血必净注射液及其组分羟基红花黄色素A对脓毒症大鼠凝血功能和生存率影响的比较研究

Comparison of effects of Xuebijing injection and its component hydroxysafflor yellow A on coagulation and survival rates of septic rats

目的探讨血必净注射液(血必净)及其组分羟基红花黄色素A对脓毒症大鼠凝血功能和生存率的影响。方法①凝血功能评估实验:将144只雄性SD大鼠按随机数字表法分为假伤组、盲肠结扎穿孔术(CLP)致脓毒症模型组(CLP组)、CLP+血必净组及CLP+羟基红花黄色素A组,每组36只。采用CLP制备脓毒症大鼠模型;假伤组大鼠仅开腹暴露盲肠后还纳腹腔,其余步骤同CLP组;CLP+血必净组及CLP+羟基红花黄色素A组大鼠术后经尾静脉注射血必净(4 mL/kg、每日2次)或羟基红花黄色素A溶液(0.378 g/L,每次298μg、每日2次)。分别于术后6、12、24 h采用全自动凝血分析仪检测外周血凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、凝血酶时间(TT)、纤维蛋白原(Fib)和D-二聚体水平;采用酶联免疫吸附试验(ELISA)检测外周血组织因子(TF)、组织因子途径抑制物(TFPI)及可溶性血栓调节蛋白(sTM)水平。②生存分析:另取120只大鼠按随机数字表法分组(组别及处理方法同凝血功能评估实验),每组30只。绘制Kaplan-Meier生存曲线,观察并记录各组大鼠术后7 d累积生存率。结果①凝血功能评估实验结果:与假伤组比较,CLP组大鼠早期即出现明显的凝血功能异常,表现为CLP术后6 h PT即明显延长(s:8.9±0.2比8.4±0.4,P<0.01),Fib、D-二聚体、TFPI及sTM水平即明显升高〔Fib(g/L):2.8±0.3比2.3±0.1,D-二聚体(ng/L):1.8±0.2比1.5±0.1,TFPI(ng/L):131.1±10.9比102.8±10.5,sTM(μg/L):27.2±1.2比19.8±2.9,均P<0.01〕;术后12 h凝血功能异常情况愈加严重,并随时间延长进一步恶化。血必净及羟基红花黄色素A在术后6 h即能显著改善脓毒症大鼠凝血功能,表现为PT较CLP组明显缩短(s:8.3±0.2、8.3±0.1比8.9±0.2,均P<0.01),Fib、D-二聚体、TFPI及sTM水平较CLP组明显降低〔Fib(g/L):2.3±0.1、2.3±0.2比2.8±0.3,D-二聚体(ng/L):1.5±0.1、1.5±0.2比1.8±0.2,TFPI(ng/L):109.5±10.2、91.5±5.0比131.1±10.9,sTM(μg/L):22.3±1.5、21.1±1.8比27.2±1.2,均P<0.01〕;但两个干预组间凝血功能指标比较差异均无统计学意义。②生存分析结果:假伤组大鼠术后7 d均存活;而CLP组大鼠术后7 d累积生存率仅36.67%(11/30)。与CLP组比较,CLP+血必净组及CLP+羟基红花黄色素A组大鼠累积生存率均明显升高〔66.67%(20/30)、66.67%(20/30)比36.67%(11/30),均P<0.05〕,且CLP+血必净组与CLP+羟基红花黄色素A组间比较差异无统计学意义。结论血必净及其组分羟基红花黄色素A均能有效改善脓毒症大鼠凝血功能障碍,提高生存率,且二者的效应无明显差异。

Objective To explore the effects of Xuebijing injection and its component hydroxysafflor yellow A on coagulation and survival rates of septic rats.Methods①Assessment of coagulation:144 male Sprague-Dawley(SD)rats were divided into four groups by random number table:sham group,cecal ligation and puncture(CLP)induced sepsis model group(CLP group),CLP+Xuebijing group,and CLP+hydroxysafflor yellow A group,with 36 rats in each group.CLP was used for reproducing septic models.The cecum of the rats in the sham group was exposed by laparotomy and then returned to the abdominal cavity without CLP,while the other steps were the same as those in the CLP group.Rats in the CLP+Xuebijing group and CLP+hydroxysafflor yellow A group were injected with Xuebijing(4 mL/kg,twice a day)or hydroxysafflor yellow A solution(0.378 g/L,298μg each time,twice a day)through caudal vein after operation.Levels of prothrombin time(PT),activated partial thromboplastin time(APTT),thrombin time(TT),fibrinogen(Fib),and D-dimer in peripheral blood were measured by automatic coagulation analyzer at 6,12,24 hours after operation.The enzyme linked immunosorbent assay(ELISA)was applied to determine levels of tissue factor(TF),tissue factor pathway inhibitor(TFPI),and soluble thrombomodulin(sTM)in peripheral blood.②Analysis of survival rates:120 rats were divided into four groups by random number table(the same groups with those in the section of assessment of coagulation),with 30 rats in each group.The Kaplan-Meier survival curve was plotted,and the cumulative survival rates were observed and recorded for 7 days after CLP surgery.Results①Results of coagulation assessment:compared with the sham group,septic rats in the CLP group showed significant dysfunction in coagulation early,as evidenced by prolonged PT at 6 hours after CLP(s:8.9±0.2 vs.8.4±0.4,P<0.01),and significantly increased levels of Fib,D-dimer,TFPI and sTM[Fib(g/L):2.8±0.3 vs.2.3±0.1,D-dimer(ng/L):1.8±0.2 vs.1.5±0.1,TFPI(ng/L):131.1±10.9 vs.102.8±10.5,sTM(μg/L):27.2±1.2 vs.19.8±2.9,all P<0.01].The coagulation dysfunction became more and more serious at 12 hours after operation,and further deteriorated with time.The use of both Xuebijing and hydroxysafflor yellow A revealed significant improvement in coagulation of septic rats at 6 hours,as shown by shortened PT(s:8.3±0.2,8.3±0.1 vs.8.9±0.2,both P<0.01),and decreased Fib,D-dimer,TFPI and sTM as compared with those in the CLP group[Fib(g/L):2.3±0.1,2.3±0.2 vs.2.8±0.3;D-dimer(ng/L):1.5±0.1,1.5±0.2 vs.1.8±0.2;TFPI(ng/L):109.5±10.2,91.5±5.0 vs.131.1±10.9;sTM(μg/L):22.3±1.5,21.1±1.8 vs.27.2±1.2;all P<0.01].However,there was no significant difference in coagulation function between the two intervention groups.②Results of survival rates analysis:the rats in the sham group all survived 7 days after operation.The 7-day cumulative survival rate of the CLP group was only 36.67%(11/30).Compared with the CLP group,the cumulative survival rates were significantly increased in rats of the CLP+Xuebijing group and CLP+hydroxysafflor yellow A group[66.67%(20/30),66.67%(20/30)vs.36.67%(11/30),both P<0.05],but no significant difference was found between the CLP+Xuebijing group and CLP+hydroxysafflor yellow A group.Conclusion Both Xuebijing and its component hydroxysafflor yellow A appear to be capable of alleviating coagulation disorders and improving survival rates of septic rats effectively,and the effects show no significant difference between them.