卫生研究

目的 研究铅染毒致高糖小鼠的神经损伤变化及其相应的机制。方法SPF级C57小鼠随机分为对照组、铅染毒组、高糖饲料组及铅染毒高糖饲料组，对照组、铅染毒组小鼠予基础饲料，高糖饲料组及铅染毒高糖饲料组予高糖饲料(50.26%碳水化合物、21.44%脂肪、17.45%蛋白质、4.21%矿物质和3.64%纤维)。对照组、高糖饲料组自由饮用纯水，铅染毒组和铅染毒高糖饲料组小鼠自由饮用250 mg/L醋酸铅水，连续染毒12周。应用糖水偏好实验、高架十字实验检测小鼠焦虑抑郁样行为变化。将小鼠小胶质细胞系BV-2细胞随机分为对照组、铅染毒组、高糖环境组及铅染毒高糖环境组，对照组不进行处理，铅染毒组给予10μmol/L醋酸铅，高糖环境组给予40 mmol/L葡萄糖，铅染毒高糖环境组给予10μmol/L醋酸铅和40 mmol/L葡萄糖，染毒24 h;应用siRNA转染在BV-2细胞建立细胞色素C氧化酶合成物1(synthesis of cytochrome C oxidase1,SCO1)敲降细胞模型(siSCO1细胞),并给予10μmol/L醋酸铅和40 mmol/L葡萄糖染毒24 h。使用ICP-MS、免疫荧光、试剂盒检测小鼠脑皮质及BV-2细胞铜、铅含量；试剂盒检测小鼠脑皮质及BV-2细胞超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)含量的变化；Western blot方法检测小鼠脑皮质及BV-2细胞SCO1、离子钙结合衔接分子1(ionized calcium binding adaptor molecule 1, Iba-1)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)蛋白表达。结果 糖水偏好结果显示，铅染毒高糖饲料组小鼠的糖水偏好率为31.00%,显著低于铅染毒组和高糖饲料组(P<0.05);高架十字结果显示，铅染毒高糖饲料组小鼠开臂进入次数百分比(27.20%)及停留时间的百分比(24.20%)显著低于铅染毒组和高糖饲料组。铅染毒高糖饲料组小鼠脑皮质中SOD含量为210.96 U/mg prot、CAT含量为108.94 U/mg prot,显著低于铅染毒组和高糖饲料组(P<0.05)。铅染毒高糖饲料组小鼠脑皮质中总铜含量为4.41μg/g,显著高于铅或高糖单独暴露组(P<0.05)。此外，铅染毒高糖饲料组小鼠脑皮质SCO1蛋白表达是对照组的1.86倍、铅染毒组的1.23倍、高糖饲料组的1.21倍(P<0.05)。铅染毒高糖饲料组小鼠脑皮质中Iba1、IL-1β及IL-6蛋白表达均显著高于铅染毒组和高糖饲料组(P<0.05)。敲降SCO1后，铅染毒高糖环境组BV-2细胞总铅平均荧光密度未见变化，但是BV-2细胞线粒体中铜含量为0.07 nmol/10⁶细胞，低于未敲降SCO1的BV-2细胞(P<0.05)。此外，敲降SCO1可导致铅和高糖联合染毒的BV-2细胞线粒体中SOD、CAT含量上升，Iba1、IL-1β蛋白表达下降(P<0.05)。结论高糖可加剧铅染毒小鼠的焦虑抑郁样行为，与SCO1增加导致小胶质细胞线粒体铜含量上升进而致小胶质细胞发生炎性活化有关。

OBJECTIVE To investigate the mechanism of neurological damage in mice induced by Pb exposure combined with high glucose.METHODS SPF C57 mice were randomly divided into a control group, a Pb group, a high-glucose diet group, and a Pb + high-glucose diet group. Mice in the control group and the Pb group were given basic feed, while those in the high-glucose diet group and the Pb + high-glucose diet group were given a high-glucose diet(50.26% carbohydrates, 21.44% fat, 17.45% protein, 4.21% minerals, and 3.64% fiber). The control group and the high-glucose diet group had free access to pure water, while the Pb group and the Pb + high-glucose diet group were given free access to 250 mg/L Pb acetate water for 12 weeks. The sucrose water preference test and elevated plus maze test were used to assess anxiety and depression-like behaviors in mice. The murine microglial cell line BV-2 was randomly divided into a control group, a Pb group, a high-glucose environment group, and a Pb + high-glucose environment group. The control group received no treatment, the Pb group was given 10 μmol/L Pb acetate, the high-glucose environment group was given 40 mmol/L glucose, and the Pb + high-glucose environment group was given 10 μmol/L Pb acetate and 40 mmol/L glucose for 24 hours. siRNA transfection in BV-2 cells was used to establish a synthesis of cytochrome C oxidase 1(SCO1) knockdown cell model(siSCO1 cells),which were exposed to 10 μmol/L Pb acetate and 40 mmol/L glucose for 24 hours. ICP-MS, immunofluorescence, and kit method were applied to detect copper and Pb content in mice cortex and BV-2 cells. Kits were used to detect superoxide dismutase(SOD) and catalase(CAT) in mice cortex and BV-2 cells. Western blot was used to measure the protein expression of SCO1, ionized calcium binding adaptor molecule 1(Iba-1), interleukin-1β(IL-1β) and interleukin-6(IL-6) in mice cortex and BV-2 cells.RESULTS The sucrose water preference test result showed that the sucrose water preference rate of mice in the Pb + high-glucose diet group was 31.00%, which was significantly lower than that in the Pb group and the high-glucose diet group(P<0.05). The elevated plus maze result showed that the percentage of entries into the open arms(27.20%) and the percentage of time spent in the open arms(24.20%) of mice in the Pb + high-glucose diet group were significantly lower than those in the Pb group and the high-glucose diet group(P<0.05). The content of SOD in the cerebral cortex of mice in the Pb + high-glucose diet group was 210.96 U/mg prot and the content of CAT was 108.94 U/mg prot, which were significantly lower than those in the Pb group and the high-glucose diet group(P<0.05). The total copper content in the cerebral cortex of mice in the Pb + high-glucose diet group was 4.41 μg/g, which was significantly higher than that in the Pb or high-glucose diet groups alone(P<0.05). Additionally, the expression of SCO1 protein in the cerebral cortex of mice in the Pb + high-glucose diet group was 1.86 times that of the control group, 1.23 times that of the Pb group, and 1.21 times that of the high-glucose diet group(P<0.05). The expression of Iba1, IL-1β, and IL-6 proteins in the cerebral cortex of mice in the Pb + high-glucose diet group was significantly higher than that in the Pb group and the high-glucose diet group(P<0.05). After knocking down SCO1, the average fluorescence density of total Pb in BV-2 cells exposed to Pb and high-glucose combined showed no change, but the copper content in the mitochondria of BV-2 cells was 0.07 nmol/10⁶ cells, which was lower than that in the non-knockdown BV-2 cells(P<0.05). Moreover, knocking down SCO1 led to an increase in the content of SOD and CAT in the mitochondria of BV-2 cells exposed to Pb and high-glucose combined, and a decrease in the expression of Iba1 and IL-1β proteins(P<0.05).CONCLISION High-glucose can exacerbate anxiety and depression-like behaviors in mice with Pb exposure, which may be related to the mitochondrial SCO1 increase, which result ing in elevated mitochondrial copper content in microglial cells, thereby causing inflammatory activation of microglial cells.