国家食用菌中心依托精准营养创新引智基地、河南省国际联合实验室平台，联合埃及国家农业中心Adel研究员对N. sativa进行了持续的研究，该成果以“The immunomodulatory effects of active ingredients from Nigella sativa in RAW264.7 cells through NF-κB/MAPK signaling pathways.”为题发表于中科院TOP 1区《Frontier in Nutrition》杂志（IF：6.576）。
Introduction
毛茛科黑种草属植物Nigella sativa原产于西南亚，主要分布在欧洲南部、亚洲西南部和北非等地区。N. sativa干燥种子具有多种药理活性，包括抗癌、抑菌、降压、保肝、抗炎、解热等，医疗上常用作膳食营养补充剂、祛风剂、利尿剂和驱虫剂。
免疫调节是N. sativa种子最有价值的特性之一。 近20年来，N. sativa种子及其主要活性成分百里醌（TQ）对免疫系统的有益作用的研究取得了显著进展。例如，TQ提高T细胞中CD8和CD4的比值，改善NK细胞的功能发挥免疫调节特性。但是，对N. sativa种子中单体化合物的免疫作用研究较少。对从N. sativa种子70%乙醇提取物分离鉴定得到的30多个化合物活性筛选时发现，两个单糖衍生物，Ethyl-α-D-galactopyranoside （EG）和Methyl-α-D-glucoside（MG）以及两个三萜类化合物，3-O-[β-D-xylopyranose-(1→3)-α-L-rhamnose-(1→2)-α-L-arabinose]-28-O-[α-L-rhamnose-(1→4)-β-D-glucopyranose-L-(1→6)-β-D-glucopyranose]-hederagenin（HXRARG）和3-O-[β-D-xylopyranose-(1→3)-α-L-rhamnose-(1→2)-α-L-arabinose]-hederagenin （HXRA）能够促进RAW264.7巨噬细胞释放NO。因此，本研究以EG、MG、HXRARG和HXRA为对象，探讨N. sativa种子的免疫增强作用和相关机制。
Fig. 1 Structures of compounds EG, MG, HXRARG and HXRA from Nigella sativa.
Results and Discussion
NO的检测
当EG浓度为60 μM时，RAW264.7分泌NO的含量远高于另外两种剂量（40和80 μM）。MG（80、60和40 μM）、HXRARG（80、60和40 μM）和HXRA（8、6和4 μM）以剂量依赖性显著提高RAW264.7细胞产生NO。
Fig. 2 Effects of EG (A), MG (B), HXRARG (C) and HXRA (D) on nitric oxide (NO) production in RAW264.7 cells. Data are expressed as the means ± SD. Compared with the control, **P<0.01, ***P<0.001.
吞噬能力检测
与空白对照组相比，EG、MG、HXRARG或HXRA刺激RAW264.7细胞后，更多的中性红进入细胞，说明EG、MG、HXRARG和HXRA可增强巨噬细胞的吞噬能力。
Fig. 3 Effects of EG, MG, HXRARG and HXRA on phagocytosis of neutral red in RAW264.7 cells. (A-E): microscope picture, (F-I): quantitative analysis picture. Data are expressed as the means ± SD. Compared with the control, **P<0.01, ***P<0.001.
iNOS，COX-2蛋白表达和基因转录
如Fig 4A-D所示，EG、 MG、 HXRARG 和 HXRA可显著增加iNOS和COX-2的蛋白表达。此外，不同浓度的EG、MG、HXRARG或HXRA可显著增加巨噬细胞中iNOS和mRNA转录（Fig 4E-F）。综上所述，在蛋白和基因水平上，EG、MG、HXRARG、HXRA均能显著促进iNOS和COX-2的合成。
Fig. 4 Effects of different concentrations of EG, MG, HXRARG and HXRA on INOS and Cox-2 protein (A-D) and mRNA (E-F) expression in RAW264.7 cells. (A-D): the left side was the protein band diagram, and the right side was the protein quantification diagram. (E): mRNA expression of iNOS. (F): mRNA expression ofCOX-2. Data are expressed as the means ± SD. Compared with the control, **P<0.01, ***P<0.001.
TNF-α，IL-6的生成及其mRNA表达
结果表明，EG、MG、HXRARG和HXRA通过促进TNF-α、IL-6 mRNA转录而刺激RAW264.7分泌更多的TNF-α和IL-6。
Fig. 5 Effects of EG, MG, HXRARG and HXRA on the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and mRNA in RAW264.7 cells. (A): secretion of IL-6 (B): secretion of TNF-α (C): TNF-α mRNA transcription (D): IL-6 mRNA transcription. Data are expressed as the means ± SD. Data are expressed as the means ±SD. Compared with the control, ***P<0.001.
