Abstract：
OBJECTIVE: To investigate the protective effect of quercetin (QR) on acute liver injury induced by diquat (DQ) poisoning in mice and its mechanism.
METHODS: Eighty healthy male C57BL/6 mice with SPF grade were randomly divided into control group, DQ model group, QR treatment group, and QR control group, with 20 mice in each group. The DQ poisoning model was established by a one-time intraperitoneal injection of DQ solution (40 mg/kg); the control and QR control groups received equivalent amounts of distilled water through intraperitoneal injection. Four hours after modeling, the QR treatment group and the QR control group received 0.5 mL QR solution (50 mg/kg) through gavage. Meanwhile, an equivalent amount of distilled water was given orally to the control group and the DQ model group. The treatments above were administered once daily for seven consecutive days. Afterwards, the mice were anesthetized, blood and liver tissues were collected for following tests: changes in the structure of mice liver tissue were observed using transmission electron microscopy; the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using enzyme linked immunosorbent assay (ELISA); the levels of glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) in liver tissues were measured using the water-soluble tetrazolium-1 (WST-1) method, the thiobarbituric acid (TBA) method, and enzymatic methods, respectively; the protein expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), Kelch-like ECH-associated protein 1 (Keap1), and activated caspase-9 in liver tissues were detected using Western blotting.
RESULTS: Severe mitochondrial damage was observed in the liver tissues of mice in the DQ model group using transmission electron microscopy, yet mitochondrial damage in the QR treatment group showed significant alleviation. Compared to the control group, the DQ model group had significantly increased levels of MDA in liver tissue, serum AST, and ALT, yet had significantly decreased levels of GSH and SOD in liver tissue. In comparison to the DQ model group, the QR treatment group exhibited significant reductions in serum levels of ALT and AST, as well as MDA levels in liver tissue [ALT (U/L): 52.60±6.44 vs. 95.70±8.00, AST (U/L): 170.45±19.33 vs. 251.10±13.09, MDA (nmol/mg): 12.63±3.41 vs. 18.04±3.72], and notable increases in GSH and SOD levels in liver tissue [GSH (μmol/mg): 39.49±6.33 vs. 20.26±3.96, SOD (U/mg): 121.40±11.75 vs. 81.67±10.01], all the differences were statistically significant (all P < 0.01). Western blotting results indicated that the protein expressions of Nrf2 and HO-1 in liver tissues of the DQ model group were significantly decreased compared to the control group. On the other hand, the protein expressions of Keap1 and activated caspase-9 were conspicuously higher when compared to the control group. In comparison to the DQ model group, the QR treatment group showed a significant increase in the protein expressions of Nrf2 and HO-1 in liver tissues (Nrf2/β-actin: 1.17±0.08 vs. 0.92±0.45, HO-1/β-actin: 1.53±0.17 vs. 0.84±0.09). By contrast, there was a notable decrease in the protein expressions of Keap1 and activated caspase-9 (Keap1/β-actin: 0.48±0.06 vs. 1.22±0.09, activated caspase-9/β-actin: 1.17±0.12 vs. 1.59±0.30), the differences were statistically significant (all P < 0.01).
CONCLUSIONS: QR may reduce acute liver injury induced by DQ poisoning in mice via activating Keap1/Nrf2 signaling pathway.
METHODS: Eighty healthy male C57BL/6 mice with SPF grade were randomly divided into control group, DQ model group, QR treatment group, and QR control group, with 20 mice in each group. The DQ poisoning model was established by a one-time intraperitoneal injection of DQ solution (40 mg/kg); the control and QR control groups received equivalent amounts of distilled water through intraperitoneal injection. Four hours after modeling, the QR treatment group and the QR control group received 0.5 mL QR solution (50 mg/kg) through gavage. Meanwhile, an equivalent amount of distilled water was given orally to the control group and the DQ model group. The treatments above were administered once daily for seven consecutive days. Afterwards, the mice were anesthetized, blood and liver tissues were collected for following tests: changes in the structure of mice liver tissue were observed using transmission electron microscopy; the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected using enzyme linked immunosorbent assay (ELISA); the levels of glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) in liver tissues were measured using the water-soluble tetrazolium-1 (WST-1) method, the thiobarbituric acid (TBA) method, and enzymatic methods, respectively; the protein expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), Kelch-like ECH-associated protein 1 (Keap1), and activated caspase-9 in liver tissues were detected using Western blotting.
