March 1, 2021

Denoch Emexicana

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Enhancement of elastin expression by transdermal administration of sialidase isozyme Neu2

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Materials and animals

Male Wistar rats (E19—12 months of age or older), male SAMP1 (24 weeks of age), and male SAMP8 (24 weeks of age) were purchased from Japan SLC (Shizuoka, Japan). The rats and mice were housed under standard laboratory conditions (23 °C ± 1 °C, 55% ± 5% humidity) and had access to tap water and diet ad libitum. The lights were automatically turned on at 8:00 and turned off at 20:00. All experiments were carried out in compliance with the guidelines established by the University of Shizuoka for the care and use of laboratory animals, and the ARRIVE guidelines. The protocols were pre-approved by the Animal Ethics Committee of the University of Shizuoka.

BTP3-Neu5Ac was synthesized according to the procedure described previously3. Geranic acid (85%), chorine bicarbonate (~ 80% in water), and dimethyl sulfoxide-d6 (DMSO-d6, 99.5 atom %D) were purchased from Merck KGaA (Darmstadt, Germany).

Sialidase activity imaging

Rats (12 weeks of age) were anesthetized with a mixture of butorphanol tartrate (Meiji Seika Pharma Co., Ltd., Tokyo, Japan; 2.5 mg/kg body weight), medetomidine hydrochloride (Fujifilm Wako Pure Chemical Co., Osaka, Japan; 0.375 mg/kg body weight) and midazolam (Fujifilm Wako Pure Chemical Co., 2 mg/kg body weight) by intraperitoneal injection. After transcardial perfusion with 200 ml of phosphate-buffered saline (PBS), lateral abdomen tissues including skin, muscle, and subcutaneous fat were quickly harvested and embedded in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan). After being frozen, the tissues were cut at − 20 °C using a cryotome into 300-μm-thick sections for staining shown in Fig. 1A,C and 100-μm-thick sections for staining shown in Fig. 2D.

Sialidase activity imaging was performed according to the procedure described previously44. Briefly, the sections were stained with BTP3-Neu5Ac (300-μm-thick sections: 1 mM for 60 min, 100-μm-thick sections: 300 μM for 30 min) in PBS or 100 mM sodium acetate buffer (pH4.6) at 27 °C. After washing with PBS, fluorescence was observed using a fluorescence microscope (Olympus IX71 or Keyence BZ-X710) with a filter set (ex/em: BP330-385/BA510IF for IX71 and BP340-380/BA500-550 for BZ-X710). In all observations with the fluorescence microscope, the gain of the microscope camera was set so as not to detect background fluorescence in a non-stained skin section. After obtaining pictures, the slices were fixed with 4% paraformaldehyde in PBS and then stained with hematoxylin–eosin. If necessary, the images were tiled together using Photoshop CS4 (Adobe Systems, San Jose, CA).

Measurement of sialidase activity

The rat skin was homogenized with sucrose (0.32 M) at 4 °C. Homogenates were transferred to a 96-well black microplate (Corning, NY, USA) and then incubated in PBS containing 4MU-Neu5Ac (10 µM, Nacalai Tesque, Kyoto, Japan) for 60 min at 27 °C. After the addition of sodium carbonate buffer (500 mM, pH 10.7), fluorescent intensities of 4-methylumbelliferone were measured using a microplate reader (ex/em, 355 nm/460 nm; Infinite M200, Tecan, Männedorf, Switzerland).

Preparation of CAGE

CAGE was prepared using a previously established method23. Two equivalents of recrystallized geranic acid (85%, 5.070 g) were added to one equivalent of choline bicarbonate (80 wt% solution, 3.109 g) in a 100 mL eggplant flask. The mixture was stirred at room temperature until no more CO2 evolved. The solvent was removed by rotary evaporation at 60 °C for 20 min, and the product was dried in a vacuum oven for 48 h at 60 °C to obtain CAGE (6.583 g). NMR assignments (collected using a JEOL ECX500, Tokyo, Japan) were in good agreement with those in a previous study23.

Measurement of the stability of sialidase in CAGE

Lyophilized sialidase from Arthrobacter ureafaciens (AUSA, Nacalai Tesque, Kyoto, Japan) was dissolved in CAGE or PBS at 10 mU/ml and then left at 27 °C for 28 days. Then the enzyme activity of sialidase was measured by mixing AUSA solution (10 μl), 4MU-Neu5Ac in PBS (100 μM, 40 μl), and PBS (50 μl).

Transdermal sialidase administration

Ionic liquid solutions of sialidase were prepared by dissolving lyophilized sialidase in CAGE. After shaving the hair, the solution of AUSA (1 U/ml) in CAGE, CAGE, or PBS was applied on the lateral abdomen skin of rats (12 weeks of age) and SAMP1 and SAMP8 twice a day for 5 days. Dermis tissues were quickly harvested and homogenized with sucrose (0.32 M) at 4 °C. Sialidase activity in the homogenate was measured with 4MU-Neu5Ac (40 µM). The level of elastin extracted from the dermis (20 mg) was measured with a quantitative dye, 5,10,15,20-tetraphenyl-21,23-porphine tetra-sulfonate, using a Fastin Elastin Assay Kit (Biocolor, Northern Ireland, UK) according to the manufacturer’s instructions.