对ROS的影响
从Fig. 6可以看出，与空白对照组相比，EG、MG、Hxrarg和Hxra处理组的峰型明显右移，说明EG、MG、Hxrarg和Hxra可以促进ROS的释放。
Fig. 6 Effect of different concentrations of EG (A), MG (B), HXRARG (C) and HXRA (D) on ROS production ability of RAW 264.7 cells.
对NF-κB信号通路的影响
NF-κB是在免疫过程中普遍存在的一类重要的核转录因子，在许多基因的启动子和增强子中都存在功能性的NF-κB结合位点，主要影响机体免疫有关的基因表达，是介导巨噬细胞成熟的一个关键通路。
EG、MG、Hxrarg和Hxra处理RAW264.7细胞后，对NF-κB P65和IκB-α的蛋白表达无明显影响，但IκB-α和P65的磷酸化蛋白表达增加，p-P65/P65，p-IκB-α/IκB-α的比值显著增大。以上结果说明，EG、MG、Hxrarg和Hxra可以激活RAW264.7细胞中的NF-κB信号通路，从而诱导RAW264.7细胞的活化。
Fig. 7 Effects of different concentrations of EG (A), MG (B), HXRARG (C) and HXRA (D) on the expression of related proteins in NF-κB signaling pathway in RAW264.7 cells. The left side was the protein band diagram, and the right side was the protein quantification diagram. Data are expressed as the means ± SD. Compared with the control, ***P<0.001.
对MAPK信号通路的影响
MAPK家族包含三个主要的激酶，即Erk1/2，JNK和P38 MAPK。 这些激酶的共同激活促进细胞增殖、迁移、侵袭、血管生成、转移和凋亡，这对巨噬细胞和T细胞的发育和活化非常重要。
EG、MG、HXRARG或HXRA刺激RAW264.7细胞后，p-P38/P38、p-Erk/Erk或p-JNK/JNK比值均显著高于空白对照组。 提示EG、MG、HXRARG或HXRA可激活MAPK信号通路，诱导免疫应答。
Fig. 8 Effects of different concentrations of EG (A), MG (B), HXRARG (C) and HXRA (D) on the expression of related proteins in MAPK signaling pathway in RAW264.7 cells. The left side was the protein band diagram, and the right side was the protein quantification diagram. Data are expressed as the means ± SD. Compared with the control, ***P<0.001.
综上所述，从N. sativa种子中分离得到的四个化合物Ethyl-α-D-galactopyranoside （EG）、Methyl-α-D-glucoside（MG）、3-O-[β-D-xylopyranose-(1→3)-α-L-rhamnose-(1→2)-α-L-arabinose]-28-O-[α-L-rhamnose-(1→4)-β-D-glucopyranose-L-(1→6)-β-D-glucopyranose]-hederagenin（HXRARG）和3-O-[β-D-xylopyranose-(1→3)-α-L-rhamnose-(1→2)-α-L-arabinose]-hederagenin （HXRA）通过激活RAW264.7细胞中NF-κB和MAPK信号通路，显著增加NO、COX-2、TNF-α、IL-6和ROS的生成，激活巨噬细胞，发挥免疫增强作用（Fig. 9）。为进一步研究N. sativa作为具有免疫调节作用的营养补充剂提供理论基础。
Fig. 9 EG, MG, HXRARG and HXRA activate NF-κB and MAPK signaling pathways to promote the activation of RAW264.7 cells.
该系列研究先后发表4篇论文：
1. Jing Dong, Qiongxin Liang, Yun Niu, Shengjun Jiang, Li Zhou, Jinmei Wang, Changyang Ma, Wenyi Kang; Effects of Nigella sativa seed polysaccharides on type 2 diabetic mice and gut microbiota. International Journal of Biological Macromolecules 2020;159:725-738 https://doi.org/10.1016/j.ijbiomac.2020.05.042 二区 IF 6.953
2. Yun Niu, Li Zhou, Lijun Meng, Sitan Chen, Changyang Ma, Zhenhua Liu, Wenyi Kang; Recent Progress on Chemical Constituents and Pharmacological Effects of the Genus Nigella. Evidence-based complementary and alternative medicine, 2020: 6756835 https://doi.org/10.1155/2020/6756835 四区，IF 2.63
3. Niu Y, Wang B, Zhou L, Ma C, Waterhouse GIN, Liu Z, Ahmed AF, Sun-Waterhouse D and Kang W (2021) Nigella sativa: A Dietary Supplement as an Immune-Modulator on the Basis of Bioactive Components. Front. Nutr. 8:722813. https://doi.org/10.3389/fnut.2021.722813 Top 1区 IF 6.576
4. Qiongxin Liang, Jing Dong, Senye Wang, Wenjing Shao , Adel F. Ahmed, Yan Zhang, Wenyi Kang（2021）Immunomodulatory effects of Nigella sativa seed polysaccharides by gut microbial and proteomic technologies. International Journal of Biological Macromolecules 184: 483–496 https://doi.org/10.1016/j.ijbiomac.2021.06.118 二区 IF 6.953