ISSN 1674-3865  CN 21-1569/R
主管：国家卫生健康委员会
主办：中国医师协会
　　　辽宁省基础医学研究所
　　　辽宁中医药大学附属医院
中国中西医结合儿科学

中国中西医结合儿科学 ›› 2022, Vol. 14 ›› Issue (1): 10-15.doi: 10.3969/j.issn.1674-3865.2022.01.004

• 实验论著 • 上一篇    下一篇

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

刘启艳, 张力文, 詹伟, 李春, 庞平, 高娜, 彭玉   

  1. 550003 贵阳，贵阳市妇幼保健院儿童康复医学科（刘启艳）；550003 贵阳，贵州中医药大学第二附属医院儿科（张力文，李春，庞平，彭玉）；551702 贵州 毕节，毕节市中医院儿科（詹伟）；554399 贵州 铜仁，铜仁市中医院儿科（高娜）

  • 收稿日期:2021-03-03 出版日期:2022-02-25 上线日期:2023-12-05
  • 通讯作者: 彭玉，E-mail:1065764916@qq.com
  • 基金资助:
    国家自然科学基金项目（81660808）；毕节市科技局联合基金项目（毕科联合字Z［2019］2号）；2020年贵州省卫生健康委科学技术基金项目（gzwjkj2020-1-144）

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

LIU Qiyan, ZHANG Liwen, ZHAN Wei, LI Chun, PANG Ping, GAO Na, PENG Yu   

  1. Children Rehabilitation Medicine Department of Guiyang Maternal and Child Health Care Hospital, Guiyang 550003, China
  • Received:2021-03-03 Published:2022-02-25 Online:2023-12-05
  • Contact: PENG Yu，E-mail:1065764916@qq.com

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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: Objective To 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.Methods Fifty 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.Results The 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).Conclusion Li-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