Abstract：
To investigate the effects of plumbagin on the proliferation and apoptosis of human hepatoma Huh-7 cells and its mechanism based on the creatine kinase B(CKB)/p53 signaling pathway. Huh-7 cells were treated with plumbagin from 1 to 12 μmol·L~(-1) for cell counting kit-8(CCK-8) assay, and 1, 3, and 6 μmol·L~(-1) were determined as low, medium, and high concentrations of plumbagin for subsequent experiments. CKB gene was knocked out in Huh-7 cells by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated proteins(Cas)-9 gene editing technology. CKB overexpression lentivirus was transfected into Huh-7 cells to up-regulate the expression of CKB. Cell proliferation and apoptosis were detected by plate cloning assay and flow cytometry. The mRNA expression of CKB was detected by quantitative real-time PCR(qRT-PCR). CKB, p53, mouse double minute 2 homolog(MDM2), B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), and caspase-3 protein were detected by Western blot(WB). The results showed that plumbagin significantly inhibited the proliferation of Huh-7 cells and induced cell apoptosis. Compared with the control group, the apoptosis level was significantly increased in the plumbagin group, while the apoptosis level was significantly decreased in the plumbagin combined with the apoptosis inhibitor group. Plumbagin significantly down-regulated the protein expression levels of CKB, Bcl-2, and MDM2 and up-regulated the protein expression levels of p53, Bax, and caspase-3. Knockdown of the CKB gene decreased the proliferative ability of Huh-7 cells, increased the apoptotic rate, decreased the expression levels of Bcl-2 and MDM2 proteins, and increased the expression levels of p53, Bax, and caspase-3 proteins. After up-regulation of CKB expression, the proliferation ability of Huh-7 cells was enhanced, and the protein expression levels of Bcl-2 and MDM2 were elevated. The protein expression levels of p53, Bax, and caspase-3 were decreased. In addition, plumbagin reversed the effect of overexpression of CKB on the proliferation and apoptosis of Huh-7 cells. In conclusion, plumbagin significantly inhibited the proliferative ability of Huh-7 cells, and the mechanism may be related to the inhibition of CKB expression, activation of the p53 signaling pathway, and regulation of the expression of mitochondrial-associated apoptotic proteins, ultimately inducing cell apoptosis.
摘要：
基于肌酸激酶B(CKB)/p53信号通路，探讨白杨素对人肝癌Huh-7细胞增殖和凋亡的影响及其机制。用1~12μmol·L~(-1)的白花素处理Huh-7细胞进行细胞计数试剂盒-8(CCK-8)检测,分别测定1、3和6μmol·L~(-1)为低，中等,和高浓度的白花精用于后续实验。通过成簇的规则间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）-9基因编辑技术，在Huh-7细胞中敲除CKB基因。将CKB过表达慢病毒转染到Huh-7细胞中以上调CKB的表达。通过平板克隆实验和流式细胞术检测细胞增殖和凋亡。实时定量PCR(qRT-PCR)检测CKBmRNA的表达。CKB,p53，小鼠双分2同源物（MDM2），B细胞淋巴瘤2（Bcl-2），Bcl-2相关X蛋白(Bax),Westernblot(WB)检测caspase-3蛋白。结果表明,白花皂甙能显著抑制Huh-7细胞的增殖,诱导细胞凋亡。与对照组相比,plumbagin组细胞凋亡水平显著升高,而plumbagin联合凋亡抑制剂组的凋亡水平显着降低。plumbagin显著下调CKB的蛋白表达水平,Bcl-2、MDM2和上调p53、Bax、和caspase-3。敲除CKB基因降低Huh-7细胞的增殖能力,增加了凋亡率，降低Bcl-2和MDM2蛋白的表达水平，并增加p53，Bax的表达水平，和caspase-3蛋白。上调CKB表达后，Huh-7细胞的增殖能力增强,Bcl-2和MDM2蛋白表达水平升高。p53、Bax、和caspase-3降低。此外,plumbagin逆转过表达CKB对Huh-7细胞增殖和凋亡的影响。总之,plumbagin显著抑制Huh-7细胞的增殖能力,其机制可能与抑制CKB表达有关,激活p53信号通路，和调节线粒体相关凋亡蛋白的表达，最终诱导细胞凋亡。
