Application of flavone compound in treating lung fibrosis
A technology of pulmonary fibrosis and composition, which is applied in the application field of neohesperidin in the treatment of pulmonary fibrosis, and can solve problems such as no neohesperidin
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Embodiment 1
[0021] Example 1 Neohesperidin inhibits TGF-β1 signaling in a dose-dependent manner
[0022] The cells used in this example are NIH3T3 mouse fibroblasts stably transfected with the CAGA-luciferase reporter gene.
[0023] The NIH3T3 cells in the logarithmic growth phase were planted in a 96-well plate, and after growing to 80%, they were treated with 0.1% serum for 24 hours, and a final concentration of 5 ng / ml TGF-β1 pure protein and different concentrations of neohesperidin were added. The final concentrations were 0.1 μmol / L, 1 μmol / L, 5 μmol / L, and 10 μmol / L (three replicate wells were set for one compound), and cultured at 37°C for 24 hours. The cells in the 96-well plate were washed twice with PBS buffer. Add 50 μL of 1×passive (Passive) lysis buffer, place the 96-well plate on a horizontal shaker at 200 rpm / min, and lyse the cells at room temperature for 30 min. Take 40 μL of cell lysate into a 96-well plate for measurement, add 35 μL of substrate, and perform lucifera...
Embodiment 2
[0027] Example 2 Neohesperidin inhibits TGF-β1-induced myofibroblast differentiation
[0028]The cells used in this example were the mouse lung fibroblast cell line Mlg obtained from the American Type Culture Collection (ATCC). After Mlg fibroblasts were overgrown, 0.1% serum was treated for 24 hours, and 5 ng / mL LTGF-β1 protein was added with a final concentration of 5 μmol / L neohesperidin or hesperidin (as a positive control). After 12 hours of treatment, RNA was collected for real-time fluorescent quantitative PCR to detect α-SMA; after 24 hours of treatment, cells were collected and Western Blotting was used to detect the expression of α-SMA protein. Real-time fluorescent quantitative PCR refers to preparing total RNA from cells using Trizol (Invitrogen), synthesizing cDNA using ThermoScript RT-PCR kit (Invitrogen), and detecting the expression of α-SMA using SYBR Green kit (Roche). Relative gene expression using 2 -Δ(ΔCT) method, and β-actin was used as an internal refe...
Embodiment 3
[0035] Example 3 Neohesperidin slows down bleomycin-induced pulmonary fibrosis
[0036] Pulmonary fibrosis animal model preparation refers to male C57BL / 6J (age 8-10 weeks) wild-type mice, anesthetized mice with 0.6mL / 100g intraperitoneal (I.P.) injection of 7.5% chloral hydrate, intratracheal injection of Bray Mycin 2.5U / kg. The specific scheme is as follows: after weighing and recording the body weight, fix the mouse on the operating table, disinfect the neck with 70% alcohol, use a scalpel to cut vertically about 1 cm in the neck of the mouse, use micro-tweezers to separate the tissue to expose the trachea, Insert the syringe into the trachea through the gap between the tracheal cartilage rings towards the heart, then slowly inject 2.5 U / kg of bleomycin saline solution in a volume appropriate to its body weight, and immediately turn the animal upright and rotate left and right to make the liquid medicine Evenly distributed in the lungs.
[0037] Neohesperidin treatment re...
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