RESULTS: Severe mitochondrial damage was observed in the liver tissues of mice in the DQ model group using transmission electron microscopy, yet mitochondrial damage in the QR treatment group showed significant alleviation. Compared to the control group, the DQ model group had significantly increased levels of MDA in liver tissue, serum AST, and ALT, yet had significantly decreased levels of GSH and SOD in liver tissue. In comparison to the DQ model group, the QR treatment group exhibited significant reductions in serum levels of ALT and AST, as well as MDA levels in liver tissue [ALT (U/L): 52.60±6.44 vs. 95.70±8.00, AST (U/L): 170.45±19.33 vs. 251.10±13.09, MDA (nmol/mg): 12.63±3.41 vs. 18.04±3.72], and notable increases in GSH and SOD levels in liver tissue [GSH (μmol/mg): 39.49±6.33 vs. 20.26±3.96, SOD (U/mg): 121.40±11.75 vs. 81.67±10.01], all the differences were statistically significant (all P < 0.01). Western blotting results indicated that the protein expressions of Nrf2 and HO-1 in liver tissues of the DQ model group were significantly decreased compared to the control group. On the other hand, the protein expressions of Keap1 and activated caspase-9 were conspicuously higher when compared to the control group. In comparison to the DQ model group, the QR treatment group showed a significant increase in the protein expressions of Nrf2 and HO-1 in liver tissues (Nrf2/β-actin: 1.17±0.08 vs. 0.92±0.45, HO-1/β-actin: 1.53±0.17 vs. 0.84±0.09). By contrast, there was a notable decrease in the protein expressions of Keap1 and activated caspase-9 (Keap1/β-actin: 0.48±0.06 vs. 1.22±0.09, activated caspase-9/β-actin: 1.17±0.12 vs. 1.59±0.30), the differences were statistically significant (all P < 0.01).
CONCLUSIONS: QR may reduce acute liver injury induced by DQ poisoning in mice via activating Keap1/Nrf2 signaling pathway.
摘要：
目的：探讨槲皮素(QR)对敌快(DQ)中毒小鼠急性肝损伤的保护作用及其机制。
方法：将80只SPF级健康雄性C57BL/6小鼠随机分为对照组，DQ模型组,QR治疗组，和QR控制组，每组20只小鼠。采用一次性腹腔注射DQ溶液(40mg/kg)建立DQ中毒模型,对照组和QR对照组腹腔注射等量蒸馏水。建模后四个小时，QR治疗组和QR对照组通过管饲法接受0.5mLQR溶液(50mg/kg).同时,对照组和DQ模型组口服等量蒸馏水。上述治疗每天施用一次,连续七天。之后,小鼠被麻醉，采血和肝组织进行以下试验:透射电镜观察小鼠肝组织结构的变化;酶联免疫吸附试验(ELISA)检测血清谷丙转氨酶(ALT)和谷草转氨酶(AST)水平;超氧化物歧化酶（SOD），和丙二醛（MDA）在肝组织中使用水溶性四唑-1（WST-1）方法测量，硫代巴比妥酸（TBA）法，和酶法，核因子2相关因子2（Nrf2）的蛋白表达，血红素加氧酶-1（HO-1），Kelch样ECH相关蛋白1（Keap1），使用蛋白质印迹法检测肝组织中活化的caspase-9。
