New medical applications of caffeol
A kind of cafestol and drug technology, applied in the application field of cafestol in the preparation of drugs for preventing or treating renal fibrosis
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Embodiment 1
[0043] Example 1 Caffeine inhibits renal fibrosis in mice induced by unilateral ureteral occlusion (UUO)
[0044] 1. Animals
[0045] SPF grade BALB / C male mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
[0046] 2. Experimental drugs and reagents
[0047] Cafestol (sigma), phenobarbital sodium (LUPI), 4% paraformaldehyde (Beijing Regen Biotechnology Co., Ltd.), OCT tissue embedding agent (SAKURA), immunostaining primary antibody dilution (Beyond), immune Fluorescence staining secondary antibody diluent (Biyuntian), modified Masson trichrome staining solution (Solarbio), neutral gum (Beijing Solarbio Technology Co., Ltd.), RIPA lysate (medium) (Biyuntian), phosphatase inhibitor tablets (Roche), protease inhibitor (Kangwei Century), 5×SDS-PAGE loading buffer (Kangrun Biotechnology Co., Ltd.), 4×SDS-PAGE separating gel buffer (Solarbio), 4×SDS-PAGE stacking gel buffer (Solarbio), 30% acrylamide (29:1) (Solarbio), PAGE gel accelerator (S...
Embodiment 2
[0056] Example 2 Cafestol can significantly increase the expression of phosphorylated YAP, reduce the amount of YAP / TAZ, and inhibit the Hippo-YAP signaling pathway.
[0057] The Western blotting method in this embodiment is carried out using common experimental procedures in the field.
[0058] In-cell Western is a high-throughput method for rapid detection of intracellular proteins, which can be directly performed on cells on cell culture plates (such as 96-well plates) based on the principle of antigen-antibody binding, using specific antibodies for target proteins and near-infrared Fluorescently labeled secondary antibodies for quantitative analysis of target proteins.
[0059] The experimental steps are as follows:
[0060] After the cell culture in the 96-well plate was completed, the culture medium was discarded, and the residual medium was washed with PBS. Fix with 4% paraformaldehyde for 10 min and wash with PBS 3 times. Permeabilize with 0.3% Triton X-100 for 15 m...
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