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目的:研究阿米福汀对苯并[a]芘(Ba P)诱导C57BL/6J小鼠腹主动脉瘤(AAA)形成的作用,并探讨相关机制。方法:体外培养RAW264.7单核巨噬细胞,分为正常对照组,溶剂对照组(即DMSO组),Ba P组,低剂量(1μmol/L)阿米福汀组,中剂量(5μmol/L)阿米福汀组及高剂量(25μmol/L)阿米福汀组,应用Western blot法检测体外培养的RAW264.7单核巨噬细胞基质金属蛋白酶(MMP)-9、MMP-12、TNF-α、NF-κB表达。48只C57BL/6J小鼠(8月龄,雄性)随机分为对照组、模型组(AngⅡ+Ba P组)、低剂量(50 mg/kg)阿米福汀组、高剂量(100 mg/kg)阿米福汀组。6周后取腹主动脉察看标本形态;行HE、Masson染色观察血管形态结构;测定腹主动脉的血管周长。Western blot、免疫组化法评价腹主动脉组织中的巨噬细胞浸润情况及MMP-9、MMP-12、TNF-α、NF-κB的表达。结果:Western blot发现Ba P刺激使巨噬细胞MMP-9、MMP-12、TNF-α、NF-κB的表达升高,而阿米福汀对此有显著抑制作用,且呈剂量依赖性(P<0.05)。动物实验可见对照组成瘤率为0,高剂量阿米福汀组成瘤率为16.67%,与模型组(58.33%)和低剂量阿米福汀组(33.33%)相比显著降低(P<0.05)。免疫组化结果显示高剂量阿米福汀组腹主动脉壁的巨噬细胞浸润程度及TNF-α、MMP-9、MMP-12、NF-κB的表达较模型组和低剂量阿米福汀组相比显著下降(P<0.05)。Western blot实验结果证实高剂量阿米福汀组腹主动脉壁TNF-α、MMP-9、MMP-12、NF-κB的表达较模型组和低剂量阿米福汀组相比显著下降(P<0.05)。结论:阿米福汀可以抑制Ba P诱导的巨噬细胞的活化,同时可以抑制Ba P诱导的小鼠腹主动脉瘤发生、发展,其机制可能跟抑制NF-κB途径、巨噬细胞浸润及MMPs、TNF-α的表达有关。
AIM: To investigate the effect of amifostine on the formation of abdominal aortic aneurysm (AAA) in C57BL / 6J mice induced by benzo [a] pyrene (Ba P) and to explore its mechanism. Methods: RAW264.7 monocyte-macrophage cells were cultured in vitro and divided into normal control group, solvent control group (DMSO group), Ba P group, low dose (1μmol / L) amifostine group, medium dose (5μmol / L) amifostine group and high dose (25μmol / L) amifostine group. Western blot was used to detect the expression of matrix metalloproteinase (MMP) -9 and MMP-12 in RAW264.7 monocyte- TNF-α, NF-κB expression. Forty eight C57BL / 6J mice (8 months old and males) were randomly divided into control group, model group (Ang Ⅱ + Ba P group), low dose (50 mg / kg) amifostine group and high dose kg) amifostine group. After 6 weeks, the abdominal aorta was taken for observation of the morphology of the specimens. The morphology of blood vessels was observed by HE and Masson staining, and the perivascular length of the abdominal aorta was measured. Western blot and immunohistochemistry were used to evaluate macrophage infiltration and expression of MMP-9, MMP-12, TNF-α and NF-κB in the abdominal aorta. Results: Western blot showed that Ba P stimulated the expression of MMP-9, MMP-12, TNF-α and NF-κB in macrophages, while amifostine had a significant inhibitory effect on the macrophages in a dose-dependent manner P <0.05). Animal experiments showed that the tumor formation rate was 0 in the control group and 16.67% in the high-dose amifostine group, significantly lower than that in the model group (58.33%) and the low-dose amifostine group (33.33%) (P <0.05 ). The results of immunohistochemistry showed that the infiltration of macrophages and the expression of TNF-α, MMP-9, MMP-12 and NF-κB in abdominal aorta of high-dose amifostine group were significantly lower than those in model group and low-dose amifostine Group decreased significantly (P <0.05). The results of Western blot showed that the expression of TNF-α, MMP-9, MMP-12 and NF-κB in abdominal aorta of high-dose amifostine group was significantly lower than that in model group and low-dose amifostine group <0.05). CONCLUSION: Amifostine can inhibit the activation of macrophages induced by Ba P and inhibit the occurrence and development of abdominal aortic aneurysms induced by Ba P in mice. The mechanism may be related to inhibition of NF-κB pathway, macrophage infiltration and MMPs, TNF-α expression.