张佳, 蒋硕, 潘坤, 宋丽莹, 赵金镯. 大气PM2.5加重2型糖尿病模型小鼠肝损伤与AMPK抑制的相关性[J]. 环境与职业医学, 2019, 36(6): 576-582. DOI: 10.13213/j.cnki.jeom.2019.19076
引用本文: 张佳, 蒋硕, 潘坤, 宋丽莹, 赵金镯. 大气PM2.5加重2型糖尿病模型小鼠肝损伤与AMPK抑制的相关性[J]. 环境与职业医学, 2019, 36(6): 576-582. DOI: 10.13213/j.cnki.jeom.2019.19076
ZHANG Jia, JIANG Shuo, PAN Kun, SONG Li-ying, ZHAO Jin-zhuo. Liver injury in type 2 diabetes mellitus model mice aggravated by ambient PM2.5 in association with AMPK inhibition[J]. Journal of Environmental and Occupational Medicine, 2019, 36(6): 576-582. DOI: 10.13213/j.cnki.jeom.2019.19076
Citation: ZHANG Jia, JIANG Shuo, PAN Kun, SONG Li-ying, ZHAO Jin-zhuo. Liver injury in type 2 diabetes mellitus model mice aggravated by ambient PM2.5 in association with AMPK inhibition[J]. Journal of Environmental and Occupational Medicine, 2019, 36(6): 576-582. DOI: 10.13213/j.cnki.jeom.2019.19076

大气PM2.5加重2型糖尿病模型小鼠肝损伤与AMPK抑制的相关性

Liver injury in type 2 diabetes mellitus model mice aggravated by ambient PM2.5 in association with AMPK inhibition

  • 摘要: 背景 有研究发现大气细颗粒物(PM2.5)暴露会导致肝纤维化和非酒精性脂肪肝。2型糖尿病(T2DM)患者本身因出现胰岛素抵抗、脂质堆积及炎症而致肝脏损伤严重。大气PM2.5暴露是否会加重T2DM患者肝损伤目前还不清楚,对该机制的研究较少。

    目的 探索腺苷酸活化蛋白激酶(AMPK)在大气PM2.5所致T2DM小鼠肝脏损伤中的作用。

    方法 将32只6周龄的db/db小鼠随机分为4组:洁净空气暴露组(FA组)、洁净空气暴露+5-氨基-4-甲酰胺咪唑核糖核苷酸(AICAR)干预组(FA-AIC组)、浓缩PM2.5暴露组(PM组)和浓缩PM2.5暴露+AICAR干预组(PM-AIC组)。小鼠在气象-环境动物暴露系统中每天暴露10 h,共12周,FA-AIC和PM-AIC组在第11周开始每天腹腔注射AICAR(250 mg/kg),共2周。采用苏木精-伊红(HE)染色法检测肝脏病理变化;采用实时荧光定量PCR法测定肝脏组织中AMPK、白细胞介素(IL)-6和肿瘤坏死因子(TNF-α)的mRNA表达水平;通过Westernblot法检测AMPK、P-AMPK、沉默信息调节因子2同源酶1(SIRT1)、P38-丝裂原活化蛋白激酶(MAPK)、P-P38-MAPK、蛋白激酶B(AKT)、P-AKT蛋白表达水平。

