الفهرس 
Ulcerative colitisOnly 14 pages are availabe for public view
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Abstract
Ulcerative colitis (UC) is an idiopathic inflammatory bowel disease (IBD) with diffuse, recurrent inflammation of the colon and rectum, which is predominantly characterized by cycles of acute inflammation, ulceration and bleeding of the colonic mucosa. The etiopathogenesis of UC remains uncertain, but the pathogenesis is likely dependent on the interaction between local immune reactions and environmental factors in genetically susceptible individuals.
Many reports using experimental animal models of acute colitis demonstrated that oxidative stress and oxidative cellular damage are hallmarks of UC, and probably play a key role in the pathogenesis of this disease. Also, the proinflammatory mediator plays an integral role in the pathogenesis of IBD.
Induction of colitis in rats using AA is a classical method used to produce an experimental model of UC. The intrarectal instillation of AA in rats caused bloody diarrhoea, shortening of the colon, mucosal ulcers and inflammatory cell infiltration that resembles and mimic the human UC.
Much research efforts have focused on the identification of flavonoids in fruits and vegetables which exerts beneficial biological effects. Among flavonoids, NG is one of the major flavonoids in citrus fruits.
MSCs are multipotent non-haematopoietic progenitor cells from adult bone marrow which can differentiate into various cells. Therefore, MSCs may be involved in tissue regeneration. Also, MSCs regulate the immune function in inflamed tissues by affecting pro-inflammatory cytokines and chemokine secretion.
The present study aimed to compare between the efficacy of naringenin (NG) and mesenchymal stem cells (MSCs), morphologically, histopathologically, histochemically, immunohistochemically and ultrastructurally, on experimentally-induced colitis model in rats by acetic acid (AA).
In this experiment, fifty adult male Wister albino rats weighing 220±10 g were used, and divided into five groups each containing 10 rats as follows:
group I (control): Rats received physiological saline once intrarectally (0.9% NaCl), parallel to the AA-treated group.
group II (NG group): Rats received NG only (50 mg/kg b.w., per os) dissolved in CMC (0.5%) daily for ten days.
group III (AA group): Rats received AA once intrarectally (1ml of 4%) and were left for ten days.
group IV (AA+NG group): Rats received AA once intrarectally (1ml of 4%), and one day after induction of UC, rats received NG (50 mg/kg b.w., p.o.) dissolved in CMC daily for ten days.
group V (AA+MSCs group): Rats received AA once intrarectally (1ml of 4%), one day after induction of UC, rats injected with MSCs via the tail vein with a single dose (1x106 cells/ml PBS/animal) and left for ten days.
The animals were sacrificed at the end of the experiment. The morphological investigation included detecting of MSCs, clinical symptoms, mean final body weight and colon length.
The morphological, histological, histochemical, immunohistochemical and ultrastructural alterations in colon tissue were examined.
The results obtained from the present investigation can be summarized as follows:
Morphological studies:
1- Detecting of the mesenchymal stem cells:
In vitro, the cell culture showed MSCs as spherical at the first two days, and fibroblastic in morphology at the 5th day.
By using flow cytometry, MSCs showed negative immune reaction to CD34 and CD45, but showed positive to CD271 and CD105.
MSCs group showed spindle-shaped, branched, and globular-shaped Prussian blue positive-stained cells in colonic tissue (in mucosa and submucosa layers).
2- Clinical symptoms:
During the period of the study, all the rats treated with AA experienced decreased food consumption, diarrhoea, rectal bleeding, weakness, unsteadiness and reduced activity. In contrast, rats received both AA+NG exhibited less signs of clinical toxicity than did the AA-treated rats. On the other hand, in rats received both AA+MSC none of these clinical symptoms were seen during the whole experimental period. Nevertheless, food intake was somewhat lower than normal.
3- Mean final body weight:
The present study showed a very highly-significant decrease in body weight in AA-treated rats compared to the control group; whereas the rats treated with AA in combination with NG or MSCs showed a highly significant and very highly significant increase, respectively , in mean body weight in comparison with the AA-treated group. On the other hand, there was a statistically-significant increase in the mean body weight in the AA+MSCs-treated group when compared to the AA+NG-treated group.
