| ObjectiveTo investigate the protective effect and mechanism of carbon monoxide release molecule-2(CORM-2)on sepsis-induced myocardial dysfunction in rats.MethodsPart ?:(1)Animals and Treatments: Seventy-two SPF male Sprague-Dawley rats were randomly divided into control group(n=28)and sepsis group(LPS group,n=44).Twenty rats were randomly selected from each group for observation of 10-day survival rate.According to the post-modeling phase,the remaining 24 of the LPS group were divided into 3 subgroups,LPS 6h group,LPS 12 h group and LPS 24 h group,with 8 rats in each group.A sepsis model was constructed by intraperitoneal injection of 10 mg/kg LPS in the LPS group,and the control group was injected with the equal volume of normal saline.(2)Observation indicators: Echocardiographic examination of cardiac function was performed at each phase of each of the LPS subgroups,and myocardial tissue and serum were collected after measuring cardiac function.Myocardial histopathological morphology was observed by optical microscope,and myocardial ultrastructure was observed by transmission electron microscopy.Serum cardiac troponin I(cTnI)and brain natriuretic peptide(BNP)concentrations were measured by ELISA.Part ?:(1)Animals and Treatments: One hundred and forty SPF male Sprague-Dawley rats were randomly divided into control group,lipopolysaccharide(LPS)-induced sepsis model group(LPS group),and CORM-2 pretreatment group(CORM-2 group),inactivated CORM-2 pretreatment group(iCORM-2 group),DMSO prevention group(DMSO group),with 28 rats in each group.The rat sepsis model was established by intraperitoneal injection of 10 mg/kg LPS.The control group was injected with the equal volume of saline.In CORM-2 group and iCORM-2 group,8mg/kg CORM-2 or iCORM-2 were injected intraperitoneally at 1 hour before LPS injection,respectively.The DMSO group was injected intraperitoneally with DMSO dissolved in the equal volume of saline at 1h before LPS injection.(2)Observation indicators: Twenty rats were randomly selected from each group to observe 10-day survival rate.Transthoracic echocardiography was performed on the remaining rats at 12 hours after establishig model.And myocardial tissue and serum were collected after measuring cardiac function.Observe the pathological changes of rat myocardium under light microscope;observe the ultrastructure of myocardial mitochondria under electron microscope.Five rats were randomly selected in each group,the electron microscope specimens of each rat were randomly selected in 5 fields of view at a magnification of 12000 X,and 20 mitochondria were randomly selected in each field for statistical analysis.The mitochondrial ultrastructure was scored according to the Flameng scoring standard,and the length and width of the mitochondria were measured using Image-Pro Plus 6.0 software to evaluate the mitochondrial morphological changes.Serum myocardial troponin I(cTnI)and brain natriuretic peptide(BNP)concentrations were measured by ELISA.Mitochondrial complex IV activity in myocardium were measured by visible spectrophotometry.And Mitochondrial protein levels of Fis1,Mfn2 and Opa1 were assessed by western blot analysis.ResultsPart ?:(1)Survival: Compared with the control group,the 10-day survival rate of the rats in the LPS group was significantly reduced [10% vs 100%,P<0.05].(2)Cardiac structure and function: Echocardiography showed that,compared with the Control group,there were no significant differences in LVEDD and LVEDV among the LPS subgroups(all P>0.05).Compared with the Control group,there was no difference in LVESD in the LPS 6h group,LVESD increased in the LPS 12 h group and the LPS 24 h group [LVESD(mm):3.51±0.63?3.88±0.97 vs 2.66±0.23,both P<0.05],and was higher than the LPS 6h group(all P<0.05).Compared with the Control group,there was no difference in LVESV in the LPS 6h group(all P>0.05),and LVESV increased in the LPS 12 h group and the LPS 24 h group[LVESV(?l):118.20±51.91?165.90±89.14 vs 50.50±13.45,all P<0.05],and higher than the LPS 6h group(all P<0.05).There was no difference in LVESD and LVESV between the LPS 12 h group and the LPS 24 h group(all P>0.05).Compared with the Control group,there was no significant change in LVEF in the LPS 6h group(P>0.05),only the LVFS decreased[LVFS(%): 41.62±4.74 vs 46.10±2.63,P<0.05];LVEF and LVFS in the LPS 12 h group and the LPS24 h group were both reduced [LVEF(%):63.59±1.65?50.80±7.91 vs82.91±2.46;LVFS(%):29.85±1.17?22.24±4.20 vs 46.10±2.63,all P<0.05],and the LVEF and LVFS in the LPS12 h group and the 24 h group were lower than in the 6h group[LVEF(%):63.59±1.65?50.80±7.91 vs 78.38±4.54;LVFS(%):29.85±1.17?22.24±4.20 vs 41.62±4.74,all P<0.05],and the Compared with the LPS12 h group,the LVEF and LVFS in the LPS 24 h group were further reduced(all P<0.05).(3)Myocardial injury markers: Compared with the Control group,the serum cTnI level increased in the LPS 6h group?LPS 12 h group and LPS 24 h group[cTnI(pg/ml):3438.88±1334.22?6396.79±2364.78?11670±3142.53 vs 320.53±111.57,all P<0.05],and the serum cTnI level increased more significantly in the LPS 24 h group(P<0.05).Compared with the Control group,serum BNPlevels increased in the LPS 6h group but the difference was not statistically significant(P>0.05),while serum BNP levels increased significantly in the LPS 12 h group and LPS 24 h group[BNP(pg/ml): 6552.51±1900.33 ? 