Immunohistochemical staining

For elastin staining, extension sections of the dermis were fixed with 4% paraformaldehyde in PBS. After blocking with 2% goat serum in tris-buffered saline with Tween 20, the sections were stained with rabbit anti-elastin polyclonal IgG (Bioss Antibodies, Woburn, MA) and FITC-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). The anti-elastin polyclonal IgG was produced using a synthetic peptide derived from human elastin, and has cross-reactivity to human, mouse and rat elastin. Counterstaining was performed using DAPI (1 µg/ml).

For Neu2 staining, lateral abdomen tissues were fixed with 4% paraformaldehyde in PBS. After blocking with 2% goat serum in tris-buffered saline with Tween 20, the sections were stained with rabbit anti-Neu2 polyclonal IgG (Rockland, Gilbertsville, PA) and HiLyte Fluor 555-conjugated goat anti-rabbit IgG (Anaspec, Fremont, CA).

Images were acquired by using a fluorescence microscope BZ-X710 with a filter set (ex/em: BP450-490/BA500-550 for FITC and BP340-380/BA435-485 for DAPI) and by using a fluorescence microscope IX71 (Olympus, Tokyo, Japan) with a filter set (ex/em: BP530-550/BA575IF for HiLyte Fluor 555). The background level of fluorescence was determined by sections stained with only the secondary antibody. All imaging was performed at least twice in different rats, and reproducibility was confirmed.

Elastin autofluorescence45

Unstained sections of the dermis were fixed with 4% paraformaldehyde in PBS. Autofluorescence was observed using a fluorescence microscope BZ-X810 (Keyence) with a filter set (ex/em: BP450-490/BA500-550).

Real-time quantitative reverse transcription-polymerase chain reaction (real-time RT-PCR)

The procedure for real-time RT-PCR was described previously44. Briefly, total RNA was isolated from rat tissues (at 12 weeks of age) such as skin and adipose in the lateral abdomen by using the guanidinium phenol reagent (TRIzol reagent, Life Technologies) according to the manufacturer’s instructions. The expression level of mRNA was evaluated using a thermal cycler system (Thermal Cycler Dice Real-Time System Lite, TaKaRa Bio), a One-Step SYBR PrimeScript PLUS RT-PCR kit (Perfect Real Time, TaKaRa Bio) and primer pairs [5′-CCCATCCCGAGTACCGAGT-3′ and 5′-CCCGGCCACAACTGGAC-3′ for Neu1, 5′-GAGCCACCAACCATGTCAAG-3′ and 5′-AAGGGACATGGATTCATGGAG-3′ for Neu2, 5-CGGAGCTGGTGAGCTGAG-3′ and 5′-CCTGCTGGAACAGAGTGCTG-3′ for Neu3, 5′-TCTGGAGAGTGCCAACTGGC-3′ and 5′-AAGGAAGTGCCTTCATCAGCAC-3′ for Neu4, 5′-GCTTAGGAGTCTCAACAGGTGC-3′ and 5′-CGGAACCTTGGCCTTGACTC-3′ for elastin, and 5′-TGAACGGATTTGGCCGTATCGG-3′ and 5′-TCAATGAAGGGGTCGTTGATGG-3′ for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. GAPDH mRNA was used as an internal standard to normalize sample variation.

Transdermal administration of Neu2

The previously established C6 rat glioma cells stably expressing C-terminal Myc-tagged rat Neu2 were cultured for 48 h in a humidified incubator with 5% CO2 at 37 °C in DMEM supplemented with 10% fetal bovine serum33. Neu2 secreted extracellularly was collected from the culture supernatant (4 ml) by ultrafiltration with the Amicon Ultra filter unit (Millipore Co., Darmstadt, Germany) at 4000 × g for 20 min. After washing with PBS, Neu2 activity was measured with 4MU-Neu5Ac in PBS (40 µM, pH 7.3). Neu2 (21.3 nmol/min/ml) was mixed with CAGE at a ratio of 3:7 (Neu2 : CAGE). After shaving, 60 µL of a mixture of Neu2 and CAGE, CAGE, or PBS was applied on the rat lateral abdomen skin (at 12 weeks of age). Alternatively, 42 µL of CAGE was applied on the skin after applying 18 µL of Neu2. The application was performed twice a day for 5 days. The level of elastin in the dermis was measured in the same manner as described above.

Statistical analysis

Statistical significance was assessed by one-way ANOVA with Bonferroni’s multiple comparison test and two-tailed unpaired t-test. If the variances between the two groups were different, the two-tailed unpaired t-test with Welch’s correction was used. F value in one-way ANOVA was denoted with the degrees of freedom for the numerator and for the denominator.

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