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isoflurane anesthesia, the serous membrane of the cecum was abraded with a swab until punctate hemorrhage occurred. The adhesion scores were evaluated ...
Accepted Manuscript Periostin antisense oligonucleotide prevents adhesion formation after surgery in mice Shinji Takai, Ph.D, Masafumi Yoshino, Kazumasa Takao, Kazunori Yoshikawa, Denan Jin PII:

S1347-8613(17)30015-4

DOI:

10.1016/j.jphs.2016.10.009

Reference:

JPHS 316

To appear in:

Journal of Pharmacological Science

Received Date: 24 August 2016 Revised Date:

5 October 2016

Accepted Date: 12 October 2016

Please cite this article as: Takai S, Yoshino M, Takao K, Yoshikawa K, Jin D, Periostin antisense oligonucleotide prevents adhesion formation after surgery in mice, Journal of Pharmacological Science (2017), doi: 10.1016/j.jphs.2016.10.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Periostin antisense oligonucleotide prevents adhesion formation after surgery in mice

Denan Jina

Affiliations.

Department of Innovative Medicine, Osaka Medical College Graduate School of

Medicine, Takatsuki, Japan AQUA Therapeutics Co., Ltd., Kobe, Japan

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Shinji Takaia,*, Masafumi Yoshinob, Kazumasa Takaob, Kazunori Yoshikawab,

*Corresponding author: Shinji Takai, Ph.D.

Address: Department of Innovative Medicine, Osaka Medical College

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Graduate School of Medicine, 2-7 Daigaku-machi, Takatsuki 569-8686, Japan.

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Telephone: +81-72-684-6021; Fax: +81-72-684-6730 E-mail: [email protected]

Number of words: 3838

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Abstract

To study the role of periostin in adhesion formation, the effect of periostin

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antisense oligonucleotide (PAO) on adhesion formation was evaluated in mice. Under anesthesia, the serous membrane of the cecum was abraded, and the adhesion score and mRNA levels of periostin and its related factors were determined after surgery. Saline,

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40 mg/kg of negative sense oligonucleotide (NSO), or 40 mg/kg of PAO were injected into the abdomen after surgery, and the adhesion score and mRNA levels were evaluated

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14 days later. Filmy adhesion formation was observed 1 day after surgery, and the adhesion score increased gradually to 14 days. The mRNA levels of periostin, transforming growth factor (TGF)-β, and collagen I increased gradually from 3 days to 14 days. The adhesion score of PAO was significantly lower than of saline or NSO 14

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days after surgery. The mRNA levels of periostin, TGF-β, and collagen I were also significantly attenuated by treatment with PAO compared with saline or NSO. Thus, these results demonstrated that the periostin mRNA level increased in the abraded

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mRNA level.

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cecum, and PAO prevented adhesion formation along with attenuation of the periostin

Keywords: Periostin, Antisense, Adhesion, Surgery, Mice

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1. Introduction

Postoperative abdominal adhesions occur frequently after abdominal surgery and

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induce serious complications, such as chronic abdominal and pelvic pain, small-bowel obstruction, infarction, and difficult re-operation (1,2). These complications increase not only morbidity, but also economic costs, and the significance of abdominal adhesions in

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general surgery has been underestimated in the clinical setting (3,4). However, there are no definitive strategies to prevent abdominal adhesions.

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Periostin has been known as osteoblast-specific factor 2 and characterized as a matricellular protein (5). Periostin is widely expressed in tissues other than osteoblasts (6). In adult healthy tissues, the expression of periostin is very low, but it is augmented at remodeling regions, such as heart tissue after myocardial infarction and with

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cutaneous wound healing (7,8). Periostin binds to the components of extracellular matrix (ECM), including fibronectin, tenacin c, and collagen I, and transmits signals from the ECM to the cellular receptors to influence tissue remodeling, such as

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fibrogenesis (9,10). Transforming-growth factor (TGF)-β is a profibrotic growth factor that is recognized as a main mediator of tissue fibrosis. Periostin is secreted from

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fibroblasts to augment collagen deposition, and the mechanism may be involved in the interaction between periostin and TGF-β (11). TGF-β is an inducer of periostin expression, but, conversely, periostin can activate TGF-β formation (5,9,12). Such interactions may promote a vicious cycle of fibrogenesis. Periostin-deficient mice show reduced fibrosis in the kidneys and lungs via attenuation of TGF-β expression (13,14). Thus, inhibition of periostin may become a useful strategy for attenuation of tissue fibrosis. However, whether periostin is involved in adhesion formation after surgery has 3

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been unclear. TGF-β is considered closely related to abdominal adhesions because it can induce abdominal adhesion formation (15,16). In contrast, anti-TGF-β antibodies prevented

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abdominal adhesions in rats (17). In the present study, the relationship between periostin expression and adhesion formation was investigated after abdominal surgery, and the effect of periostin antisense oligonucleotide (PAO) on abdominal adhesions was

2.1. Reagents

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2. Materials and Methods

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evaluated.

