UC is a chronic inflammatory condition of the intestinum crassum whose etiology remains unknown. Cytokines are crucial components of the inflammatory pathways that take place during the active and chronic phases of UC.
NF-
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of transcription factors. The antidiabetic drugs thiazolidinediones (TZDs) are ligands for the
We conducted rats model of UC induced by the mixture solution of 2,4,6-trinitrobenzenesulfonic acid solution (TNBS) and ethanol through enema, and after these rats were treated with rosiglitazone, a kind of TZDs, by gastric lavage, the colonic mucosal expression of PPAR
Healthy male SD rats weighing 180–220 g and aged 4–8 weeks were supplied by the SPF laboratory animal center of Dalian Medical University. TNBS was purchased from Sigma. Rosiglitazone maleate tablets were supplied by GlaxoSmithKline. PPAR
A total of 72 SD rats were randomly divided into three groups: a control group, a rosiglitazone treatment group, and a UC model group, each group had 24 rats. The SD rat model of UC was established by administering the mixture solution of TNBS (100 mg/kg) and 50% ethanol (0.25 mL) by enema. The control group was subject to enema and gastric lavage with normal saline. For the rosiglitazone treatment group, TNBS was administered by enema and rosiglitazone was administered by gastric lavage (8 mg/kg once a day). The UC model group was subjected to TNBS enema and the gastric lavage was saline control. Rats from all groups were sacrificed on days 7, 14, 21, and 35, 6 rats per group were sacrificed each day after the enema and colonic tissue, 2.0–10.0 cm from the anus, was collected for HE staining, RT-PCR and immunohistochemistry analysis. Rats were kept in a normally controlled breeding room with standard laboratory food and water for one week before the experiments. The rats were maintained in accordance with internationally accepted principles for laboratory animal use.
The rats were weighed and checked for behavior, stool consistency, and the presence of gross blood in the stool every day. The scores were assigned as follows: percentage of body weight reduction (0, no change; 1, 1–5%; 2, 6–10%; 3, 11–15%; 4, >15%); stool consistency (0, normal; 2, loose; 4, diarrhea); and the presence of fecal blood (0, normal; 2, positive occult blood test; 4, visible bleeding) [
Rats were sacrificed at the timepoints indicated and the entire colon was excised from the cecum to the anus and opened longitudinally. Macroscopic damage was evaluated using a validated (CMDI) scoring system with slight modifications [
Paraffin-embedded and formalin-fixed colon samples were cut into 4-
mRNA was extracted from colonic tissue samples using Trizol according to the manufacturer’s protocols (Invitrogen) and reverse transcription polymerase chain reaction (RT-PCR) was done according to the instructions of the Takara RNA PCR kit3.0 (AMV). An equal amount of cDNA from each sample was amplified using primers specific to each gene (Table
Oligonucleotide of primers of target genes.
mRNA species | mRNA | PCR product | |
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PPAR- |
sense | 5′-CTT CGG AAT CAG CTC TGT GGA C-3′ | 352 bp |
antisense | 5′-GCA TCC TTC ACA AGC ATG GAC TC-3′ | ||
NF- |
sense | 5′-ATG GAC GAT CTGTTT CCC CTC ATC-3′ | 254 bp |
antisense | 5′-ATT GGG TGC GTC TTA GTG GTA TCT-3′ | ||
TNF- |
sense | 5′-CTC CAG CTG GAA GAC TCC TCC CAG-3′ | 171 bp |
antisense | 5′-CCC GAC TAC GTG CTC CTC ACC-3′ | ||
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sense | 5′-AAG CCT AAG GCC AAC CGT GAA AAG-3′ | 241 bp |
antisense | 5′-TCA ATG AGG TAG TCT GTC AGG T-3′ |
Immunohistochemistry was done according to the Max Vision kit protocol. Image analysis software (Image-pro plus 6.0) was used to measure the light density of positive control cells in which the cytoplasm was tan-yellow or brown after 3,3’-diaminobenzidine (DAB) staining. For each section, the positive integrated optical density (IOD) and total area of five representative visual fields without overlap were observed under high-power microscope (×400). The ratio of IOD and total area represents the mean value of optical density, with a higher ratio indicating a higher level of protein expression.
Data showed a normal distribution and are expressed as means
The DAI and CMDI of the UC model group were significantly higher than that of the control group (
DAI and CMDI of three groups on different time (mean
Time | Control group | Model group | Rosiglitazone-treatedgroup | |||
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DAI | CMDI | DAI | CMDI | DAI | CMDI | |
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35th day |
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Note: *
As shown in Figure
Pathological change of control group, UC model group, and rosiglitazone treatment group on day 14 under light microscope (HE×200). The colonic mucosa structure was intact in the control group. Mucosa and submucosa defects could be seen with infiltrations of inflammatory neutrophils and lymphocytes in the lamina propria of the model group on day 14. The degree of mucosal inflammation relieved in the rosiglitazone treatment group on day 14. Regenerated epithelia covered the surface of ulcer and the mucosa tended to be complete. The base of ulcer was filled with tissues and scars.
In the control group, expression of PPAR
Colonic mucosal mRNA expression of PPAR
Time | Control group | Model group | Rosiglitazone-treated group | ||||||
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PPAR |
NF- |
TNF- |
PPAR |
NF- |
TNF- |
PPAR |
NF- |
TNF- |
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In the control group, expression of PPAR
Colonic mucosal protein expression of PPAR
Time | Control group | Model group | Rosiglitazone-treated group | ||||||
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PPAR |
NF- |
TNF- |
PPAR |
NF- |
TNF- |
PPAR |
NF- |
TNF- |
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The colonic mucosal PPAR
Colonic mucosal NF-
Colonic mucosal TNF-
Correlation studies showed that the expression of PPAR
The NF-
UC is a chronic inflammatory condition of the intestinum crassum that etiology remains unknown. Cytokines are crucial components of the inflammatory pathways that take place during the active and chronic phases of UC. UC likely results from deregulation of colonic mucosal immunity and breakdown of delicate balance of proinflammatory and anti-inflammatory cytokines.
NF-
PPAR
Our results demonstrated that the colonic mucosal expression of PPAR
The antidiabetic drugs thiazolidinedione (TZDs) are ligands for the
Furthermore, we used Spearman correlation analysis to study the relationship between PPAR
In conclusion, rosiglitazone can alleviate colonic mucosal inflammatory response and has the protective role on UC by upregulating PPAR
J. Mao and H. Tang contributed equally to this work.
The authors thank Wan-Jing Zou and Gong-Jun Wang for their excellent technical assistance for help with immunohistochemistry. They also thank the SPF laboratory animal center of Dalian Medical University. The work was supported by Grants from the First Affiliated Hospital of Dalian Medical University.