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Inflammation Research (2018) 67:663–670 https://doi.org/10.1007/s00011-018-1155-6

ORIGINAL RESEARCH PAPER

Adiponectin administration alleviates DSS-induced colonic inflammation in Caco-2 cells and mice

Qin Zhao1 · Yang Liu2 · Lei Tan3 · Liyong Yan4 · Xiuli Zuo5

Received: 11 April 2018 / Revised: 27 April 2018 / Accepted: 3 May 2018 / Published online: 15 May 2018 © The Author(s) 2018

AbstractBackground Adiponectin, a protein hormone produced by adipose tissues, exhibits anti-inflammatory functions in various models. This study was investigated the effects of adiponectin on dextran sodium sulfate (DSS)-colonic injury, inflammation, apoptosis, and intestinal barrier dysfunction in Caco-2 cell and mice.Materials and methods The results showed that DSS caused inflammatory response and intestinal barrier dysfunction in vitro and in vivo. Adiponectin injection alleviated colonic injury and rectal bleeding in mice. Meanwhile, adiponectin downregu-lated colonic IL-1β and TNF-α expressions and regulated apoptosis relative genes to attenuate DSS-induced colonic inflam-mation and apoptosis. Adiponectin markedly reduced serum lipopolysaccharide concentration, a biomarker for intestinal integrity, and enhanced colonic expression of tight junctions (ZO-1 and occludin). The in vitro data further demonstrated that adiponectin alleviated DSS-induced proinflammatory cytokines production and the increased permeability in Caco-2 cells.Conclusion Adiponectin plays a beneficial role in DSS-induced inflammation via alleviating apoptosis and improving intes-tinal barrier integrity.

Keywords Adiponectin · Inflammation · Apoptosis · Barrier

Background

Inflammatory bowel diseases (IBD), characterized by rectal bleeding, diarrhea, intestinal motility disorder, and colonic shortening, are linked with colonic chronic inflammation [1, 2]. Recently, Intestinal macrophages and dendritic cells have been indicated to involve in the initiation of inflammation

in IBD through inappropriate responses to enteric microbial stimuli, inefficient clearance of microbes from host tissues, and impaired transition from appropriate proinflammatory responses to anti-inflammatory responses [3–5]. Chronic inflammatory response further contributes to the pathogen-esis of IBD as highlighted by recent genome-wide associa-tion studies [6–9]. Therefore, anti-inflammatory drugs serve as a potential therapy for IBD patients.

Adiponectin is a protein hormone produced by adipose tissues and has various protective properties. The molecular mechanism of adiponectin is associated with its receptors, including adiponectin receptor 1, 2 and T-cadherin. Activa-tion of adiponectin receptors inhibits inflammatory response and oxidative stress [10, 11]. Compelling evidence has dem-onstrated the anti-inflammatory function of adiponectin in various inflammatory models, such as skin inflammation [12], peripheral inflammation [13], LPS-induced inflamma-tory response in adipocytes [14]. However, the anti-inflam-matory effect of adiponectin on colonic inflammation is still unknown. Thus, in this study, effects of adiponectin on dex-tran sodium sulfate (DSS)-colonic inflammation and injury were investigated in Caco-2 cell lines and Kunming mice.

Inflammation Research

Responsible Editor: John Di Battista.

* Xiuli Zuo zuoxiuli_s@163.com

1 Department of Gastroenterology, Taian City Central Hospital, Taian, Shandong, People’s Republic of China

2 Department of Medicine, Beijing 316 Hospital, Beijing, People’s Republic of China

3 Department of Cerebral Surgery, Taian City Central Hospital, Taian, Shandong, People’s Republic of China

4 Department of Stomatology, Taian City Central Hospital, Taian, Shandong, People’s Republic of China

5 Department of Gastroenterology, Qilu Hospital, Shandong University, 107 Wenhua West Road, Jinan 250012, People’s Republic of China

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Materials and methods

Animal model and groups

120 female Kunming mice (20.44 ± 0.83 g) were randomly divided into four groups: control group (Cont, n = 30), DSS-treated group (DSS, n = 30), adiponectin group (AG, n = 30), and DSS plus adiponectin group (DA, n = 30). Mice in DSS and DA groups received 4% DSS (average molecular weight 5000, Sigma-Aldrich) instead for tap water to establish IBD model [15]. Mice in AG and DA groups were intraperito-neally administrated with full-length adiponectin (2 µg/g, 0.2 ml/mouse) [16]. The control and untreated challenged animals received the same volume of saline alone.

