Hypaconitine inhibits TGF-β1-induced epithelial–mesenchymal transition and suppresses adhesion,migration,and invasion of lung cancer A549 cells

Epithelial-mesenchymal transition(EMT) has been implicated in tumor invasion and metastasis and provides novel strategies for cancer therapy. Hypaconitine(HpA), a diester-diterpenoid alkaloid isolated from the root of the Aconitum species, exhibits anti-inflammatory, analgesic, and especially, cardiotoxic activities. Here, we reported the anti-metastatic potentials of HpA in transforming growth factor-β1(TGF-β1)-induced EMT in lung cancer A549 cells. The cytotoxic effect of HpA was determined by MTT assay. A549 cells were treated with TGF-β1 with or without HpA co-treatment, and the morphological alterations were observed with a microscopy. The expression of E-cadherin, N-cadherin, and NF-κB was determined by both Western blotting and immunofluorescence analyses. The adhesion, migration, and invasion were detected with Matrigel, wound-healing, and transwell assays, respectively. The expression of Snail was determined by Western blotting. The expression of NF-κB p65, IκBα, and p-IκBα in nuclear and cytosolic extracts was assessed by Western blotting. The results showed that low concentration of HpA(<16 μmol×L^(–1)) had no obvious cytotoxicity to A549 cells. Morphologically, TGF-β1 treatment induced spindle-shaped alteration in the cells. The upregulation of N-cadherin, NF-κB, and Snail and the downregulation of E-cadherin were detected after TGF-β1 treatment. The adhesion, migration and invasion abilities were also increased by TGF-β1. Besides, TGF-β1 induced expression of Snail in a time-dependent manner. Furthermore, TGF-β1 induced nuclear translocation of NF-κB p65. All these alterations were dramatically inhibited by HpA co-treatment. In addition, the NF-κB inhibitor PDTC showed similar inhibitory effect. In conclusion, these results showed that HpA inhibited TGF-β1-induced EMT in A549 cells, which was possibly mediated by the inactivation of the NF-κB signaling pathway, providing an evidence for anti-cancer effect of HpA.

Protective mechanisms of hypaconitine and glycyrrhetinic acid compatibility in oxygen and glucose deprivation injury

This study investigated the protective effect of the compatibility of hypaconitine （HA） and glycyrrhetinic acid （GA） on H9c2 cells under oxygen and glucose deprivation （OGD）-induced injury, and the possible mechanisms. We found that HA＋GA significantly improved pathology and morphology of the nucleus and ultrastructure of H9c2 cells under OGD as determined by Hoechst 33342 staining and transmission electron microscopy （TEM） tests. It also reduced the releases of lactate dehydrogenase （LDH）, creatine kinase-myocardial band isoenzyme （CK-MB）, and aspartate transaminase （AST） from the cultured supernatant of H9c2 cells, which were tested by enzyme-linked immune sorbent assay （ELISA） kits. In addition, it lessened the apoptotic rate as determined by a fluorescein isothiocyanate-annexin V/propidium iodide （FITC-AV/PI） double staining assay. It was also found that HA＋GA might regulate the protein expression associated with the phosphatidylinositol 3-kinase （PI3K）/Akt signaling pathway. Overall, the study demonstrated that HA＋GA protected H9c2 cells against OGD-induced injury, and the signaling mechanism might be related to the PI3K/Akt signaling pathway.

超高效液相色谱串联四极杆质谱联用分析动物血样中乌头类生物碱

An ultra performance liquid chromatography-tandem mass spectrum method has been developed for the determination of three aconitum alkaloids (aconitine,hypaconitine and mesaconitine).The three alkaloids were analyzed simultaneously with an Waters Acquity BHE C18 column by gradient elution using 0.05% aqueous ammonia-acetonitrile as mobile phase and detected by the MRM mode of the mass spectrum.The recoveries of the SPE method were between 68.79%-95.65% (RSD<7.94%,n=4),and all the alkaloids showed good linearity (r>0.998) in a relatively wide concentration range.The LOD reached 0.0516,0.0640,0.0744 ng/mL of aconitine,hypaconitine and mesaconitine,respectively.The analysis time was within 3 min which could meet the high-throughput detection.The results indicated that contents of alkaloids in animal blood can be well detected;and this method can be used in quality control of aconitum drugs and pharmacokinetics study.

Analysis of the Aconitine Alkaloids in Chuanwu by Electrospray Ionization/Tandem Mass Spectrometry

An electrospray ionization / tandem mass spectrometric (ESI/MS/MS) method was developed for the simultaneous identification and analysis of three aconitine alkaloids [ mesacontine (MA), hypaconitine (HA), and aconitine (A)] as intact molecules at low nanogram level in Chinese traditional medicine Chuanwu decoction as well as in human whole blood extract without chromatographic separation.