马尾松针提取物对镉毒16HBE细胞保护作用
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摘要
目的研究马尾松松针提取液对氯化镉(CdCl2)致16HBE细胞毒性的保护作用。方法用萃取法提取马尾松松针乙醇提取物(PMNE),分别用1、100和500μg/mL的PMNE(低、中、高浓度组)预处理16HBE细胞6 h,随后用25μmol/L CdCl2毒染至48 h,同时设阴性对照组(正常培养16HBE细胞)及空白对照组(无细胞组),用CCK8法检测细胞相对生存率,硫代巴比妥酸(TBA)法检测各组细胞内丙二醛(MDA)含量,qPCR法检测各组细胞凋亡相关基因Bcl-2及Bax相对表达量。结果与阴性对照组相比,CdCl2单独作用组细胞相对生存率下降(P <0.05),细胞内MDA表达量升高(P <0.05);低、中、高浓度PMNE预处理组细胞与CdCl2单独处理组相比,细胞相对生存率均升高(P <0.05),MDA含量均降低(P <0.05),且浓度越高细胞相对生存率越高(P <0.05),MDA含量越低(P <0.05)。与CdCl2单独作用组相比,低、中、高浓度PMNE预处理组抑凋亡基因Bcl-2相对表达量均上调(P <0.05),促凋亡基因Bax相对表达量均下调(P <0.05),且浓度越高Bcl-2相对表达量越高(P <0.05)。结论 PMNE对CdCl2所致的细胞毒性有一定程度的保护作用。
Objective To study the protective effect of Pinus massoniana pine needle extract(PMNE) on cadmium chloride-induced cytotoxicity in human bronchial epithelial cells(16 HBE). Methods Ethanol extraction method was used to prepare PMNE. The 16 HBE cells of low-, moderate-, and high-dose groups were treated with 1, 100, and 500 μg/mL PMNE for 6 hours; then the 16 HBE cells were exposed to 25 μmol/L cadmium chloride(CdCl2) for 48 hours. A negative control group(normally cultured 16 HBE cells) and a blank control group(culture medium only) were also established. Cell Counting Kit-8(CCK8) method was used to detect relative survival rate of the cells. Thiobarbituric acid(TBA) method was adopted to measure intracellular content of malondialdehyde(MDA). Quantitative PCR(qPCR) was applied to determine relative cellular expressions of apoptosis-related genes of B cell lymphoma 2(Bcl-2) and Bcl-2-associated X protein.Results Compared with that of negative control cells, the relative survival rate of cells treated with CdCl2 alone was decreased significantly(P < 0.05) but the intracellular MDA expression was increased(P < 0.05). The relative survival rate of the cells treated with low-, moderate-, and high-dose PMNE were increased but the intracellular MDA of the cells was decreased, both in a significant dose-response manner, in comparison with those of the cells treated with CdCl2 alone significantly(P < 0.05 for all). Significantly increased relative expression of anti-apoptotic gene Bcl-2 and decreased relative expression of pro-apoptotic gene Bax, both in a significant dose-response manner, were observed in the cells with low-,moderate-, and high-dose PMNE treatment in contrast to those of the cells treated with CdCl2 alone(all P < 0.05).Conclusion Ethanol extract of Pinus massoniana pine needles has a certain protective effect against chloride-induced cytotoxicity in human bronchial epithelial cells in vitro.
Objective To study the protective effect of Pinus massoniana pine needle extract(PMNE) on cadmium chloride-induced cytotoxicity in human bronchial epithelial cells(16 HBE). Methods Ethanol extraction method was used to prepare PMNE. The 16 HBE cells of low-, moderate-, and high-dose groups were treated with 1, 100, and 500 μg/mL PMNE for 6 hours; then the 16 HBE cells were exposed to 25 μmol/L cadmium chloride(CdCl2) for 48 hours. A negative control group(normally cultured 16 HBE cells) and a blank control group(culture medium only) were also established. Cell Counting Kit-8(CCK8) method was used to detect relative survival rate of the cells. Thiobarbituric acid(TBA) method was adopted to measure intracellular content of malondialdehyde(MDA). Quantitative PCR(qPCR) was applied to determine relative cellular expressions of apoptosis-related genes of B cell lymphoma 2(Bcl-2) and Bcl-2-associated X protein.Results Compared with that of negative control cells, the relative survival rate of cells treated with CdCl2 alone was decreased significantly(P < 0.05) but the intracellular MDA expression was increased(P < 0.05). The relative survival rate of the cells treated with low-, moderate-, and high-dose PMNE were increased but the intracellular MDA of the cells was decreased, both in a significant dose-response manner, in comparison with those of the cells treated with CdCl2 alone significantly(P < 0.05 for all). Significantly increased relative expression of anti-apoptotic gene Bcl-2 and decreased relative expression of pro-apoptotic gene Bax, both in a significant dose-response manner, were observed in the cells with low-,moderate-, and high-dose PMNE treatment in contrast to those of the cells treated with CdCl2 alone(all P < 0.05).Conclusion Ethanol extract of Pinus massoniana pine needles has a certain protective effect against chloride-induced cytotoxicity in human bronchial epithelial cells in vitro.
引文
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[2]Jin Y,Liu L,Zhang S,et al.Cadmium exposure to murine macrophages decreases their inflammatory responses and increases their oxidative stress[J].Chemosphere,2016,144:168-175.
[3]张超,刘卉,徐媛媛,等.马尾松松针提取物清除自由基作用的研究[J].中华中医药学刊,2013,31(9):1946-1948.
[4]刘莉莉,林岚,殷霄,等.镉毒性研究进展[J].中国职业医学,2012,39(5):445-447.
[5]潘炘,刘亚群,陈顺伟,等.马尾松松针提取物抗氧化能力研究[J].浙江林业科技,2009,29(1):44-46.
[6]杨丽华.马尾松松针抗氧化活性研究及其抗氧化功能饮料研制[D].广州:广州医科大学,2011.
[7]杨杰波,李明,谢菁菁,等.松针提取物α-蒎烯对肝癌HepG2细胞miR-221及其下游靶基因表达的影响[J].中国中药杂志,2016,41(21):3996-3999.
[8]郑晓珂,周微,王小兰.松针不同提取部位体外抗肿瘤作用的实验研究[J].现代预防医学,2009,36(9):1749-1754.
[9]Tsikas D,Rothmann S,Schneider JY,et al.Development,validation and biomedical applications of stable-isotope dilution GC-MSand GC-MS/MS techniques for circulating malondialdehyde(MDA)after pentafluorobenzyl bromide derivatization:MDA as a biomarker of oxidative stress and its relation to 15(S)-8-isoprostaglandin F2αand nitric oxide(NO)[J].J Chromatogr B Analyt Technol Biomed Life Sci,2016,1019:95-111.
[10]Chen D,Zheng X,Kang D,et al.Apoptosis and expression of the Bcl-2 family of proteins and P53 in human pancreatic ductal adenocarcinoma[J].Med Princ Pract,2012,21(1):68-73.