Full Text:   <1172>

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CLC number: Q25

On-line Access: 2015-12-04

Received: 2015-04-25

Revision Accepted: 2015-09-14

Crosschecked: 2015-11-24

Cited: 4

Clicked: 2166

Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Hai-sheng Xu

http://orcid.org/0000-0002-3190-3777

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Journal of Zhejiang University SCIENCE B 2015 Vol.16 No.12 P.971-979

http://doi.org/10.1631/jzus.B1500098


Effect of lipopolysaccharide on the hemocyte apoptosis of Eriocheir sinensis


Author(s):  Hai-sheng Xu, Sun-jian Lyu, Jie-hao Xu, Bin-jie Lu, Jing Zhao, Song Li, Yi-qun Li, Yu-yin Chen

Affiliation(s):  College of Animal Sciences, Zhejiang University, Hangzhou 310058, China

Corresponding email(s):   hsxu@zju.edu.cn, chenyy@zju.edu.cn

Key Words:  Hemocyte, Eriocheir sinensis, Lipopolysaccharide, Apoptosis, Reactive oxygen species


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Hai-sheng Xu, Sun-jian Lyu, Jie-hao Xu, Bin-jie Lu, Jing Zhao, Song Li, Yi-qun Li, Yu-yin Chen. Effect of lipopolysaccharide on the hemocyte apoptosis of Eriocheir sinensis[J]. Journal of Zhejiang University Science B, 2015, 16(12): 971-979.

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author="Hai-sheng Xu, Sun-jian Lyu, Jie-hao Xu, Bin-jie Lu, Jing Zhao, Song Li, Yi-qun Li, Yu-yin Chen",
journal="Journal of Zhejiang University Science B",
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number="12",
pages="971-979",
year="2015",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1500098"
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Abstract: 
In the present study, we investigated the possible toxicity mechanism of lipopolysaccharide (LPS) extracted from Gram-negative bacteria in Eriocheir sinensis hemocytes. Apoptotic hemocytes and reactive oxygen species (ROS) production induced by the LPS were monitored by the combination of flow cytometry and microscope observation. It was shown that LPS induced serious damage on the DNA and morphological changes in hemocytes, including cell shrinkage, fracture of nucleus membrane, margination, condensation and fragmentation of chromatin, and formation of apoptotic bodies indicating obvious hemocyte apoptosis. As compared with the control group, the apoptotic cell ratio increased to 30.61% and 39.01% after 1-h exposure and 57.72% and 75.01% after 2-h exposure to 1 and 10 μg/ml LPS, respectively (P<0.05). Significant outburst of ROS production was observed in LPS-treated hemocytes with approximately 176.6% of relative dichlorofluorescein mean fluorescence at 1-h exposure, followed by a drastic decline (P<0.05). These results indicated that LPS would induce oxidative stress on hemocytes from E. sinensis and cause ROS burst, DNA damage, and subsequently apoptosis. The process of ROS-mediated apoptosis might be one of the potential toxicity mechanisms of LPS on crustacean hemocytes.

脂多糖对中华绒螯蟹血细胞凋亡影响的研究

目的:评估细菌脂多糖(LPS)在体外环境下对中华绒螯蟹血细胞凋亡的影响,并探讨其诱导血细胞凋亡的机制。
创新点:首次在体外条件下实验证明了LPS可诱导中华绒螯蟹血细胞的凋亡,并确定了活性氧自由基在细胞凋亡过程中的变化和作用。
方法:在体外26 °C条件下,培养中华绒螯蟹血细胞。用浓度1 µg/ml 的LPS处理血细胞0、4、8和16 h后,利用4’,6-二脒基-2-苯基吲哚(DAPI)染色、荧光显微镜和透射电子显微镜观察血细胞的形态变化,用DNA Ladder试剂盒检测脱氧核糖核酸(DNA)的损伤程度。分别用浓度1和10 µg/ml的LPS处理血细胞0、1和2 h,经膜联蛋白 V-异硫氰酸荧光素(V-FITC)/碘化丙啶(PI)染色后,用流式细胞仪检测血细胞凋亡数量。收集血细胞制成细胞悬浮液,加入LPS至终浓度为10 µg/ml,同时加入2’,7’-二氯荧光黄双乙酸盐(DCFH-DA)探针至终浓度为1 µmol/L;对照组加入等量生理盐水和DCFH-DA探针。在26 °C孵育不同时间后,用流式细胞仪和激光共聚焦显微镜检测血细胞活性氧的产生情况。
结论:LPS可诱导中华绒螯蟹血细胞出现典型的凋亡特征,包括染色质浓缩和边聚、核膜破裂、线粒体肿胀、凋亡小体形成等(图1和2),形成明显的DNA梯形条带(图3),且细胞凋亡数量与LPS浓度呈正相关,大多数凋亡细胞处于凋亡晚期(图4)。活性氧检测结果显示:经LPS刺激后,细胞内活性氧自由基信号显著增强,随后又迅速减弱(图5和6)。综上所述,体外LPS可诱导中华绒螯蟹血细胞产生明显的凋亡反应,且凋亡过程伴随着显著的活性氧爆发。

关键词:血细胞;中华绒螯蟹;脂多糖;凋亡;活性氧自由基

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