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补肾活血通络汤含药血清对多发性骨髓瘤KM3细胞增殖的影响及其机制

[Effects and mechanisms of the kidney-reinforcing and blood circulation-activating and collateral dredging decoction metabolites on the proliferation of multiple myeloma KM3 cells].

作者信息

Shi J B, Li C N, Wei W J, Ding J Y, Ma G D, Li L L, Wang Y R, Lu Y T, Xu J, Zheng W, Wang Y, Wang J Y, Xu R R, Cui S Y

机构信息

The First School of Clinical Medicine, Shandong University of Traditional Chinese Medicine, Jinan 250014, China.

Department of Hematology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250014, China.

出版信息

Zhonghua Xue Ye Xue Za Zhi. 2025 Jul 14;46(7):647-654. doi: 10.3760/cma.j.cn121090-20241209-00547.

Abstract

To evaluate the effects and underlying mechanisms of metabolites derived from the kidney-reinforcing, blood circulation-activating, and collateral dredging decoction on the proliferation of multiple myeloma (MM) KM3 cells. MM KM3 cells in the logarithmic growth phase were treated with 3%, 6%, 9%, or 12% metabolites of kidney-reinforcing, blood circulation-activating, and collateral dredging decoction. Cell viability was assessed using the CCK-8 assay. Apoptosis and necrosis were evaluated using flow cytometry and TUNEL staining. Mitochondrial and cellular ultrastructural changes were examined using transmission electron microscopy. mRNA and protein expression levels of dynamin-related protein 1 (Drp1), mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (MFF), PTEN-induced kinase 1 (Pink1), and E3 ubiquitin ligase (Parkin) were determined through quantitative real-time PCR and western blotting. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) combined with network pharmacology, was utilized for reverse verification of the pharmacodynamic mechanisms and therapeutic targets underlying the anti-MM activity of this decoction. The metabolites of the kidney-reinforcing, blood circulation-activating, and collateral dredging decoction inhibited KM3 cell proliferation and induced apoptosis in a dose-dependent manner. Transmission electron microscopy revealed increased mitochondrial fission and autophagic structures, with effects intensifying at higher metabolite concentrations. mRNA and protein expression of Drp1, Fis1, MFF, Pink1, and Parkin were significantly upregulated in treatment groups compared to controls (<0.05), with the most pronounced effects observed in the 12% metabolite group (<0.01). HPLC-MS/MS identified 121 bioactive compounds in BHTF, which shared 474 overlapping targets with MM. Enrichment analysis suggested that BHTF exerts antitumor effects primarily through apigenin, palmatine, and other key components by modulating TNF, NF-κB, and mitophagy pathways. The kidney-reinforcing and blood circulation-activating and collateral dredging decoction suppresses the proliferation of MM KM3 cells, potentially through mechanisms involving the regulation of mitochondrial dynamics and induction of autophagy.

摘要

评估补肾活血通络方含药血清对多发性骨髓瘤(MM)KM3细胞增殖的影响及其潜在机制。对数生长期的MM KM3细胞分别用3%、6%、9%或12%的补肾活血通络方含药血清处理。采用CCK-8法检测细胞活力。通过流式细胞术和TUNEL染色评估细胞凋亡和坏死情况。运用透射电子显微镜观察线粒体和细胞超微结构变化。通过定量实时PCR和蛋白质印迹法检测发动蛋白相关蛋白1(Drp1)、线粒体分裂蛋白1(Fis1)、线粒体分裂因子(MFF)、PTEN诱导激酶1(Pink1)和E3泛素连接酶(Parkin)的mRNA和蛋白表达水平。采用高效液相色谱-串联质谱(HPLC-MS/MS)结合网络药理学,对该方抗MM活性的药效学机制和治疗靶点进行反向验证。补肾活血通络方含药血清可抑制KM3细胞增殖并诱导其凋亡,且呈剂量依赖性。透射电子显微镜显示线粒体分裂和自噬结构增加,且在较高含药血清浓度下作用增强。与对照组相比,各给药组Drp1、Fis1、MFF、Pink1和Parkin的mRNA和蛋白表达均显著上调(<0.05);其中12%含药血清组作用最为明显(<0.01)。HPLC-MS/MS鉴定出补肾活血通络方中的121种生物活性成分,这些成分与MM共有474个重叠靶点。富集分析表明,补肾活血通络方主要通过芹菜素、巴马汀等关键成分调节TNF、NF-κB和线粒体自噬通路发挥抗肿瘤作用。补肾活血通络方可抑制MM KM3细胞增殖,其机制可能与调节线粒体动力学和诱导自噬有关。

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