Inhibitor of apoptosis‐stimulating p53 protein protects against inflammatory bowel disease in mice models by inhibiting the nuclear factor kappa B signaling

Summary

Drugs and therapies available for the treatment of inflammatory bowel disease (IBD) are not satisfactory. Our previous study has established the inhibitor of apoptosis‐stimulating p53 protein (iASPP) as an oncogenic regulator in colorectal cancer by forming a regulatory axis or feedback loop with miR‐124, p53, or p63. As iASPP could target and inhibit nuclear factor kappa B (NF‐κB) activation, in this study the role and mechanism of iASPP in IBD was investigated. The aberrant up‐regulation of iASPP in IBD was subsequently confirmed, based on online data sets, clinical sample examinations and 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐ and dextran sulfate sodium (DSS)‐induced colitis mice models. TNBS or DSS stimulation successfully induced colon shortness, body weight loss, mice colon oxidative stress and inflammation. In both types of colitis mice models, iASPP over‐expression improved, whereas iASPP knockdown aggravated TNBS or DSS stimulation‐caused colon shortness, body weight loss and mice colon oxidative stress and inflammation. Meanwhile, in both types of colitis mice models, iASPP over‐expression inhibited p65 phosphorylation and decreased the levels of tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6, C‐X‐C motif chemokine ligand (CXCL)1 and CXCL2 in mice colons, whereas iASPP knockdown exerted opposite effects.

iASPP overexpression improved, whereas iASPP knockdown aggravated TNBS or DSS stimulation‐caused colon shortness, bodyweight loss, and mice colon oxidative stress and inflammation.

INTRODUCTION

One of the critical pathogeneses of inflammatory bowel disease (IBD), including Crohn’s disease (CD) (1) and ulcerative colitis (UC) (2), is chronic progressive inflammation. The initiation and progression of IBD could be attributed to genetic, environmental and microbial factors and immune response disorders. IBDs are characterized by intestinal damage (barrier destruction, the influx of microbiota) and massive inflammation (1, 2, 3). Combined, these factors can cause an increased risk of colitis‐related cancer and increase cancer incidence by up to 20% (4). Colitis‐related cancer could account for up to one‐sixth of all deaths in UC patients (5, 6, 7). However, present medications and treatment regimens are not deemed satisfactory. This has created an urgent need for the creation and devising of novel treatment regimens.

PPP1R13L gene‐encoded inhibitor of apoptosis‐stimulating p53 protein (iASPP) is an evolutionarily conserved inhibitor of p53 and p63 (8, 9). Our previous studies reported iASPP as an oncogenic regulator in colorectal cancer by forming a regulatory axis or feedback loop with microRNA (miR)‐124, p53 or p63 (10, 11, 12, 13). Interestingly, iASPP also participates in inflammatory pathways (14, 15). Initially, iASPP was a known binding partner for nuclear factor kappa B (NF‐κB), manifested as a yeast two‐hybrid assay (15). Hu et al. (16) also demonstrated that a caspase‐cleaved 80 kDa fragment of iASPP could translocate from the cytoplasm to the nucleus, binding and inhibiting RelA/p65 efficiently. Correspondingly, iASPP could also potentially regulate human immunodeficiency virus type 1 expression in a RelA/p65‐dependent manner (17), and iASPP‐deficiency in both patients and mice leads to up‐regulation of inflammation‐related genes, most of which are NF‐κB targets (14, 18). Given the central role of p65 in inflammation signaling, iASPP might also play a role in IBD pathogenesis.

Activation of RelA/P65 usually induces proliferative and anti‐apoptotic signal transduction. Constitutively activated RelA/P65 signaling is known to be linked to the development and progression of many cancers, as well as drug resistance [2]. Considering the central role of p65 in inflammation signaling, constitutive NF‐κB activation and deregulated cytokine production foreseeably occur in IBD patients (19, 20). Biopsy of inflamed intestine has shown increased NF‐κB activation in macrophages and epithelial cells (21). Correspondingly, several studies have reported the involvement of epithelial NF‐κB signaling in IBD mice models (19, 22, 23). Therefore, NF‐κB signaling has become an attractive target for IBD therapies, and many current drugs used to treat IBD directly or indirectly affect NF‐κB signaling.

