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

作　　者：刘启艳 张力文 詹伟 李春 庞平 高娜 彭玉 LIU Qiyan;ZHANG Liwen;ZHAN Wei;LI Chun;PANG Ping;GAO Na;PENG Yu(Children Rehabilitation Medicine Department of Guiyang Maternal and Child Health Care Hospital, Guiyang 550003, China)

机构地区：[1]贵阳市妇幼保健院儿童康复医学科,贵阳550003 [2]贵州中医药大学第二附属医院儿科,贵阳550003 [3]毕节市中医院儿科,贵州毕节551702 [4]铜仁市中医院儿科,贵州铜仁554399

出　　处：《中国中西医结合儿科学》2022年第1期10-15,共6页Chinese Pediatrics of Integrated Traditional and Western Medicine

基　　金：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)。

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。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 7^(th) 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 o

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 

分 类 号：R322.81[医药卫生—人体解剖和组织胚胎学]