苹果多糖的制备及对脂多糖诱导RAW 264.7细胞凋亡的影响
|
摘要
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 polysaccharides(AP) on lipopolysaccharide(LPS)-induced RAW 264.7 apoptosis.Methods:AP was extracted from apple pomace by the method of water extraction and alcohol precipitation,and the sugar content and molecular weight were determined.The effect of AP on LPS-induced RAW 264.7 apoptosis was studied by using flow cytometry.The effects of AP on Bcl-2 and Bax protein expression in LPS-induced RAW 264.7 cells were studied by using Western Blot.Results:The sugar content of AP was 91.3%,and the molecular weight was 184613 Da.The results of flow cytometry test showed that LPS-induced RAW 264.7 apoptosis was obviously higher than that of the normal control group(P<0.01).Compared with LPS group,the RAW 264.7 apoptosis rate obviously decreased after the treatment with AP at the concentration of 0.5 mg·ml~(-1)(P<0.01) for 72 h,and the RAW 264.7 apoptosis rate obviously decreased after the treatment with AP at the concentration of 1.0mg·ml~(-1)(P<0.01) for 48 h and 72 h.The results of Western Blot test showed that the expression of Bcl-2 protein in LPS-induced RAW 264.7 cells obviously increased after 48 h and 72 h,and the difference was statistically significant(P<0.01) when compared with the normal control group.Compared with that in LPS group,the expression of Bcl-2 protein in LPS-induced RAW 264.7 cells increased and that of Bax protein decreased after the treatment with AP at the concentration of 1.0 mg·ml~(-1),and the difference was statistically significant(P<0.01).Conclusion:AP can inhibit LPS-induced RAW 264.7 apoptosis,and the mechanism may be related with increasing the expression of Bcl-2 protein and decreasing that of Bax protein in LPS-induced RAW 264.7 cells.
Objective:To observe the effect and mechanism of apple polysaccharides(AP) on lipopolysaccharide(LPS)-induced RAW 264.7 apoptosis.Methods:AP was extracted from apple pomace by the method of water extraction and alcohol precipitation,and the sugar content and molecular weight were determined.The effect of AP on LPS-induced RAW 264.7 apoptosis was studied by using flow cytometry.The effects of AP on Bcl-2 and Bax protein expression in LPS-induced RAW 264.7 cells were studied by using Western Blot.Results:The sugar content of AP was 91.3%,and the molecular weight was 184613 Da.The results of flow cytometry test showed that LPS-induced RAW 264.7 apoptosis was obviously higher than that of the normal control group(P<0.01).Compared with LPS group,the RAW 264.7 apoptosis rate obviously decreased after the treatment with AP at the concentration of 0.5 mg·ml~(-1)(P<0.01) for 72 h,and the RAW 264.7 apoptosis rate obviously decreased after the treatment with AP at the concentration of 1.0mg·ml~(-1)(P<0.01) for 48 h and 72 h.The results of Western Blot test showed that the expression of Bcl-2 protein in LPS-induced RAW 264.7 cells obviously increased after 48 h and 72 h,and the difference was statistically significant(P<0.01) when compared with the normal control group.Compared with that in LPS group,the expression of Bcl-2 protein in LPS-induced RAW 264.7 cells increased and that of Bax protein decreased after the treatment with AP at the concentration of 1.0 mg·ml~(-1),and the difference was statistically significant(P<0.01).Conclusion:AP can inhibit LPS-induced RAW 264.7 apoptosis,and the mechanism may be related with increasing the expression of Bcl-2 protein and decreasing that of Bax protein in LPS-induced RAW 264.7 cells.
