目的 研究大黃素對人肝癌HepG2細(xì)胞線粒體凋亡的影響。方法 培養(yǎng)人肝癌HepG2細(xì)胞，與5、10、20、40、60、80、100 μmol/L的大黃素作用24、48 h，MTS法檢測細(xì)胞增殖；40、80、160 μmol/L大黃素作用HepG2細(xì)胞24 h，AO/EB雙熒光染色法觀察細(xì)胞凋亡的形態(tài)學(xué)改變；Annexin V/PI染色經(jīng)流式細(xì)胞儀檢測細(xì)胞凋亡；分光光度法檢測caspase 3活性；ATP試劑盒檢測細(xì)胞ATP含量，不同熒光探針加載后流式細(xì)胞儀測定大黃素對HepG2細(xì)胞內(nèi)活性氧（ROS）含量、Ca²⁺濃度、線粒體膜電位（MMP）變化的影響。結(jié)果 大黃素抑制HepG2細(xì)胞生長，且呈時間、濃度相關(guān)性，半數(shù)抑制濃度（IC₅₀）為（77.42±1.25）μmol/L；隨著大黃素濃度升高，AO/EB雙染觀察到細(xì)胞核濃縮、碎裂、凋亡小體等凋亡形態(tài)；與對照組比較，大黃素40、80、160 μmol/L作用于HepG2細(xì)胞24 h后細(xì)胞凋亡率顯著增加，caspase 3活性顯著增強，ROS水平、Ca²⁺濃度明顯增加（P<0.05、0.01、0.001），80、160 μmol/L組線粒體膜電位明顯降低，ATP含量顯著下降（P<0.05、0.01、0.001）。結(jié)論 大黃素造成HepG2細(xì)胞內(nèi)ROS堆積，ATP合成功能障礙，線粒體膜電位明顯下降，進而誘導(dǎo)線粒體通透轉(zhuǎn)運孔開放，導(dǎo)致鈣離子和細(xì)胞色素C外流，活化caspase蛋白家族，導(dǎo)致細(xì)胞凋亡。

Objective To study the effect of emodin on mitochondrial apoptosis in human hepatocellular carcinoma cell line (HepG2). Methods HepG2 cells were treated with 5, 10, 20, 40, 60, 80, and 100 μmol/L of emodin for 24 and 48 h, then the anti-proliferative effect was assessed by MTS assay. HepG2 cells were treated with 40, 80, 160 μmol/L of emodin for 24 h, and another control group was also established; The morphological changes of apoptosis were observed by AO/EB double fluorescence staining. Apoptosis was detected by flow cytometry (FCM) with Annexin V/PI staining. Caspase3 activity was detected by spectrophotometry. ATP assay kit was used to detect the content of ATP. Flow cytometry was used to determine the Ca²⁺ concentration, the content of ROS, and mitochondrial membrane potential (MMP) of emodin in HepG2 cells after loading with different fluorescence probes. Results Emodin could significantly inhibit the proliferation of HepG2 cells and showed a time and concentration correlation. The IC₅₀ values were determined as (77.42±1.25) μmol/L. With the increase of emodin concentration, the apoptotic morphology of nucleus condensation, fragmentation and apoptotic bodies were observed by AO/EB double staining. Compared with the control group, after HepG2 cells treated with 40, 80, and 160 μmol/L emodin for 24 h, the apoptosis rate increased significantly, the activity of caspase3 increased significantly, and the level of ROS and the concentration of Ca²⁺ increased significantly (P<0.05, 0.01 and 0.001). Mitochondrial membrane potential decreased significantly in 80 and 160 μmol/L emodin group, and ATP content decreased significantly (P<0.05, 0.01, and 0.001). Conclusion Emodin causes the accumulation of ROS in HepG2 cells, the dysfunction of ATP synthesis, the decrease of mitochondrial membrane potential, which leads to the opening of the mitochondrial permeable transport pores, resulting in the efflux of calcium ions and cytochrome C, activating the caspase protein family and promoting cell apoptosis.

國家科技重大新藥創(chuàng)制項目（2015ZX09501004）；天津市科技計劃項目（16PTGCCX00090）