4- Colon length:
The current work showed a very highly-significant decrease in colon length in AA-treated rats compared to the control group; whereas the rats treated with AA in combination with NG or MSCs showed a significant and a highly-significant increase, respectively, in colon length in comparison with the AA-treated group. On the other hand, there was a significant increase in the colon length in the AA+MSCs-treated groups when compared to the AA+NG groups.
Microscopic studies:
1- Light microscopic study:
A- Histological study:
a- Haematoxlylin and eosin (H&E) stained sections:
Histopathological examination of distal colon sections from the rats administered AA revealed severe damage of mucosa and submucosa with distortion of crypts and abscess formation, complete necrosis as well as loss of epithelium integrity with ulceration. Besides, severe diffuse inflammatory cell infiltration of mucosa was shown in the mucosal layer.
The histopathological examination of distal colon sections from the rats administered AA+NG revealed moderate healing of colonic wall with some areas of the mucosa with lost crypts, mild inflammatory cell infiltration and mucosal oedema. While, distal colon sections from the rats administered AA+MSCs exhibited almost completely restored surface columnar epithelium with intact regularly arranged closely related crypts that exhibit numerous goblet cells with minimal inflammatory cell infiltration and mild congestion of blood vessels.
b- Masson’s trichrome stained sections:
By Masson’s trichrome stain, the colonic tissue of AA-treated rat showed obvious damage of the colon tissue with marked increase in collagen fibres, as compared to the control group, throughout the colon wall, mainly in between the affected mucosal crypts and in the submucosa. Colon sections obtained from the therapeutic group that was treated with AA then followed by NG revealed moderate decrease in collagen fibres in the lamina propria and the submucosa. While, the AA+MSCs group showed nearly normal few fine collagen fibres in the lamina propria between the crypts and in the submucosa.
B- Histochemical study:
Alcian blue stained sections:
By Alcian blue stain, the colonic tissue of AA-treated rat revealed severe depletion of many mucin secreting goblet cells with massive reduction in the intensity of Alcian blue-positive reaction for acidic mucins.
Colon sections of the distal colon of rats treated with AA then NG showed moderate increase in the number of acidic mucin secreting goblet cells as well as moderate increase in the intensity of Alcian blue-positive mucins. While, rats treated with AA then MSCs demonstrated a preserved colonic structure with a staining affinity for acidic mucins of secreting cells that showed marked increase in goblet cells that appeared more distended with mucins.
C- Immunohistochemical study
TNF-α is produced by various immune and non-immune cells in the inflamed intestines, including macrophages and epithelial cells. In the control group, healthy colon revealed slight production of TNF-α in mucosal epithelia and infiltrated inflammatory cells. But, in rats treated with AA, the colonic tissue showed more intense TNF-α-positive immunostaining in the form of scattered fine brown granules mainly in the cytoplasm of mucosal epithelia and infiltrated inflammatory cells. Colon sections obtained from the therapeutic group that were treated with AA, then followed by NG showed a moderate reduction of TNF-α immunoreactivity and infiltrated inflammatory cells in colonic mucosa and submucosa. While, colon sections obtained from the therapeutic group that was treated with AA, then followed by MSCs showed weak immunostaining in colonic mucosa and submucosa.
2- Electron microscopic study:
Electron micrographs obtained from colonic tissue of AA-treated rats revealed ultrastructural impairments including, distorted crypt with deformed nuclei of epithelial and goblet cells, dilated, fragmented rough endoplasmic reticulum, swollen mitochondria with disrupted cristae and cytoplasmic vacuoles. In addition, the connective tissue stroma of the mucosa showed increased number of active myofibroblasts, increase collagen fibres, numerous fat droplets, extravasation of erythrocytes and severe inflammatory cell infiltration.
Ultrastructural observations of the colon of rats treated with AA+NG showed moderate degree of restoration in the structure of the cells of the mucosal layer. There were almost normal nuclei, relatively intact desmosomes and partially ordered microvilli. However, some vacuolated mitochondria and fragmented rER were still seen. Also, the connective tissue stroma showed relatively normal inflammatory cells.
Ultrastructural observations of the colon of rats treated with AA+MSCs showed a considerable degree of restoration in the structure of the mucosal layer. Most of the crypt epithelium preserved the normal ultrastructure of cellular components and intact intercellular spaces. However, only slight mitochondrial swelling, some dilated rough endoplasmic reticulum were still seen. The connective tissue stroma appeared normal and had predominantly lymphocytes and plasma cells.