51 research outputs found

    Table1_Screening of anti-heart failure active compounds from fangjihuangqi decoction in verapamil-induced zebrafish model by anti-heart failure index approach.DOCX

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    Heart failure is the end stage of various cardiovascular diseases. Fangjihuangqi Decoction (FJHQD) is a famous traditional Chinese medicine (TCM) formula, which is clinically effective in the treatment of chronic heart failure. However, the anti-heart failure ingredients of FJHQD have not been clarified, and the related mechanisms of action are rarely studied. In the present study, through quantification analysis of heart rate and ventricular area changes, a heart failure model and cardiac function evaluation system in cardiomyocytes-labelled Tg (cmlc2: eGFP) transgenic zebrafish larvae were constructed, and the anti-heart failure index (AHFI) that can comprehensively evaluate the cardiac function of zebrafish was proposed. Based on this model, FJHQD, its mainly botanical drugs, components and ingredients were evaluated for the anti-heart failure effects. The results showed that FJHQD and its botanical drugs exhibited potent anti-heart failure activity. Furthermore, total alkaloids from Stephania tetrandra S. Moore, total flavonoids from Astragalus mongholicus Bunge and total flavonoids from Glycyrrhiza uralensis Fisch. ex DC. were identified to be the main components exerting the anti-heart failure activity of FJHQD. Then, we screened the main ingredients of these components, and glycyrrhizic acid, licochalcone A and calycosin were found to exhibit excellent cardioprotective effects. Finally, we found that FJHQD, glycyrrhizic acid, licochalcone A and calycosin may improve cardiac function in zebrafish by regulating oxidative stress, inflammatory response and apoptosis-related pathways. Taken together, our findings offer biological evidences toward the anti-heart failure effect of FJHQD, and provide guidance for the clinical application of FJHQD.</p

    HSP40 Interacts with Pyruvate Kinase M2 and Regulates Glycolysis and Cell Proliferation in Tumor Cells

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    <div><p>Pyruvate kinase M2 (PKM2) is predominantly expressed in cancers, which is considered as a key regulator of the Warburg effect. In this study, HSP40 was identified as a novel binding partner of PKM2. HSP40-PKM2 association destabilized PKM2 protein through HSC70. In the presence of HSP40, PKM2 protein level and PKM2-mediated PDK1 expression were down-regulated. Moreover, HSP40 was involved in regulating glucose metabolism on PKM2 dependent way and at the mean time had an effect on mitochondrial oxygen respiration. In line with inhibition effect of HSP40 on glycolysis, the growth of cancer cells was inhibited by HSP40.Our data provided a new regulation mechanism of PKM2, which suggested a new therapeutic target for cancer therapy.</p></div

    Cellular consequences followed by HSP40 binding to PKM2.

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    <p>HSP40 interacts with PKM2, inhibits PKM2 protein level and pyruvate kinase activity. Consequently, glucose uptake from medium decreases and lactate consumption drops as well and mitochondrial oxygen respiration rate ascends at the same time. Interestingly, the transcriptional co-factor function of PKM2 was also impacted. As a result of all these cellular PKM2 functions change, the proliferation of tumor cells was impacted to some degree. Red arrows and dashed lines indicated a decrease and green arrow indicated an increase.</p

    Specific Turn-On Fluorescent Probe with Aggregation-Induced Emission Characteristics for SIRT1 Modulator Screening and Living-Cell Imaging

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    SIRT1 is an important protein that catalyzes the nicotinamide adenine dinucleotide (NAD)<sup>+</sup>-dependent deacetylation reaction, which is regarded as a novel target to treat metabolic disorders and aging-related diseases. However, there is lack of appropriate approach for SIRT1 modulator screening and bioimaging of SIRT1 in living cells. We designed and synthesized a “turn-on” fluorescent probe by connecting a specifically recognized peptide to tetraphenylethene core. It exhibits excellent selectivity and sensitivity in homogeneous measurement of SIRT1 activity for screening both SIRT1 inhibitors and activators. 20­(S)-ginsenoside Rg<sub>3</sub> and ophiopogonin D′ were found to activate SIRT1. It was also successfully applied to monitor SIRT1 modulation in the cardiomyocytes as well as in the wild-type and SIRT1<sup>–/–</sup> mesenchymal stem cells

    PKM2 interacts with HSP40/DNAJB1 in vivo and in vitro.