Animal models are not remotely able to effectively and completely mimic human IBD. However, multiple models are able to replicate the mechanical process of the chronic inflammation process in IBD. For example, dextran sulfate sodium (DSS) oral administration could induce colitis in mice; a 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‑ethanol enema also evokes immune responses in mice and results in colitis (24). In this study, to investigate the specific role of iASPP in IBD and the underlying mechanism, TNBS‐ and DSS‐induced colitis, respectively, was established. The iASPP expression in these two models was monitored. After the achieved iASPP over‐expression or iASPP knockdown in vivo, the effects of iASPP over‐expression or iASPP knockdown on colitis and p65 signaling were examined.

MATERIALS AND METHODS

Clinical specimens

A total of 12 cases of ulcerative colitis tissues and 12 paired control tissues from the distal end of colon ulcers were harvested through surgical resection from patients diagnosed with IBD at the Second Xiangya Hospital. This study was approved by the Ethics Committee of Second Xiangya Hospital, and informed consent was obtained from each patient enrolled. The diagnosis of CD or UC meets the Copenhagen criteria (25). All harvested tissue samples were fixed or frozen at −80°C immediately after sampling pending laboratory analysis. The clinical characters are listed in Table 1.

TABLE 1.

Clinical characteristics of patients with Crohn’s disease and ulcerative colitis

Sample	Age (years)	Sex	Sampling site	Disease extent	Medication (active intervention)
UC1	30	M	Ileum	Pancolitis	5ASA.PSL
UC2	25	M	Colon	Pancolitis	5ASA.PSL
UC3	44	F	Colon	Pancolitis	5ASA.PSL
UC4	52	F	Colon	Pancolitis	5ASA
UC5	30	M	Colon	Pancolitis	5ASA.PSL
UC6	56	F	Colon	Pancolitis	5ASA
UC7	41	M	Colon	Pancolitis	5ASA.PSL.AZA
UC8	40	M	Colon	Pancolitis	5ASA.PSL
CD1	40	M	Colon	Ileocolon	5ASA.PSL
CD2	31	F	Colon	Ileocolon	5ASA.PSL.AZA
CD3	27	F	Ileum	Ileum	AZA
CD4	30	M	Ileum	Ileum	5ASA.MTX.IFX

CD = Crohn’s disease; UC = ulcerative colitis; 5ASA = 5 aminosalicylic acid; PSL = prednisolone; AZA = azathioprine; MTX = methotrexate; IFX = infliximab.

Experimental animals

C57BL/6 male mice used in this study were obtained from Hunan SJA Laboratory Animal Co., Ltd (Changsha, China). Mice aged 6~7 weeks were used for TNBS‐colitis model establishment, while mice aged 8~12 weeks were used for DSS‐colitis model establishment. The mice were housed in the SPF facility at the experimental animal center of the Second Xiangya Hospital for at least 1 week before inclusion in experiments. During this period, mice were given free access to food and drink water. All the studies were performed following the Second Xiangya Hospital Animal Care and Use Committee guidelines.

Colitis model induced by TNBS or DSS in mice

For TNBS induction of colitis, 5 μl/g body weight of TNBS (2.5%; Sigma‐Aldrich, St Louis, Missouri, USA) was dissolved in 50% ethanol (total volume, 100 μl) and administered to mice under mild anesthesia (methoxyflurane) through intrarectal injection. The mice in the control group received 50% ethanol without TNBS. On day 6 after TNBS administration, mice were anesthetized and euthanized for colon tissue harvesting. Colon length, body weight and the disease activity index (DAI) of mice were measured at indicated time‐points.

For DSS induction of acute colitis, mice received 3% (w/v) DSS (Sigma‐Aldrich) in drinking water for 7 days. On day 8, mice were anesthetized and euthanized for colon tissue harvesting. Colon length, body weight and the DAI of mice were measured at indicated time‐points.

Measurements of DAI

DAI score is calculated based on weight loss, stool consistency and bleeding, as described previously (26, 27, 28). DAI was scored daily throughout the entirety of the study for a period of 21 days. The standards are listed in Table 2.

TABLE 2.

Standard for DAI scoring

	0	1	2	3	4
Weight loss	No loss	5–10% weight loss	10‐15% weight loss	15–20% weight loss	20% weight loss
Stool	Normal	˗	Loose stool	˗	Diarrhea
Bleeding	No blood	˗	Presence	˗	Gross blood

In ‐vivo knockdown or over‐expressing of mouse iASPP

In‐vivo knockdown of iASPP in mice colon tissues (either TNBS‐induced or DSS‐induced) was achieved by intracolonic administration of 50 μl (50 × 10⁹ IU) adenovirus containing iASPP [negative control (NC)/iASPP] or short hairpin (sh) RNA for mouse iASPP (sh‐NC/sh‐iASPP) on days 1, 3 and 5 (GeneChem, Shanghai, China).