Ten mice from each group were killed at days 4, 7, and 10 (n = 10). The length and weight of colonic tissues were determined in each mouse. Then, one piece of colon tissues in each mouse was collected and immediately frozen in liq-uid nitrogen for RT-PCR and western blotting analyses. All experiments involving animals in this study were approved by the animal welfare committee of Shandong University.

Clinical evaluation of DSS colitis

Diarrhea ration from mice were recorded at days 4, 7, and 10. A diarrhea score was used to evaluate diarrhea ratio after DSS exposure as follows: 0 means no diarrhea was noticed; 2 points mean little diarrhea with pasty and semiformed stools; and 4 points mean serious diarrhea with liquid stools [17]. Stool blood concentrations were determined through haemoccult tests (Beckman Coulter) and a bleeding score was used to evaluate the DSS colitis as follows: 0 means no blood was noticed; 2 points mean positive haemoccult; and 4 points mean gross bleeding in mice.

Serum lipopolysaccharide (LPS) determination

Blood samples were collected through eyes orbiting and serum was separated by centrifugation at 3000×g for 10 min and under 4 °C. Serum LPS were determined by A Mouse LPS ELISA kits (Wuhan Cusabio, China).

Cell lines and cell culture

Human epithelial Caco-2 cells were grown in Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 10% FBS (HyClone, Logan, UT) and 50 U/mL penicil-lin–streptomycin and maintained at 37 °C in a humidified chamber of 5% CO2 [18]. Confluent cells (85–90%) were incubated with different concentrations of adiponectin (AD, 10, 50, 100, and 200 µM) and 2% DSS for 4 days to establish inflammatory model [19].

Cellular proinflammatory cytokine measurement

Cellular proinflammatory cytokines (IL-1β, IL-17, and TNF-α) were determined according ELISA kits (CUSABIO, Wuhan, China).

Trans‑epithelial electrical resistance (TEER) measurements

Caco-2 cells were grown in a 12-well Transwell system and the changes of TEER were determined using an epithelial voltohmmeter ERS-2 (Merck Millipore, USA). When the filter-grown Caco-2 monolayers reached epithelial resistance of at least 500 Ω cm2, the cells were incubated in different dosages of AD with 2% DSS treatment. Electrical resist-ance was measured until similar values were recorded on three consecutive measurements. Values were corrected for background resistance due to the membrane insert and cal-culated as Ω cm2.

Paracellular marker FD‑4 (FITC‑Dextran 4 kDa) flux measurements

Paracellular permeability was estimated via FD-4 flux. Briefly, Caco-2 cells were seeded in a 12-well Transwell system to reach monolayers. After treatment with AD and DSS, cells were incubated in the upper chamber with Hank’s balanced salt solution for 2 h, which contains 1 mg/mL FD-4 solution. FD-4 signal was determined via Synergy H2 micro-plate reader (Biotek Instruments, USA).

Real‑time PCR

Total RNA was isolated with TRIZOL regent (Invitrogen, USA) and reverse transcribed into the first strand (cDNA) using DNase I, oligo (dT) 20 and Superscript II reverse transcriptase (Invitrogen, USA). Primers were designed with Primer 5.0 according to the gene sequence of mouse to produce an amplification product. The primer sets used as followed: β-Actin, F:GTC CAC CTT CCA GCA GAT GT, R:GAA AGG GTG TAA AAC GCA GC; IL-1β, F:CTG TGA CTC GTG GGA TGA TG R:GGG ATT TTG TCG TTG CTT GT; IL-17, F:TAC CTC AAC CGT TCC ACG TC, R:TTT CCC TCC GCA TTG ACA C; TNF-α, F:AGG CAC TCC CCC AAA AGA T, R:TGA GGG TCT GGG CCA TAG AA; p53, F: GAG GTT CGT GTT TGT GCC TG, R: CTT CAG GTA GCT GGA GTG AGC; Bcl-2, F: GAA CTG GGG GAG GAT TGT GG, R: GCA TGC TGG GGC CAT ATA GT; Bax, F: CTG GAT CCA AGA CCA GGG TG, R: CCT TTC CCC TTC CCC CAT TC. Relative expression was normalized and expressed as a ratio to the expression in control group [20–22].

