Chronic liver disease mediated by activation of hepatic stellate cells (HSCs) and Kupffer cells (KCs) leads to liver fibrosis. Here, we aimed to investigate the molecular mechanism and define the cell type involved in mediating the sphingosine kinase (SphK)1‐dependent effect on liver fibrosis. The levels of expression and activity of SphK1 were significantly increased in fibrotic livers compared with the normal livers in human. SphK1 was coexpressed with a range of HSC/KC markers including desmin, α‐smooth muscle actin (α‐SMA) and F4/80 in fibrotic liver. Deficiency of SphK1 (SphK1−/−) resulted in a marked amelioration of hepatic injury, including transaminase activities, histology, collagen deposition, α‐SMA and inflammation, in CCl4 or bile duct ligation (BDL)‐induced mice. Likewise, treatment with a specific inhibitor of SphK1, 5C, also significantly prevented liver injury and fibrosis in mice induced by CCl4 or BDL. In cellular levels, inhibition of SphK1 significantly blocked the activation and migration of HSCs and KCs. Moreover, SphK1 knockout in KCs reduced the secretion of CCL2, and SphK1 knockout in HSCs reduced C‐C motif chemokine receptor 2 ([CCR2] CCL2 receptor) expression in HSCs. CCL2 in SphK1−/− mice was lower whereas microRNA‐19b‐3p in SphK1−/− mice was higher compared with wild‐type (WT) mice. Furthermore, microRNA‐19b‐3p downregulated CCR2 in HSCs. The functional effect of SphK1 in HSCs on liver fibrosis was further strengthened by the results of animal experiments using a bone marrow transplantation (BMT) method. Conclusion: SphK1 has distinct roles in the activation of KCs and HSCs in liver fibrosis. Mechanistically, SphK1 in KCs mediates CCL2 secretion, and SphK1 in HSCs upregulates CCR2 by downregulation of miR‐19b‐3p. (Hepatology 2018).
The usage of traditional Chinese medicines has expanded globally, but the data about authentication, efficacy, and safety is far from sufficient to meet the criteria supporting their use worldwide due to complexity in the composition. Fingerprinting describes integral characterization and reflects interactive aspects of complex components; therefore, it can offer the possibility of evaluating quality of traditional Chinese medicines following the overall principle. Chemometric techniques introduce multivariate analytical methods into fingerprinting to obtain more information that is useful, which is consistent with the holistic thought and plays an important role in research on the substantial basis. In this review, we will start with three aspects to expound the quality evaluation of traditional Chinese medicines based on fingerprints. The analytical techniques used in developing fingerprints including chromatographic methods, spectroscopic methods, and capillary electrophoresis are introduced. Strategies for fingerprints analysis usually based on chemometric methods including unsupervised and supervised pattern recognition are described. Applications of fingerprints for multi‐component quantification, quality control, screening of bioactive components, and fingerprint‐efficacy relationship study are also outlined. Finally, we propose challenges and future perspectives of fingerprints in quality evaluation to promote the development of modernization and internationalization of traditional Chinese medicines.
Multidrug resistance protein 7 (MRP7, ABCC10) is a recently discovered member of the ATP-binding cassette (ABC) family which are capable of conferring resistance to a variety of anticancer drugs, including taxanes and nucleoside analogs, in vivo. MRP7 is highly expressed in non-small cell lung cancer cells, and Mrp7-KO mice are highly sensitive to paclitaxel, making MRP7 an attractive chemotherapeutic target of non-small cell lung cancer. However, only a few inhibitors of MRP7 are currently identified, with none of them having progressed to clinical trials. We used MRP7-expressing cells to investigate whether tariquidar, a third generation inhibitor of P-glycoprotein, could inhibit MRP7-mediated multidrug resistance (MDR). We found that tariquidar, at 0.1 and 0.3 µM, significantly potentiated the sensitivity of MRP7-transfected HEK293 cells to MRP7 substrates and increased the intracellular accumulation of paclitaxel. We further demonstrated that tariquidar directly impaired paclitaxel efflux and could downregulate MRP7 protein expression in a concentration- and time-dependent manner after prolonged treatment. Our findings suggest that tariquidar, at pharmacologically achievable concentrations, reverses MRP7-mediated MDR through inhibition of MRP7 protein expression and function, and thus represents a promising therapeutic agent in the clinical treatment of chemoresistant cancer patients.