Quantitative reverse transcription–polymerase chain reaction (qRT‐PCR)

RNAiso Plus, the total RNA extraction reagent, was obtained from TaKaRa (Dalian, China) and used to extract total RNA from scraped colonic mucosa. Total RNA was reverse‐transcribed into cDNA, using the synthesized 2 mol/l cDNA. The expression levels were measured using a SYBR Green qPCR assay (TaKaRa) following the methods described previously (29). β‐actin was used as the housekeeping gene. The threshold cycle (Ct) value reflects the concentration of cDNA in each sample, and the threshold cycle is compared with the relative quantification method. The relative expression levels were calculated using the 2^–ΔΔCq method. Protein expressions were normalized to the control group (including normal tissue, TNBS/DSS + NC and TNBS/DSS + sh‐NC groups). The primers are listed in Supporting information, Table S1.

Immunoblotting

iASPP, tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6, C‐X‐C motif chemokine ligand (CXCL)1, CXCL2, p65, phospho (p)‐p65, IκB and p‐IκB protein levels were determined by immunoblotting as carried out previously (29), using primary antibodies against iASPP (1 : 500, CSB‐PA290837; Cusabio, Wuhan, China), TNF‐α (1 : 500, 17590‐1‐AP; Proteintech, Wuhan, China), IL‐1β (1 : 500, 26048‐1‐AP; Proteintech), IL‐6 (1 : 1000, 66146‐1‐immunoglobulin (Ig); Proteintech), CXCL1 (ab86436; Abcam, Cambridge, Massachusetts, USA), CXCL2 (ab9950; Abcam, Cambridge, Massachusetts, USA), p65 (1 : 1000, 10745‐1‐AP; Proteintech), p‐p65 (1 : 1000, ab194726; Abcam), IκB (1 : 1000, ab32518; Abcam) and p‐IκB (1 : 10000, ab133462; Abcam) and horseradish peroxidase (HRP)‐conjugated secondary antibody. Signal visualization was achieved using enhanced chemiluminescent (ECL) substrates (Millipore, Massachusetts, USA), taking β‐actin level as an internal reference. Protein expression was normalized to both the β‐actin and control groups (including normal tissue, TNBS/DSS + NC and TNBS/DSS + sh‐NC groups).

Histopathological evaluation

For histopathological evaluation, colonic tissues were fixed with 4% paraformaldehyde in phosphate‐buffered saline (PBS). Tissues were embedded in paraffin and subsequently cut into 5‐μm thick sections, and the sections were stained with hematoxylin and eosin (H&E).

For immunohistochemical (IHC) staining for iASPP, the sections were deparaffinized with xylene for 10 min and rehydrated with ethanol. For antigen retrieval, the sections were put into 0.01 M citrate puffer and heated in a microwave for 10 min. For blocking non‐specific bindings, the sections were incubated with 5% goat serum in TBS for 20 min. The sections were finally incubated with anti‐iASPP (1 : 50, CSB‐PA290837; Cusabio), p‐p65 (1 : 50, ab194726; Abcam) or rabbit IgG polyclonal‐isotype (negative control, 1 : 50, ab37415; Abcam) overnight at 4°C and subsequently incubated with poly‐IgG‐HRP antibody (SV0004; Boster, Wuhan, China) for 30 min at 37°C. The section was stained by a diaminobenzidine (DAB) kit (AR1022; Boster) for 10 min. Isotype antibodies were used as the negative control for IHC staining (Supporting information, Figure S1).

Myeloperoxidase activity

The myeloperoxidase (MPO) activity of colonic tissue samples representing colonic mucosa neutrophil infiltration was evaluated using an MPO colorimetric activity assay kit (MAK068‐1KT; Sigma‐Aldrich) as before (26).

Superoxide dismutase (SOD), glutathione peroxidase (GSH‐px), malondialdehyde (MDA), and catalase (CAT) assay

According to the methods of the total SOD activity detection kit, GSH peroxidase detection kit and lipid peroxidation MDA detection kit (all from Beyotime, Shanghai, China), protein samples from colon tissues were prepared for these determinations. The expression unit of SOD and GSH‐px is per mg protein. One unit is defined as the activity required to degrade 1 mmol hydrogen peroxide/mg of total protein/min at 25°C.

