理脾复方制剂含药血清对大鼠下丘脑神经元细胞瘦素、神经肽Y、八肽胆囊收缩素蛋白与基因的影响

摘要/Abstract

摘要： 目的观察理脾复方制剂含药血清对大鼠下丘脑神经元细胞瘦素(LP)、神经肽Y(NPY),八肽胆囊收缩素(CCK-8)等蛋白及基因的调控影响，探讨其治疗厌食的作用机制。方法 50只大鼠随机分为对照组，低、中、高剂量理脾复方制剂组，硫酸锌组，每组10只。对照组灌胃给予等体积饮用水；硫酸锌组灌胃给予20 mg/kg硫酸锌口服溶液；低、中、高理脾复方制剂组分别灌胃给予2.4、4.8和9.8 g/kg理脾复方制剂，各组每日灌胃1次，连续7 d,末次灌胃后股动脉采血制备含药血清。体外培养大鼠下丘脑神经元细胞，MAP2免疫荧光鉴定细胞。采用CCK-8法筛选对照组、理脾复方制组(低、中、高剂量组),硫酸锌组在0、2、4、6、8、12、24、36、48 h对下丘脑神经元活性的影响，发现在24 h后时，各含药血清组对下丘脑神经元细胞活性开始增强并具有区别性。ELISA检测下丘脑神经元细胞上清液中LP、NPY、CCK-8的含量；Western Blot法及实时荧光定量PCR法分别检测下丘脑神经元细胞LP、NPY、CCK-8蛋白及基因相对表达量。结果细胞鉴定为下丘脑神经元细胞。与对照组比较，低、中、高剂量理脾复方制剂组中下丘脑神经元细胞上清液中LP、NPY、CCK-8含量降低，差异具有统计学意义(P<0.05);中、高剂量理脾复方制剂组下丘脑神经元细胞中LP、NPY、CCK-8蛋白相对表达量降低，差异具有统计学意义(P<0.05);高剂量理脾复方制剂组下丘脑神经元细胞中LP基因相对表达量降低，差异具有统计学意义(P<0.05);低、中、高剂量理脾复方制剂组下丘脑神经元细胞中NPY、CCK-8基因相对表达量降低，差异具有统计学意义(P<0.05)。结论 理脾复方制剂治疗可能通过综合调节下丘脑神经元中LP、NPY、CCK-8蛋白及基因表达，实现改善厌食相关症状，但其影响作用与剂量呈负相关性。

关键词: 理脾复方制剂, 下丘脑, 神经元细胞, 原代培养, LP, NPY, CCK-8

Abstract: ObjectiveTo observe the regulating effects of drug-containing serum of Li-pi compound preparation on LP,NPY and CCK-8 proteins and genes in rat hypothalamic neuron cells and to explore the mechanism of its treatment for anorexia.MethodsFifty rats were randomly divided into control group, low-, medium- and high-dose Li-pi compound preparation group and zinc sulfate group, with 10 in each group. Control group was given equal volume of drinking water by gavage. Zinc sulfate group was given 20 mg/kg zinc sulfate oral solution. Low-, medium- and high-dose Li-pi compound groups were given 2.4,4.8 and 9.8 g/kg of the compound, respectively. Each group was given gavage once a day for 7 days in a row, and the femoral artery blood was collected on the 7th day after gavage to prepare the drug-containing serum. Rat hypothalamic neuron cells were cultured in vitro and identified by MAP2 immunofluorescence. The hypothalamic neurons were randomly divided into control group, low-, medium- and high-dose Li-pi compound preparation group, and zinc sulfate group by CCK-8, and effect on the activity of hypothalamic neuron cells was assessed at 0 h, 2 h,4 h,6 h,8 h,12 h,24 h,36 h and 48 h. After 24 hours of the corresponding serum, the activity of hypothalamic neurons in each drug-containing serum group began to increase and became different. ELISA was used to detect LP, NPY and CCK-8 content in supernatant of hypothalamic neuron cells; Western Blot and real-time fluorescence quantitative PCR were used to detect the relative expression of LP, NPY and CCK-8 protein and genes in hypothalamic neuronal cells.ResultsThe cells were identified as hypothalamic neuronal cells. Compared with the control group, the LP,NPY and CCK-8 content in supernatant of hypothalamic neuron cells was decreased in the supernatant of hypothalamic neuron cells of low-，medium- and high-dose Li-pi compound preparation, and the differences were statistically significant(P<0.05);the relative expression of LP,NPY and CCK-8 protein in hypothalamic neuron cells in Li-pi compound preparation of medium and high doses was decreased, and the differences were statistically significant(P<0.05); the relative expression of LP gene in hypothalamic neuron cells in high-dose Li-pi compound preparation group was decreased, and the difference was statistically significant(P<0.05); the relative expression of NPY and CCK-8 genes in hypothalamic neuron cells in low-, medium- and high-dose Li-pi compound preparation groups was decreased, and the differences were statistically significant(P<0.05).ConclusionLi-pi compound preparation may improve anorexia-related symptoms by comprehensively regulating the expression of LP, NPY and CCK-8 proteins and genes in hypothalamic neurons, but its effect is negatively correlated with dose.

Key words:

Li-pi compound preparation, Hypothalamus, Neuron cells, Primary culture, LP, NPY, CCK-8

刘启艳, 张力文, 詹伟, 李春, 庞平, 高娜, 彭玉. 理脾复方制剂含药血清对大鼠下丘脑神经元细胞瘦素、神经肽Y、八肽胆囊收缩素蛋白与基因的影响[J]. 中国中西医结合儿科学, 2022, 14(1): 10-15.

LIU Qiyan, ZHANG Liwen, ZHAN Wei, LI Chun, PANG Ping, GAO Na, PENG Yu.. Effect of drug-containing serum of Li-pi compound preparation on LP,NPY and CCK-8 proteins and genes of rat hypothalamic neuron cells[J]. Chinese Pediatrics of Integrated Traditional and Western Medicine, 2022, 14(1): 10-15.

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理脾复方制剂含药血清对大鼠下丘脑神经元细胞瘦素、神经肽Y、八肽胆囊收缩素蛋白与基因的影响

Effect of drug-containing serum of Li-pi compound preparation on LP,NPY and CCK-8 proteins and genes of rat hypothalamic neuron cells

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