Mechanism of cucurbitacin B suppresses proliferation of hepatocellular carcinoma Huh-7 cells via inducing ferroptosis / 中国药理学通报

Aim To study the effects of cucurbitacin B (Cu B) on proliferation of hepatocellular carcinoma Huh-7 cells and its mechanism. Methods CCK-8 was used to detect the survival rate of Huh-7 cells with different concentrations of Cu B. Huh-7 cells were treated with Cu B (0. 5, 1, 2 njnol; L

Saikosaponin A Changes Drug Resistance of A549/DDP Cells Through Wnt/β-Catenin Pathway / 中国实验方剂学杂志

Objective:To investigate the effect and mechanism of saikosaponin A （SSA） on the reversal of cisplatin （DDP） resistance in human lung cancer cell line A549/DDP. Methods:The resistance of A549 and A549/DDP cells to DDP and the inhibitory effects of SSA against the proliferation of A549 and A549/DDP cells were detected using cell counting kit-8 （CCK-8）. The apoptosis rates of A549/DDP cells treated with SSA or DDP or SSA combined with DDP and the changes in reactive oxygen species （ROS） were determined by flow cytometry. The mRNA expression levels of C-myc， B-cell lymphoma 2 （Bcl-2） and cysteinyl aspartate-specific protease-3 （Caspase-3） were detected by real-time polymerase chain reaction （Real-time PCR）， followed by the determination of <italic>β</italic>-catenin transcriptional activity using the TopFish dual-luciferase reporter assay system and the measurement of <italic>β</italic>-catenin protein expression in A549/DDP cells by Western blot. Results:The results of CCK-8 assay showed that the DDP resistance of A549/DDP cells was 12.82 times that of A549 cells （<italic>P</italic><0.05）. SSA inhibited the viability of A549 cells with the half maximal inhibitory concentration （IC₅₀） being 34.9 μmol·L^-1， and also suppressed the viability of A549/DDP cells in a concentration-dependent manner. Since the inhibition rate of 20 μmol/L SSA against A549/DDP cells was less than 10%， the reversal concentration was set at 20 μmol/L. Flow cytometry revealed that compared with the control， DDP alone increased the apoptosis rate of A549/DDP cells （<italic>P</italic><0.05）， stimulated the accumulation of intracellular ROS （<italic>P</italic><0.05）， down-regulated the mRNA expression levels of C-myc and Bcl-2 in A549/DDP cells， up-regulated Caspase-3 mRNA expression， and reduced the transcriptional activity of <italic>β</italic>-catenin （<italic>P</italic><0.05）. Compared with the DDP group， the SSA+DDP group exhibited obviously increased apoptosis of A549/DDP cells， enhanced accumulation of intracellular ROS， down-regulated C-myc and Bcl-2 mRNA expression， up-regulated Caspase-3 mRNA expression （<italic>P</italic><0.05）， and weakened <italic>β</italic>-catenin transcription （<italic>P</italic><0.05）. DDP combined with SSA better decreased the <italic>β</italic>-catenin protein expression in contrast to that of control or DDP （<italic>P</italic><0.05）. Conclusions:SSA enhances the sensitivity of A549/DDP cells to DDP possibly by inhibiting the activation of Wnt/<italic>β</italic>-catenin pathway.

Effect of Licochalcone A on Apoptosis in Human Breast Cancer MDA-MB-231 Cells / 中国实验方剂学杂志