CAT activity was detected following Aebi’s method (30) by measuring the decrease of hydrogen peroxide as before (31). One unit was defined as the activity required to degrade 1 mmol of hydrogen peroxide/min/mg of total protein in the sample at 25°C.

Statistical analysis

Data from results of at least three independent experiments were processed using SPSS version 17.0 (IBM, Armonk, New York, USA) and presented as the mean ± standard deviation (SD). Student’s t‐test was applied for analysis between two groups. One‐way analysis of variance (anova) followed by Tukey’s post‐hoc test was applied for analysis among more than two groups; *p < 0.05; **p < 0.01.

RESULTS

iASPP expression is up‐regulated in IBD

To verify the expression levels of iASPP, GEO data sets were analyzed. According to GSE112366 and GSE96665, iASPP expression was significantly up‐regulated in IBD specimens compared with the normal control group (Figure 1a). In harvested IBD tissues, iASPP levels were also significantly higher than those in collected healthy controls (Figure 1a). The protein levels of iASPP were increased in IBD tissues compared to those in healthy controls (Figure 1c). The histopathological characteristics and the degree of intestinal mucosal inflammation were evaluated through H&E staining, as illustrated in Figure 1d; cellular infiltration and structural damage were observed in IBD tissues. Moreover, IHC staining also showed higher iASPP levels in IBD tissues (Figure 1d). These data confirm the aberrant up‐regulation of iASPP in IBD.

FIGURE 1.

Inhibitor of apoptosis‐stimulating p53 protein (iASPP) expression is up‐regulated in inflammatory bowel disease (IBD). (a) Expression of iASPP in healthy control and IBD tissues, according to GSE112366 and GSE96665. (b) Expression of iASPP in healthy control (n = 12) and IBD (n = 12) tissues was determined by quantitative reverse transcription–polymerase chain reaction (qRT–PCR). (c) Protein levels of iASPP in healthy controls and IBD tissues were determined by immunoblotting. Representative images were photographed. (d) Histopathological characteristics and content of iASPP in normal control and IBD tissues were examined through hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining, respectively; **p < 0.01 compared to the normal group

TNBS‐ and DSS‐induced colitis model in mice and iASPP expression in models

Prior to investigating the specific effects of iASPP on IBD, TNBS‐ or DSS‐induced colitis models in mice was first established. Modeling and assay time‐points are depicted in Figure 2a. As illustrated in Figure 2b,c, both TNBS and DSS significantly shortened the colon length of mice compared to control mice. Meanwhile, the body weight of mice in DSS and TNBS groups decreased (Figure 2d), whereas the DAI score increased (Figure 2e) compared to control mice. Regarding oxidative stress, the CAT, GSH‐px, SOD, MPO and MDA were examined; in TNBS‐ and DSS‐induced mice, CAT, GSH‐px and SOD significantly decreased, whereas MPO and MDA increased compared to control mice (Figure 2f). TNBS and DSS, respectively, induced a remarkable increase in the release of the proinflammatory cytokines, including TNF‐α, IL‐1β, IL‐6, CXCL1 and CXCL2 in colon tissues in mice compared with control mice (Figure 2g,h). Meanwhile, protein levels and mRNA expression of iASPP were significantly up‐regulated in TNBS‐ and DSS‐induced mice colon compared to control mice (Figure 2g,h). In both TNBS‐ and DSS‐induced mice colon, H&E staining showed cellular infiltration and structural damage, similar to that in IBD tissues (Figure 2i). Together, these indices indicate the successful establishment of the TNBS‐ and DSS‐induced colitis model in mice, and iASPP was up‐regulated in the colitis model.

FIGURE 2.

