目的 探討蔓性千斤拔素A通過細(xì)胞外調(diào)節(jié)蛋白激酶（ERK）/哺乳動物雷帕霉素靶蛋白（mTOR）通路對非小細(xì)胞肺癌細(xì)胞自噬的影響。方法 將A549細(xì)胞分為對照組及蔓性千斤拔素A低、中、高濃度（20、25、30 μmol·L^-1）組或?qū)φ战M、蔓性千斤拔素A 30 μmol·L^-1、SCH772984 10 μmol·L^-1+蔓性千斤拔素A 30 μmol·L^-1和SCH772984 10 μmol·L^-1濃度組，對照組細(xì)胞培養(yǎng)液為含1%胎牛血清的RMPI 1640培養(yǎng)基，其余各組分別加入蔓性千斤拔素A或SCH772984（預(yù)處理細(xì)胞1 h）使達(dá)到相應(yīng)濃度，連續(xù)培養(yǎng)12 h。MTT法檢測細(xì)胞活力；細(xì)胞集落形成實(shí)驗(yàn)檢測細(xì)胞集落形成的情況；細(xì)胞自噬染色檢測試劑盒檢測細(xì)胞內(nèi)自噬的情況；免疫熒光檢測細(xì)胞內(nèi)LC3B蛋白表達(dá)水平的變化；Western blotting實(shí)驗(yàn)檢測細(xì)胞內(nèi)磷酸化的細(xì)胞外調(diào)節(jié)蛋白激酶（p-ERK）、磷酸化的哺乳動物雷帕霉素靶標(biāo)（p-mTOR）、自噬相關(guān)蛋白Beclin 1（Beclin 1）、螯合體1（P62）蛋白表達(dá)水平的變化。結(jié)果 與對照組比較，MTT法檢測結(jié)果顯示各濃度（20、25、30 μmol·L^-1）蔓性千斤拔素A能夠顯著抑制A549細(xì)胞活力（P＜0.05、0.001），SCH772948 （10 μmol·L^-1）能夠顯著逆轉(zhuǎn)蔓性千斤拔素A誘導(dǎo)的A549細(xì)胞死亡（P＜0.01）；細(xì)胞集落形成實(shí)驗(yàn)顯示20、25、30 μmol·L^-1蔓性千斤拔素A能顯著抑制A549細(xì)胞集落的形成（P＜0.001）；細(xì)胞自噬染色檢測試劑盒結(jié)果顯示20、25、30 μmol·L^-1蔓性千斤拔素A能顯著抑制A549細(xì)胞自噬，SCH772948 10 μmol·L^-1能夠顯著逆轉(zhuǎn)蔓性千斤拔素A介導(dǎo)的A549細(xì)胞自噬抑制；免疫熒光結(jié)果顯示蔓性千斤拔素A 30 μmol·L^-1能夠顯著減弱LC3B的熒光，SCH772948 10 μmol·L^-1能夠顯著逆轉(zhuǎn)蔓性千斤拔素A導(dǎo)致的LC3B熒光減弱；Western blotting實(shí)驗(yàn)結(jié)果表明，20、25、30 μmol·L^-1蔓性千斤拔素A能夠使A549細(xì)胞內(nèi)p-ERK、p-mTOR、P62蛋白表達(dá)水平顯著升高及Beclin 1蛋白表達(dá)水平顯著降低（P＜0.001），SCH772948 10 μmol·L^-1能夠顯著逆轉(zhuǎn)蔓性千斤拔素A導(dǎo)致的A549細(xì)胞內(nèi)p-ERK、p-mTOR、P62蛋白表達(dá)水平升高及Beclin 1蛋白降低（P＜0.001）。結(jié)論 蔓性千斤拔素A可通過ERK/mTOR通路抑制非小細(xì)胞肺癌細(xì)胞自噬并導(dǎo)致細(xì)胞死亡。

Objective To investigate the effect of flemiphilippinin A on autophagy in non-small cell lung cancer (NSCLC) cells through the ERK/mTOR signaling pathway. Methods A549 cells were divided into control, flemiphilippinin A low, medium and high concentration (20, 25 and 30 μmol·L^-1) groups or control, flemiphilippinin A 30 μmol·L^-1, SCH772984 10 μmol·L^-1 + flemiphilippinin A 30 μmol·L^-1 and SCH772984 10 μmol·L^-1 concentration groups. The cells in the control group were cultured in RMPI 1640 medium containing 1% fetal bovine serum, and the cells in the other groups were pretreated with flemiphilippinin A or SCH772984 for 1 h to achieve the corresponding concentration, and the cells were continuously cultured for 12 h. Cell viability was detected by the MTT assay. Cell colony formation assay was used to detect cell colony formation. Autophagy staining detection kit was used to detect autophagy in cells. The expression of the LC3B protein was detected by immunofluorescence. The protein expression levels of p-ERK, p-mTOR, Beclin 1, and P62 were detected by Western blotting. Results The results of MTT assay showed that each concentration of flemiphilippinin A could significantly inhibit the viability of A549 cells (P < 0.05, 0.001). SCH772948 (10 μmol·L^-1) could reverse the inhibition of flemiphilippinin A-induced proliferation of A549 cells (P < 0.01). The cell colony formation assay showed that flemiphilippinin A (20, 25, and 30 μmol·L^-1) could inhibit the colony formation of A549 cells (P < 0.01). The results of the autophagy staining assay kit showed that flemiphilippinin A (20, 25, and 30 μmol·L^-1) could inhibit autophagy in A549 cells, and SCH772948 10 μmol·L^-1 could reverse the flemiphilippinin A-mediated autophagy inhibition in A549 cells. Immunofluorescence results showed that the fluorescence of LC3B induced by flemiphilippinin A (30 μmol·L^-1) was decreased, and SCH772948 (10 μmol·L^-1) could reverse the decrease of LC3B fluorescence induced by flemiphilippinin A. The results of Western blotting showed that the expression levels of p-ERK, p-mTOR, and P62 proteins in A549 cells were significantly increased, and the expression level of Beclin 1 protein was significantly decreased of flemiphilippinin A (20, 25, and 30 μmol·L^-1) (P < 0.001). 10 μmol·L^-1 SCH772948 could significantly reverse the increase of p-ERK, p-mTOR, and P62 protein expression levels and the decrease of Beclin 1 protein in A549 cells induced by flemiphilippinin A (P < 0.001). Conclusion Flemiphilippinin A can inhibit autophagy and cause cell death in non-small-cell lung cancer cells through the ERK/mTOR pathway.

R285.5

廣西中醫(yī)藥大學(xué)2022年引進(jìn)博士科研啟動基金項(xiàng)目（2022BS008）;2023年度廣西高校中青年教師科研基礎(chǔ)能力提升項(xiàng)目（2023KY0303）;廣西中醫(yī)藥大學(xué)-柳藥集團(tuán)青年科技創(chuàng)新能力提升計(jì)劃項(xiàng)目（YXY0000305）;廣西研究生教育創(chuàng)新計(jì)劃項(xiàng)目（YCSW2023402）