Objective:To investigate the effect of licochalcone A （LCA） on apoptosis in human breast cancer MDA-MB-231 cells， and to explore its possible mechanism. Method:MDA-MB-231 cells were treated with LCA of different concentrations， and<italic> </italic>cell counting kit-8 （CCK-8） assay was used to detect the cell viability. The cells were treated with LCA （10， 20， and 40 μmol·L^-1） for 24 h， and apoptosis was detected by Annexin V staining with fluorescein isothiocyanate （FITC） and propidium iodide （PI） （Annexin V-FITC/PI）. The level of intracellular reactive oxygen species （ROS） was detected by 2′，7′-dichlorodihydrofluorescein diacetate （DCFA-DA） fluorescent probe. Mitochondrial membrane potential （MMP） was detected by 5， 5′， 6， 6′-tetrachloro-1， 1′， 3， 3′-tetraethyl-imidacarbocyanine （JC-1） fluorescence probe. Western blot was used to detect the expression of cell apoptosis-related proteins， such as B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax）， and endoplasmic reticulum （ER） stress-related proteins， such as C/EBP homologous protein （CHOP）， activating transcription factor 4 （ATF4）， protein kinase R-like ER kinase （PERK）， p-PERK， eukaryotic translation initiation factor 2 alpha （eIF2<italic>α</italic>）， and p-eIF2<italic>α</italic>. Result:With the increase in the drug concentration （starting from 5 μmol·L^-1）， the cell viability decreased （<italic>P<</italic>0.05） with IC₅₀of 19.05 μmol·L^-1 as compared with the normal group. Additionally， the apoptosis rates of the LCA groups （10， 20， 40 μmol·L^-1） significantly increased （<italic>P</italic><0.05）， which reached 30.2% （<italic>P</italic><0.05） at LCA concentration of 40 μmol·L^-1. LCA （10， 20， and 40 μmol·L^-1） decreased the expression of Bcl-2 （<italic>P<</italic>0.05） and increased Bax expression （<italic>P<</italic>0.05） in a dose-dependent manner. Besides， the intracellular ROS level was elevated （<italic>P<</italic>0.05） and mitochondrial MMP was reduced （<italic>P<</italic>0.05） after LCA （10， 20， and 40 μmol·L^-1） treatment in a dose-dependent manner， leading to mitochondrial dysfunction. LCA （10， 20， and 40 μmol·L^-1） induced ER stress to up-regulate the expression of CHOP， ATF4， p-PERK， and p-eIF2<italic>α</italic> （<italic>P<</italic>0.05） in a dose-dependent manner. Conclusion:LCA can induce MDA-MB-231 cell apoptosis by increasing intracellular ROS level and reducing MMP to trigger mitochondrial dysfunction and ER stress.

Study on Mechanism of Reducing Excess Fire of Liver and Gallbladder of Bile Processed Coptidis Rhizoma Based on UPLC-Q-Orbitrap HRMS and Network Pharmacology / 中国实验方剂学杂志

Objective:Based on UPLC-Q-Orbitrap HRMS and network pharmacology, the material basis, processing principle and molecular mechanism of bile processed Coptidis Rhizoma (BPRC) for reducing excess fire of liver and gallbladder were elucidated.Method:The chemical ingredients of BPRC were analyzed by UPLC-Q-Orbitrap HRMS. Chromatographic separation was achieved with 0.1% formic acid solution (A)-acetonitrile (B) as the mobile phase in gradient elution (0-20 min, 5%-80%B; 20-30 min, 80%-95%B; 30-30.1 min, 95%-5%B; 30.1-35 min, 95%-5%B). The flow rate was 0.2 mL·min-1, electrospray ionization (ESI) was applied and operated in positive and negative ion modes, the acquisition range was m/z 100-1 500. Based on the clinical manifestations and pathogenic factors of excess fire of liver and gallbladder, the potential effective ingredients, targets and functional characteristics of BPRC were predicted and analyzed by online database. Based on the characteristics of the new active ingredients after processing, the processing principle of BPRC was investigated by network pharmacology.Result:A total of 19 ingredients in BPRC were identified, six of which were newly added cholic acids after processing. It was determined that the alkaloids, including worenine, epiberberine, jatrorrhizine, coptisine, berberrubine, berberine, palmatine and cholic acids, including glycohyodeoxycholic acid, taurohyodeoxycholic acid, glycochenodeoxycholic acid, hyodeoxycholic acid and taurochenodeoxycholic acid, were identified as material basis of BPRC. A total of 66 targets of reducing excess fire of liver and gallbladder of BPRC were screened. There were 16 common targets and multiple same signaling pathways between cholic acids and alkaloids of BPRC, and many lesions of excess fire of liver and gallbladder were target organs of cholic acids. By acting on some targets, including albumin (ALB), Caspase-3 (CASP3), mitogen-activated protein kinase 14 (MAPK14), glucocorticoid receptor (NR3C1) and other targets and some signaling pathways, including interleukin (IL)-17, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt), MAPK and other pathways, BPRC could reduce excess fire of liver and gallbladder.Conclusion:BPRC has the characteristics of multi-component, multi-target and multi-pathway on reducing excess fire of liver and gallbladder. Bile and Coptidis Rhizoma have synergistic effect and bile can enhance the intensity of BPRC in lesions, which confirms the processing theory that the effect of BPRC on excess fire of liver and gallbladder enhance after being processed by bile.