2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐ and dextran sodium sulfate (DSS)‐induced colitis model in mice and inhibitor of apoptosis‐stimulating p53 protein (iASPP) expression in models. (a) TNBS‐ and DSS‐induced colitis models were established in mice, respectively. Modeling and assay time‐points are shown. (b) Representative schematic of the colon samples in control, DSS and TNBS groups. (c) Colon length of mice in control, DSS and TNBS groups. (d) Body weight of mice in control, DSS and TNBS groups. (e) Disease activity index (DAI) of mice in controls, DSS and TNBS groups. (f) Catalase (CAT), glutathione peroxidase (GSH‐px), superoxide dismutase (SOD), myeloperoxidase (MPO) and malondialdehyde (MDA) of mice in the control, DSS and TNBS groups. (g) Protein levels of iASPP, tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6, C‐X‐C motif chemokine ligand (CXCL)1 and CXCL2 in colon tissues from mice in the control, DSS and TNBS groups were determined by immunoblotting. (h) RNA expression levels of iASPP, TNF‐α, IL‐1β, IL‐6, CXCL1 and CXCL2 in colon tissues from mice in controls, DSS and TNBS groups were determined through quantitative reverse transcription–polymerase chain reaction (qRT–PCR). (i) Histopathological characteristics of colon tissues from mice in the controls, DSS and TNBS groups were determined by hematoxylin and eosin (H&E) staining; **p < 0.01, compared between normal and DSS group; ##p < 0.01, compared between normal and TNBS groups

Determination of iASPP over‐expression and knockdown by intracolonic administration of specific adenovirus in mice

Since iASPP aberration was observed in IBD, in‐vivo over‐expression and knockdown of iASPP in mice were achieved, respectively (Supporting information, Figure S2A,B). Over‐expression or knockdown of iASPP did not affect the colon length in healthy mice (Supporting information, Figure S2B). Over‐expression and knockdown of iASPP were confirmed by examination of the protein level of iASPP using immunoblotting and IHC staining (Supporting information, Figure S2C,D). As shown in the results, intracolonic injection of iASPP or sh‐iASPP adenovirus successfully over‐expressed or knockdown iASPP in mice colon tissues.

Effects of iASPP over‐expression or knockdown on DSS‐induced colitis

In‐vivo knockdown of iASPP was subsequently achieved in the TNBS‐ and DSS‐induced colitis models in mice, respectively (Figure 3a), to investigate the specific role of iASPP in colitis pathogenesis. Over‐expression and knockdown of iASPP were confirmed in the DSS‐induced colitis model through examination of the protein level of iASPP using immunoblotting (Figure 3b). DSS‐induced colon shortening was improved by iASPP administration compared with NC administered mice, and even aggravated by sh‐iASPP administration compared with sh‐NC administrated mice (Figure 3c,d). DSS‐induced body weight loss and DAI increase were improved by iASPP administration, whereas it was aggravated by sh‐iASPP administration (Figure 3e,f). Consistent with these indices, DSS‐induced cellular infiltration and structural damage were improved by iASPP administration, whereas it was aggravated by sh‐iASPP administration (Figure 3g). Moreover, iASPP administration increased the levels of CAT, GSH‐px and SOD and decreased MPO and MDA; in contrast, sh‐iASPP administration decreased CAT, GSH‐px and SOD, while MPO and MDA increased (Figure 3h).

FIGURE 3.

Effects of inhibitor of apoptosis‐stimulating p53 protein (iASPP) over‐expression or knockdown on dextran sodium sulfate (DSS)‐induced colitis. (a) In‐vivo knockdown or over‐expression of iASPP was achieved in the DSS‐induced colitis mice model by intracolonic injection of 50 μl (50 × 10⁹ IU) adenovirus containing iASPP [negative control (NC)/iASPP] or short hairpin RNA for mouse iASPP (sh‐NC/sh‐iASPP) on days 1, 3 and 5. (b) Over‐expression and knockdown of iASPP were confirmed by examining the protein level of iASPP using immunoblotting. (c) Representative schematic of the colon samples in the NC, iASPP, sh‐NC and sh‐iASPP groups. (d) Colon length of mice in the NC, iASPP, sh‐NC and sh‐iASPP groups. (e) Body weight of mice in the NC, iASPP, sh‐NC and sh‐iASPP groups. (f) The disease activity index (DAI) of mice in the NC, iASPP, sh‐NC and sh‐iASPP groups. (g) Histopathological characteristics of colon tissues from mice in the NC, iASPP, sh‐NC and sh‐iASPP groups were determined by hematoxylin and eosin (H&E) staining. The content of iASPP in colon tissues from mice in the NC, iASPP, sh‐NC and sh‐iASPP groups was determined by immunohistochemical (IHC) staining. (h) Catalase (CAT), glutathione peroxidase (GSH‐px), superoxide dismutase (SOD), myeloperoxidase (MPO) and malondialdehyde (MDA) of mice in the NC, iASPP, sh‐NC and sh‐iASPP groups; **p < 0.01, compared to the NC group; ##p < 0.01, compared to the sh‐NC group

