复方依达拉奉注射液抗脓毒症作用及机制研究

Anti-sepses Effect of Compound of Edaravone and Its Possible Mechanism

目的:研究复方依达拉奉注射液对脓毒症体内及体外模型的影响。方法:将130只SD大鼠随机分为7组:假手术组(Sham)、模型组(Model)、依达拉奉低剂量组(3 mg·kg-1)、依达拉奉高剂量组(6 mg·kg-1)、复方依达拉奉低剂量组(3.75 mg·kg-1)、复方依达拉奉高剂量组(7.5mg·kg-1)、冰片组(给予2-莰醇1.5 mg·kg-1);除假手术组10只外,每组均为20只。Sham组仅开腹不结扎,其余组均采用盲肠结扎穿孔术建立大鼠脓毒症模型。术后30 min、4 h、12 h分别交叉循环尾静脉注射相应的药物,假手术组与模型组给予生理盐水。术后6 h,检测血清肿瘤坏死因子α(TNF-α)、白介素6(IL-6)、谷草转氨酶(AST)、碱性磷酸酶(ALP)和总胆红素(TBIL)水平,并观察动物24 h生存率。采用LPS刺激小鼠RAW 264.7巨噬细胞建立炎症模型,在不同浓度的依达拉奉注射液(E3:333μM),复方依达拉奉注射液(E3B3:依达拉奉333μM加冰片333μM、E3B4:依达拉奉333μM加冰片1000μM)和冰片(B3:333μM、B4:1000μM)作用下,检测白介素6(IL-6)的浓度及环氧酶2(COX-2)蛋白的表达。结果:对比于假手术组、模型组动物血清炎症因子(TNF-α、IL-6)水平显著升高(P<0.01),肝功能指标(AST、ALP、TBIL)也明显增加(P<0.05);复方依达拉奉注射液能明显降低血清TNF-α、IL-6、AST、ALP水平(P<0.05),对血清TBIL水平也具减缓作用。依达拉奉、冰片和复方依达拉奉均可降低细胞上清液IL-6、COX-2蛋白水平,以复方依达拉奉最优。结论:复方依达拉奉注射液可能通过抑制COX-2,降低促炎因子的产生,缓解肝脏的损伤而起到抗脓毒症的作用,且优于单方依达拉奉和冰片。

Objective: To explore the Compound of Edaravone injection(C.EDA) on sepsis in vivo and vitro models. Methods: One hundred and thirty SD rats were randomly divided into seven groups: sham operation group, model group, low-EDA group(L-EDA, 3 mg·kg-1), high-EDA group(H-EDA, 6 mg·kg-1),low-C.EDA group(L-C.EDA, 3.75 mg·kg-1), high- C.EDA group(H-C.EDA, 7.5 mg·kg-1) and borneol group(BOR, 1.5 mg·kg-1) with 20 rats in each group except the sham group. The septic rat models were established with cecal ligation and puncture except the sham group, the later accepted only laparotomy without ligation. At 30 min, 4 h and 12 h after the surgery, drugs were administered alternately and circularly by tail vein injection, rats in the sham and model groups were given physiological saline. The concentrations of serum tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), aspartate aminotransferase(AST), alkaline phosphatase(ALP) and total bilirubin(TBIL) were measured 6 h after the surgery, and the mortality of each group were recorded 24 h after the surgery too. RAW 264.7 cells were pretreated with LPS 1 μg to set up the inflammatory model, various concentrations of EDA(E3: edaravone 333 μM), C.EDA(E3B3:edaravone 333 μM plus borneol 333 μM; E3B4: edaravone 333 μM plus borneol 1000 μM) or borneol(B3:borneol 333 μM; B4: borneol 1000 μM) were administered to explore their effects on the production of IL-6 and the expression of COX-2 in these LPS-stimulated cells. Results: Compared with the sham operation group, the levels of serum inflammatory factors(TNF-α, IL-6) increased significantly(P<0.01), the liver function(AST, ALP, TBIL) was also raised(P<0.05) in the model group. The levels of serum TNF-α, IL-6, AST and ALP in the C.EDA group were notably lower than those in the model group(P<0.05), the level of serum TBIL decreased slowly compared with the model group. EDA, BOR and C.EDA could decrease the activity of IL-6 and COX-2 in cell supernatant, and C.EDA was the best. Conclusion: C.EDA has antisepsis effects possibly by inhibiting the production of COX-2 and proinflammatory factors and attenuating hepatic injury, and it is superior to EDA and BOR.