick and thin myocardial myofilaments are arranged regularly and neatly.Individual bands appeared relatively clear.Normal sarcomere structure and large amounts of mitochondria in normal size and oval shape were distributed between myofilaments.The crest was dense and parallel-aligned and the matrix contained a large amount of dense matrix granules.Compared with the NC group,there was no significant difference in the Sham group.In the LPS group,myocardial myofilaments appeared disordered and mitochondria appeared signifificantly swollen:some mitochondria showed vacuole-like changes,unclear boundaries,decreased matrix density,and non-uniformity at 6,12 and 24 h.At 12 hours after modeling,the mitochondrial changes in the LPS group were the most severe.After SFI treatment,the damage to mitochondria of cardiomyocytes was significantly alleviated:vacuole-like changes were significantly reduced,and mitochondrial swelling was also reduced,especially in the "LPS+SFI-High" group.6.Western-blot analysis showed that compared with the NC group,the expression of Bcl-2 in the myocardium of mice was significantly inhibited after LPS modeling(P<0.0001),and the expression of Bid,tBid,and Caspase-9 was significantly up-regulated(P<0.0001).The expression of Bcl-2 was significantly higher than that of the LPS group,and the difference was statistically meaningful(P=0.001).At the same time,the expression of Bid,tBid,and Caspase-9 in the SFI groups was lower than that of the LPS group.The difference in tBid and Caspase-9 was statistically significant(P=0.002,0.006).7.It suggested that the levels of CK-MB,CTnI were positively correlated with mitochondrial swelling.Conclusion:1.Sepsis can cause ultrastructural damage to myocardial mitochondria.2.High dose of SFI protects against the sepsis-induced injury in the myocardial mitochondria.3.SFI inhibits the sepsis-induced mitochondrial apoptosis to preserve the function of myocardium through upregulateing the protein expression of Bcl-2 and downregulating the protein expression of Bid,t-Bid and caspase-9.