炎症因子IL-6、IL-1β与趋化因子CXCR4在氟化钠诱导肝细胞中的表达

Expression of inflammatory cytokines IL-6,IL-1β and chemokine CXCR4 in hepatocytes induced by sodium fluoride

目的通过培养人胚胎肝细胞(HHL-5)并经氟化钠染毒,酶联免疫吸附法(ELISA)检测人胚胎肝细胞中趋化因子CXCR4、炎症因子白介素-6(IL-6)与白介素1-β(IL-1β)的表达水平,为氟中毒肝脏致病机理提供依据。方法用倒置显微镜观察HHL-5细胞第一代、第二代、第三代在培养48 h后的细胞生长状态,在第三代HHL-5细胞设置不同浓度梯度氟化钠(0、1、2、3 mmol/L NaF)共4组进行染氟,每组设3~5个复孔;用CCK-8法检测HHL-5细胞存活率,并筛选NaF作用浓度作为模型组;ELISA检测模型组与对照组细胞上清液中炎症因子IL-6、IL-1β与趋化因子CXCR4含量。结果HHL-5细胞经染氟成功造模后CCK-8结果显示,1 mmol/L NaF组存活率为(64.78%±1.79%)、2 mmol/L NaF组存活率为(62.54%±3.54%)、3 mmol/L NaF组存活率为(50.73%±1.55%),经单因素方差分析组间比较具有统计学差异(F=297.983,P<0.001);经Dunnett-t检验各染氟模型组与对照组(0 mmol/L NaF)细胞存活率相比均具有显著性差异(P<0.05)。在本实验条件下氟化钠浓度为3 mmol/L时,细胞抑制率最接近LD50,故选用氟化钠浓度为3 mmol/L作为模型实验组。经ELISA检测结果显示模型组中趋化因子CXCR4表达水平(188.37±7.10)ng/mL显著高于对照组(164.07±7.71 ng/mL,t=4.016,P<0.05);模型组中炎症因子IL-6表达水平(8.59±0.20 pg/mL)显著低于对照组(9.68±0.64 pg/mL,t=2.78,P<0.05);模型组中炎症因子IL-1β表达水平(13.94±0.72 pg/mL)与对照组比较无显著性差异(13.40±0.39 pg/mL,t=-1.468,P>0.05)。结论HHL-5细胞经染氟后成功造模,不同NaF浓度可干扰肝细胞存活率;趋化因子受体4(CXCR4)在模型组中的表达水平显著升高,炎症因子IL-6模型组中的表达水平降低,提示趋化因子CXCR4与炎症因子IL-6在NaF诱导肝脏损伤的表达中具有重要意义。

Objectives Human embryonic hepatocytes(HHL-5) were cultured and exposed to sodium fluoride.The expression levels of chemokine CXCR4,inflammatory factors interleukin-6(IL-6) and interleukin-1β(IL-1β) in human embryonic hepatocytes were detected by Enzyme-Linked Immunodeficient Assay(ELISA),which provided a basis for the pathogenesis of fluorosis liver.Methods The growth status of the first, second and third generations of HHL-5 cellsafter 48 hours of culture was observed by inverted microscope.Four groups of different concentration gradients of sodium fluoride(0,1,2,3 mmol/L NaF) were set up in the third generation of HHL-5 cells for fluoride staining, and each group had 3 to 5 holes.The survival rate of HHL-5 cells was detected by CCK-8 method, and the concentration of NaF was selected as the model group.The contents of inflammatory factors IL-6,IL-1β and chemokine CXCR4 in the supernatant of model group and control group were detected by ELISA.Results CCK-8 results showed survival rate of 1mmol/L NaF group was(64.78%±1.79%),survival rate of 2 mmol/L NaF group was(62.54%±3.54%),and survival rate of 3 mmol/L NaF group was( 50.73% ± 1.55%).There was significant difference between the two groups by one-way analysis of variance(F=297.983,P0.001).The Dunnett-t test showed that there was a significant difference in cell viability between fluoride model group and control group(0 mmol/LNaF)(P0.05).Under the experimental conditions, when the concentration of sodium fluoride was 3 mmol/L,the cell inhibition rate was closest to half of the lethal dose, and the concentration of sodium fluoride was 3 mmol/L as the model experimental group.ELISA results showed expression of CXCR4 in model group(188.37 ng/mL±7.10 ng/mL) was significantly higher than that in control group(164.07 ng/mL±7.71 ng/mL,t=4.016,P0.05).Expression of IL-6 in model group(8.59 pg/mL ± 0.20 pg/mL) was significantly lower than that in control group(9.68 pg/mL±0.64 pg/mL,t=2.78,P0.05).The expression level of inflamma-tory factor IL-1β in model group(13.94 pg/mL ± 0.72 pg/mL) was not significantly different from that in control group(13.40 pg/mL±0.39 pg/mL,t=-1.468,P0.05).Conclusion HHL-5 cells were successfully modeled after fluoride exposure, and different NaF concentrations could interfere with the survival rate of hepatocytes.The expression level of chemokine receptor 4(CXCR4) in model group was significantly increased, and the expression level of inflammatory factor IL-6 in model group was decreased, suggesting that chemokine CXCR4 and inflammatory factor IL-6 play an important role in the expression of NaF-induced liver injury.