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目的:通过c-Jun氨基末端激酶(c-jun N-terminal kinase,JNK)信号通路来研究蹄叶橐吾醇化乙酸乙酯萃取物的抗炎作用机制。方法:采用噻唑蓝比色法(MTT法)测定倍比稀释的蹄叶橐吾醇化乙酸乙酯萃取物(浓度区间为0~640 mg·L-1)作用于密度为5×104个/mL的RAW264.7细胞24 h后的细胞活力,计算出药物对细胞的无毒浓度;测定JNK抑制剂SP600125(终浓度分别为25,12.5,6.25μmol·L-1)作用于细胞密度为5×104个/mL的RAW264.7细胞24 h后的细胞活力,计算出SP600125作用的安全有效浓度;测定蹄叶橐吾醇化乙酸乙酯萃取物(终质量浓度分别为5,2.5,1 mg·L-1)作用于LPS诱导细胞密度为5×104个/mL的RAW264.7细胞12,24,36,48 h后的细胞活力,计算出药物抑制细胞增殖的浓度。采用蛋白质印迹(Western-blot)法测定蹄叶橐吾醇化乙酸乙酯萃取物(5 mg·L-1)作用于LPS诱导的RAW264.7细胞24 h后p-JNK(磷酸化c-Jun氨基末端激酶)蛋白、JNK蛋白和环氧化酶-2(COX-2)蛋白的表达变化。结果:蹄叶橐吾醇化乙酸乙酯萃取物作用于LPS诱导的RAW264.7细胞,测定p-JNK,JNK,COX-2相对灰度值分别为:0.12±0.03,0.48±0.03,0.18±0.04,无药物作用的LPS诱导的RAW264.7细胞,测定p-JNK,JNK,COX-2,相对灰度值分别为:0.68±0.05,0.83±0.04,0.58±0.04,两组数据比较具有明显差异(P<0.01)。蹄叶橐吾醇化乙酸乙酯萃取物抑制LPS诱导的RAW264.7细胞增殖,下调p-JNK,JNK,COX-2等炎性蛋白的表达。结论:蹄叶橐吾醇化乙酸乙酯萃取物抗炎机制通过JNK信号通路来完成。
OBJECTIVE: To investigate the anti-inflammatory mechanism of alcohol extract of Eupatorium Lustris by ethyl c-Jun N-terminal kinase (JNK) signaling pathway. Methods: MTT assay was used to determine the effect of ethyl acetate extract (concentration range 0-640 mg · L-1) on the ratio of RAW264 with density of 5 × 104 / mL .7 cells after 24 h of cell viability, calculate the drug non-toxic concentration of cells; determination of JNK inhibitor SP600125 (final concentrations were 25,12.5,6.25μmol·L-1) on the cell density of 5 × 104 / mL of RAW264.7 cells 24 h after the cell viability, calculate the safe and effective concentration of SP600125 role; measured leaf hoofed alcoholic ethyl acetate extract (final concentration of 5,2.5,1 mg · L-1) role The cell viability of RAW264.7 cells with LPS-induced cell density of 5 × 104 cells / mL for 12, 24, 36 and 48 h was calculated. The concentration of drug that inhibits cell proliferation was calculated. The protein expression of p-JNK (phosphorylated c-Jun N-terminal kinase) in LPS-induced RAW264.7 cells was assayed by Western-blot after treated with 5 mg · L-1 alcoholic ethyl acetate extract ) Protein, JNK protein and cyclooxygenase-2 (COX-2) protein expression changes. Results: The relative gray values of p-JNK, JNK and COX-2 were 0.12 ± 0.03, 0.48 ± 0.03 and 0.18 ± 0.04, respectively, in RAW264.7 cells induced by LPS. Drug-induced LPS-induced RAW264.7 cells, the relative gray values of p-JNK, JNK and COX-2 were 0.68 ± 0.05, 0.83 ± 0.04 and 0.58 ± 0.04, respectively <0.01). Lactobacillus plantarum ethanol extract inhibited LPS-induced RAW264.7 cell proliferation and down-regulated the expression of inflammatory proteins such as p-JNK, JNK and COX-2. Conclusion: The anti-inflammatory mechanism of alcoholic ethyl acetate extract of Ligustrum lucidum is accomplished through JNK signaling pathway.