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Western bolt

Proteins were extracted with protein extraction reagents (Thermo Fisher Scientific Inc., USA). Proteins (30 µg) were separated by SDS–polyacrylamide gel electrophore-sis and electrophoretically transferred to apolyvinylidene difluoride (PVDF) membrane (BioRad, Hercules, CA, USA). Membranes were blocked and then incubated with the following primary antibodies: ZO-1 (ab59720), Claudin1 (ab115225), and Occludin (ab31721) (Abcam, Inc., USA). Mouse β-actin antibody (Sigma) was used for protein loading control. After primary antibody incuba-tion, membranes were washed, incubated with alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgG antibodies (Promega, Madison, WI, USA), and quantified and digitally analyzed using the image J program (NIH).

Statistical analysis

All statistical analyses were performed using SPSS 17.0 software. Group comparisons were performed using the one-way analysis of variance (ANOVA) to test homoge-neity of variances via Levene’s test and followed with Tukey’s multiple comparison test. Values in the same row with different superscripts are significant (P < 0.05), while values with same superscripts are not significant different (P > 0.05).

Results

Adiponectin alleviated DSS‑induced colonic injury

At days 7 and 10, DSS markedly reduced body weight and colonic length (P < 0.05) but adiponectin failed to alleviate the reduction (P > 0.05) (Fig. 1). From days 4 to 10, DSS exposure significantly increased rectal bleeding score and diarrhea score compared with the control group (P < 0.05). Adiponectin injection attenuated DSS-caused rectal bleeding at days 7 and 10 (P < 0.05).

Adiponectin alleviated DSS‑induced colonic inflammation

DSS enhanced colonic expressions of IL-1β and TNF-α and day 4 and IL-1β, IL-17, and TNF-α at days 7 and 10 (P < 0.05) (Fig. 2), suggesting that DSS exposure mark-edly caused colonic inflammation. Although adiponectin injection failed to alleviate colonic inflammatory response at day 4, expressions of IL-1β and TNF-α at days 7 and 10

were markedly lower in DSS plus adiponectin group than that in DSS group (P < 0.05).

Adiponectin alleviated DSS‑induced colonic apoptosis

At day 4, DSS treatment upregulated colonic Bax com-pared with the control group (P < 0.05) (Table 1). At day 7, DSS upregulated p53 and Bax genes and adiponectin injection significantly alleviated the overexpression of Bax caused by DSS (P < 0.05). At day 10, p53 and Bax were upregulated in DSS group, while downregulated in DSS + adiponectin group (P < 0.05). Meanwhile, bcl-2 was significantly lower in DSS group (P < 0.05) but adiponec-tin failed to attenuate the inhibitory effect (P > 0.05).

Adiponectin alleviated DSS‑induced colonic barrier dysfunction

ZO-1, claudin1, and occludin are three major tight junc-tion proteins for intestinal barrier. At day 4, DSS tended to inhibit ZO-1 and occludin, but the difference was insig-nificant (P > 0.05) (Fig. 3). At day 7, ZO-1 and occludin were significantly downregulated in DSS group and adi-ponectin enhanced colonic ZO-1 and occludin expressions after DSS exposure (P < 0.05). At day 10, ZO-1, claudin1, and occludin were inhibited in DSS group and adiponec-tin enhanced colonic ZO-1 and occludin expressions after DSS exposure (P < 0.05).

Blood LPS concentration has been widely used as a biomarker for intestinal barrier. In this study, we found that DSS treatment significantly increased serum LPS abundance at days 7 and 10 (P < 0.05) (Fig. 4), suggesting that DSS caused colonic injury and enhanced colonic per-meability. Meanwhile, adiponectin exhibited a protective role in DSS-induced colonic injury evidenced by the lower serum LPS concentrations (P < 0.05).

