异功散调节巨噬细胞铁代谢的机制研究

Mechanism of Yigong Powder on regulation of iron metabolism in macrophages

目的:研究异功散调节慢性病贫血(ACD)铁代谢的作用机制。方法:①体外培养小鼠巨噬细胞系RAW 264.7细胞,分为空白组和异功散干预组(0.01、0.1、1、1.25、2、2.5、3.75 mg/ml),各组给予相应浓度干预,孵育24 h、48 h和72 h后CCK8检测细胞活性,筛选有效浓度。②取RAW 264.7细胞分为空白组、脂多糖(LPS)组、异功散低剂量(0.1 mg/ml)组、异功散高剂量(1.0 mg/ml)组、LPS+异功散低剂量(0.1 mg/ml)组和LPS+异功散高剂量(1.0 mg/ml)组。预先配置含不同浓度异功散的DMEM培养基,对应加入各药物干预组。预处理1 h后再加入0.1μg/ml LPS刺激。细胞培养24 h、48 h后,比色法检测细胞内外铁含量,PCR检测细胞HAMP和膜铁转运蛋白(FPN)的基因表达水平,Western blot检测细胞信号转导转录激活因子3(STAT3)和磷酸化STAT3(p-STAT3)的蛋白表达水平。③取人肝癌HepG2细胞,在上述分组基础上增加抑制剂+LPS组、LPS+抑制剂+异功散低剂量(0.1 mg/ml)组和LPS+抑制剂+异功散高剂量(1.0 mg/ml)组。预先配置含不同浓度异功散的DMEM培养基,对应加入各药物干预组。预处理1 h后,各抑制剂干预组加入STAT3阻断剂(10μmol)处理10 min,再加入0.1μg/ml LPS刺激。细胞培养48 h后,PCR检测细胞HAMP基因表达水平。结果:①0.01～3.75 mg/ml异功散干预RAW 264.7细胞48 h内无细胞毒性作用,且能促进细胞增殖。选取0.1 mg/ml和1.0 mg/ml为有效浓度。②无LPS刺激状态下,细胞培养24 h、48 h后,异功散低、高剂量组的细胞外铁含量较空白组显著降低(P<0.05),但细胞内铁含量无明显变化。LPS刺激状态下,细胞培养24 h、48 h后,LPS组细胞外铁含量较空白组显著降低(P<0.05),而细胞内铁含量明显升高(P<0.05);与LPS组比较,LPS+异功散低剂量组细胞外铁含量无明显变化,细胞内铁含量降低(P<0.05),LPS+异功散高剂量组细胞外铁含量升高(P<0.05),细胞内含量铁降低(P<0.05)。③无LPS刺激状态下,与空白组相比,异功散低、高剂量组细胞培养24 h、48 h后HAMP mRNA表达无明显变化,异功散高剂量组细胞培养48 h后FPN mRNA表达量显著升高(P<0.01),且高剂量组细胞FPN mRNA表达水平较低剂量组显著升高(P<0.01)。LPS刺激状态下,细胞培养24 h、48 h后,与空白组相比,LPS组细胞HAMP mRNA表达显著增多(P<0.05),FPN mRNA表达明显降低(P<0.05,P<0.01);与LPS组相比,LPS+异功散高剂量组细胞HAMP mRNA表达显著降低(P<0.05),FPN mRNA表达显著升高(P<0.05,P<0.01);且LPS+异功散高剂量组细胞FPN mRNA表达水平较LPS+异功散低剂量组显著升高(P<0.05,P<0.01)。④无LPS刺激状态下,细胞培养24 h、48 h后,与空白组相比,异功散低、高剂量组细胞STAT3和p-STAT3蛋白表达无明显变化。LPS刺激状态下,细胞培养24 h、48 h后,与空白组相比,LPS组细胞STAT3蛋白表达无明显变化,p-STAT3蛋白表达有上调趋势;与LPS组相比,LPS+异功散低剂量组和LPS+异功散高剂量组细胞p-STAT3蛋白表达有降低趋势,STAT3蛋白表达无明显变化。⑤LPS刺激状态下,细胞培养48 h后,与LPS组相比,LPS+抑制剂组细胞HAMP mRNA表达显著降低(P<0.01);与LPS+抑制剂组比较,LPS+抑制剂+异功散高剂量组细胞HAMP mRNA表达显著下调(P<0.05)。结论:异功散可通过降低STAT3磷酸化水平,抑制HAMP mRNA过表达,上调FPN mRNA水平,从而促进巨噬细胞内铁的外流,改善LPS诱导的RAW 264.7细胞铁代谢异常,且异功散与STAT3阻断剂在调控HAMP表达方面具有协同作用。

