腹腔注射内毒素制备脓毒症心肌抑制大鼠模型的验证

Validation of a septic myocardial inhibition model by intraperitoneal injection of endotoxin in rats

目的通过心脏超声方法验证脓毒症心肌抑制大鼠模型的建立。方法按随机数字表法将成年雄性SD大鼠分为对照组和模型组,每组10只。以腹腔注射内毒素10 mg/kg制备脓毒症心肌抑制动物模型;对照组则给予相应体积生理盐水。制模后8 h进行心脏超声检测,分别测量左室舒张期末内径(LVDd)、左室舒张期末容积(LVEDV)、左室收缩期末内径(LVDs)、左室收缩期末容积(LVESV)、左室射血分数(LVEF)、右室舒张期末内径(RVDd)、右室收缩期末内径(RVDs)、心率(HR)、肺动脉正向流速、主动脉流速。随后处死大鼠,获取血清和心肌组织,采用酶联免疫吸附试验(ELISA)测定血清肿瘤坏死因子-α(TNF-α)、核转录因子-?κB(NF-κB)、白细胞介素-1(IL-1)、心肌肌钙蛋白I(cTnI)、B型脑钠肽(BNP)水平;采用实时荧光定量聚合酶链反应(real-time PCR)检测心肌组织TNF-α、IL-1、NF-κB mRNA表达;采用苏木素-伊红(HE)染色,于光镜下观察心肌组织病理学改变。结果与对照组比较,模型组大鼠心肌抑制明显,表现为心脏整体外形扩大,HR明显增快(次/min:449.0±21.1比356.7±23.3,P<0.01),左心和右心功能均有影响,以左心功能受损为主,LVDd、LVDs、LVEDV、LVESV均扩大〔LVDd(mm):10.03±0.95比7.04±0.71,LVDs(mm):5.95±0.71比3.07±0.05,LVEDV(mL):2.11±0.53比0.81±0.21,LVESV(mL):0.51±0.16比0.07±0.01,均P<0.05〕,LVEF明显下降(0.760±0.046比0.901±0.025,P<0.01),RVDd明显增大(mm:4.48±0.58比3.22±0.20,P<0.05),肺动脉正向流速明显下降(cm/s:64.2±9.3比89.0±0.8,P<0.05)。与对照组相比,模型组血清NF-κB、TNF-α、IL-1、BNP、cTnI水平均明显升高〔NF-κB(ng/L):103.84±6.55比57.29±41.34,TNF-α(ng/L):1?198.32±164.07比835.45±24.01,IL-1(ng/L):1?089.90±221.96比746.19±165.83,BNP(ng/L):1?097.36±293.84比454.71±197.79,cTnI(ng/L):6?938.59±1?400.21比3?731.90±1?349.31,均P<0.01〕,心肌组织TNF-α、NF-κB、IL-1的mRNA表达也明显升高(2-ΔΔCT:1.50±0.42比0.71±0.40,1.10±0.17比0.63±0.06,1.77±0.67比0.10±0.03,均P<0.05)。光镜下观察,对照组心肌组织结构正常,心肌纤维排列整齐,细胞间结构清晰;模型组心肌组织间结构较疏松、模糊,细胞肿胀,具有明显的病理学改变。结论经心脏超声、炎性因子表达、心肌标志物测定、病理学观察等评估心功能,验证腹腔注射10 mg/kg内毒素可成功制备脓毒症心肌抑制大鼠模型。

Objective To establish septic myocardial inhibition rat model by echocardiography.Methods Twenty adult male Sprague-Dawley(SD)rats were divided into control group and model group according to the random number table method,with 10 rats in each group.The rat model of septic myocardial inhibition was reproduced by intraperitoneal injection of 10 mg/kg lipopolysaccharide,while the control group was given the same volume of saline.The left ventricular end-diastolic diameter(LVDd),left ventricular end-diastolic volume(LVEDV),left ventricular end-systolic diameter(LVDs),left ventricular end-systolic volume(LVESV),left ventricular ejection fraction(LVEF),right ventricular end-diastolic diameter(RVDd),right ventricular end-systolic diameter(RVDs),heart rate(HR),positive pulmonary artery flow rate and aortic flow rate were measured at 8 hours after model establishment by echocardiography.Then the rats were sacrificed to harvest serum and myocardial tissue.The levels of serum tumor necrosis factor-α(TNF-α),nuclear factor-κB(NF-κB),interleukin-1(IL-1),cardiac troponin I(cTnI)and B-type brain natriuretic peptide(BNP)were measured by enzyme linked immunosorbent assay(ELISA).The mRNA expressions of TNF-α,IL-1 and NF-κB in myocardium were detected by real-time polymerase chain reaction(real-time PCR).The pathological changes of myocardium were observed by hematoxylin-eosin(HE)staining under light microscope.Results Compared with control group,myocardial inhibition was obviously observed in model group,manifesting as enlargement of overall shape of heart,and prominent increase of HR(bpm:449.0±21.1 vs.356.7±23.3,P<0.01);left ventricular and right ventricular functions were affected,LVDd,LVDs,LVEDV,LVESV were enlarged[LVDd(mm):10.03±0.95 vs.7.04±0.71,LVDs(mm):5.95±0.71 vs.3.07±0.05,LVEDV(mL):2.11±0.53 vs.0.81±0.21,LVESV(mL):0.51±0.16 vs.0.07±0.01,all P<0.05],LVEF was significantly decreased(0.760±0.046 vs.0.901±0.025,P<0.01),RVDd was significantly increased(mm:4.48±0.58 vs.3.22±0.20,P<0.05),and positive pulmonary artery velocity was significantly decreased(cm/s:64.2±9.3 vs.89.0±0.8,P<0.05).Compared with control group,the levels of serum NF-κB,TNF-α,IL-1,BNP and cTnI in model group were significantly increased[NF-κB(ng/L):103.84±6.55 vs.57.29±41.34,TNF-α(ng/L):1 198.32±164.07 vs.835.45±24.01,IL-1(ng/L):1 089.90±221.96 vs.746.19±165.83,BNP(ng/L):1 097.36±293.84 vs.454.71±197.79,cTnI(ng/L):6 938.59±1 400.21 vs.3 731.90±1 349.31,all P<0.01],the mRNA expressions of TNF-α,NF-κB and IL-1 in myocardial tissue were significantly increased(2-ΔΔCT:1.50±0.42 vs.0.71±0.40,1.10±0.17 vs.0.63±0.06,1.77±0.67 vs.0.10±0.03,all P<0.05).It was shown by HE staining that the structure of myocardial tissue in control group was distinct,the arrangement of myocardial fibers was neat,and transverse was clear;the structure of myocardial tissue in model group was loose,blurred,and the cells were swollen,with obvious pathological changes.Conclusions Cardiac function was assessed by echocardiography,expression of inflammatory factors,myocardial markers and pathological changes.It was verified that intraperitoneal injection of 10 mg/kg endotoxin could successfully prepare a rat model of septic myocardial inhibition.