结果：透射电镜观察到DQ模型组小鼠肝组织线粒体严重损伤，然而,QR治疗组的线粒体损伤表现出显著缓解.与对照组相比,DQ模型组肝组织MDA含量显著升高,血清AST,ALT,但肝组织中GSH和SOD水平显著降低。与DQ模型组相比，QR治疗组显示ALT和AST的血清水平显着降低，以及肝组织中的MDA水平[ALT（U/L）：52.60±6.44vs.95.70±8.00，AST（U/L）：170.45±19.33vs.251.10±13.09，MDA（nmol/mg）：12.63±3.41vs.18.04±3.72],肝组织中GSH和SOD水平显着增加[GSH（μmol/mg）：39.49±6.33vs.20.26±3.96，SOD（U/mg）：121.40±11.75vs.81.67±10.01]，差异均有统计学意义(均P<0.01)。Westernblotting结果显示，与对照组相比，DQ模型组肝组织中Nrf2和HO-1的蛋白表达明显降低。另一方面,与对照组相比，Keap1和活化的caspase-9的蛋白表达明显高于对照组。与DQ模型组相比，QR治疗组肝组织中Nrf2和HO-1的蛋白表达显着增加（Nrf2/β-肌动蛋白：1.17±0.08vs.0.92±0.45，HO-1/β-肌动蛋白：1.53±0.17vs.0.84±0.09）。相比之下,Keap1和活化的caspase-9的蛋白表达显着降低（Keap1/β-肌动蛋白：0.48±0.06vs.1.22±0.09，激活的caspase-9/β-肌动蛋白：1.17±0.12vs.1.59±0.30),差异均有统计学意义(均P<0.01)。
结论：QR可能通过激活Keap1/Nrf2信号通路减轻DQ中毒小鼠急性肝损伤。
方法：将80只SPF级健康雄性C57BL/6小鼠随机分为对照组，DQ模型组,QR治疗组，和QR控制组，每组20只小鼠。采用一次性腹腔注射DQ溶液(40mg/kg)建立DQ中毒模型,对照组和QR对照组腹腔注射等量蒸馏水。建模后四个小时，QR治疗组和QR对照组通过管饲法接受0.5mLQR溶液(50mg/kg).同时,对照组和DQ模型组口服等量蒸馏水。上述治疗每天施用一次,连续七天。之后,小鼠被麻醉，采血和肝组织进行以下试验:透射电镜观察小鼠肝组织结构的变化;酶联免疫吸附试验(ELISA)检测血清谷丙转氨酶(ALT)和谷草转氨酶(AST)水平;超氧化物歧化酶（SOD），和丙二醛（MDA）在肝组织中使用水溶性四唑-1（WST-1）方法测量，硫代巴比妥酸（TBA）法，和酶法，核因子2相关因子2（Nrf2）的蛋白表达，血红素加氧酶-1（HO-1），Kelch样ECH相关蛋白1（Keap1），使用蛋白质印迹法检测肝组织中活化的caspase-9。
结果：透射电镜观察到DQ模型组小鼠肝组织线粒体严重损伤，然而,QR治疗组的线粒体损伤表现出显著缓解.与对照组相比,DQ模型组肝组织MDA含量显著升高,血清AST,ALT,但肝组织中GSH和SOD水平显著降低。与DQ模型组相比，QR治疗组显示ALT和AST的血清水平显着降低，以及肝组织中的MDA水平[ALT（U/L）：52.60±6.44vs.95.70±8.00，AST（U/L）：170.45±19.33vs.251.10±13.09，MDA（nmol/mg）：12.63±3.41vs.18.04±3.72],肝组织中GSH和SOD水平显着增加[GSH（μmol/mg）：39.49±6.33vs.20.26±3.96，SOD（U/mg）：121.40±11.75vs.81.67±10.01]，差异均有统计学意义(均P<0.01)。Westernblotting结果显示，与对照组相比，DQ模型组肝组织中Nrf2和HO-1的蛋白表达明显降低。另一方面,与对照组相比，Keap1和活化的caspase-9的蛋白表达明显高于对照组。与DQ模型组相比，QR治疗组肝组织中Nrf2和HO-1的蛋白表达显着增加（Nrf2/β-肌动蛋白：1.17±0.08vs.0.92±0.45，HO-1/β-肌动蛋白：1.53±0.17vs.0.84±0.09）。相比之下,Keap1和活化的caspase-9的蛋白表达显着降低（Keap1/β-肌动蛋白：0.48±0.06vs.1.22±0.09，激活的caspase-9/β-肌动蛋白：1.17±0.12vs.1.59±0.30),差异均有统计学意义(均P<0.01)。
结论：QR可能通过激活Keap1/Nrf2信号通路减轻DQ中毒小鼠急性肝损伤。
