该成果以“Effects of two triterpenoids from Nigella sativa seeds on insulin resistance of 3T3-L1 adipocytes”为题发表在中科院TOP 1区期刊Frontiers in Nutrition (IF 6.576)
胰岛素抵抗（Insulin resistance，IR）是一种生理异常，在胰岛素未能激活靶器官中的信号转导通路时发生。研究发现用口服抗糖尿病药物补充Nigella sativa种子可以通过增强β细胞活性和减轻IR来改善血糖。然而，N. sativa种子的植物化学物质的活性和相关机制尚未得到彻底研究。在本研究中，对两种三萜类化合物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) and 3-O-[β-D-xylopyranose-(1→3)-α-L-rhamnose-(1→2)-α-L-arabinose]hederagenin (Hxra)，通过3T3-L1脂肪细胞模型研究IR。
Fig. 1 Structures of Hxrarg (A) and Hxra (B) from Nigella sativa.
实验结果
1.Hxrarg和Hxra对3T3-L1前脂肪细胞活力的影响
结果表明，Hxrarg浓度从0增加到100 µM时，3T3-L1前脂肪细胞的存活率大于95%；Hxra在浓度低于50 μM时，存活率均在90%以上。
Fig. 2 Viability effect of Hxrarg and Hxra on 3T3-L1 preadipocytes. Compared with control group, ∗P < 0.05, ∗∗∗P < 0.001.
2. 3T3-L1前脂肪细胞的分化和鉴定
3T3-L1前脂肪细胞的细胞质中没有脂滴，呈不规则的多边形或纺锤形（图3A）。加入分化培养基I和II并用油红O染色后，在细胞质中检测到脂滴。超过80％的细胞具有成熟的脂肪细胞特征（图3B），表明3T3-L1前脂肪细胞已成功分化。
Fig. 3 Microscopic view of 3T3-L1 preadipocytes differentiated to adipocytes. (A) 0th day; (B) oil red O staining in the 8th day.
3. Hxrarg和Hxra对3T3-L1细胞脂质积累的影响
用Hxrarg或Hxra培养后，脂滴的产生被抑制，从而降低了诱导过程中的脂滴量并抑制了细胞分化，同时不同浓度的Hxrarg和Hxra处理组减少了3T3-L1细胞的脂质积累。
Fig. 4 Inhibition of lipid droplet formation by Hxrarg (A) and Hxra (B). Effects of Hxrarg (C) and Hxra (D) on lipid accumulation during 3T3-L1 differentiation. Compared with control group, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
4. Hxrarg和Hxra对甘油三酯含量的影响
从图5中可以看出，Hxrarg和Hxra处理组中，TG含量低于对照组，表明Hxrarg和Hxra可以显著降低TG含量并阻断细胞分化。
Fig. 5 Effects of Hxrarg (A) and Hxra (B) on TG contents in 3T3-L1 cells differentiation. Compared with control group, ∗∗∗P < 0.001.
5. IR-3T3-L1脂肪细胞模型的建立
结果表明，IR脂肪细胞模型的最佳时间是Dex治疗后72 h，IR细胞模型构建成功60 h后稳定。72 h后模型组上清液葡萄糖含量较对照组降低。因此，选择48 h作为给药时间来研究Hxrarg和Hxra对IR-3T3-L1的影响。
Fig. 6 Establishment of IR-3T3-L1 adipocyte model. (A) Glucose consumption test during insulin resistance induced by Dex; (B) dex induced insulin resistance in adipocytes glucose consumption difference; (C) Stability of insulin resistance model.
6. Hxrarg和Hxra对IR-3T3-L1细胞葡萄糖摄取的影响
3T3-L1细胞在用Hxrarg和Hxra处理48 h后消耗更多的葡萄糖，使葡萄糖含量显著下降，表明Hxrarg和Hxra能够促进IR-3T3-L1脂肪细胞的葡萄糖摄取并改善IR状态。
Fig. 7 Glucose uptake effect of Hxrarg (A) and Hxra (B) on IR-3T3-L1 adipocytes. Compared with control group, ###P < 0.001; compared with model group, ∗P < 0.05, ∗∗∗P < 0.001.
7. PI-3K/AKT信号通路相关蛋白的表达
PI-3K/AKT信号通路受损，阻止胰岛素信号继续向下游转导或降低传输效率。Dex处理72 h后IR细胞模型成功建立，在不同浓度下，Hxrarg和Hxra对IRS和AKT蛋白表达没有影响，但会上调p-IRS、p-AKT、PI-3K和GLUT4的表达。这表明，Hxrarg和Hxra刺激PI-3K/AKT通路以增强胰岛素信号转导，最终改善IR。
Fig. 8 The effects of Hxrarg (A) and Hxra (B) on protein expression in IR adipocyte signaling pathway. Compared with the control, ###P < 0.001; compared with the model group, ∗P < 0.05, ∗∗∗P < 0.001.
8.胰岛素受体底物、AKT、PI-3K和GLUT4的mRNA表达
结果表明，在不同浓度的Hxrarg和Hxra组中，IRS、AKT、PI-3K和GLUT4 mRNA的表达上调。
Fig. 9 The effects of Hxrarg (A–D) and Hxra (E–H) on mRNA transcription. (A,E) IRS mRNA; (B,F) AKT mRNA; (C,G) PI-3K mRNA; and (D,H) GLUT4 mRNA. Compared with control group, ##P < 0.01, ###P < 0.001; compared with model group, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
综上所述，从Nigella sativa种子中分离出的两个化合物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) and 3-O-[β-D-xylopyranose-(1→3)-α-L-rhamnose-(1→2)-α-L-arabinose]hederagenin (Hxra)，能够通过激活脂肪组织中胰岛素信号转导通路IRS/AKT/PI-3K上的相关蛋白，从而促进脂肪细胞对葡萄糖的摄取。
Fig. 10 Hxrarg and Hxra activate PI-3K/AKT signaling pathways to promote the translocation of GLUT4.
该系列研究先后发表5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
5. Wei J, Wang B, Chen Y, Wang Q, Ahmed AF, Zhang Y, Kang W. The Immunomodulatory Effects of Active Ingredients From Nigella sativa in RAW264.7 Cells Through NF-κB/MAPK Signaling Pathways. Front Nutr. 9:899797. https://doi.org/10.3389/fnut.2022.899797 Top 1区 IF 6.576