Martius Gesa, Cameron Silke, Rave-Fränk Margret, Hess Clemens F, Wolff Hendrik A, Malik Ihtzaz A
Department of Gastroenterology and Endocrinology, University Medical Center Goettingen, Robert-Koch-Strasse 40, Goettingen 37075, Niedersachsen, Germany.
Department of Radiotherapy and Radiooncology, University Medical Center Goettingen, Robert-Koch-Strasse 40, Goettingen 37075, Niedersachsen, Germany.
Int J Mol Sci. 2015 Mar 2;16(3):4682-97. doi: 10.3390/ijms16034682.
Previously, we reported a radiation-induced inflammation triggering fat-accumulation through fatty-acid-translocase/cluster of differentiation protein 36 (FAT/CD36) in rat liver. Furthermore, inhibition of radiation-induced FAT/CD36-expression by anti-tumor necrosis factor-α (anti-TNF-α) (infliximab) was shown in vitro. The current study investigates fat-accumulation in a mouse-model of single-dose liver-irradiation (25-Gray) and the effect of anti-TNF-α-therapy on FAT/CD36 gene-expression. Mice livers were selectively irradiated in vivo in presence or absence of infliximab. Serum- and hepatic-triglycerides, mRNA, and protein were analyzed by colorimetric assays, RT-PCR, Immunofluorescence and Western-Blot, respectively. Sudan-staining was used demonstrating fat-accumulation in tissue. In mice livers, early (1-3 h) induction of TNF-α-expression, a pro-inflammatory cytokine, was observed. It was followed by elevated hepatic-triglyceride level (6-12 h), compared to sham-irradiated controls. In contrast, serum-triglyceride level was decreased at these time points. Similar to triglyceride level in mice livers, Sudan staining of liver cryosections showed a quick (6-12 h) increase of fat-droplets after irradiation. Furthermore, expression of fat-transporter-protein FAT/CD36 was increased at protein level caused by radiation or TNF-α. TNF-α-blockage by anti-TNF-α showed an early inhibition of radiation-induced FAT/CD36 expression in mice livers. Immunohistochemistry showed basolateral and cytoplasmic expression of FAT/CD36 in hepatocytes. Moreover, co-localization of FAT/CD36 was detected with α-smooth muscle actin (α-SMA+) cells and F4/80+ macrophages. In summary, hepatic-radiation triggers fat-accumulation in mice livers, involving acute-phase-processes. Accordingly, anti-TNF-α-therapy prevented early radiation-induced expression of FAT/CD36 in vivo.
此前,我们报道过辐射诱导的炎症通过脂肪酸转运蛋白/分化簇蛋白36(FAT/CD36)在大鼠肝脏中引发脂肪堆积。此外,体外实验表明抗肿瘤坏死因子-α(抗TNF-α,英夫利昔单抗)可抑制辐射诱导的FAT/CD36表达。本研究调查了单剂量肝脏照射(25格雷)小鼠模型中的脂肪堆积情况以及抗TNF-α治疗对FAT/CD36基因表达的影响。在有或没有英夫利昔单抗的情况下,对小鼠肝脏进行体内选择性照射。分别通过比色法、逆转录聚合酶链反应(RT-PCR)、免疫荧光和蛋白质印迹法分析血清和肝脏甘油三酯、mRNA和蛋白质。采用苏丹染色法显示组织中的脂肪堆积。在小鼠肝脏中,观察到促炎细胞因子TNF-α表达的早期(1 - 3小时)诱导。随后,与假照射对照组相比,肝脏甘油三酯水平升高(6 - 12小时)。相比之下,这些时间点的血清甘油三酯水平降低。与小鼠肝脏中的甘油三酯水平相似,肝脏冰冻切片的苏丹染色显示照射后脂肪滴迅速增加(6 - 12小时)。此外,辐射或TNF-α导致脂肪转运蛋白FAT/CD36的蛋白质水平表达增加。抗TNF-α阻断TNF-α显示可早期抑制小鼠肝脏中辐射诱导的FAT/CD36表达。免疫组织化学显示FAT/CD36在肝细胞的基底外侧和细胞质中表达。此外,还检测到FAT/CD36与α平滑肌肌动蛋白(α-SMA+)细胞和F4/80+巨噬细胞的共定位。总之,肝脏辐射会引发小鼠肝脏中的脂肪堆积,涉及急性期过程。因此,抗TNF-α治疗可在体内预防早期辐射诱导的FAT/CD36表达。