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目的:研究新型抗肿瘤药物6'-羟基爵床素A(6'-hydroxy justicidin A,JR6)对HepG2细胞的内质网应激及胞内钙离子浓度变化的影响及其意义。方法:HepG2细胞经过终浓度分别为0,0.615,2.45,9.8μmol.L-1的JR6处理24h后采用实时定量RT-PCR技术、蛋白印迹(Western blot)法检测细胞中X-盒结合蛋白1(XBP-1)mRNA,IRE1α(inositol-requiring enzyme-1α),GRP78/BiP(glucose regulated protein/BiP),及Bcl-2(B cell leukemia 2)蛋白表达量的变化。激光扫描共聚焦显微镜测JR6作用24h后细胞内静息钙浓度变化及IP3R钙泵抑制剂光溜海绵素C干预后JR6的作用变化。结果:IRE1,XBP-1mRNA,Bip,Bcl-2蛋白的表达量均与JR6剂量负相关。高剂量组的各蛋白表达量与空白组相比,均下降了40%左右。共聚焦显微镜观察结果显示JR6引起细胞内钙离子浓度升高了2倍左右;加入IP3R钙泵抑制剂后,JR6不能引起细胞内钙升高。结论:JR6通过内质网钙泵IP3R升高细胞内的钙浓度,破坏细胞内的钙稳态;同时引起了内质网应激,并抑制了未折叠蛋白反应的相关因子。
Objective: To study the effect and significance of 6'-hydroxy justicidin A (JR6), a novel anti-tumor drug, on the changes of endoplasmic reticulum stress and intracellular calcium concentration in HepG2 cells. METHODS: HepG2 cells were treated with JR6 at the final concentrations of 0, 0.615, 2.45 and 9.8 μmol·L-1, respectively, for 24 h. The real-time quantitative RT-PCR was used to detect the expression of X-box binding protein 1 (XBP-1) mRNA, IRE1α (inositol-requiring enzyme-1α), GRP78 / BiP (glucose regulated protein / BiP) and Bcl-2 (B cell leukemia 2) Changes of resting intracellular Ca2 + concentration after 24h with JR6 laser confocal microscopy and the effect of JR6 after intervention of IP3R Ca2 + pumpkin with hamamatin C were observed. Results: The expression of IRE1, XBP-1mRNA, Bip, Bcl-2 protein were negatively correlated with the dose of JR6. Compared with the blank group, the protein expression of high-dose group decreased about 40%. Confocal microscopy showed that JR6 caused a 2-fold increase in intracellular calcium concentration. After addition of IP3R calcium pump inhibitor, JR6 did not induce intracellular calcium elevation. CONCLUSION: JR6 increases intracellular Ca2 + concentration through endoplasmic reticulum calcium pump IP3R and disrupts intracellular calcium homeostasis. At the same time, JR6 induces endoplasmic reticulum stress and inhibits unfolded protein responses.