白芍(Radix paeoniae alba)提取物对中华绒螯蟹(Eriocheir sinensis)造血组织发育及造血因子Astakine的影响

血淋巴细胞在甲壳动物免疫反应中起着非常重要的作用,其主要由造血组织(Hematopoietic tissue, HPT)生成并随着开管式循环分布到身体各处。到目前为止,还没有相关研究表明甲壳动物造血组织是否受草本植物的影响。本研究以中华绒螯蟹(Eriocheir sinensis)为对象,利用荧光定量PCR、免疫荧光和组织化学等技术,初步探索了白芍(Radix paeoniae alba)提取物对中华绒螯蟹造血组织和造血因子Astakine的影响。结果显示,在原代培养的造血组织细胞中添加终浓度为0.85mg/mL的白芍提取物后,造血因子Astakine mRNA的表达量显著上调(P0.05)。进一步制备含1%白芍提取物的中华绒螯蟹饲料并进行饲喂实验,结果显示饲喂含白芍提取物饲料的中华绒螯蟹造血组织的Astakine mRNA的表达量较饲喂前上调了10.5倍,要高于对照组。免疫荧光结果同样表明,饲喂含白芍提取物饲料后中华绒螯蟹造血组织的Astakine蛋白荧光信号也要强于对照组。通过造血组织的形态学观察和造血组织体重百分比的检测,我们发现白芍组中华绒螯蟹造血组织含有较多卵形小叶,且造血组织体重百分比较饲喂前增加了6.64%,而对照组仅增加1.6%。免疫保护率实验显示,在脂多糖急性刺激下,白芍添加组的中华绒螯蟹的存活率(77.5%)要高于对照组(57.5%)。综上所述,白芍提取物可以促进中华绒螯蟹造血因子的表达和造血组织的发育,从而提高中华绒螯蟹的免疫调节功能。

EFFECTS OF RADIX PAEONIAE ALBA EXTRACTION ON HEMATOPOIETIC TISSUE AND HEMATOPOIETIC FACTOR ASTAKINE IN ERIOCHEIR SINENSIS

Hematopoietic tissue (HPT) is responsible for crustacean haemocytes proliferation, and it can be generally released into hemolymph. At present, no research about the effect of plant herbs on HPT development has been reported. Among widely used immunopotentiator candidate of plant herbs, Radix paeoniae alba (RPA) is a major species that can be used for the preparation of blood nourishment medication. Therefore, we studied the effects of RPA on the Eriocheir sinensis HPT in combination with analysis of HPT cytokine Astakine and HPT development. The results indicate that the final RPA concentration of 0.85 mg/mL significantly up-regulated the Astakine mRNA expression in the HPT cells (P<0.05). In addition, RPA-containing commercial feed up-regulated the Astakine mRNA expression of HPT to 10.5 folds as compared to its initial level. In the immunofluorescence assay, we observed the expression of Astakine protein in HPT in RPA group. As indicated by the morphological observation and hematopoietic tissue body percentage evaluation, the HPT in RPA-fed crab contained more ovoid lobules and the hematopoietic tissue body percentage was increased by 6.64%, while only by 1.6% in the control. Furthermore, as shown in the lipopolysaccharide challenge experiment, the survival rate (77.5%) in RPA feeding group was higher that than that of the control group (57.5%). Therefore, the RPA could boost the Astakine expression, promote the HPT development, and further improve the immune system function.