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    <p>(A) Growth of yeast expressing HSP40/DNJB1-GalAD and PKM2-GalBD on nutritional deficient SD medium (-Trp-Leu or -Trp-Leu-Ade-His). Vector pGADT7-T and pGBKT7-p53 performed as positive control, pGADT7-T and pGBKT7-Lam performed as negative control. (B) Cell lysates of HEK293T cells transfected with HA-PKM2 and Flag-HSP40 were immunoprecipitated with anti-HA antibody, the bound proteins were detected by anti-Flag antibodies. (C) Cell lysates of HEK293T cells transfected with HA-PKM2 and Flag-HSP40 were immunoprecipitated with anti-Flag antibody, the bound proteins were detected by anti-HA antibodies. (D) HeLa cells were lysed and Co-IP was performed with anti-PKM2 antibody, then western blot was analyzed using anti-PKM2 and anti-HSP40 antibodies. (E) HeLa cells were lysed and Co-IP was performed with anti-HSP40 antibody, then western blot was analyzed using anti-PKM2 and anti-HSP40 antibodies. (F) Immunofluorescence analysis reveals that HSP40 and PKM2 co-localize with each other in nucleus in HeLa cells.</p

    HSP40 regulates glycolysis and mitochondrial respiration in a PKM2 dependent way.

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    <p>(A and B) HeLa, A549 and HepG2 cells were transfected with the siRNAs of negative control (NC), PKM2 (si-PKM2), HSP40 (si-HSP40) or both. The media were collected for analysis of glucose consumption and lactate production (mean ± S.D., n = 3). (C and D) HeLa, A549 and HepG2 cells were transfected with empty vector, HA-PKM2 or both HA-PKM2 and Flag-HSP40. The media were collected to analysis of glucose consumption and lactate production (mean ± S.D., n = 3). (E) Basal OCR of HeLa, A549 and HepG2 cells after transfected with NC or si-HSP40 were detected by seahorse XF24 extracellular flux analyzer. (F) HeLa cells were transfected with NC or si-HSP40. 24 h after transfection, the cells were replanted to detect O<sub>2</sub> consumption rate (OCR) by seahorse XF24 extracellular flux analyzer (mean ± S.D., n = 5). (mean ± S.D., n = 5).</p

    HSP40 impairs PKM2 stability and functions in HeLa cells.

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    <p>(A, B and C) HeLa, A549 and HepG2 cells were transfected with Flag-HSP40, after 48 h incubation, endogenous PKM2 protein levesl were detected by anti-PKM2 antibody. (D) HeLa cells were transfected with HSP40 siRNA, after 48 h incubation, PKM2 protein level was analyzed using anti-PKM2 antibody. (E) Cell lysates of HEK293T cells transfected with HA-HSC70 and Flag-HSP40/DNAJB1 were immunoprecipitated with anti-Flag antibody, the bound proteins were detected by anti-HA antibody. (F) HeLa cells transfected with HA-HSC70 or both HA-HSC70 and Flag-HSP40 were lysed, then western blot was analyzed using anti-PKM2, anti-HA and anti-Flag antibodies. (G) Pyruvate kinase activity of extracts prepared from HeLa cells transfected with HSP40 siRNA. The pyruvate kinase activity is detected by PK assay kit and normalized by protein measurement (mean ± S.D., n = 3). (H and I) HeLa cells were transfected with HSP40 siRNA or Flag-HSP40, after 24 h normal incubation followed with 24 hr anaerobic incubation, RT-PCR was performed to analyze PDK1 mRNA level (mean ± S.D., n = 3).</p

    HSP40 impacts growth of HeLa cells via regulating PKM2 protein.

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    <p>(A, C and D) HeLa, A549 or HepG2 cells were transfected with HA-PKM2 or Flag-HSP40 or both. 24 h after transfection, cells were replanted and cell numbers were counted every 24 h for analysis of cell proliferation (mean ± S.D., n = 3). (B, D and E) HeLa, A549 or HepG2 cells were transfected with si-PKM2 or si-HSP40 or both. 24 h after transfection, cells were replanted and cell numbers were counted every 24 h for analysis of cell proliferation (mean ± S.D., n = 3).</p
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