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目的:观察苹果多糖(AP)对脂多糖(LPS)诱导RAW 264.7细胞凋亡的影响及其机制。方法:采用水提醇沉法从苹果果渣中提取苹果多糖,并测定其糖含量和分子量。采用流式细胞仪考察AP对LPS诱导RAW 264.7细胞凋亡的作用。采用Western Blot法考察AP对LPS诱导RAW 264.7细胞中Bcl-2、Bax蛋白表达的影响。结果:经水提醇沉法提取纯化得到苹果多糖的糖含量为91.3%,重均分子量为184613 Da。流式细胞仪检测结果显示,LPS诱导RAW 264.7细胞凋亡明显高于正常对照组(P<0.01);与LPS组比较,浓度为0.5 mg·ml~(-1)的AP作用72 h后RAW 264.7细胞凋亡率显著降低(P<0.01),浓度为1.0mg·ml~(-1)的AP作用48,72 h后RAW 264.7细胞凋亡率显著降低(P<0.01)。Western Blot检测结果显示,与正常对照组比较,LPS诱导RAW 264.7细胞中的Bcl-2蛋白在48,72 h表达明显升高,差异具有统计学意义(P<0.01);与LPS组比较,浓度为1 mg·ml~(-1)的AP作用后,升高了LPS诱导的RAW 264.7细胞Bcl-2蛋白的表达,降低其Bax蛋白的表达,差异具有统计学意义(P<0.01)。结论:AP抑制LPS诱导RAW 264.7细胞凋亡,其机制可能是通过提高RAW 264.7细胞中Bcl-2蛋白的表达,降低其Bax蛋白表达。
Objective: To observe the effect and mechanism of apple polysaccharide (AP) on lipopolysaccharide (LPS) -induced RAW 264.7 cell apoptosis. Methods: The apple polysaccharide was extracted from apple pomace by water extraction and alcohol precipitation, and its sugar content and molecular weight were determined. Flow cytometry was used to investigate the effect of AP on LPS-induced RAW 264.7 cell apoptosis. The effects of AP on Bcl-2 and Bax protein expression in RAW 264.7 cells induced by LPS were investigated by Western Blot. Results: The sugar content of apple polysaccharide extracted and purified by water extraction and alcohol precipitation was 91.3% and the weight average molecular weight was 184613 Da. Flow cytometry results showed that LPS induced RAW 264.7 cell apoptosis was significantly higher than the normal control group (P <0.01); compared with the LPS group, the concentration of 0.5 mg · ml -1 AP at 72 h RAW (P <0.01). The apoptotic rate of RAW 264.7 cells was significantly decreased at the concentration of 1.0mg · ml -1 for 48 and 72 h (P <0.01). The results of Western Blot showed that compared with the normal control group, the expression of Bcl-2 protein in RAW 264.7 cells induced by LPS was significantly increased at 48 and 72 h (P <0.01). Compared with LPS group, the concentration 1 mg · ml ~ (-1) AP increased the Bcl-2 protein expression and decreased the Bax protein expression in LPS-induced RAW 264.7 cells, the difference was statistically significant (P <0.01). CONCLUSION: AP inhibits LPS-induced apoptosis of RAW 264.7 cells by decreasing the expression of Bcl-2 protein and decreasing the expression of Bax protein in RAW 264.7 cells.