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目的了解氯化镧(LaCl_3)对内毒素/脂多糖(LPS)刺激的巨噬细胞诱导型一氧化氮合酶(iNOS)表达的影响,并探讨其机制。方法将小鼠巨噬细胞株RAW 264.7分为空白对照组、LaCl_3组、LPS组和LaCl_3+LPS组。前3组细胞分别用常规培养液、含2.5μmol/L LaCl_3的培养液、含1 mg/L LPS的培养液培养24 h,LaCl_3+LPS组用含2.5μmol/L LaCl_3的培养液培养24 h后,换为含1 mg/L LPS的培养液培养24 h。采用免疫细胞化学染色法检测iNOS在各组细胞中的表达强度;蛋白质印迹法检测iNOS的蛋白表达水平;反转录-PCR测定iNOS的mRNA表达水平;硝酸还原酶法测定各组细胞培养上清液中一氧化氮(NO)含量。结果免疫细胞化学染色结果显示,iNOS主要分布于各组细胞的胞质中,空白对照组和LaCl_3组荧光强度极弱;LPS组荧光强度最强,阳性细胞百分率为44.4%,明显高于LaCl_3+LPS组(11.8%,P<0.05)。LPS组iNOS蛋白及其mRNA表达量和细胞培养上清液中NO含量均高于其余各组(P<0.05)。结论LaCl_3可在mRNA水平和蛋白水平抑制LPS诱导的iNOS过度表达,减少NO生成,提示LaCl_3能拮抗LPS诱导的iNOS过度活化。
Objective To investigate the effect of lanthanum chloride (LaCl_3) on the expression of inducible nitric oxide synthase (iNOS) in macrophages stimulated by lipopolysaccharide (LPS) and its mechanism. Methods Macrophage cell line RAW 264.7 was divided into blank control group, LaCl_3 group, LPS group and LaCl_3 + LPS group. The cells in the first 3 groups were cultured in the medium containing 2.5μmol / L LaCl_3 and the medium containing 1 mg / L LPS for 24 h, and the LaCl_3 + LPS group were cultured in the medium containing 2.5μmol / L LaCl_3 for 24 h After that, the medium containing 1 mg / L LPS was cultured for 24 h. Immunocytochemical staining was used to detect the expression of iNOS in each group of cells. The protein expression of iNOS was detected by Western blotting. The mRNA expression of iNOS was detected by reverse transcription-PCR. Nitric acid reductase Fluid nitric oxide (NO) content. Results The results of immunocytochemical staining showed that iNOS mainly distributed in the cytoplasm of each group, and the fluorescence intensity of the blank control group and LaCl_3 group was extremely weak. The fluorescence intensity of LPS group was the strongest, the percentage of positive cells was 44.4%, which was significantly higher than that of LaCl_3 + LPS group (11.8%, P <0.05). The expression of iNOS protein and its mRNA in LPS group and NO in cell culture supernatant were higher than those in the other groups (P <0.05). Conclusion LaCl_3 can inhibit LPS-induced iNOS overexpression at mRNA and protein levels and decrease NO production, suggesting that LaCl_3 can antagonize LPS-induced iNOS overexpression.