超氧化物歧化酶抑制精液来源的病毒增强因子形成的研究

doi:10.3969/j.issn.1674-8115.2021.04.008

摘要/Abstract

摘要：

目的·探究超氧化物歧化酶（superoxide dismutase，SOD）对精液来源的病毒增强因子（semen-derived enhancer of viral infection， SEVI）淀粉样纤维形成的抑制作用。方法·将440 μmol/L的前列腺酸性磷酸酶248-286（prostatic acid phosphatase 248-286，PAP248-286）多肽溶液或精液分别与不同活性水平的SOD混合孵育，于不同的时间点取样，用硫磺素T染色法、圆二色谱法、透射电子显微镜检测SOD对SEVI形成的影响；用病毒感染增强实验，检测SOD对SEVI促进人类免疫缺陷病毒1型（human immunodeficiency virus-1，HIV-1）感染能力的影响。灭活SOD并检测其对SEVI形成及病毒感染能力的影响。总SOD活性检测试剂盒检测5份健康志愿者精液中SOD的活性水平。结果·硫磺素T染色法结果显示，48 h时，46 U/mL和92 U/mL的SOD在体外可以抑制PAP248-286溶液形成SEVI淀粉样纤维；圆二色谱结果表明SOD可以抑制SEVI β-折叠结构的形成；透射电子显微镜结果也表明，SOD对SEVI的形成具有抑制作用。精液中的硫磺素T染色分析结果显示，SOD可抑制精液中的SEVI形成。病毒感染增强实验结果显示，SOD能够抑制SEVI促进HIV-1感染能力的作用。SOD灭活后上述作用均基本消失。5份健康男性精液中SOD活性分别为74.87、68.69、85.46、113.29、109.53 U/mL。结论·SOD在体外可以抑制SEVI淀粉样纤维的形成及其促进HIV-1感染的作用。

关键词: 超氧化物歧化酶, 精液来源的病毒增强因子, 淀粉样纤维, 人类免疫缺陷病毒, 性传播, 精液

Abstract:

Objective·To explore the inhibitory effect of superoxide dismutase (SOD) on the formation of semen-derived enhancer of viral infection (SEVI) amyloid fibrils.

Methods·Prostatic acid phosphatase 248-286 (PAP248-286) at 440 μmol/L or seminal fluid was incubated with SOD at different activity levels, and at different time points, aliquots were taken from each sample for thioflavin T (ThT) staining, circular dichroism (CD) and transmission electron microscope (TEM) to detect the effect of SOD on the formation of SEVI. The effect of SOD on the ability of SEVI to promote human immunodeficiency virus-1 (HIV-1) infection was detected by virus infection enhancement experiment. The effects of inactivated SOD on the formation of SEVI and the ability of SEVI to promote virus infection were also detected. The SOD activity levels in the semen of five healthy volunteers were detected by using total SOD activity detection kit.

Results·The results of ThT staining showed that 46 U/mL and 92 U/mL SOD could inhibit the formation of SEVI fibrils in the PAP248-286 solution in vitro at 48-h time point. The results of CD showed that SOD could inhibit the formation of β-sheet structure of SEVI. The results of TEM also showed that SOD could inhibit the formation of SEVI. The ThT staining in the semen also demonstrated the inhibitory effect of SOD on the formation of SEVI. As shown in the virus infection enhancement experiment, SOD could inhibit the promoting effect of SEVI on HIV-1 infection. When SOD was inactivated, the effects above almost disappeared. The SOD activities in the semen of the five healthy males were 74.87, 68.69, 85.46, 113.29, and 109.53 U/mL, respectively.

Conclusion·SOD can inhibit the formation of SEVI fibrils in vitro and the promoting effect of SEVI on HIV-1 infection.

Key words: superoxide dismutase (SOD), semen-derived enhancer of viral infection (SEVI), amyloid fibril, human immunodeficiency virus (HIV), sexual transmission, semen

中图分类号:

R512.91

邱梦婕, 李钊锋, 黎奕斌, 陈玉柳, 程宏彦, 刘叔文, 谭穗懿. 超氧化物歧化酶抑制精液来源的病毒增强因子形成的研究[J]. 上海交通大学学报(医学版), 2021, 41(4): 467-472.

Meng-jie QIU, Zhao-feng LI, Yi-bin LI, Yu-liu CHEN, Hong-yan CHENG, Shu-wen LIU, Sui-yi TAN. Inhibition of semen-derived enhancer of viral infection by superoxide dismutase[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(4): 467-472.

图/表 5

图1 SOD对淀粉样纤维SEVI形成的抑制作用Note： A. The effect curves of SOD for inhibiting SEVI formation by using ThT fluorescence assay. ①P=0.041, ②P=0.026, compared with the pure PAP248-286. B. β-Sheet formation of SEVI fibrils monitored by CD spectroscopy at 4 h and 48 h. The experiment was repeated twice, and the results were similar. C. SEVI fibrils in the presence or absence of SOD at 4 h and 48 h under TEM. Scale bars indicate 200 nm.

Fig 1 Inhibitory effect of SOD on the assembly of SEVI amyloid fibrils

图2 SOD对精液中淀粉样纤维形成的抑制作用Note: ①P=0.015, ②P=0.023, ③P=0.012, ④P=0.010, compared with the pure SE-F at the same time point.

Fig 2 Inhibitory effect of SOD on the assembly of amyloid fibrils in SE-F

图3 SOD对SEVI促进HIV-1感染能力的抑制作用Note: ①P=0.045, ②P=0.033, compared with the pure PAP248-286.

Fig 3 Inhibition of SOD on the ability of SEVI to promote HIV-1 infection

图4 灭活的SOD对淀粉样纤维SEVI形成的作用Note: A. The effect curve of inactivated SOD for inhibiting SEVI formation by using ThT fluorescence assay. ①P=0.732, ②P=0.029, compared with PAP248-286+inactivated SOD. B. β-Sheet formation of SEVI fibrils monitored by CD spectroscopy at 4 h and 48 h. The experiment was repeated twice, and the results were similar.

Fig 4 Effect of inactivated SOD on the assembly of SEVI amyloid fibrils

图5 灭活SOD对SEVI促进病毒感染能力的作用Note: ①P=0.045, compared with PAP248-286+inactivated SOD.

Fig 5 Effect of inactivated SOD on the ability of SEVI to promote HIV-1 infection

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