iASPP functions on the p65 signaling pathway in DSS‐induced colitis

Considering the close link of p65 signaling to inflammation and its critical role in IBD (32, 33, 34), changes in p65 phosphorylation in colon tissues from mice, the NC, iASPP, sh‐NC and sh‐iASPP groups were subsequently monitored. Both IHC staining and immunoblotting revealed that iASPP administration hindered the phosphorylation of p65, whereas sh‐iASPP administration had an opposite effect (Figure 4a,d). Meanwhile, IκB phosphorylation was suppressed by iASPP administration and promoted by sh‐iASPP administration (Figure 4a,d). Consistently, iASPP administration decreased, whereas sh‐iASPP administration increased the mRNA expression and protein levels of TNF‐α, IL‐1β, IL‐6, CXCL1 and CXCL2 in colon tissues from mice (Figure 4b,c). Therefore, iASPP administration improves inflammation in mice colon induced by DSS.

FIGURE 4.

Inhibitor of apoptosis‐stimulating p53 protein (iASPP) functions on the p65 signaling pathway in dextran sodium sulfate (DSS)‐induced colitis. (a) p65 phosphorylation in colon tissues from mice the negative control (NC), iASPP, short hairpin (sh)‐NC and sh‐iASPP groups were determined by immunohistochemical (IHC) staining. (b) mRNA expression levels of iASPP, tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6, C‐X‐C motif chemokine ligand (CXCL)1 and CXCL2 in colon tissues from mice in the NC, iASPP, sh‐NC and sh‐iASPP groups were determined by quantitative reverse transcription–polymerase chain reaction (qRT–PCR). (c) Protein levels of iASPP, TNF‐α, IL‐1β, IL‐6, CXCL1 and CXCL2 in colon tissues from mice in the NC, iASPP, sh‐NC and sh‐iASPP groups were determined by immunoblotting. (d) Protein levels of p‐p65, p65, p‐IκB and IκB in colon tissues from mice in the NC, iASPP, sh‐NC and sh‐iASPP groups were determined by immunoblotting; **p < 0.01, compared to the NC group; ##p < 0.01, compared to the sh‐NC group

Effects of iASPP over‐expression or knockdown on TNBS‐induced colitis

Similarly, the above‐mentioned indices were also monitored in TNBS‐induced colitis in mice. In‐vivo over‐expression or knockdown of iASPP was achieved and confirmed in the TNBS‐induced colitis mice model (Supporting information, Figure S3A,B). In TNBS‐induced colitis, iASPP over‐expression improved, whereas iASPP knockdown aggravated TNBS‐induced colon shortening (Supporting information, Figure S3C,D), body weight loss (Supporting information, Figure S3E) and DAI increase (Supporting information, Figure S3F). In colon tissues, TNBS‐induced cellular infiltration and structural damage were improved by iASPP over‐expression, while it was aggravated by iASPP knockdown (Supporting information, Figure S3G). Regarding oxidative stress, iASPP over‐expression increased CAT, GSH‐px and SOD, and decreased MPO and MDA, while iASPP knockdown exerted opposite effects (Supporting information, Figure S3H).

Furthermore, p65 phosphorylation in colon tissues from the TNBS‐induced colitis mice model was monitored. Both IHC staining and Immunoblotting revealed that iASPP administration hindered the phosphorylation of p65 in TNBS‐induced colitis, whereas sh‐iASPP administration had an opposite effect (Supporting information, Figure S4A,D). Meanwhile, IκB phosphorylation was suppressed by iASPP administration and promoted by sh‐iASPP administration (Supporting information, Figure S4A,D). Consistently, iASPP administration decreased, whereas sh‐iASPP administration increased the mRNA expression and protein levels of TNF‐α, IL‐1β, IL‐6, CXCL1 and CXCL2 in colon tissues from TNBS‐induced mice (Supporting information, Figure S4B,C). Therefore, iASPP administration also improves the inflammation in mice colon induced by TNBS.

