Mechanism of Rhodiola rosea-Euonymus alatus drug pair against rheumatoid arthritis: Network pharmacology and experimental validation.

PURPOSE: The present study aimed to explore the potential components and mechanisms of Rhodiola rosea-Euonymus alatus drug pair (TY) that ameliorate rheumatoid arthritis (RA). METHODS: The main active components, core targets, and important pathways of TY against RA were predicted by network pharmacology analysis. The binding activity between the main active components and the core targets was verified by the molecular docking technique. Collagen-induced arthritis (CIA) rat model and tumor necrosis factor (TNF)-α-induced fibroblast-like synovial cells in human RA (HFLS-RA) model were established, respectively. The core targets were verified by cell counting kit-8 assay, hematoxylin eosin, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot analysis, and the therapeutic effect of TY was evaluated. RESULTS: A total of 18 possible components and 34 core targets were obtained by network pharmacology, among which inflammatory response, phosphatidylinositide 3-kinases (PI3K)-AKT and MAPK pathways were involved in the therapeutic effect of TY on RA. The results of molecular docking showed that kaempferol and quercetin had high binding affinity to interleukin (IL)-1ß, IL-6, matrix metalloproteinase (MMP)9, and TNF-α. In vivo and in vitro experiments showed that TY dose-dependently inhibited the proliferation of HFLS-RA cells induced by TNF-α, and significantly reduced the paw swelling and arthritis scores in CIA rats. At the same time, TY inhibited the production of inflammatory factors in CIA rat serum and TNF-α-induced HFLS-RA cells. It also decreased the expression of PI3K, phospho-protein kinase B, MMP1, MMP3, MMP9, and increased the protein and mRNA levels of tissue inhibitors of MMPs (TIMP)1 in synovial tissue. CONCLUSION: TY can inhibit the PI3K/AKT signaling pathway and regulate the balance between MMPs and TIMP, thus playing a therapeutic role in RA.

Protective effects of combined micronutrients on islet beta-cells of streptozotocin-induced diabetic mice.

There is a tendency for the incidence of diabetes in a population to increase with an improvement in living standards. This would imply the involvement of nutritional factors in the development of diabetes, and so nutritional considerations could be a key aspect in the research and development of an effective remedy for diabetes. In this study, combined micronutrients (selenium, vitamin E, vanadium, and chromium) were orally supplemented to streptozotocin-induced diabetic mice. Results showed that combined micronutrients could decrease the high blood glucose levels (p<0.05 or p<0.01) of diabetic mice. The protective effects of combined micronutrients on structures of beta-cells in pancreatic islets of diabetic mice were observed histopathologically and ultrastructurally. In addition, the supplementation of combined micronutrients increased insulin expression by beta-cells in pancreatic islets of diabetic mice at both translational and transcriptional levels. The immune molecular mechanisms involved were preliminarily regarded as downregulation of the expression of pathogenic T-helper 1 lymphocyte (Th1) cytokine tumor necrosis factor-alpha (TNF-alpha) (p<0.01) along with upregulation of the expression of protective T-helper 2 lymphocyte Th2 cytokine interleukin 10 (IL-10) (p<0.01) which ameliorates the Th1/Th2 imbalance in diabetes. In conclusion, supplementation of combined micronutrients to diabetic mice could effectively improve disordered glucose metabolism, protect islet structures, and improve the function of beta-cells in pancreatic islets, which are affected by differential regulation of the expression of Th1/Th2 cytokines involved in the pathogenesis of diabetes.

Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors.

BACKGROUND: RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR). METHODS: The two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student's t tests. P < 0.05 was considered statistically significant. RESULTS: In MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P < 0.05), 30.1% (P < 0.01) (transient transfection) and 37.6% (P < 0.05), 28.0% (P < 0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P < 0.05), 54-fold (P < 0.01) (transient transfection) and to 108-fold (P < 0.05), 50-fold (P < 0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells. CONCLUSIONS: shRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.

[Regulatory effects of micronutrient complex on the expression of Th1 and Th2 cytokines in diabetic C57BL mice].

OBJECTIVE: To investigate the regulatory effects of micronutrients complex (MC) on the expression of Th1 and Th2 cytokines in diabetic mice for exploring the molecular mechanisms of MC in treating for diabetes. METHODS: IDDM mice model was made by the injection of multiple low dose of streptozotocin (MLDS). The composition of Selenium (Se), Vitamin E (VE), Vanadium (V) and Chrome (Cr) was supplemented. The percentage of Th1 cytokines(TNF-alpha, IFN-gamma) and Th2 cytokines (IL-4, IL-10) positive lymphocytes were detected by flow cytometer (FCM). RESULTS: The expression of TNF-alpha and IFN-gamma of peripheral blood lymphocytes of MLDS group significantly increased (P < 0.01), while the IL-10 expression of blood and spleen lymphocytes of MLDS group obviously decreased (P < 0.01). Combined supplementation of MC markedly decreased blood lymphocytes TNF-alpha expression (P < 0.01) and increased blood lymphocytes IL-10 expression (P < 0.01) and spleen lymphocytes IL-4 expression (P < 0.05) of IDDM mice respectively. CONCLUSION: MC may prevent from the onset and development of IDDM by down-regulating Th1 cytokines genes expression and up-regulating Th2 cytokines genes expression of IDDM mice.