Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
Department of Endocrinology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
BMC Complement Med Ther. 2020 Dec 9;20(1):373. doi: 10.1186/s12906-020-03166-1.
4-Hydroxyisoleucine (4-HIL) is an active ingredient extracted from Trigonella foenum-graecum L., a Chinese traditional herbal medicine, which exerts the efficacy of anti-obesity and anti-diabetes. We previously reported that 4-HIL potentiates anti-inflammatory and anti-insulin resistance effects through down-regulation of TNF-α and TNF-α converting enzyme (TACE) in 3 T3-L1 adipocytes and HepG2 cells. In the present study, we further investigate the effects and mechanisms of 4-HIL on obesity-induced inflammation in RAW264.7 macrophages and 3 T3-L1 adipocytes co-culture system.
RAW264.7 macrophages and 3 T3-L1 adipocytes were co-cultured to mimic the microenvironment of adipose tissue. siRNA-iRhom2 transfection was performed to knockdown iRhom2 expression in RAW264.2 macrophages. The mRNA and protein expression of iRhom2 and TACE were measured by real-time quantitative PCR (RT-qPCR) and western blotting. The production of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), IL-6 and IL-10 were evaluated by ELISA. The ratio of M2/M1 was detected by flow cytometry.
4-HIL significantly repressed the mRNA and protein levels of iRhom2 and TACE in RAW264.7 macrophages after LPS stimulated. Meanwhile, the levels of pro-inflammatory cytokines, including TNF-α, MCP-1, and IL-6, were substantially suppressed by 4-HIL in the co-culture system. Moreover, the level of anti-inflammatory cytokine IL-10 was increased significantly by 4-HIL in the co-culture system after LPS stimulation. Additionally, the ratio of M2/M1 was also increased by 4-HIL in the co-culture system after LPS stimulation. Finally, these effects of 4-HIL were largely enhanced by siRNA-iRhom2 transfection.
Taken together, our results indicated that obesity-induced inflammation was potently relieved by 4-HIL, most likely through the iRhom2-dependent pathway.
4-羟基异亮氨酸（4-HIL）是从中药鹰嘴豆芽中提取的一种活性成分，具有抗肥胖和抗糖尿病的功效。我们之前的研究表明，4-HIL 通过下调 3T3-L1 脂肪细胞和 HepG2 细胞中的 TNF-α 和 TNF-α 转化酶（TACE）来增强抗炎和抗胰岛素抵抗作用。在本研究中，我们进一步研究了 4-HIL 对 RAW264.7 巨噬细胞和 3T3-L1 脂肪细胞共培养系统中肥胖诱导的炎症的影响及其机制。
RAW264.7 巨噬细胞和 3T3-L1 脂肪细胞共培养以模拟脂肪组织的微环境。用 siRNA-iRhom2 转染 RAW264.2 巨噬细胞以敲低 iRhom2 表达。用实时定量 PCR（RT-qPCR）和 Western blot 测定 iRhom2 和 TACE 的 mRNA 和蛋白表达。用 ELISA 测定肿瘤坏死因子-α（TNF-α）、单核细胞趋化蛋白-1（MCP-1）、IL-6 和 IL-10 的产生。用流式细胞术检测 M2/M1 比值。
4-HIL 显著抑制 LPS 刺激后 RAW264.7 巨噬细胞中 iRhom2 和 TACE 的 mRNA 和蛋白水平。同时，4-HIL 显著抑制共培养系统中包括 TNF-α、MCP-1 和 IL-6 在内的促炎细胞因子的水平。此外，LPS 刺激后共培养系统中 4-HIL 显著增加抗炎细胞因子 IL-10 的水平。另外，LPS 刺激后共培养系统中 4-HIL 也增加了 M2/M1 的比值。最后，siRNA-iRhom2 转染大大增强了 4-HIL 的这些作用。
综上所述，我们的研究结果表明，4-HIL 强烈缓解肥胖诱导的炎症，可能通过 iRhom2 依赖性途径。
