The Regulation of VEGF Signal Axis by DATS and the Improvement of Formation of Placental Angiogenesis in URSA Mice
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摘要:
目的 探讨二烯丙基三硫醚（diallyl trisulfide，DATS）对不明原因反复自然流产（unexplained recurrent spontaneous abortion，URSA）小鼠胎盘血管生成的影响，并基于血管内皮细胞生成因子（vascular endothelial growth factor，VEGF）/血管内皮细胞生长因子受体2（vascular endothelial growth factor receptor 2 ，VEGFR-2）信号通路探索其作用机制。 方法 将16只URSA小鼠随机分为对照组与实验组，每组8只，实验组常规饲喂的同时给予纯度为98%的DATS，按照50 mg/（kg·d）配制成200 μL悬浊液进行灌胃；对照组常规饲喂的同时给予 200 μL PBS液灌胃。2组孕鼠连续给药18 d后，使用分光光度计检测胎盘组织中的硫化氢（hydrogen sulfide，H2S）含量，使用反转录酶-聚合酶链反应（RT-qPCR）检测胎盘组织中血小板-内皮细胞粘附分子（platelet endothelial cell adhesion molecule-1，CD31）、血管内皮生长因子A（vascular endothelial growth factor A，VEGFA）与VEGFR2的 mRNA相对表达量，并使用蛋白免疫印迹（Western blot）法检测胎盘组织中VEGFA与VEGFR2的蛋白表达水平。 结果 分光光度计检测结果显示：实验组（777.4922±72.9759） nmol/g H2S水平显著高于对照组（529.1824±99.7489） nmol/g （P < 0.05）；RT-qPCR结果显示：实验组CD31的相对表达量（0.0042±0.0006）显著高于对照组（0.0020±0.0004）（P < 0.05）、实验组VEGFA的相对表达量（0.7073±0.0677）显著高于对照组（0.5200±0.0946）（P < 0.05）、实验组VEGFR2的相对表达量（0.7304±0.1262）显著高于对照组（0.3984±0.047）（P < 0.05）；Western blot结果显示，DATS可显著提高胎盘组织中VEGFA和VEGFR2的蛋白表达水平（P < 0.05）。 结论 DATS可显著提高URSA胎盘组织中的H2S水平及胎盘组织中血管形成因子CD31、VEGFA及VEGFR2的相对表达量，并通过VEGF信号轴改善URSA小鼠胎盘血管的形成，将为DATS的临床应用提供一定的科学依据和参考价值。 Abstract:Objective The aim of this study is to investigate the effect of Diallyl trisulfide （DATS） on placental angiogenesis in mice with unexplained recurrent spontaneous abortion （URSA） and to explore its mechanism based on the vascular endothelial growth factor （VEGF）/vascular endothelial growth factor receptor-2 （VEGFR-2） signaling pathway. Methods Sixteen URSA mice selected were randomly divided into the control group and the experimental group, with 8 mice in each group. The experimental group was given 98% pure DATS （200 μL suspension prepared according to 50 mg/（kg·d) by gavage. The rats in the control group were fed conventionally and given 200 μL PBS solution by gavage. After 18 days of continuous administration, Hydrogen sulfide （H2S） content in the placental tissue was detected by spectrophotometer, and the relative expression of CD31, VEGFA and VEGFR2 mRNA in the placental tissue was detected by reverse transcriptase-polymerase chain reaction （RT-qPCR）. The protein expression levels of VEGFA and VEGFR2 in placenta were detected by Western blot. Results The spectrophotometer detection showed that the H2S level in the experimental group （777.4922±72.9759） was significantly higher than that in the control group （529.1824±99.7489） （P < 0.05）; The RT-qPCR results showed that the relative expression of CD31 in the experimental group （0.0042±0.0006） was significantly higher than that in the control group （0.0020±0.0004） （P < 0.05）, the relative expression of VEGFA in the experimental group （0.7073±0.0677） was significantly higher than that in the control group （0.5200±0.0946）（P < 0.05）, and the relative expression of VEGFR2 in the experimental group （0.7304±0.1262） was significantly higher than that in the control group （0.3984±0.047） （P < 0.05）; Western blot results showed that DATS significantly increased the protein expression levels of VEGFA and VEGFR2 in placental tissue （P < 0.05）. Conclusion DATS can significantly increase the level of H2S in URSA placental tissue and the relative expression levels of angiogenic factors CD31, VEGFA, and VEGFR2 in placental tissue. Through the VEGF signal axis, it can improve the formation of placental blood vessels in URSA mice. The relevant research results will provide scientific basis and reference value for the clinical application of DATS. -
Key words:
- DATS /
- VEGF signaling axis /
- URSA mice /
- Placental vessels
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图 1 2组胎盘组织中H2S含量比较
与实验组比较，***P < 0.001。
Figure 1. Comparison of H2S content in two groups of placental tissue
图 2 2组胎盘组织中CD31的蛋白相对表达量分析
与实验组比较，***P < 0.001。
Figure 2. Analysis of the relative expression level of CD31 protein between two groups of placental tissues
图 3 2组胎盘组织中VEGFA及VEGFR2的蛋白相对表达量分析
与实验组比较，***P < 0.001。
Figure 3. Analysis of the relative expression levels of VEGFA and VEGFR2 proteins between two groups of placental tissue
表 1 引物序列
Table 1. Primer sequences
基因名称 序列（5′→3′） 长度
（bp）PCR产物
长度（bp）ZNF470-F2 CCCCGGCAATCATAATGGAAA 21 95 ZNF470-R2 CTCCCTCTCAAACAAGTCTTCAC 23 ZNF545-F2 GACCTTTAGCCGTGGTTATCATC 23 88 ZNF545-R2 GGCTTTCCAGCATTCCTTACAT 22 GAPDH-F GGACCTGACCTGCCGTCTAG 20 100 GAPDH-R GTAGCCCAGGATGCCCTTGA 20 F：上游引物、正向；R：下游引物、反向。 
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