Shikonin inhibits colitis-associated colorectal dysplasias in a mouse model of azoxymethane/dextran sulfate sodium colitis

Shikonin (Shk) inhibits the development of colorectal dysplasia and the colitis induced in a mouse model of azoxymethane (AOM)/dextran sulfate sodium (DSS), as demonstrated both by macroscopic and biochemical determinations. The oral administration of shikonin prevents weight loss and colorectum shortening. Histological analysis revealed a decrease both in the severity and extent of inflammation, together with an amelioration of the colonic architecture and the protection from the appearance of dysplasia. This was associated reduction in MPO activity together with the inhibition of COX-2 and iNOS expression. A possible mechanism of action which would be responsible for this protection is the inhibition of NF-κB activation, since we demonstrated the inhibition of the translocation of NF-κB-p65 subunit to the nucleus in the colon homogenate. As a positive control we examined the effect of oral administration of sulfasalazine (Ssz).

Group	Blank	Controls		AOM-groups				DSS
		AOM	DSS	DSS	DSS-Ssz	DSS-Shk 3.5	DSS-Shk 7	Shk 7
AOM (7.5mg/kg i.p.)	-	✔✓	-	✓✔	✓✔	✓✔	✔✓	-
1.5% DSS	-	-	✓	✓	✓	✓	✓	✓✔
Treatment	-	-	-	-	100mg/kg Ssz	3.5mg/kg Shk	7mg/kg Shk

Introduction:

The biomedical development and the general improvement of the living conditions in Western societies has led over the last century to a change in epidemiological patterns of the most relevant diseases. Thus, allergies, autoimmune disorders, chronic inflammatory diseases, such as inflammatory bowel disease (IBD) and cancerous processes, have replaced infectious diseases as the main cause of mortality. [1]

Patients with a long-term established IBD have a higher risk of developing colorectal cancer (CRC), which depends on the preexisting colitis, its duration, anatomic extent and degree of inflammation. Thus, the risk of colon cancer is between 2 and 3 times higher than that of the general population, showing an increased incidence of 2, 8 and 18% after 10, 20 and 30 years of suffering from IBD, respectively. [2]

Experimental approach: The Azoxymethane (AOM)/dextran sodium sulfate (DSS) model of colitis-associated CRC employed in this study was based on that of Tanaka et al. (2003). Female Balb/C mice were distributed in 8 groups (6–10 animals/group) as following:

Group	Blank	AOM	DSS	AOM DSS	AOM DSS SSZ	AOM DSS Sh 3,5	AOM DSS Sh 7	DSS Sh 7
AOM (7.5mg/kg i.p.)		✔		✔	✔	✔	✔
1,5% DSS			✔	✔	✔	✔	✔	✔
Treatment					100mg/kg sulfasalazine	3,5mg/kg shikonin	7mg/kg shikonin

The experimental protocol is outlined in the following table:

DSS, sulfasalazine and shikonin were administered in the drinking water of the corresponding groups.

Body weights were recorded at the time of AOM injection and every day until the end of the experiment. Animals were sacrificed at the end of the experiment. At that time the entire colon and rectum were measured, excised, cut longitudinally, and rinsed in PBS. Following groß examination, some colons were frozen in liquid nitrogen and processed in their entirety for histopathologic evaluation. Others were processed for myeloperoxidase activity analysis and for COX-2, iNOS and p65 determination by Western blot.

RESULTS:

[1] Danese S, Fiocchi C. Etiopathogenesis of inflammatory bowel diseases. World J Gastroenterol. 2006;12:4807–12.