DISCUSSION

Aberrant up‐regulation of iASPP in IBD was confirmed by this study, based on online data sets and clinical sample examination and TNBS‐ and DSS‐induced colitis mice models. TNBS or DSS stimulation successfully induced colon shortness, body weight loss and mice colon oxidative stress and inflammation. In both types of colitis mice models iASPP over‐expression improved, whereas iASPP knockdown aggravated TNBS or DSS stimulation caused colon shortness, body weight loss and mice colon oxidative stress and inflammation. Meanwhile, iASPP over‐expression inhibited p65 phosphorylation and decreased levels of TNF‐α, IL‐1β, IL‐6, CXCL1 and CXCL2 in mice colons, whereas iASPP knockdown exerted opposite effects in both types of colitis mice models.

DSS‐ and TNBS‐induced colitis models are widely used for selecting therapeutic compounds for colitis, as both models mimic immunological and histological parameters in human IBD (35, 36, 37). The TNBS‐induced colitis model is widely used to study various aspects potentially related to CD (38), while oral administration of DSS is widely used to study various aspects potentially related to the characteristics of human UC flare (39). To completely delineate the role of iASPP in IBD, both types of colitis models were established in mice. Consistent with previous studies, colon shortness, body weight loss, DAI increase and colon oxidative stress and inflammation were observed in mice challenged by both stimuli. Moreover, aberrant up‐regulation of iASPP was first confirmed in colon tissues harvested from both types of mice models, suggesting the potential role of iASPP in UC and CD.

iASPP has been previously reported as an oncogene in colorectal cancer. By inhibiting p53, iASPP protects colorectal cancer against p53‐dependent cell apoptosis, contributing to colorectal cancer cell survival and resistance to PDT treatment (10, 11, 12, 13). Interestingly, iASPP is associated with inflammation signaling through inhibiting p65 activity via binding (14, 16, 18). However, the previously reported association between iASPP and inflammation signaling was based on other disorders than colitis. In the present study, for the first time we examined the specific functions of iASPP on TNBS‐ and DSS‐induced colitis mice models. As we have mentioned, in both types of colitis model mice, colon shortness, body weight loss and DAI increase were observed; after over‐expressing iASPP in model mice, these symptoms were significantly improved.

Meanwhile, iASPP over‐expression also increased the levels of SOD, CAT and GSH‐px and decreased MPO and MDA, suggesting that iASPP over‐expression protects against oxidative stress injury. Moreover, TNBS‐ and DSS‐increased inflammatory factors, including TNF‐α, IL‐1β, IL‐6, CXCL1 and CXCL2, were reduced by iASPP over‐expression. Conversely, iASPP knockdown even aggravated TNBS‐ and DSS‐induced symptoms in both colitis mice models. CD is known as an inappropriate immune response to the T helper type 1 (Th1) pathway and the involvement of various mechanisms at different stages of disease development (40). UC is related to impaired intestinal barrier integrity (41), mucosal inflammatory and immune responses and increased oxidative stress (42, 43). Macrophage activation and increased production of proinflammatory cytokines such as TNF‐α, IFN‐γ, IL‐1β, IL‐6 and IL‐12 are major characteristics of UC (44, 45). Therefore, the in‐vivo findings in both mice models point towards the protective effects of iASPP over‐expression against TNBS‐ and DSS‐induced colitis through attenuation of oxidative stress and colon inflammation.

NF‐κB signaling is a central player in inflammation and, thus, has a critical role in IBD (32, 33, 34). Many tumor‐promoting cytokines activate NF‐κB signaling, or these cytokines are activated via NF‐κB (46). According to a previous study, iASPP could inhibit NF‐κB activation; herein, the alteration in the NF‐κB signaling in response to iASPP over‐expression or knockdown in both types of colitis models was also monitored. Consistent with the previous study, iASPP over‐expression inhibited, whereas iASPP knockdown enhanced, the phosphorylation of p65 in colon tissues in both types of model mice. In conclusion, in TNBS‐ and DSS‐induced colitis mice models, iASPP over‐expression improves the symptoms, colon oxidative stress and colon inflammation. Meanwhile, NF‐κB signaling in mice colons could also be inhibited by iASPP over‐expression. iASPP administration could potentially be a novel strategy in the treatment of IBD. However, further clinical investigations need to be applied prior to market implementation.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

Investigation and writing: Ke Qian and Yong Kuang; clinical samples collection and analysis: Lianwen Yuan and Shalong Wang; investigation and data validation: Qianqian Jin, Dongju Long, Yuhong Jiang and Hua Zhao. Supervision, designation of the study and editing the manuscript: Kuijie Liu and Hongliang Yao.

Supporting information

Supplementary Material

DATA AVAILABILITY STATEMENT

All available data are presented in the manuscript and supplementary files.