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Aim To explore the effect of ZLY18 on angiotensin II-induced cardiac fibrosis and the underlying mechanism. Methods Ang II was used to induce cardiac fibrosis in vitro and in vivo. Cardiac fibroblasts were divided into blank control group, model group and medicine group. The medicine group was subdivided into ZLY18(L)group, ZLY18(M)group and ZLY18(H)group. Compound ZLY18 was given 1, 2, 5 μmol·L-1 respectively. C57BL/6 mice were randomly divided into control group, model group and medicine group. The medicine group were subdivided into ZLY18(L)group, ZLY18(M)group and ZLY18(H)group. Compound ZLY18 was given 10,20 and 50 mg·kg-1 respectively. Both the model group and the medicine group were given with Ang II to induce cardiac fibrosis. The changes of protein levels were detected by Western blot and immunofluorescence. The changes of cardiac function indexes in C57BL/6 mice were detected by small animal echocardiography. The morphology, cell arrangement and collagen fibers of cardiac fibroblasts were observed by tissue section staining and other methods. Results The model of Ang II-induced myocardial fibrosis was successfully established at the cell and animal levels, and ZLY18 treatment improved the elevated fibrosis-related protein caused by Ang II and abnormal cardiac function in mice. Moreover, ZLY18 was able to inhibit the increased phosphorylation of TGF-1 and Smad3 caused by Ang II and increased Smad2/3 nuclear entry, suggesting that the antifibrotic effect of ZLY18 might be related to the activation of TGF-1/Smads signaling pathway. Conclusions ZLY18 has a protective effect on Ang II-induced cardiac fibrosis. ZLY18 may inhibit TGF-β/Smads signaling pathway activation to exert anti-fibrotic effects.
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Objective To investigate the detailed molecular mechanism of matrix stiffness regulating cell drug resistance. Methods Polyacrylamide hydrogels of soft substrate (10 kPa), hard substrate (38 kPa) and rigid substrate (57 kPa) with different matrix stiffness were configured to simulate the physical matrix stiffness at different stages of breast cancer in vivo. Results The cell proliferation rate of the hard substrate was significantly higher than that of the soft and rigid substrates. The intracellular endocytosis was significantly lower on the hard substrate. The YAP nucleus translocation increased significantly on the hard substrate, compared with the soft and the rigid substrates, indicating that YAP was a key molecule involved in drug resistance of tumor cells. Conclusions Matrix stiffness could regulate the drug resistance of breast cancer cells through YAP activation. This study not only provides a new direction for elucidating the mechanism of drug resistance, but also lays a new foundation for the drug delivery system of breast cancer treatment.
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OBJECTIVE: To establish a stably-transfected cell line with adr adrenergic receptorα2B-AR) and enhanced green fluorescent protein (EGFP) labeled nucleus factor of activated T cells 2 (NFAT2) (EGFP-NFAT2) in U20S. METHODS U20S cells that stably expressed EGFP-NFAT2(U20S-EGFP-NFAT2 cells) were transfected with pcDNA3.1 expressing hygromycin B (Hygro) resistance gene and α2B-AR gene (pcDNA3.1-Hygro-α2B-AR) recombinant plasmid. The transfected cells were selected by Hygro (200 mg · L-1) for 10 d before being screened on the nucleus translocation assay of EGFP-NFAT2. Then, the selected cells were evaluated by Z' factor for functional stability and azrAR expression in the selected cells was analyzed by quantitative real-time PCR (qRT-PCR) and Western blotting. Finally, the activity of α2B-AR on NFAT2 nucleus translocation was evaluated by α2-AR agonist dexme-detomidine hydrochloride (DMED) or antagonist atipamezole hydrochloride (ATI) treatment. RESULTS Recombinant plasmid pcDNA3.1-Hygro-α2B-AR was established and transfected to U20S cells stably expressing EGFP-NFAT2. In the stably-transfected No.12 cell strain, the relative nuclear translocation index (0.445) was the highest among the selected cell lines, and Z' factors on three independent experiments were 0.664, 0.533 and 0.634, respectively. The mRNA expression of α2B-AR of No. 12 cell strain was detected by qRT-PCR and was about 140 times that of U20S-EGFP-NFAT2 cells (P<0.01) in 20 generations. The protein band of α2B-AR was also detected in No. 12 cell strain whereas no band of α2B-AR was detected in U20S-EGFP-NFAT2 cell by Western blotting. DMED concentration-depend-ently increased the relative translocation nuclear index in U20S-EGFP-NFAT2-α2B-AR cell [EC50= (2.616±0.121) nmol·L-1]. ATI concentration-dependency decreased the relative nuclear translocation index in U20S-EGFP-NFAT2-α2B-AR cell [IC50 =(89.05±0.22) nmol-L-1]. CONCLUSION The stably-transfected U20S-EGFP-NFAT2-α2B-AR cell line is established and can be used for high throughout screening of biased chemicals and the study on the mechanism of α2B-AR.