Adiponectin alleviated DSS‑induced cellular inflammation in Caco‑2 cells

Cells incubated with DSS exhibited marked inflammatory response evidenced by the increased IL-1β, IL-17, and TNF-α generation (P < 0.05) (Table 2). Adiponectin (100 and 200 nM) significantly alleviated DSS-induced TNF-α generation (P < 0.05), and the protective effect exhibited a dosage-dependent manner. Adiponectin also tended to affect IL-1β and IL-17 production, but the difference was insignificant (P > 0.05).

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Adiponectin alleviated DSS‑induced increase in cellular permeability in Caco‑2 cells

TEER and FD-4 flux were used to estimate the cellular per-meability after DSS exposure (Fig. 5). The results showed that DSS markedly enhanced cellular permeability evidenced by the decreased TEER and increased FD-4 flux (P < 0.05) at days 2–4. Meanwhile, adiponectin alleviated DSS-induced increase in cellular permeability (P < 0.05), and the protec-tive effect exhibited a dosage-dependent manner.

Discussion

Adiponectin is an important adipokine and previous reports suggest that adiponectin serves as a protective mechanism in inflammatory response and related diseases [23, 24]. Adi-ponectin deficiency promotes diarrhea, stool hemoccult, and weight loss in DSS-induced colitis and contributes to inflammation-induced colon cancer [25–27], indicating that adiponectin treatment may play a beneficial role in colonic inflammation. In this study, we used DSS to induce colonic

Fig. 1 Effects of adiponectin on DSS-induced colonic injury in mice. Data are presented as mean ± SEM. The values having different super-script letters were significantly different (P < 0.05; n = 10)

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inflammation and we found that adiponectin alleviated rectal bleeding and colonic injury.

In this study, DSS induced colonic inflammation and caused colonic injury, which is similar with previous reports [28–30]. Adiponectin has been widely demonstrated to mediate inflammatory response and exert an anti-inflamma-tory effect. In DSS-induced colonic inflammation, adiponec-tin deficiency exacerbated inflammatory response tumori-genesis [26]. Meanwhile, mice with higher adiponectin had lower expression of proinflammatory cytokines (TNF and IL-1β), adipokines, and cellular stress and apoptosis mark-ers [31]. Shibata et al. reported that the anti-inflammatory function of adiponectin might be associated with suppress-ing IL-17 production from γδ-T cells [12]. In this study, adi-ponectin markedly alleviated DSS-induced IL-1β and TNF-α overexpression in mice, which is further demonstrated in Caco-2 cells that adiponectin alleviated DSS-caused TNF-α production.

Fig. 2 Effects of adiponectin on colonic expression of proinflammatory cytokines in mice. Data are presented as mean ± SEM. The values hav-ing different superscript letters were significantly different (P < 0.05; n = 10)

Table 1 Effects of adiponectin on colonic expression of apoptosis relative genes in mice

Data are presented as mean ± SEM. The values having different superscript letters were significantly different (P < 0.05; n = 10)

Item Cont DSS Adiponectin DSS + Adi-ponectin

Day 4 p53 1.00 ± 0.13ab 1.34 ± 0.17a 0.85 ± 0.07b 1.23 ± 0.12a

Bcl-2 1.00 ± 0.12 0.92 ± 0.13 0.96 ± 0.12 0.91 ± 0.09 Bax 1.00 ± 0.09b 1.65 ± 0.16a 0.94 ± 0.06b 1.55 ± 0.13a

Day 7 p53 1.00 ± 0.13b 1.56 ± 0.14a 1.02 ± 0.09b 1.27 ± 0.13ab

Bcl-2 1.00 ± 0.13 1.01 ± 0.13 1.23 ± 0.16 1.28 ± 0.20 Bax 1.00 ± 0.16bc 1.87 ± 0.13a 0.74 ± 0.13c 1.40 ± 0.13b

