经方丹栀逍遥丸对代谢相关脂肪性肝病小鼠巨噬细胞增殖活性及防炎癌作用的影响及机制研究

Influence and Mechanism of Classical Prescription Danzhi Xiaoyao pill(丹栀逍遥丸) on Macrophage Cytoactive and Anti-Inflammation and Anti-Tumor Effect in Mice with Metabolic Associated Fatty Liver Disease

目的 评价经方丹栀逍遥丸(JF-DZXYW)含药血清对代谢相关脂肪性肝病小鼠巨噬细胞增殖活性及防炎癌转化的调控,并阐述其诱导免疫系统发挥抗炎、抗肿瘤作用。方法 40只体质量18~22 g雄性昆明种小鼠,随机分为对照组(生理盐水组)、JF-DZXYW低剂量组(125μg/mL)、JF-DZXYW中剂量组(250μg/mL)和JF-DZXYW高剂量组(500μg/mL),灌胃0.4 mL/次,1次/d,共3 d。第4天灌胃后行小鼠腹主动脉取血,分离血清。采用CCK-8法检测各剂量组含药血清对小鼠巨噬细胞株Raw 264.7细胞增殖的影响,并筛选出最佳给药浓度。酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)法检测细胞因子:肿瘤坏死因子-α(tumor necrosis factor, TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)、干扰素-β(interferon-β,IFN-β)、白细胞介素-12(interleukin-12p40,IL-12p40)、白细胞介素-10(interleukin-10,IL-10)含量。运用MTT法分析HepG2肝癌细胞被巨噬细胞杀伤的效果。结果 小鼠巨噬细胞培养12 h后,JF-DZXYW各剂量组含药血清处理组巨噬细胞增殖速度与对照组相比无显著升高(P>0.05);细胞培养24 h后,JF-DZXYW各剂量组含药血清处理组巨噬细胞增殖速度与对照组相比显著升高(P<0.01)。JF-DZXYW中、高剂量组含药血清可显著提升细胞因子TNF-α、IL-1β含量,与对照组相比差异有统计学意义(P<0.01);JF-DZXYW低、中、高剂量组含药血清可显著提升细胞因子IL-6含量,与对照组相比差异有统计学意义(P<0.01);JF-DZXYW中剂量组含药血清可显著提升细胞因子IL-12p40、IFN-β含量,与对照组相比差异有统计学意义(P<0.05);JF-DZXYW高剂量组含药血清可显著提升细胞因子IL-12p40、IFN-β含量,与对照组相比差异有统计学意义(P<0.01);JF-DZXYW高剂量组含药血清可显著提升细胞因子IL-10含量,与对照组相比差异有统计学意义(P<0.01);JF-DZXYW低、中剂量组含药血清可显著提升细胞因子IL-10含量,与对照组相比差异有统计学意义(P<0.05)。JF-DZXYW各剂量组含药血清细胞杀瘤率显著升高,与对照组相比差异有统计学意义(P<0.01)。结论 JF-DZXYW可以通过上调巨噬细胞增殖活性、诱导免疫系统发挥抗炎、抗肿瘤作用。

Objective To evaluate the regulatory effect of the serum containing the classic formula Danzhi Xiaoyao Pill(丹栀逍遥丸,JF-DZXYW) on macrophage proliferation activity and anti-inflammatory cancer transformation in mice with metabolic related fatty liver disease and elucidate its induction of anti-inflammatory and anti-tumor effects in the immune system. Methods Forty male Kunming breed mice weighing 1822 g were randomly divided into a control group(saline group),JF-DZXYW low dose group(125 μg/mL),JF-DZXYW medium dose group(250 μg/mL) and JF-DZXYW high dose group(500 μg/mL) by gavage with the dose of 0.4 mL/time, 1 time/d for 3 d. After gavage on the 4th day, the blood was collected from the abdominal aorta of mice and the serum was separated. The effect of each dose group on the proliferation of mouse macrophage Raw264.7 cells was detected by the CCK-8 method, and the optimal administration concentration was screened. Enzyme linked immunosorbent assay(ELISA) was used to detect cytokines: tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6),interferon-β(IFN-β),interleukin-12p40(IL-12p40),interleukin-10(IL-10). The effect of the killing HepG2 hepatocellular carcinoma cells by macrophages was analyzed using the MTT method. Results After 12 h of mouse macrophage culture, the proliferation rate of macrophages in the JF-DZXYW drug-containing serum treatment groups did not increase significantly compared with that of the control group(P>0.05). After 24 h of cell culture, the proliferation rate of macrophages in the JF-DZXYW drug-containing serum treatment groups increased significantly compared with that of the control group(P<0.01). The JF-DZXYW medium and high dose drug-containing serum groups significantly increased the contents of TNF-α and IL-1β compared with the control group(P<0.01). The JF-DZXYW low, medium and high dose drug-containing serum groups significantly increased the content of IL-6 compared with the control group(P<0.01). The JF-DZXYW medium dose drug-containing serum group significantly increased the contents of IL-12p40 and IFN-β compared with the control group(P<0.05). JF-DZXYW high dose drug-containing serum group could significantly enhance the contents of IL-12p40 and IFN-β compared with the control group(P<0.01). JF-DZXYW high dose drug-containing serum group could significantly enhance the cytokine IL-10 content compared with the control group(P<0.01). JF-DZXYW low and medium dose drug-containing serum groups significantly elevated IL-10 content compared with the control group(P<0.05). The cell killing rate of serum containing JF-DZXYW was significantly higher in each dose group compared with the control group(P<0.01). Conclusion JF-DZXYW can exert anti-inflammatory and anti-tumor effects by up-regulating macrophage proliferative activity and inducing the immune system.