目的 探究氧化苦參堿誘導(dǎo)結(jié)腸癌HCT116細胞死亡的作用機制。方法 將結(jié)腸癌HCT116細胞隨機分為對照組和氧化苦參堿（2、4、8、12、16、20、30、40 mmol·L^-1）組，通過CCK-8法檢測各濃度氧化苦參堿作用24、48 h對結(jié)腸癌細胞存活率的影響；光學(xué)顯微鏡下觀察氧化苦參堿（15、20、25 mmol·L^-1）對HCT116細胞形態(tài)的影響；通過Hoechst染色法和Annexin V/PI雙染流式細胞術(shù)法觀察氧化苦參堿對HCT116細胞凋亡的影響；JC-1染色觀察細胞線粒體膜電位的變化；Western blotting法檢測各組細胞成熟體半胱氨酸蛋白酶-3（cleaved Caspase-3）、B淋巴細胞瘤-2（Bcl-2）、BCL2關(guān)聯(lián)X蛋白（Bax）、細胞色素C （Cyt-C）、消化道皮膚素E （GSDME）、細胞周期蛋白1（cyclin-D1）、骨髓細胞瘤病毒癌基因（c-Myc）、聚腺苷二磷酸核糖聚合酶（PARP）、β-連環(huán)素（β-catenin）、生存素（survivin）的蛋白表達情況。結(jié)果 與對照組比較，氧化苦參堿可以明顯降低HCT116細胞的存活率，且存在劑量及時間相關(guān)性，作用24、48h的半數(shù)抑制濃度（IC₅₀）分別為18.80、10.75 mmol·L^-1；氧化苦參堿組形狀正常的HCT116細胞明顯減少，均出現(xiàn)部分類圓球形的細胞，并且折光率明顯降低；經(jīng)Hoechst染色后氧化苦參堿組HCT116細胞的藍色熒光明顯加深，且相對集中，流式細胞術(shù)結(jié)果顯示細胞凋亡率顯著增加（P<0.05、0.01）；JC-1染色結(jié)果表明氧化苦參堿可以降低HCT116細胞線粒體膜電位；氧化苦參堿組Bcl-2、β-catenin、c-Myc、cyclin D1和survivin蛋白表達水平顯著下降（P<0.05、0.01），Bax、cleaved Caspase-3、cleaved PARP、Cyt-C、GSDME蛋白表達水平及Bax/Bcl-2顯著上升（P<0.05、0.01）。結(jié)論 氧化苦參堿可能通過下調(diào)Wnt/β-catenin信號通路，促進線粒體依賴的細胞內(nèi)源性凋亡和細胞焦亡發(fā)揮抗結(jié)腸癌作用。

Objective To explore the mechanism of oxymatrine inducing colon cancer HCT116 cell death.Methods Colon cancer cells were randomly divided into control group, oxymatrine(2, 4, 8, 12, 16, 20, 30, and 40 mmol·L^-1) group.The CCK-8 method was used to detect the effect of different concentrations of oxymatrine on colon cancer cells survival after 24 and 48 h.The effects of oxymatrine(15, 20, 25 mmol·L^-1) on the morphological changes of HCT116 cells were observed by observation under optical microscope.Hoechst staining and Annexin V/PI double staining flow cytometry were used to observe the effect of oxymatrine on apoptosis of HCT116 cells.JC-1 staining was used to observe the changes of mitochondrial membrane potential.Western blotting method was used to detect the effect of oxymatrine on expressions of cleaved Caspase-3, B lymphocytoma-2(Bcl-2), BCL2-associated X protein(Bax), cytochrome C(Cyt-C), gastrointestinal dermatin E(GSDME), Cyclin 1(cyclin-D1), myeloma virus oncogene(c-Myc), poly adenosine diphosphate-ribose polymerase(PARP), β-catenin, survivin in colon cancer cells.Results Compared with control group, oxymatrine could significantly reduce the survival rate of HCT116 cells, and there was a dose and time correlation.The half inhibition concentration(IC50) of 24 and 48 h after treatment was 18.80 and 10.75 mmol·L^-1, respectively.In oxymatrine group, HCT116 cells with normal shape were significantly reduced, with some spherical like cells, and the refractive index was significantly reduced.After Hoechst staining, the blue fluorescence of HCT116 cells in oxymatrine group was significantly deepened and concentrated.Flow cytometry showed that the apoptosis rate was significantly increased(P<0.05, 0.01).JC-1 staining showed that oxymatrine could reduce the mitochondrial membrane potential of HCT116 cells.In oxymatrine group, the expression levels of Bcl-2, β-catenin, c-Myc, cyclin D1 and survivin protein decreased significantly(P<0.05, 0.01), while the expression levels of Bax, cleaved caspase-3, cleaved PARP, Cyt-C, GSDME protein and Bax/Bcl-2 protein increased significantly(P<0.05, 0.01).Conclusion Oxymatrine may play an anti-tumor effect by downregulating the Wnt/β-catenin signaling pathway and promoting mitochondria-dependent endogenous apopto sis and pyroptosis.

R285.5

江蘇衛(wèi)生健康職業(yè)學(xué)院院級重點課題（JKB202005）；江蘇省大學(xué)生創(chuàng)新創(chuàng)業(yè)訓(xùn)練計劃項目（202014255002Y）；江蘇衛(wèi)生健康職業(yè)學(xué)院院級重點課題（JKB2021005）；江蘇衛(wèi)生健康職業(yè)學(xué)院重大項（JKA202004）