9115.11±1472.71 vs 1262.75±800.66,all P<0.05].Compared with the LPS 12 h group,the serum BNP level in the LPS 24 h group increased,but the difference was not statistically significant(P>0.05).(4)Myocardial histopathology and ultrastructure: Compared with the Control group,the myocardial pathological morphology and ultrastructure of the LPS 12 h group and the LPS 24 h group showed obvious damage.Part ?:(1)Survival: All rats in Control group survived;compared with Control group,the 10-day survival rate of LPS group,CORM-2 group,iCORM-2 group and DMSO group was significantly decreased [10%?70%?25%?15% vs 100%,all P<0.05];However,the10-day survival rate in the CORM-2 group was significantly higher than that in the LPS group,iCORM-2 group and DMSO group(all P<0.05).(2)Cardiac structure and function:Compared with the Control group,echocardiography showed that LVEDD and LVEDV in the LPS group,CORM-2 group,iCORM-2 group,and DMSO group had no significant changes after 12 hours of LPS treatment(all P>0.05).Compared with the Control group,LVESD and LVESV were significantly increased in the LPS group,iCORM-2 group,and DMSO group(all P<0.05);while the LVESD and LVESV in the CORM-2 group were higher than the Control group,but there was no significant difference.(all P>0.05),and the LVESD and LVESV of the CORM-2 group were lower than those of the other groups treated by LPS(all P<0.05).Compared with the Control group,the LVEF and LVFS of the four groups of LPS treatment were all reduced(all P<0.05),but the LVEF and LVFS of the CORM-2 group were higher than that of the LPS group,iCORM-2 group and DMSO group [LVEF(%):75.54±2.59 vs 63.58±1.66?63.30±5.09?64.15±5.57;LVFS(%):38.90±2.16 vs 29.85±1.17?29.80±3.24?30.45±3.62,? P<0.05].(3)Myocardial injury markers: Compared with the Control group,the serum cTnI and BNP levels in the remaining four groups of rats treated with LPSincreased(all P<0.05);while the cTnI and BNP levels in the CORM-2 group were lower than those in the LPS group,iCORM-2 group and DMSO group [cTnI(pg/ml): 3283.54±803.50 vs6449.18±1105.10? 5919.21±1068.27 ? 6349.80±1153.08;BNP(pg/ml): 3456.62±905.85 vs6070.18±1287.62 ? 5581.13±1161.17 ? 5974.89±988.89,all P<0.05].(4)Myocardial histopathology and ultrastructure: Optical microscope showed that the pathological changes of myocardial tissue in the LPS group,iCORM-2 group and DMSO group were severe;Transmission electron microscopy showed mitochondrial swelling,and some vacuoles changed.But the myocardial pathological morphology and mitochondrial ultrastructural integrity of the CORM-2 group were significantly better than those of the other groups treated with LPS.Compared with the Control group,the mitochondrial Flamen score of the four groups of LPS treatment increased(all P<0.05);the mitochondrial Flameng score of the CORM-2 group was lower than that of the LPS group,iCORM-2 group and DMSO group[Flameng score: 1.27±0.82 vs 3.14±0.62,3.10±0.69,3.11±0.72,all P<0.05].Compared with the Control group,the mitochondrial length and aspect ratio of the LPS group,iCORM-2group,and DMSO group all decreased(all P<0.05);the mitochondrial length and aspect ratio of the CORM-2 group were not different from those of the Control group(P>0.05),but increased compared with the LPS group,iCORM-2 group,and DMSO group [mitochondrial length(?m): 1.19±0.31 vs 1.02±0.28,1.03±0.26,1.01±0.32;mitochondrial aspect ratio:1.67±0.46 vs 1.48±0.47,1.48±0.39,1.51±0.38,all P<0.05].(5)mitochondrial complex IV activity: Compared with the Control group,the mitochondrial complex IV activity in the remaining four groups of LPS treatment decreased(all P<0.05).The mitochondrial complex IV activity in the CORM-2 group was significantly higher than that in the LPS group and the DMSO group [U/g: 100.65±12.98 vs 74.25±34.12 ? 75.90±15.73,all P<0.05].The mitochondrial complex IV activity in the CORM-2 group was higher than that of iCORM-2group,but there was no statistical significance(P>0.05).Compared with LPS group,iCORM-2 group and DMSO group,there was no statistically significant difference in mitochondrial complex IV activity(P>0.05).(6)fusion-fission protein expression:Compared with the Control group,the expression of Fis1 in the four groups of LPS treatment was significantly increased;while the expression level of Fis1 in the CORM-2 group was lower than that of the LPS group,iCORM-2 group,and DMSO group [Fis1:0.586±0.031 vs0.920±0.054?0.856±0.036?0.924±0.053,all P<0.05].Compared with the Control group,the expressions of Mfn2 and Opal in the LPS group,iCORM-2 group,and DMSO group decreased;while the expression levels of Mfn2 and Opal in the CORM-2 group were higher than those of the other three groups treated with LPS [Mfn2:0.919±0.063 vs 0.459±0.069?0.550±0.104?0.400±0.077;Opa1:0.767±0.062 vs 0.526±0.047?0.518±0.092?0.496±0.077,all P<0.05].There was no statistically significant difference in the expression of the above three proteins in the LPS group,iCORM-2 group and DMSO group(all P>0.05).Conclusion1.CORM-2 can improve myocardial dysfunction,reduce myocardial damage,and improve survival rate in septic rats.2.CORM-2 can inhibit excessive fission of myocardial mitochondria,promote mitochondrial fusion,and improve mitochondrial respiratory function in septic rats,which may be one of its protective mechanisms. |
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