引文
1 Fan Y,Liu B.Expression of Toll-like receptors in the mucosa of patients with ulcerative colitis[J].Exp Ther Med,2015,9(4):1455-1459
2 Cheng Y,Zhu Y,Huang X,et al.Association between TLR2 and TLR4 Gene Polymorphisms and the Susceptibility to Inflammatory Bowel Disease:A Meta-Analysis[J].Plo S One,2015,10(5):e0126803
3 Munkholm P.The incidence and prevalence of colorectal cancer in inflammatory bowel disease[J].Aliment Pharmacol Ther,2003,Suppl2:1-5
4 Feng J,Guo C,Zhu Y,et al.Baicalin down regulates the expression of TLR4 and NFk B-p65 in colon tissue in mice with colitis[J].Int J Clin Exp Med,2014,7(11):4063-4072
5 Kumar S,Ahuja V,Sankar MJ,et al.Curcumin for maintenance of remission in ulcerative colitis[J].Cochrane Database Syst Rev,2012,10 :CD008424
6 Mehrlnder K,Dietrich H,Sembries S,et al.Structural characterization of oligosaccharides and polysaccharides from apple juices produced by enzymatic pomace liquefaction[J].J Agric Food Chem,2002,50(5):1230-1236
7 Dou J,Meng Y,Liu L,et al.Purification,characterization and antioxidant activities of polysaccharides from thinned-young apple[J].Int J Biol Macromol,2015,72:31-40
8 Aggarwal BB,Kumar A,Bharti AC,et al.Anticancer potential of curcumin:preclinical and clinical studies[J].Anticancer Res,2003,23(1A):363-398
9 GrosséF,Guyot S,Roussi S,et al.Chempreventive properties of apple procyanidins on human colon cancer-derived metastatic SW620 cells and in rat model of colon carcinogensis[J].Cancinogenesis,2005,26(7):1291-1295
10 江艳,王浩,吕龙,等.贡空孢子多糖LZPS-1的化学研究及其总多糖的抗肿瘤活性[J].药学学报,2005,40(4):347-350
11 Biancheri P,Foster MR,Fyfe MC,et al.Effect of Narrow Spectrum Versus Selective Kinase Inhibitors on the Intestinal Proinflammatory Immune Response in Ulcerative Colitis[J].Inflamm Bowel Dis,2016,22(6):1306-1315
12 吉登波,崔景荣.肿瘤的发生、发展及治疗与炎症的关系[J].中国新药杂志,2010,19(17):1551-1555
13 刘真,肖斌,毛旭虎,等.炎症与肿瘤的关系研究进展[J].现代生物医学进展,2009,3(9):591-594
14 杨连君.bcl-2,bax与肿瘤细胞凋亡[J].中国肿瘤生物治疗杂志,2003,10(3):232-234
2 Cheng Y,Zhu Y,Huang X,et al.Association between TLR2 and TLR4 Gene Polymorphisms and the Susceptibility to Inflammatory Bowel Disease:A Meta-Analysis[J].Plo S One,2015,10(5):e0126803
3 Munkholm P.The incidence and prevalence of colorectal cancer in inflammatory bowel disease[J].Aliment Pharmacol Ther,2003,Suppl2:1-5
4 Feng J,Guo C,Zhu Y,et al.Baicalin down regulates the expression of TLR4 and NFk B-p65 in colon tissue in mice with colitis[J].Int J Clin Exp Med,2014,7(11):4063-4072
5 Kumar S,Ahuja V,Sankar MJ,et al.Curcumin for maintenance of remission in ulcerative colitis[J].Cochrane Database Syst Rev,2012,10 :CD008424
6 Mehrlnder K,Dietrich H,Sembries S,et al.Structural characterization of oligosaccharides and polysaccharides from apple juices produced by enzymatic pomace liquefaction[J].J Agric Food Chem,2002,50(5):1230-1236
7 Dou J,Meng Y,Liu L,et al.Purification,characterization and antioxidant activities of polysaccharides from thinned-young apple[J].Int J Biol Macromol,2015,72:31-40
8 Aggarwal BB,Kumar A,Bharti AC,et al.Anticancer potential of curcumin:preclinical and clinical studies[J].Anticancer Res,2003,23(1A):363-398
9 GrosséF,Guyot S,Roussi S,et al.Chempreventive properties of apple procyanidins on human colon cancer-derived metastatic SW620 cells and in rat model of colon carcinogensis[J].Cancinogenesis,2005,26(7):1291-1295
10 江艳,王浩,吕龙,等.贡空孢子多糖LZPS-1的化学研究及其总多糖的抗肿瘤活性[J].药学学报,2005,40(4):347-350
11 Biancheri P,Foster MR,Fyfe MC,et al.Effect of Narrow Spectrum Versus Selective Kinase Inhibitors on the Intestinal Proinflammatory Immune Response in Ulcerative Colitis[J].Inflamm Bowel Dis,2016,22(6):1306-1315
12 吉登波,崔景荣.肿瘤的发生、发展及治疗与炎症的关系[J].中国新药杂志,2010,19(17):1551-1555
13 刘真,肖斌,毛旭虎,等.炎症与肿瘤的关系研究进展[J].现代生物医学进展,2009,3(9):591-594
14 杨连君.bcl-2,bax与肿瘤细胞凋亡[J].中国肿瘤生物治疗杂志,2003,10(3):232-234