Effects of oxidative stress, PSMB5, TFEB, and lysosomes on sodium arsenite-induced liver injury in rats
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
PSMB5、 TFEB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
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
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.
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.
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
PSMB5and 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 PSMB5and TFEBin 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.