Application of beta-sitosterol to preparation of medicine for treating or preventing influenza A
A technology of influenza A and influenza A virus, which is applied in the field of medicine, can solve the problems that the effect of β-sitosterol has not been reported, and achieve the effect of meeting the needs of large-scale production, easy quality control, and stable chemical properties
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Embodiment 1
[0022] Example 1 Effect of β-sitosterol on Influenza Virus Induced Host Signaling Pathway
[0023] 1. Materials: cells and viruses
[0024] A549 cells were purchased from ATCC and kept in our laboratory; A / PR8 / 3 / 4(H1N1) were kept in our laboratory.
[0025] 2. Chemical reagents and antibodies
[0026] Trisbase was purchased from Ameresco; sodium chloride (NaCl) was purchased from Guangzhou Chemical Reagent Factory; potassium chloride (KCl) was purchased from Guangzhou Chemical Reagent Factory; potassium dihydrogen phosphate (KH 2 PO 4 ) was purchased from Guangzhou Chemical Reagent Factory; disodium hydrogen phosphate (Na 2 HPO 4 12H 2 O) was purchased from Guangzhou Chemical Reagent Factory; Glycine was purchased from Guangzhou Chemical Reagent Factory; Sodium Dodecyl Sulfonate (SDS) was purchased from American Sigma Company; Tween Tween-20 was purchased from Guangzhou Chemical Reagent Factory; BCA protein concentration determination Kit was purchased from American Ther...
Embodiment 2
[0050] Example 2 Effect of β-sitosterol on the stimulation signal transduction of exogenous interferon (IFN-β)
[0051] A549 cells were seeded into 6-well plates and placed in an incubator. After the cells adhered to the wall after culturing for 12 hours, the original medium was discarded, washed twice with PBS, and then added with influenza virus A / PR8 / 3 / 4 (H1N1) without Serum DMEM / DF12 (1:1) medium adsorbed the cells for 2 hours; then, the cells were washed with PBS to remove unadsorbed virus, and different concentrations of β-sitosterol (200 μg / ml, 400 μg / ml, 600 μg / ml) were added , continue to incubate for 4 hours. After adding 20ng / ml IFN-β to stimulate for 15min, the total protein of the cells was extracted for western blot (the specific process can be carried out by referring to the corresponding steps in Example 1). In this example, rabbit anti-human primary antibodies P-STAT1 and P-STAT3 antibodies were used to incubate the antibodies To detect P-STAT1 and P-STAT3 mo...
Embodiment 3
[0052] Example 3 Effect of β-sitosterol on Influenza Virus Infection Induced Inflammatory Mediators
[0053] A549 cells were planted into 6-well plates, placed in an incubator, and cultured for 12 hours. After the cells adhered to the wall, the original medium was discarded, washed twice with PBS, and then added with influenza virus A / PR8 / 3 / 4 (H1N1) without The DMEM / DF12 (1:1) medium of serum adsorbed the cells for 2 hours; then, the cells were washed with PBS to remove unadsorbed viruses. Using drug treatment mode, drug intervention group added different concentrations of β-sitosterol (150μg / ml, 300μg / ml, 450μg / ml). 300 μg / ml of β-sitosterol was added to the drug group alone, and the A549 cells in the drug group alone were not adsorbed by influenza virus. After continuing to culture for 24 hours, the cell culture supernatant was collected, centrifuged to remove cell debris and filtered to remove virus particles. Then the treated culture medium was added to stimulate another...
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