    结果 PM组肝脏脂肪变性分数、肿大分数、小叶和门管区炎症分数分别为1.50±0.42、1.10±0.35、0.80±0.12、0.70±0.16;PM-AIC组肝脏脂肪变性分数、肿大分数、小叶和门管区炎症分数分别为1.40±0.31、1.20±0.29、0.70±0.22、0.60±0.18,均大于相应的FA组(P < 0.05);FA-AIC组肝脏肿大分数和肝小叶炎症分数均小于FA组(P < 0.05);PM-AIC组和PM组肝脏脂肪变性分数、肿胀分数、小叶和门管区炎症分数的差异不具有统计学意义。结果显示,PM2.5抑制了肝脏AMPK mRNA的表达,促进了IL-6TNF-α mRNA的表达。无论是暴露于浓缩PM2.5还是清洁空气,注射AICAR后均能增加AMPK的mRNA表达(P < 0.05)。此外,暴露于浓缩PM2.5的小鼠肝脏SIRT1蛋白表达水平以及AMPK和AKT的磷酸化水平与暴露于清洁空气的小鼠相比,差异均具有统计学意义(P < 0.05)。在注射了AICAR后,暴露于洁净空气的小鼠肝脏AMPK和AKT的磷酸化水平增加(P < 0.05),但暴露于浓缩PM2.5的小鼠在注射AICAR后,肝脏内这些蛋白表达水平变化均无统计学意义(P > 0.05)。

    结论 大气PM2.5加重2型糖尿病模型小鼠肝损伤与AMPK抑制相关。

     

    Abstract: Background Studies have found that exposure to ambient fine particulate matters (PM2.5) could lead to liver fibrosis and non-alcoholic fatty liver disease. Type 2 diabetes mellitus (T2DM) patients have severe liver damage due to insulin resistance, lipid accumulation, and inflammation in the liver. It is not clear whether ambient PM2.5 exposure would aggravate liver damage in T2DM patients. Currently, there are few studies on its mechanism.

    Objective This experiment is designed to explore the role of AMP-activated protein kinase (AMPK) in liver injury of T2DM mice caused by ambient PM2.5.

    Methods Thirty-two six-week-old db/db mice were randomly divided into four groups:filtered air (FA) group, filtered air plus 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) (FA-AIC) group, concentrated PM2.5 (PM) group, and concentrated PM2.5 plus AICAR (PM-AIC) group. The mice were exposed using Shanghai Meteorological and Environmental Animal Exposure System (ShanghaiMETAS) 10 h per day for 12 weeks. The FA-AIC and PM-AIC groups received daily intraperitoneal injection of AICAR (250 mg/kg) at week 11 for a total of 2 weeks. The pathological changes of liver were determined after hematoxylin-eosin (HE) staining. The mRNA expression levels of AMPK, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were determined by real-time fluorescent quantitative PCR. The protein expression levels of AMPK, P-AMPK, silent mating type information regulation 2 homolog 1 (SIRT1), P38-mitogen-activated protein kinase (MAPK), P-P38-MAPK, protein kinase B (AKT), and P-AKT were detected by Western blot.

    Results The scores of the PM group of liver steatosis, ballooning, lobular inflammation, and portal inflammation were 1.50±0.42, 1.10±0.35, 0.80±0.12, and 0.70±0.16, respectively; the scores of the PM-ACI group were 1.40±0.31, 1.20±0.29, 0.70±0.22, and 0.60±0.18, respectively, which were all higher than those of the FA group (P < 0.05); the FA-AIC group showed lower scores of liver ballooning and lobular inflammation than the FA group (P < 0.05); there were no differences in the above four indicators between the PM-AIC and the PM groups (P > 0.05). The expression of AMPK mRNA in liver was inhibited by PM2.5 exposure; however, the expressions of IL-6 and TNF-α mRNA in liver were elevated by PM2.5 exposure. The mRNA expression of AMPK was increased after AICAR injection in both groups treated with concentrated PM2.5 and filtered air (P < 0.05). The protein expressions of SIRT1 and phosphorylation of AMPK and AKT in the mice exposed to concentrated PM2.5 were significantly lower than those in the mice treated with filtered air (P < 0.05). After AICAR injection, the phosphorylation levels of AMPK and AKT in liver of the mice treated with filtered air increased significantly (P < 0.05), but there was no significant difference in the PM-AIC group (P > 0.05).

    Conclusion AMPK inhibition is in association with liver injury in type 2 diabetes mellitus model mice aggravated by ambient PM2.5.

     

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