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The sequences of PAO and nonsense oligonucleotide (NSO) were 5’ caccaCTGTTCGTAAuuugg3’ and 5’cgacaTCGTGCGTCGuauau3’, respectively. ; the small letters, a, u, g and c, show 2’-O-methyl modified phosphorothioated

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ribonucleotides and the capital letters, A, T, G, and C, show phosphorothioated deoxyribonucleotides. Nucleobases indicated by a/A, u, T and g/G are unmodified

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adenine, uracil, thymine and guanine, respectively, and those indicated by c and C are 5-methylcytosine residues.

2.2. Animals

Twelve-week-old male C57BL/6 mice (n=48) were obtained from Japan SLC (Shizuoka, Japan) and housed in a temperature-, humidity-, and light-controlled room. 4

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All procedures involving animals were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals at Osaka Medical College. The surgical procedure was done according to our previous studies (18,19). Under

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isoflurane anesthesia, the serous membrane of the cecum was abraded with a swab until punctate hemorrhage occurred. The adhesion scores were evaluated 1, 3, 7, and 14 days after surgery, and then the cecum was removed for histological analysis and

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measurement of mRNA levels.

The effect of PAO on adhesion formation was evaluated by subcutaneous

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administration of saline, 40 mg/kg of NSO, or 40 mg/kg of PAO after cecal abrading, and the adhesion scores were evaluated at 1 and 14 days later (each group: n=6).

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2.3. Scoring of Adhesions

The adhesions were scored blindly according to a modified classification of Hulka et al. (20): 0, no adhesions; 1, mild adhesions; 2, localized moderate adhesions;

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moderate and wide adhesions; and 4, severe, impossible to separate adhesions.

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2.4. Histological analysis

The tissue specimens were fixed overnight with Carnoy’s fixative in 10%

methanol. The fixed tissues were embedded in paraffin and then cut at a thickness of 4 µm. The sections were mounted on silanized slides (Matsunami Glass Ind., Kishiwada, Japan) and deparaffinized with xylene and ethanol. The cecum and its surroundings were stained using Mallory-Azan staining; a 5

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blue-stained lesion was defined as a fibrotic lesion (21). The procedure for immunohistochemical analysis of periostin has been previously described (22). Sections were incubated with anti-periostin antibody (Santa Cruz

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Biotechnology, Dallas, TX), followed by a reaction with appropriate reagents from a streptavidin-biotin peroxidase kit (Dako LSAB kit; Dako Co., Carpinteria, CA) and 3-amino-9-ethylcarbazole, which was used for color development; a brown-stained

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region was defined as periostin-positive cells. The sections were lightly counterstained

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with hematoxylin.

2.5. Real-time polymerase chain reaction (RT-PCR)

Total cecal RNA was extracted using the Trizol reagent (Life Technologies,

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Rockville, MD) and subsequently dissolved in RNase-free water (Takara Bio Inc. Otsu, Japan) (23). Total RNA (1 µg) was transcribed into cDNA with Superscript VIRO (Invitrogen, Carlsbad, CA). Levels of mRNA were measured by RT-PCR on a

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LightCycler with software (Roche Diagnostics, Tokyo, Japan) using TaqMan fluorogenic probes. Primers and probes for RT-PCR of periostin, transforming-growth

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factor (TGF)-β, collagen I, and 18S ribosomal RNA (rRNA) were designed by Roche Diagnostics. The primers were as follows: 5’-atcaggggtcgggatcag-3’ (forward) and 5’-ggagctgaagtatttctttttggt-3’ (reverse) for periostin; 5’-tggagcaacatgtggaactc-3’ (forward) and 5’-cagcagccggttaccaag-3’ (reverse) for TGF-β; 5’-catgttcagctttgtggacct-3’ (forward) and 5’-gcagctgacttcagggatgt-3’ (reverse) for collagen I; and 5’-gcaattattccccatgaacg-3’ (forward) and 5’-gggacttaatcaacgcaagc-3’ (reverse) for 18S rRNA. The probes were as follows: 5’-ctccagca-3’ for periostin; 5’-ttcctggc-3’ for 6

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TGF-β; 5’-tcctgctc-3’ for collagen I; and 5’-ttcccagt-3’ for 18S rRNA. The mRNA levels of periostin, TGF-β, and collagen I were normalized to that of 18S rRNA.

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2.6. Statistical analysis

Data are expressed as means ± standard error of the mean (SEM). Significant

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differences among mean values of multiple groups were evaluated using one-way analysis of variance followed by Fisher’s test. Values of P