丹参多酚酸盐抑制库普弗细胞活化减轻肝脏再灌注损伤

Salvianolate ameliorates warm hepatic ischemia reperfusion injury by inhibiting Kupffer cells activation

目的研究丹参多酚酸盐(SV)对肝脏缺血再灌注(IR)损伤的保护作用及其机制。方法选择健康、雄性的C57BL/6小鼠24只,随机分为假手术组、0.9%氯化钠溶液(NS)组和SV组,每组8只。假手术组小鼠仅接受开腹及关腹操作,NS组和SV组小鼠建立IR模型。IR术前1h,SV组小鼠经尾静脉注射SV60mg/kg,NS组小鼠注射等量NS。再灌注6h,检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)水平,并观察肝组织形态学的变化。采用酶联免疫吸附试验(ELISA)及荧光定量反转录-聚合酶链反应(RT-PCR)检测血清及肝组织内肿瘤坏死因子(TNF)-α和白细胞介素(IL)-6的表达水平;应用免疫组织化学技术检测各组肝脏标本内库普弗细胞特异性抗原F4/80的表达情况,采用荧光定量RT-PCR检测库普弗细胞表面分子CD11bmRNA的表达水平。分离小鼠腹腔巨噬细胞,以脂多糖(LPS)作为活化剂,TNF-α作为巨噬细胞活化指标,检测体外培养体系中SV能否抑制巨噬细胞活化。结果与NS组相比,SV组再灌注6h的血清ALT、AST水平显著降低(P值均<0.01),血清TNF-α和IL-6表达水平显著降低(P值分别<0.05、0.01),肝组织内TNF-αmRNA和IL-6mRNA表达水平亦显著降低(P值均<0.05),肝组织内F4/80阳性库普弗细胞的浸润显著减少(P<0.05),肝组织内库普弗细胞表面分子CD11bmRNA的相对表达水平显著降低(P<0.05)。SV体外抑制腹腔巨噬细胞活化实验显示,SV组的上清液内TNF-α水平显著低于NS组(P<0.05)。结论 SV对肝脏IR损伤具有保护作用,其机制可能与抑制肝脏再灌注后库普弗细胞的活化有关。

Objective To evaluate the protective effect of salvianolate against warm hepatic ischemia reperfusion （IR） in mice and to study the related mechanism. Methods Twenty-four healthy male C57BL/6 mice were randomly divided into three groups with eight mice in each group：sham operation group,normal saline group and salvianolate group. Mice in the salvianolate group were injected with 60 mg/kg salvianolate via tail vein 1 h before IR,those in the normal saline group were given normal saline in the same manner,and those in the sham group only received sham operation. At 6 h after reperfusion,the serum levels of alanine transiminase （ALT） and aspartate transaminase （AST） were determined and hepatic histological changes were also examined. The expression of tumor necrosis factor （TNF）-α and IL-6 in serum and liver samples were determined by enzyme linked immunosor bant assay （ELISA） and real-time reverse transcription-polymerase chain reaction （RT-PCR）. The infiltration of F4/80 positive Kupffer cells in liver samples was detected by immunohistochemical analyses,and mRNA levels of CD11b were also examined. Furthermore,peritoneal macrophages were isolated and co-cultured with salvianolate or saline. After lipopolysaccharides （LPS） stimulation,the activation of macrophages was determined by measuring the expression of TNF-α. Results Compared with normal saline group,6 h after reperfusion mice in salvianolate group exhibited significantly lower levels of ALT and AST （P0.01）,lower levels of TNF-α and IL-6 （P0.05 or 0.01） in the serum and liver samples （P0.05）,less F4/80 positive Kupffer cell infiltration （P0.05）,and lower expression of CD11b molecule （P0.05）. Furthermore,macrophage activation test showed that supernatant TNF-α level in salvianolate group was significantly lower than that in the normal saline group （P0.05）. Conclusion Salvianolate can ameliorate the warm hepatic IR injury,which might be associated with the inhibition of Kupffer cell activation.