血红素氧合酶-1对老龄脓毒症大鼠微循环及肝脏功能的影响

Effects of heme oxygenase-1 on microcirculation and hepatic function in aged septic rats

目的研究血红素氧合酶-1(HO-1)对老龄脓毒症大鼠微循环及肝脏功能的影响及其作用机制。方法采用盲肠结扎穿孔术(CLP)制备老龄脓毒症大鼠模型,72只老龄Wistar雄性大鼠按随机数字表法分为四组:假手术组(sham组)、脓毒症组(CLP组)、氯化血红素组(CLP+hemin组)及锌卟啉原组(CLP+znpp组),分时相检测各组丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、凝血酶原时间(PT)、活化部分凝血活酶时间(APTT),ELISA法检测血浆活化蛋白C(APC)、蛋白C(PC)、肿瘤坏死因子(TNF)-α水平变化;应用MSB(Martius-Scarlet-Blue)染色对比各组肝脏组织血栓形成情况;应用免疫荧光共聚焦显微镜比较各组大鼠肝脏PC表达情况;Western印迹检测肝脏组织HO-1、PC表达水平。结果与对照组相比,CLP组大鼠血浆APC、PC水平下降(P<0.05),TNF-α水平上升(P<0.05),PT、APTT缩短(P<0.05),肝脏微血栓形成增加(P<0.05),ALT、AST升高(P<0.05),肝脏PC表达下降(P<0.05),此变化在CLP+znpp组中更为明显;而与CLP组相比,CLP+hemin组大鼠血浆APC、PC水平上升(P<0.01),TNF-α水平下降(P<0.05),PT、APTT延长(P<0.05),肝脏微血栓明显减少(P<0.05),ALT、AST下降(P<0.01),肝脏PC表达上调(P<0.01)。结论 HO-1可以上调老龄脓毒症大鼠肝脏中PC表达,提高血浆中PC、APC水平,通过PC途径抑制老龄脓毒症大鼠肝脏微血栓形成,改善微循环障碍,保护肝脏功能。

Objective To investigate the effect and mechanism of heme oxygenase-1 （HO-1） on microcirculation and hepatic func- tion in aged septic rats. Methods Aged septic rat was induced by cecal ligation and puncture （CLP）. Seventy-two Wistar rats were ran- domly assigned into sham, CLP, CLP ＋ heroin （heroin, an HO-1 inducer）, and CLP ＋ znpp （zinc protoporphyrin IX, an HO-1 inhibitor） groups. At each of the indicated time points, the differentiations of alanine amino transferase（ALT） , aspartate amino transferase （AST）, prothrombin time （FF） and activated partial thromboplastin time （APTF） in each group were analyzed. The plasma levels of tumor necrosis factor alpha （TNF-α）, protein C （PC） and activated protein C （aPC） was assayed by ELISA. Histopathology examination was performed by Martius-Searlet-Blue（ MSB ）staining method. The expressions of HO-1 and PC in liver tissue were tested by Western blot assay and immunofluorescence was used to determine PC expression in liver tissues. Results Compared with those of sham group, the plasma levels of PC and APC were significantly decreased （P 〈 0. 01 ） ,and the expression of TNF-ct was significantly increased （P 〈 0. 05 ） in CLP group; PT and AFTT were shortened （ P 〈 0. 05 ） , the number of mierothrombus was significantly increased（ P 〈 0. 05 ） , the plasma levels of ALT and AST were increased （P 〈0. 01 ） , and the expression of PC in liver tissues was deereased（P 〈0. 01 ） in CLP group . Administration of heroin increased the plasma levels of PC and APC, decreased the plasma levels of TNF-α （ P 〈 0. 05 ） , prolonged PT and APTF （ P 〈 0. 05 ） , decreased the number of microthrombus （P 〈 0. 05 ）, decreased the plasma levels of ALT and AST （P 〈 0. 01 ） , and increased the expression of PC in liver tissues. Whereas znpp aggravated them. Conclusions HO-1 could increase the expression of PC in liver tissue and the plas- ma levels of PC and APC, accordingly restraining the formation of microthrombus and improving microcirculation and hepatic function via the protein C system in aged septic rats.