Doxycycline HCl

Kinase Assay [1]
Gelatin (0.1% (w/v) is added to standard LaemmLi acrylamide polymerization mixture. Tissue extract is mixed 1:2 with sample buffer [250 mM Tris-Cl pH 6.8, 10% (w/v) SDS, 20% (v/v) glycerol, 0.005% (w/v) bromphenol blue]. Serum is diluted 1:10 with electrophoresis buffer (2.5 mM Tris, 20 mM glycine, 0.005% SDS) and mixed 1:2 with sample buffer. Twenty μLs are loaded after 10-min incubation at room temperature without boiling. After electrophoresis at 90 V, the gels are soaked in 2.5% (w/v) Triton X-100, incubated 2 to 3 days at 37°C in gelatin digestion buffer [50 mM Tris-Cl, pH 8.0, 8 mM CaCl2, 10 mM ZnSO2, 0.02% (w/v) NaN3], stained in 0.05% Coomassie blue R-250 in acetic acid/methanol/water (1:4.5:4.5 by volume), destained in 10% acetic acid and 5% methanol, and scanned for lysis band intensity. The lysis band intensity is proportional to gelatinase activity and is quantified densitometrically by using One-Dimensional Scan software. The result, a number between 0.07 and 3.75, is normalized to the protein content by dividing the densitometry result with the relative optical density from the BCA protein assay kit result. The result is used for the analysis as the arbitrary unit. For the total MMP activity results of lysis bands of pro-MMP-9, active MMP-9, pro-MMP-2, and active MMP-2 are added. A protein size marker is used to determine the correct size.

Cell Assay [4]
All in vitro treatments are performed in SMC cultures at 90% confluence, when ECM synthesis in SMC starts to be evident. Doxycycline (20 nM) is diluted in culture medium at a concentration of 10 μg/mL (20 nM), at which no toxicity or variation in primary cultured SMC proliferation has been reported, as well as in other cell lines and the incubation time is 48 h. SMC-C and SMC-Ch are seeded at equal cell density in 6-well plates and, when confluence reaches 90%, 1 mL culture medium is added to each well containing 3.7×104 Bq L-[5-H3]-proline (9.62×1011 Bq/mmol). After 48 h incubation, cells are lysed with 0.5 mL 0.5 mol/L NaOH for 1 h. The resulting solution is neutralized with an equal amount of 0.5 mol/L HCl, and 50 μL are used to measure total proteins with the Bradford method. One volume of 10% TCA is added to the remaining 250 μL and centrifuged at 13,000 g for 15 min at 4°C. The resulting precipitate is dissolved in 100 μL 0.2 mol/L NaOH, and then neutralized with 1 mol/L HCl. The solution is incubated with collagenase buffer (Tris-HCl, pH 7.6 20 mM, and CaCl2 250 mM final concentration) and 10 units of collagenase at 37°C overnight. Then, 150 μL 10% TCA are added and centrifuged at 13000 g for 15 min at 4°C. The resulting supernatant is added to 4 mL of scintillation fluid and the radioactivity is measured in a liquid scintillation counter LS 600 TA.

Animal Administration [1]
Two groups of 6-month-old female heterozygous (HT) Col3a1 knockout mice are treated for 3 months with doxycycline. Treatment is provided with food containing 200 or 800 mg/kg of doxycycline. Because preliminary measured food intake of these mice is averaged at 3.5 g/day and the average body weight of animals is 25 g, the average drug dose for low- and high-dose groups is 25 (Doxy25) or 100 (Doxy100) mg/kg per day, respectively. Untreated (wild type) WT and HT mice are maintained on a regular diet (NIH-07 mouse/rat diet) and served as controls. After 3 months, under general inhalation anesthesia (2% of isoflurane in oxygen) and aseptic conditions, the abdominal aortas are surgically exposed and stressed by the following technique: the blood flow is stopped by occluding the abdominal aorta against the spinal column with a sterile cotton-tip applicator pressed at the level of the renal arteries. After 30 s a second applicator is pressed at the level of iliac bifurcation and the first applicator is abruptly released, followed by release of the second applicator. The abdominal incision is sutured closed, and mice are returned to home cages. The treatment is continued after the intervention. One week after intervention mice are euthanized by an overdose of isoflurane. Blood is collected, and aortas and segments of colon and skin are harvested.

References:
[1]. Wilfried Briest, et al. Doxycycline ameliorates the susceptibility to aortic lesions in a mouse model for the vascular type of Ehlers-Danlos syndrome. J Pharmacol Exp Ther. 2011 Jun;337(3):621-7. [2]. Zhang N, et al. The PK/PD Interactions of Doxycycline against Mycoplasma gallisepticum. Front Microbiol. 2016 May 4;7:653. [3]. Trajano VC, et al. Osteogenic activity of cyclodextrin-encapsulated doxycycline in a calcium phosphate PCL and PLGA composite. Mater Sci Eng C Mater Biol Appl. 2016 Jul 1;64:370-5. [4]. Palomino-Morales R, et al. Inhibition of extracellular matrix production and remodeling by doxycycline in smooth muscle cells. J Pharmacol Sci. 2016 Mar 25. Epub ahead of print