基于自噬途径探讨半枝莲总黄酮抑制肿瘤细胞NLRP3炎症小体表达的机制研究

陈明+王举涛+高华武+雷升萍+朱永恒

[摘要] 通過研究半枝莲总黄酮（total flavonoids in Scutellaria barbata，TF-SB）对肿瘤细胞自噬以及炎症小体NLRP3的影响，探讨自噬与炎症小体NLRP3激活的关系，为进一步探讨TF-SB的抗肿瘤机制提供实验依据。该研究拟采用人工接种B16-F1细胞株致小鼠黑色素瘤模型，随机均分为模型对照组、阳性对照组（Rap，1.5 mg·kg-1）、TF-SB高、中、低（200，100，50 mg·kg-1）剂量组，另取健康C57BL/6J小黑鼠作为正常组以备血清检测对照，每组10只，每日给药1次，连续2周，末次药后30 min处理动物。采用HE染色检测肿瘤组织病理形态学改变；采用Western blot法检测肿瘤组织内LC3-Ⅱ/LC3-Ⅰ以及炎症小体NLRP3，caspase-1，IL-1β，IL-18的蛋白表达水平；采用ELISA法检测血清炎症因子IL-1β，IL-18含量。结果显示：模型组肿瘤细胞呈明显异型性及恶性增殖的特点，阳性对照组及TF-SB各剂量组肿瘤组织向周围侵犯情况明显减轻、肿瘤组织坏死范围明显增大、炎性反应明显减轻。阳性对照组及TF-SB各剂量组LC3-Ⅱ/LC3-Ⅰ明显增加，NLRP3，caspase-1，IL-1β，IL-18蛋白表达明显减少。模型组小鼠血清IL-1β，IL-18含量明显升高，与正常对照组比较有极显著性差异（P<0.001），阳性对照组及TF-SB各剂量组血清IL-1β，IL-18含量明显减少，上述指标各药物组与模型组比较均有显著性差异（P<0.05，P<0.01或P<0.001）。实验结果表明半枝莲总黄酮可通过抑制NLRP3炎症小体的表达、改变肿瘤生长微环境，从而产生抗肿瘤作用，其机制可能与其诱导肿瘤细胞发生自噬有关。

[关键词] 半枝莲总黄酮； NLRP3炎症小体； 自噬

[Abstract] This paper was aimed to investigate the relationship between autophagy and NLRP3 inflammasome activation by studying the effect oftotal flavonoids in Scutellaria barbata （TF-SB） on autophagy in tumor cells and NLRP3 inflammasome， and to provide experimental evidence for further study of the anti-tumor mechanism of TF-SB. Mielanoma models were established by inoculating B16-F1 cell line to mice， and then were randomly divided into 5 groups （n=10 in each group）： model control， positive control control（Rap， 1.5 mg·kg-1）， and TF-SB low， middle and high groups （50， 100 and 200 mg·kg-1）. Meanwhile， healthy C57BL/6J mice were used as normal control group （n=10）. The drugs were given once daily for 2 weeks consecutively. Thirty minutes after last treatment， the determinations at endpoint were performed； pathological changes of tumor tissue were evaluated by using HE staining； protein expressions of LC3-II/LC3-I or NLRP3inflammasome/caspase-1/IL-1β and IL-18 in tumor tissues were detected by using Western-blot； and serum levels of IL-1β and IL-18 were detected by using Elisa kit. The results showed that the tumor cells in model group showed obvious atypia and malignant proliferation； the invasion of tumor tissue was significantly reduced， the tumor necrosis area was significantly increased， and the inflammatory reaction was significantly alleviated in positive control group and various TF-SB groups. As compared with model control group， LC3-II/LC3-I was significantly increased， while NLRP3/caspase-1/IL-1βand IL-18 protein expressions were significantly decreased in positive control group and TF-SB groups. Serum IL-1β and IL-18 levels in model control group were found higher than those in control group （P<0.001）， but they were significantly lowered in positive control group and TF-SB groups （P<0.05， P<0.01 or P<0.001）. Taken together， total flavonoids in S. barbata could effectively alter the tumor growth micro-environment by inhibiting the expression of NLRP3 inflammasome， and its anti-tumor effect may be associated with the induction of tumor cell autophagy.endprint