Cancer metastasis is the leading cause of high mortality and disease recurrence in breast cancer. In this study, a novel tumor microenvironment charge reversal polymetformin (PMet)-based nanosystem co-delivering doxorubicin (DOX) and plasmid encoding IL-12 gene (pIL-12) was developed for chemo− gene combination therapy on metastatic breast cancer. Cationic PMet was readily self-assembled into micelles for DOX physical encapsulation and pIL-12 complexation, and a hyaluronidasesensitive thiolated hyaluronic acid (HA-SH) was then collaboratively assembled to the pIL-12/DOX-PMet micelleplexes, abbreviated as HA/pIL-12/DOX-PMet. DOX/pIL-12 loaded in HA/pIL-12/DOX-PMet micelleplexes presented prolonged circulation in blood, efficient accumulation in tumors, and internalization in tumor cells via CD44 receptor-mediated tumor specific-targeting, and DOX/pIL-12 was co-released in endo/lysosomes tumor microenvironment followed by HAase-triggered HA-SH deshielding from HA/pIL-12/DOX-PMet micelleplexes. Moreover, HA/PMet micelleplexes displayed excellent pIL-12 transfection and IL-12 expression in tumors of 4T1 tumor-bearing mice. Importantly, HA/pIL-12/DOX-PMet micelleplexes synergistically enhanced the NK cells and tumor infiltrated cytotoxic T lymphocytes and modulated the polarization from protumor M2 macrophages to activated antitumor M1 macrophages, with concomitant decreasing of the immunosuppressive regulatory T (Treg) cells, accompanied by an increase in the cytokines expression of IL-12, IFN-γ and TNF-α, consequently showing an improved antitumor and antimetastasis activity in 4T1 breast cancer lung metastasis mice model. In conclusion, the tumor microenvironment charge reversal HA/PMet nanosystem holds great promise for DOX/pIL-12 co-delivery and exploitation in chemo−gene combination therapy on metastatic breast cancer.
Reactive oxygen species (ROS) are known to not only mediate the damage of cellular constituents but also to regulate cellular signaling. Analysis of ROS is essential if we wish to understand the mechanisms of cellular alterations. In this paper, a microfluidic chip-based approach to the determination of ROS in single erythrocyte was developed by using a simple crossed-channel glass chip with integrated operational functions, including cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser-induced fluorescence (LIF) detection. Non-fluorescent dihydrorhodamine 123 (DHR 123), which can be oxidized intracellularly by ROS to the fluorescent rhodamine 123 (Rh 123), was used as the fluorogenic reagent. The effect of pH on the migration time of Rh 123 and detection sensitivity was discussed. The present method minimized dilution of intracellular ROS during reaction with DHR 123 and determination. As a result, an extremely low detection limit of 0.8 amol has been achieved. The time required for complete analysis of one human erythrocyte was less than 3 min. A migration time precision of 4.1% RSD was obtained for six consecutively-injected cells. Upon stimulation with 4 mmol/l H2O2 for 10 min, the intracellular ROS concentration was found to increase on average by about a factor of 8.4.
Summary Phosphodiesterase type 5 (PDE5) inhibitors are widely used in the treatment of male erectile dysfunction and in improving the breathing efficiency in pulmonary hypertension. Recently, several groups have evaluated the ability of PDE5 inhibitors for their anticancer activities. Previously, we have shown that sildenafil, vardenafil and tadalafil could reverse P-glycoprotein (P-gp, ABCB1)-mediated multidrug resistance (MDR). In the present study, we determined whether these PDE5 inhibitors have the potential to reverse multidrug resistance protein 7 (MRP7, ABCC10)-mediated MDR. We found that sildenafil and vardenafil dose-dependently enhanced the sensitivity of MRP7-transfected HEK293 cells to paclitaxel, docetaxel and vinblastine, while tadalafil had only minimal effect. Accumulation and efflux experiments demonstrated that sildenafil and vardenafil increased the intracellular accumulation of [3H]-paclitaxel by inhibiting the efflux of [3H]-paclitaxel in HEK/MRP7 cells. In addition, immunoblot and immunofluorescence analyses indicated that no significant alterations of MRP7 protein expression and localization in plasma membranes were found after treatment with sildenafil, vardenafil or tadalafil. These results demonstrated that sildenafil and vardenafil reverse MRP7-mediated MDR through inhibition of the drug efflux function of MRP7. Our findings indicated a potentially novel use of PDE5 inhibitors as an adjuvant chemotherapeutic agent in clinical practice.
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