Day 10 p53 1.00 ± 0.09c 1.76 ± 0.16a 0.84 ± 0.18c 1.44 ± 0.14b

Bcl-2 1.00 ± 0.05b 0.56 ± 0.08c 1.43 ± 0.09a 0.53 ± 0.11c

Bax 1.00 ± 0.11b 1.92 ± 0.11a 0.68 ± 0.08c 1.37 ± 0.16b

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DSS-induced colonic inf lammation is commonly accompanied with apoptosis via influencing apoptosis relative proteins, such as p53, bax, and bcl-2 [32–34]. p53, a tumor suppressor, can directly execute apoptosis in response to various cellular stresses, such as inflammation and oxidative stress [35–37]. Meanwhile, p53 involves in the apoptotic mechanisms in the mitochondria by regulat-ing bcl-2 family proteins (bax and bcl-2) [38–40]. In this

study, DSS induced colonic apoptosis by upregulating p53 and bax and inhibiting bcl-2 expression. Meanwhile, adi-ponectin played a protective role in DSS-induced apopto-sis through influencing p53 and bax expressions. Similarly, Long et al. reported that adiponectin treatment prevented palmitate-induced apoptosis by inducing an upregulation of bcl-2 and a downregulation of bax [41]. Adiponectin also has been demonstrated to inhibit neutrophil apoptosis via activation of AMP kinase, PKB, and ERK 1/2 MAP kinase [42].

Intestinal barrier disturbances subsequent with the increased permeability plays a crucial role in the patho-genesis of IBD [43, 44]. In this study, intestinal perme-ability was significantly increased and tight junctions (ZO-1, claudin1, and occludin) were downregulated in DSS-induced colonic inflammation. Interestingly, adi-ponectin injection improved intestinal permeability evi-denced by decreasing serum LPS and enhancing colonic expressions of tight junctions in mice. The high level of serum LPS is considered to be the consequence of the increased intestinal permeability [45]. Thus, the in vivo results suggested a beneficial role of adiponectin in barrier integrity, which was further demonstrated in Caco-2 cells that adiponectin alleviated DSS-induced the decreased TEER and increased FD-4 flux.

Fig. 3 Effects of adiponectin on colonic protein abundances of tight junctions in DSS-challenged mice at days 4 (a), 7 (b), and 10 (c). Data are presented as mean ± SEM. The values having different superscript letters were significantly different (P < 0.05; n = 5)

Fig. 4 Effects of adiponectin on serum LPS level (ng/ml). Data are presented as mean ± SEM. The values having different superscript letters were significantly different (P < 0.05; n = 10)

Table 2 Effects of adiponectin on cellular proinflammatory cytokines in Caco-2 cells

Data are presented as mean ± SEM. The values having different superscript letters were significantly different (P < 0.05; n = 3)

Item Cont DSS 10AD 50AD 100AD 200AD

IL-1β 63.82 ± 5.28b 117.32 ± 10.37a 123.98 ± 12.57a 108.25 ± 10.38ab 97.23 ± 8.49ab 89.93 ± 89.76ab

IL-17 177.56 ± 22.37b 234.12 ± 25.71a 239.28 ± 24.38a 222.17 ± 22.19a 219.29 ± 32.16a 211.35 ± 28.44a

TNF-α 337.76 ± 28.93c 421.15 ± 43.29a 413.28 ± 44.23a 408.77 ± 37.18ab 391.65 ± 27.39b 375.27 ± 23.15b

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Conclusions

Adiponectin alleviated colonic injury, inflammatory response, and apoptosis in mice. Meanwhile, adiponec-tin improved intestinal integrity in DSS-challenged mice evidenced by the lowered serum LPS enhanced colonic expressions of tight junctions (ZO-1 and occludin). The in vitro data further demonstrated that adiponectin allevi-ated DSS-induced proinflammatory cytokines production and the increased permeability in Caco-2 cells. Together, adiponectin plays a beneficial role in DSS-induced inflam-mation via alleviating apoptosis and improving intestinal barrier integrity.

Compliance with ethical standards

Conflict of interest The authors declare that they have no competing interests.

Open Access This article is distributed under the terms of the Crea-tive Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribu-tion, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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