Inhibitory effect of astaxanthin on autophagy and apoptosis of AGS cell infected by Helicobacter pylori
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摘要:目的 探讨虾青素对幽门螺杆菌（Hp）感染AGS细胞自噬和凋亡的影响。方法 选取胃上皮细胞株AGS细胞进行Hp感染，根据虾青素的浓度分为对照组[二甲基亚砜（DMSO）处理]、10 μmol/L组、20 μmol/L组、50 μmol/L组。分组处理96 h后，采用免疫荧光标记检测细胞Hp外膜蛋白（OMP）表达，实时荧光定量PCR（qRT-PCR）检测相关基因微管相关蛋白1轻链3-Ⅱ（LC3-Ⅱ）、LC3-Ⅰ、Beclin1、B细胞淋巴瘤/白血病-2（Bcl-2）、Bcl-2相关X蛋白（Bax）mRNA表达，Western blot法检测细胞自噬和凋亡相关蛋白的表达，TUNEL法检测细胞凋亡情况。结果 Hp在AGS细胞质中弥散分布，Hp感染AGS细胞成功。虾青素可抑制Hp的增殖，10、20、50 μmol/L组细胞凋亡率均低于对照组，差异有统计学意义（P < 0.05）。Beclin1、LC3-Ⅱ与LC3-Ⅰ比值（LC3-Ⅱ/LC3-Ⅰ）、Bax蛋白及mRNA的表达随着虾青素浓度的增高而逐渐降低，Bcl-2蛋白及mRNA表达随着虾青素浓度的增高而逐渐升高，与对照组相比，差异有统计学意义（P < 0.01）。虾青素10、20、50 μmol/L组白细胞介素-17（IL-17）含量均低于对照组，差异有统计学意义（P < 0.05），且随着虾青素浓度的增高，IL-17含量逐渐下降。结论 随着虾青素浓度的增高，Hp感染的AGS细胞的自噬及凋亡能力减弱。Abstract:Objective To investigate the effect of astaxanthin on autophagy and apoptosis of AGS cell infected by Helicobacter pylori (Hp).Methods The AGS cells of gastric epithelial cell line were infected by Hp. The infected cells were divided into control group[processed by dimethyl sulfoxide (DMSO)], 10 μmol/L astaxanthin group, 20 μmol/L astaxanthin group and 50 μmol/L astaxanthin group. After 96 hours of grouping, expression of outer membrane protein (OMP) of Hp of cells was detected by immunofluorescence. The mRNA expressions of microtubule associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ), LC3-Ⅰ, Beclin1, B-cell lymphoma/leukemia-2 (Bcl-2) and Bcl-2 related X protein (Bax) were detected by quantitative real-time PCR (qRT-PCR). The expressions of autophagy and apoptosis related proteins were detected by Western blot, and apoptosis was detected by TUNEL.Results Hp was dispersed in AGS cytoplasm, and Hp was able to infect AGS cells successfully. Astaxanthin was able to inhibit the proliferation of Hp, and the apoptosis rates of 10, 20 and 50 μmol/L groups were significantly lower than those of the control group (P < 0.05). The protein and gene expressions of Beclin1, ratio of LC3-Ⅱ to LC3-Ⅰ (LC3-Ⅱ/LC3-Ⅰ) and Bax protein decreased gradually with the increase of astaxanthin concentration, while protein and gene expressions of Bcl-2 protein increased gradually with the increase of astaxanthin concentration, and there were significant differences when compared to control group (P < 0.01). The contents of interleukin-17 (IL-17) in 10, 20 and 50 μmol/L astaxanthin groups were significantly lower than that in the control group (P < 0.05), and the content of IL-17 decreased gradually with the increase of astaxanthin concentration.Conclusion With the increase of astaxanthin concentration, the autophagy and apoptosis of AGS cells infected by Hp decrease.
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Keywords:
- astaxanthin /
- Helicobacter pylori /
- gastric epithelial cell line /
- autophagy /
- apoptosis
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图 1 激光共聚焦观察Hp感染AGS细胞(放大倍数40倍)
A: 细胞核DAPI染色(蓝色); B: Hp OMP蛋白染色(绿色); C: A、B合并图; D: Hp在AGS细胞内大量增殖的完整细胞图。
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图 6 各组Beclin1、LC3-Ⅱ/LC3-Ⅰ蛋白、mRNA的表达
A: Western blot结果; B: 蛋白表达量; C: mRNA表达量。与对照组比较, **P < 0.01。
表 1 引物序列
引物名称 序列 LC3-Ⅱ 上游 5′-AAGCAACTCTGGATGGGATT-3′ 下游 5′-GCAGCCACAGGACGA AAC-3′ LC3-Ⅰ 上游 5′-GGAAGAGCACCGUGAUCAAdTdT-3′ 下游 5′-AACACGCCCGUGCCCAUGAACdTdT-3′ Beclin 1 上游 5′-AGCATTCCGTCTGACCATCA-3′ 下游 5′-ACTCAGACCCTGAGGCTCAAAG-3′ Bcl-2 上游 5′-GATACGAAGGGAGGGTGTACCA-3′ 下游 5′-CTCGGCCAGGGTGTTGAA-3′ Bax 上游 5′-CTGAAATTTATTGACAAACAGC-3′ 下游 5′-GCATCGAGAGAAGAGCAGGGACAT-3′ GAPDH 上游 5′-GCAAATTCCATGGCACCGT-3′ 下游 5′-TCGCCCCACTTGATTTTGG-3′ 
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