汪红铃, 时明阳, 毕顶念, 支海燕, 胡倩, 胡勇. 氧化应激、PSMB5、TFEB和溶酶体在亚砷酸钠致大鼠肝损伤中的作用[J]. 环境与职业医学, 2022, 39(10): 1134-1139. DOI: 10.11836/JEOM22128
引用本文: 汪红铃, 时明阳, 毕顶念, 支海燕, 胡倩, 胡勇. 氧化应激、PSMB5、TFEB和溶酶体在亚砷酸钠致大鼠肝损伤中的作用[J]. 环境与职业医学, 2022, 39(10): 1134-1139. DOI: 10.11836/JEOM22128
WANG Hongling, SHI Mingyang, BI Dingnian, ZHI Haiyan, HU Qian, HU Yong. Effects of oxidative stress, PSMB5, TFEB, and lysosomes on sodium arsenite-induced liver injury in rats[J]. Journal of Environmental and Occupational Medicine, 2022, 39(10): 1134-1139. DOI: 10.11836/JEOM22128
Citation: WANG Hongling, SHI Mingyang, BI Dingnian, ZHI Haiyan, HU Qian, HU Yong. Effects of oxidative stress, PSMB5, TFEB, and lysosomes on sodium arsenite-induced liver injury in rats[J]. Journal of Environmental and Occupational Medicine, 2022, 39(10): 1134-1139. DOI: 10.11836/JEOM22128

氧化应激、PSMB5、TFEB和溶酶体在亚砷酸钠致大鼠肝损伤中的作用

Effects of oxidative stress, PSMB5, TFEB, and lysosomes on sodium arsenite-induced liver injury in rats

  • 摘要: 背景

    地方性砷中毒肝损伤较为严重。研究表明,氧化应激、蛋白酶体β5亚基(PSMB5)、调节转录因子EB(TFEB)和溶酶体与肝损伤有关,但它们与砷中毒肝损伤的具体联系尚不清楚。

    目的

    利用课题组前期建立的亚砷酸钠(NaAsO2)致大鼠肝损伤模型,检测肝组织中PSMB5、TFEB和溶酶体关联膜蛋白1(LAMP1)的表达。

    方法

    24只SPF级Wistar大鼠随机分为对照组,低、中、高浓度组,每组6只,雌雄各半;染毒浓度分别为0、25、50、100 mg·L−1 NaAsO2水溶液,染毒24周。染毒结束后,麻醉大鼠取肝脏。采用化学比色法检测肝组织碱性磷酸酶(ALP)、谷丙转氨酶(ALT)、总胆汁酸(TBA)、过氧化氢酶(CAT)水平;采用酶联免疫吸附实验(ELISA)检测肝组织脂质过氧化物(LPO)、4-羟基壬烯醛(4-HNE)、LAMP1、组织蛋白酶D(CTSD)水平;采用实时荧光定量PCR法检测肝组织PSMB5TFEB转录表达水平;采用免疫组化检测肝组织PSMB5、TFEB、磷酸化TFEB(p-TFEB)蛋白表达情况。

    结果

    化学比色法和ELISA法结果显示:与对照组相比,各染砷组大鼠肝匀浆ALP、TBA、LAMP1,中、高浓度组ALT、LPO,高浓度组4-HNE、CTSD水平均升高,而各染砷组CAT活性均降低(P<0.05)。实时荧光定量PCR结果显示:各染砷组肝组织PSMB5、TFEB转录水平与对照组相比均下降(P<0.05)。免疫组化结果显示:与对照组比较,各染砷组肝组织PSMB5,中、高浓度组TFEB蛋白表达均下降,而各染砷组p-TFEB蛋白表达均升高(P<0.05);随染砷浓度增加,TFEB蛋白表达在胞核逐渐减弱,p-TFEB蛋白表达在细胞质中逐渐增强,p-TFEB在胞核未见表达。Pearson相关性分析结果显示:肝组织PSMB5与CAT呈正相关(r=0.818,P<0.05),与4-HNE、p-TFEB呈负相关(r=−0.582,r=−0.899;P<0.05);TFEB与CTSD、LAMP1均呈负相关(r=−0.457,r=−0.564;P<0.05);CTSD与ALT、ALP均呈正相关(r=0.529、r=0.485;P<0.05)。

    结论

    NaAsO2长期暴露可诱导氧化应激发生,抑制PSMB5和TFEB表达,促使细胞质中p-TFEB累积增多,活性TFEB入核减少,溶酶体受损,引起肝脏损伤。

     

    Abstract: Background

    Liver damage presented in endemic arsenic poisoning is usually serious. Studies have shown that oxidative stress, proteasome beta 5 subunit (PSMB5), regulatory transcription factor EB (TFEB), and lysosomes are associated with liver injury, but their specific links to arsenic-induced liver injury remain unclear.

    Objective

    Using a sodium arsenite (NaAsO2)-induced rat liver injury model established earlier by the research group, the expressions of PSMB5, TFEB, and lysosomal associated membrane protein 1 (LAMP1) in liver tissues were detected.

    Methods

    Twenty-four SPF Wistar rats were randomly divided into control group, and low, medium, and high dose groups, with 6 rats in each group, half male and half female. The exposure concentrations were 0, 25, 50, and 100 mg·L−1 NaAsO2 solutions for 24 weeks. At the end of the experiment, liver was dissected after rats were anesthetized. The levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), total bile acid (TBA), and catalase (CAT) in liver tissues were detected by chemical colorimetry, and the levels of lipid peroxide (LPO), 4-hydroxynonenal (4-HNE), LAMP1, and cathepsin D (CTSD) in liver tissues were detected by enzyme-linked immunosorbent assay (ELISA); the transcriptional expression levels of PSMB5 and TFEB in liver tissues were detected by real-time fluorescence quantitative PCR (RT-qPCR), and the protein expressions of PSMB5, TFEB, and phosphorylated TFEB (p-TFEB) in liver tissues were detected by immunohistochemistry.

    Results

    The results of chemical colorimetry and ELISA showed that compared with the control group, the liver homogenate levels of ALP, TBA, and LAMP1 of each arsenic-exposed group, the ALT and LPO in the medium and high concentration groups, the 4-HNE and CTSD in the high concentration group were increased, while the CAT activity of each arsenic-exposed group was decreased (P<0.05). The results of real-time fluorescence quantitative PCR showed that the transcription levels of PSMB5 and TFEB in the liver tissues of each arsenic-exposed group were decreased compared with those of the control group (P<0.05). The results of immunohistochemistry showed that compared with the control group, the expression of PSMB5 of each arsenic-exposed group were decreased, the expression of TFEB in the medium and high concentration groups was decreased, while the expression of p-TFEB of each arsenic-exposed group was increased (P<0.05). The expression of TFEB protein gradually decreased in the nucleus, while the expression of p-TFEB protein gradually increased in the cytoplasm, but no expression of p-TFEB was found in the nucleus. The results of Pearson correlation analysis showed that PSMB5 in liver tissues was positively correlated with CAT (r=0.818, P<0.05), and negatively correlated with 4-HNE and p-TFEB (r=−0.582, r=−0.899; P<0.05); TFEB was negatively correlated with CTSD and LAMP1 (r=−0.457, r=−0.564; P<0.05); CTSD was positively correlated with ALT and ALP (r=0.529, r=0.485; P<0.05).

    Conclusion

    Long-term exposure to NaAsO2 can induce oxidative stress, inhibit the expression of PSMB5 and TFEB, promote the accumulation of p-TFEB in the cytoplasm, decrease the nuclear entry of active TFEB, damage the lysosome, and cause liver damage.

     

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