Objective: To study the mechanism of Yigong Powder regulating iron metabolism in anemia of chronic diseases(ACD). Methods:① Mouse macrophage RAW 264.7 cell line was cultured in vitro, and divided into the blank group and Yigong Powder intervention groups(0.01, 0.1, 1, 1.25, 2, 2.5, 3.75 mg/ml). Each group was treated with the corresponding concentration, and after incubated for 24, 48 and 72 hours, the cell activity was detected by CCK8 assay to screen the effective concentration.② RAW 264.7 cells were divided into the blank group, LPS group, Yigong Powder group with low-dose(0.1 mg/ml), Yigong Powder group with high-dose(1.0 mg/ml), LPS+Yigong Powder low-dose(0.1 mg/ml) group, and LPS+Yigong Powder high-dose(1.0 mg/ml) group. The DMEM medium containing Yigong Powder at different concentrations was prepared in advance, and then added into the corresponding drug intervention groups. After pretreatment for one hour, 0.1 μg/ml LPS was added for stimulation. After cell culture for 24 and 48 hours, the intracellular and extracellular contents of iron were detected by colorimetry, the gene expression levels of HAMP and ferroportin(FPN) were detected by PCR, and the protein expression levels of signal transducer and activator of transcription 3(STAT3) and phosphorylated STAT3(p-STAT3) were detected by Western blot.③ Human liver cancer HepG2 cells were taken. On the basis of above groups, the inhibitor+LPS group, LPS+inhibitor+ Yigong Powder low-dose(0.1 mg/ml) group and LPS+inhibitor+ Yigong Powder high-dose(1.0 mg/ml) group were added. The DMEM medium containing Yigong Powder at different concentrations was prepared in advance, and then added into the corresponding drug intervention groups. After pretreatment for one hour, STAT3 blocker(10 μmol)were added to the inhibitor-treated groups. After 10-minute treatment, 0.1 μg/ml LPS was added for stimulation. After cell culture for 48 hours, the expression level of HAMP gene was detected by PCR. Results:① Intervention within 48 hours, Yigong Powder at 0.01-3.75 mg/ml showed no cytotoxicity on RAW 264.7 cells, and could promote the cell proliferation. The effective concentrations were selected as 0.1 mg/ml and 1.0 mg/ml.② Without LPS stimulation, after cell culture for 24 and 48 hours, the extracellular iron content in the Yigong Powder groups with low-and high-dose was significantly lower than that in the blank group(P<0.05), but there was no significant change in the intracellular iron content. With LPS stimulation, after cell culture for 24 and 48 hours, the extracellular iron content in the LPS group was significantly lower than that in the blank group(P<0.05), while the intracellular iron content was significantly increased(P<0.05);compared with the LPS group, there was no significant change in the extracellular iron content in the LPS+Yigong Powder low-dose group, but the intracellular iron content was decreased(P<0.05), while the extracellular iron content was increased(P<0.05) and the intracellular iron content was decreased(P<0.05) in the LPS+Yigong Powder high-dose group.③ Without LPS stimulation, compared with the blank group, there was no significant change in the expression of HAMP mRNA in the Yigong Powder groups with low-and high-dose after cell culture for 24 and 48 hours;the expression of FPN mRNA was significantly increased in the Yigong Powder group with high-dose after cell culture for 48 hours, and the expression of FPN mRNA in the high-dose group was significantly higher than that in the low-dose group(P<0.01). With LPS stimulation, after cell culture for 24 and 48 hours, compared with the blank group, the expression of HAMP mRNA was significantly increased(P<0.05) and the expression of FPN mRNA was significantly decreased in the LPS group(P<0.05, P<0.01);compared with the LPS group, the expression of HAMP mRNA was significantly decreased in the LPS+Yigong Powder high-dose group(P<0.05), and the expression of FPN mRNA was significantly increased(P<0.05, P<0.01), and the expression of FPN mRNA in the LPS+Yigong Powder high-dose group was significantly higher than that in the LPS+ Yigong Powder low-dose group(P<0.05, P<0.01).④ Without LPS stimulation, after cell culture for 24 and 48 hours, compared with the blank group, there were no significant changes in the protein expressions of STAT3 and p-STAT3 in the Yigong Powder groups with low-and high-dose. With LPS stimulation, after cell culture for 24 and 48 hours, compared with the blank group, there was no significant change in the protein expression of STAT3 in the LPS group, and the protein expression of p-STAT3 showed the tendency of up-regulation;compared with the LPS group, the protein expression of p-STAT3 showed the decreasing trend in the LPS+Yigong Powder low-dose group and LPS+Yigong Powder high-dose group, and the protein expression of STAT3 showed no significant change.⑤ With LPS stimulation, after cell culture for 48 hours, compared with the LPS group, the expression of HAMP mRNA was significantly reduced in the LPS+ inhibitor group(P<0.01);compared with the LPS+ inhibitor group, the expression of HAMP mRNA was significantly down-regulated in the LPS+inhibitor +Yigong Powder high-dose group(P<0.05). Conclusion: Yigong Powder can reduce the phosphorylation level of STAT3, inhibit the over-expression of HAMP mRNA, and up-regulate the level of FPN mRNA, so as to promote the iron outflow in macrophages and improve the abnormal iron metabolism of RAW 264.7 cells induced by LPS. Moreover, Yigong Powder and STAT3 blocker show the synergistic effect on the regulation of HAMP expression.