Application of C188-9, Venetoclax and Bumetanide in drugs for fibrotic diseases
A fibrotic disease, fibrosis technology, applied in sexual diseases, blood diseases, bone diseases, etc., can solve problems such as affecting collagen synthesis and increasing the stability of collagen mRNA
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Embodiment 1
[0048] Example 1 Screening C188-9, three compounds of Venetoclax and Bumetanide as a process for treating fibrous diseases
[0049] The main implementation process involved in this embodiment is as follows:
[0050] 1) Fifty small molecule compounds with strong affinity to LARP6-RRM were screened out by computer simulation;
[0051] 2) Treat NIH 3T3 fibroblasts with the small molecule compound screened in 1), and detect the changes of collagen mRNA by Real-Time PCR;
[0052] 3) Using the small molecular compound that affects collagen synthesis screened in 2), the direct combination of the small molecular compound and collagen is detected by micro-thermophoresis;
[0053] The main experimental parts involved in this embodiment are as follows:
[0054] 1. Screen out small molecular compounds with strong affinity to LARP6-RRM
[0055] The experimental steps are as follows:
[0056] Using Taosu Biology's classic known activity library (Cat. No.: L4000), 5,370 compounds with bi...
Embodiment 2
[0098] Example 2 Bumetanide inhibits bleomycin-induced pulmonary fibrosis in mice.
[0099] The main experimental parts involved in this embodiment are as follows:
[0100] 1. Detection of collagen gene expression by RIP-PCR
[0101] 1) Treat the cells with Bumetanide or solvent control.
[0102] 2) The cells in a 10 cm culture dish were cross-linked in ice, and irradiated with 400 mJ / cm2 UV for 5 minutes under the action of 1 ml of cold PBS.
[0103] 3) Discard PBS, add 500ul cold wash buffer (1×PBS, 0.1% SDS, 0.5% NP-40, 0.5% deoxycholic acid) containing 200U / ml RNase inhibitor (Takara) and protease inhibitor cocktail (Roche) Sodium), apply ice for 10 minutes.
[0104]4) Scrape off cells and put into 1.5ml tube.
[0105] 5) Centrifuge at 16000 g for 20 minutes at 4°C.
[0106] 6) Add RQ I (Promega) to a final concentration of 1 U / mol / l, incubate in a 37°C water bath for 5 minutes, and then cool on ice for 5 minutes.
[0107] 6) For co-immunoprecipitation, incubate over...
Embodiment 3
[0138] Example 3 Bumetanide inhibits collagen synthesis in human fibroblasts.
[0139] The main experimental parts involved in this embodiment are as follows:
[0140] 1. RIP-PCR detection of LARP6 binding collagen mRNA level
[0141] Method is detected with RIP-PCR in embodiment 2, and the results are shown in Figure 7A -C.
[0142] 2. RT-PCR detection of the effect of Bumetanide on the production of collagen mRNA
[0143] Method is detected with RT-PCR in embodiment 1, and the results are shown in Figure 8A -C.
[0144] Summary: The above results show that Bumetanide also has the function of inhibiting collagen production in human fibroblasts.
[0145] Based on the above-mentioned embodiments, the present invention starts from the library of natural active compounds, and through computer simulation screening, repeated research at the level of cell animals, it is found that in C188-9, Venetoclax and Bumetanide can be used as a new potential clinical treatment of fibrot...
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