RETRACTED ARTICLE: Adiponectin administration alleviates DSS-induced colonic inflammation in Caco-2 cells and mice

Background 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 downregulated colonic IL-1β and TNF-α expressions and regulated apoptosis relative genes to attenuate DSS-induced colonic inflammation 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 intestinal barrier integrity.


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][4][5]. Chronic inflammatory response further contributes to the pathogenesis of IBD as highlighted by recent genome-wide association studies [6][7][8][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. Activation of adiponectin receptors inhibits inflammatory response and oxidative stress [10,11]. Compelling evidence has demonstrated the anti-inflammatory function of adiponectin in various inflammatory models, such as skin inflammation [12], peripheral inflammation [13], LPS-induced inflammatory response in adipocytes [14]. However, the anti-inflammatory effect of adiponectin on colonic inflammation is still unknown. Thus, in this study, effects of adiponectin on dextran sodium sulfate (DSS)-colonic inflammation and injury were investigated in Caco-2 cell lines and Kunming mice.

Animal model and groups
120 female Kunming mice (20.44 ± 0.83 g) were randomly divided into four groups: control group (Cont, n = 30), DSStreated 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 intraperitoneally 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 liquid 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).

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 cm 2 , the cells were incubated in different dosages of AD with 2% DSS treatment. Electrical resistance was measured until similar values were recorded on three consecutive measurements. Values were corrected for background resistance due to the membrane insert and calculated as Ω cm 2 .

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 microplate 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  [20][21][22].

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 homogeneity 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).

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 markedly 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 compared 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 adiponectin failed to attenuate the inhibitory effect (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 permeability. 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 increase in cellular permeability in Caco-2 cells
TEER and FD-4 flux were used to estimate the cellular permeability 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 protective 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]. Adiponectin deficiency promotes diarrhea, stool hemoccult, and weight loss in DSS-induced colitis and contributes to inflammation-induced colon cancer [25][26][27], indicating that adiponectin treatment may play a beneficial role in colonic inflammation. In this study, we used DSS to induce colonic In this study, DSS induced colonic inflammation and caused colonic injury, which is similar with previous reports [28][29][30]. Adiponectin has been widely demonstrated to mediate inflammatory response and exert an anti-inflammatory effect. In DSS-induced colonic inflammation, adiponectin deficiency exacerbated inflammatory response tumorigenesis [26]. Meanwhile, mice with higher adiponectin had lower expression of proinflammatory cytokines (TNF and IL-1β), adipokines, and cellular stress and apoptosis markers [31]. Shibata et al. reported that the anti-inflammatory function of adiponectin might be associated with suppressing IL-17 production from γδ-T cells [12]. In this study, adiponectin 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.  DSS-induced colonic inflammation is commonly accompanied with apoptosis via influencing apoptosis relative proteins, such as p53, bax, and bcl-2 [32][33][34]. p53, a tumor suppressor, can directly execute apoptosis in response to various cellular stresses, such as inflammation and oxidative stress [35][36][37]. Meanwhile, p53 involves in the apoptotic mechanisms in the mitochondria by regulating bcl-2 family proteins (bax and bcl-2) [38][39][40]. In this study, DSS induced colonic apoptosis by upregulating p53 and bax and inhibiting bcl-2 expression. Meanwhile, adiponectin played a protective role in DSS-induced apoptosis 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 pathogenesis of IBD [43,44]. In this study, intestinal permeability was significantly increased and tight junctions (ZO-1, claudin1, and occludin) were downregulated in DSS-induced colonic inflammation. Interestingly, adiponectin injection improved intestinal permeability evidenced 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.

Conclusions
Adiponectin alleviated colonic injury, inflammatory response, and apoptosis in mice. Meanwhile, adiponectin 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 alleviated DSS-induced proinflammatory cytokines production and the increased permeability in Caco-2 cells. Together, adiponectin plays a beneficial role in DSS-induced inflammation via alleviating apoptosis and improving intestinal barrier integrity.

Compliance with ethical standards
Conflict of interest The authors declare that they have no competing interests.
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