基于自噬途徑探討半枝蓮總黃酮抑制腫瘤細(xì)胞NLRP3炎癥小體表達(dá)的機(jī)制研究

陳明+王舉濤+高華武+雷升萍+朱永恒

[摘要] 通過(guò)研究半枝蓮總黃酮（total flavonoids in Scutellaria barbata，TF-SB）對(duì)腫瘤細(xì)胞自噬以及炎癥小體NLRP3的影響，探討自噬與炎癥小體NLRP3激活的關(guān)系，為進(jìn)一步探討TF-SB的抗腫瘤機(jī)制提供實(shí)驗(yàn)依據(jù)。該研究擬采用人工接種B16-F1細(xì)胞株致小鼠黑色素瘤模型，隨機(jī)均分為模型對(duì)照組、陽(yáng)性對(duì)照組（Rap，1.5 mg·kg-1）、TF-SB高、中、低（200，100，50 mg·kg-1）劑量組，另取健康C57BL/6J小黑鼠作為正常組以備血清檢測(cè)對(duì)照，每組10只，每日給藥1次，連續(xù)2周，末次藥后30 min處理動(dòng)物。采用HE染色檢測(cè)腫瘤組織病理形態(tài)學(xué)改變；采用Western blot法檢測(cè)腫瘤組織內(nèi)LC3-Ⅱ/LC3-Ⅰ以及炎癥小體NLRP3，caspase-1，IL-1β，IL-18的蛋白表達(dá)水平；采用ELISA法檢測(cè)血清炎癥因子IL-1β，IL-18含量。結(jié)果顯示：模型組腫瘤細(xì)胞呈明顯異型性及惡性增殖的特點(diǎn)，陽(yáng)性對(duì)照組及TF-SB各劑量組腫瘤組織向周圍侵犯情況明顯減輕、腫瘤組織壞死范圍明顯增大、炎性反應(yīng)明顯減輕。陽(yáng)性對(duì)照組及TF-SB各劑量組LC3-Ⅱ/LC3-Ⅰ明顯增加，NLRP3，caspase-1，IL-1β，IL-18蛋白表達(dá)明顯減少。模型組小鼠血清IL-1β，IL-18含量明顯升高，與正常對(duì)照組比較有極顯著性差異（P<0.001），陽(yáng)性對(duì)照組及TF-SB各劑量組血清IL-1β，IL-18含量明顯減少，上述指標(biāo)各藥物組與模型組比較均有顯著性差異（P<0.05，P<0.01或P<0.001）。實(shí)驗(yàn)結(jié)果表明半枝蓮總黃酮可通過(guò)抑制NLRP3炎癥小體的表達(dá)、改變腫瘤生長(zhǎng)微環(huán)境，從而產(chǎn)生抗腫瘤作用，其機(jī)制可能與其誘導(dǎo)腫瘤細(xì)胞發(fā)生自噬有關(guān)。

[關(guān)鍵詞] 半枝蓮總黃酮； NLRP3炎癥小體； 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