Optimization of extraction process of Compound Guizhi Shaoyao Zhimu Granules by combination of response surface methodology with entropy weight method / 中草药

Objective: To optimize the extraction process parameters of Guizhi Shaoyao Zhimu Granules (GSZG). Methods: Using high performance liquid chromatography and Elisa, the extraction rate of trans-cinnamaldehyde, paeoniflorin, mangiferin, glycyrrhizic acid, the dry extract yield of extracted herbs and inflammatory factor IL-6 were comprehensively evaluated. The information entropy weighting method was used to determine the objective weight of each index, and response surface methodology was adopted to optimize the extraction process parameters of GSZG. Results: The method had good linear relationship within the range of 3.27-104.64 μg/mL for trans-cinnamaldehyde (r = 0.999 9), 8.16-261.03 μg/mL for paeoniflorin (r = 1.000 0), 1.39-89.27 μg/mL for mangiferin (r = 0.999 9), and 1.00-33.00 μg/mL for glycyrrhizic acid ammonium salt (r = 0.999 9). Guizhi-Shaoyao-Zhimu extraction had the effect of inhibiting the proliferation of MH7A cells (molded by TNF-α) with IC50 of 1.02 mg/mL, and had anti-inflammatory activity. Entropy method was more scientific, reasonable and stable. According to the comprehensive scoring results and practical situation, it was determined that the best extraction process of the preparation was to add 16 times the amount of water, and decocted three times for 1 h each time. The RSD of the predicted value was less than 3% compared with the measured value. Conclusion: The preferred process has high extraction rate, with good stability and repeatability, which is suitable for the mass production of GSZG.

Spectrum-effect relationship between HPLC fingerprint and free radicals scavenging in Guizhi Shaoyao Zhimu Decoction / 中国中药杂志

To establish the spectrum-effect relationship between HPLC fingerprint and free radicals activity scavenging in Guizhi Shaoyao Zhimu Decoction( GSZD),and provide a basis for the quality evaluation and modernization of classical prescriptions. Shimadsu GL-science C18 column( 4. 6 mm×250 mm,5 μm) was used with acetonitrile-0. 1% formic acid solution as the mobile phase for gradient elution. The detective wave length was 254 nm; the column temperature was set at 32 ℃; the injection volume was 20 μL; and the flow rate was 1. 0 m L·min-1.10 batches of primary standard samples of GSZD were detected,and their HPLC fingerprint was established by using the similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine( TCM). The activity of scavenging free radicals was studied by 1,1-diphenyl-2-trinitrophenylhydrazine( DPPH) method,and the spectrum-effect relationship was studied by Pearson bivariate correlation analysis. The common mode of GSZD fingerprints was established,and 26 common peaks were marked,with similarities ranging from 0. 929 to 0. 998. Eight of the chromatographic peaks were identified by using the control comparison method: gallic acid,mangiferin,paeoniflorin,glycyrrhizin,asparagus,5-O-methylvisamicin,cinnamic acid,and ammonium glycyrrhetate. Among them,the content changes of No. 14( paeoniside),20,12( mangiferin),13 and 23( cinnamic acid) common peaks were negatively correlated with free radical scavenging activity. The fingerprint showed high precision,repeatability and stability,and the common peaks were well separated,so it can be used for the quality evaluation of GSZD,and could provide reference for further studies on the material basis of GSZD.