الفهرس 
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Abstract
Ulcerative colitis is a chronic inflammatory bowel disease which is characterized by chronic and relapsing inflammation of the colon (Sartor, 2006; Danese and Fiocchi, 2011). The etiology of UC is not clearly understood and it is strongly dependent on cellular immune reaction and exaggerated inflammatory response due to genetic, immune and environmental factors (Shih and Targan, 2009; Nagahori et al., 2010) . . The pathogenesis of UC is multifactorial, including disorders of immune responses, genetic susceptibility, epithelial barrier dysfunction and environmental factors (Ungaro et al., 2017). Inflammatory bowel disease is associated with increased intestinal permeability and decreased expression of tight junction proteins in the inflamed mucosa (Poritz et al., 2007). In UC, TNF-α exerts its actions on intestinal mucosa via activation of sphingosine kinase (Xia et al., 1998) which is highly conserved lipid kinase that phosphorylates sphingosine to form S1P (Maines et al., 2008) .SphK1 and SphK2 are the two main isoforms acting as major regulators of the “sphingolipid rheostat”(Hait et al., 2006) . SphK1 is highly expressed in many tissues especially in brain, heart and colon (Fukuda et al., 2003). Activation of SPHK1 by TNF-α in colonic mucosa increases the expression of adhesion molecules, activating NOS producing nitric oxide and increasing neutrophil and macrophage infiltration into the colonic mucosa leading to the production of superoxide and other free radicals that induced further inflammation and destruction to colonic mucosa (MacKinnon et al., 2002; Itagaki and Hauser, 2003) Sphingosine-1phosphate is considered a key regulator of important physiological functions, including cell growth, survival, angiogenesis, cell motility and migration (Strub et al., 2010). These effects are mediated by binding to a family of five S1PRs. S1P also serves as a major activator of the IL-6/STAT3 pathway involved in the pathogenesis of inflammatory bowel disease and colon cancer (Degagné and Saba, 2014). In addition, it has been shown to induce the expression of adhesion molecules as ICAM-1 or VCAM-1 in various cell types (Milara et al., 2009; Costello et al., 2011) and thus exaggerate the inflammatory responses. Intracellular cell adhesion molecule-1 plays an important role in the pathogenesis of IBD. It is proposed to be involved in adhesion of neutrophils to endothelial cells, directing the movement of captured neutrophils to intercellular gaps in the endothelium and macrophage , lymphocyte aggregation in the lamina propria and at epithelial lesions (Vainer, 2005). Prokes et al investigated that ICAM-1 deficiency protects mice against severe forms of experimentally induced colitis which clarified the critical role of ICAM-1 in pathogenesis of UC (Prokes et al., 2000). The importance of the SphK1/S1P/S1PR1 amplification loop was confirmed by the findings that FTY720, which inhibit SphK1 and subsequently prevents immune cells recruitment, pro inflammatory cytokine production (especially IL-6 and TNF-α), and persistent STAT3 activation leading to inhibition of colitis and CAC (Liang et al., 2013) . Consequently SphK1/S1P signaling pathway has strong association with inflammation process in ulcerative colitis. It was also proved that SphK1 deficient mice had significantly reduced systemic inflammation and less colon cancer compared to wild type animals (Snider et al., 2010; Liang et al., 2013). It was also reported that the UC pathogenesis also related to abnormal apoptosis. Several evidences suggested that the count of apoptotic epithelial cells increased with the development of UC which may cause the breakdown of the epithelial barrier function, with subsequent pathogenic bacteria infiltration (Yao et al., 2016) Interleukin-10 is a potent anti-inflammatory cytokine essential for protecting the host against excessive inflammatory and immune responses and its levels are strongly down regulated in UC (Iyer and Cheng, 2012). A close association between IL10 and intestinal mucosal homeostasis became clear with the discovery that mice deficient in IL10 and its receptor develop intestinal inflammation spontaneously. For these reasons the anti-inflammatory properties of IL-10 have been emerged as a very promising target for IBD therapy (Shouval et al., 2014). Understanding the pathogenesis and finding effective treatment for UC is a critical topic of current research, and it is one of the urgent medical problems needed to be resolved (Gao et al., 2019). Traditional treatments for UC include aminosalicylates, steroids, immuno suppressants and colectomy for patients with refractory disease or colonic tumors (Ungaro et al., 2017). Mesalazine is a synthetic formulation of 5-ASA which displayed significant efficacy for the treatment of mild to moderate UC (Sutherland and MacDonald, 2003; Sutherland and Macdonald, 2006). However, such traditional treatments for UC are still compromised by their adverse effects and insufficient potency to prevent remission for long-term periods. So, new treatment strategies that have the potential to provide more efficacy and safety for UC needed to be developed. Statins exert numerous pleiotropic effects including anti-inflammatory, antioxidant properties, endothelial function improvement and immunomodulation independent of their basic lipid-lowering properties (Kwak et al., 2000). Metformin is a biguanide has been widely used for the treatment of type 2 diabetes mellitus. It is shown that metformin exerts anti-inflammatory effects in vitro and in vivo (Hattori et al., 2006; Son et al., 2014) . Specifically, Koh et al and Lee et al reported that metformin can attenuate intestinal inflammation in experimental colitis (Koh et al., 2014; Lee et al., 2015). Accordingly, the major aim of this study is to explore the role of SPHK1/S1P signaling pathway on pathogenesis of UC and the possible modulatory effects of atorvastatin and metformin on SPHK1/S1P pathway. Also to examine the combination of mesalazine with atorvastatin or metformin for their potential effects on enhancing anti-inflammatory effects and ameliorate the progression of UC. Ulcerative colitis was induced in white albino rats by oxazolone as described by Zhang et al. (2009). In brief pre sensitization of the skin was initiated by topical application of oxazolone in a dose of 300μL of 5% (w/v) in absolute alcohol to induce an allergic reaction on a shaved area on the back of each animal followed by intra rectal administration of 450μL of 5% oxazolone in 50% ethanol solution on 5th and 7th days using fine rubber catheter inserted in the colon through the rectum to about 8cm proximal to the anal verge under light ether anesthesia .The animals were kept in a vertical position for 45 seconds after intra rectal administration to ensure even distribution of oxazolone solution through the colon . Rats were then randomly divided into eight equal groups (10 rats each) according to the treatment as follows: Control group which were normal untreated rats. Control saline group received vehicle of intra rectal 50% ethanol in the 5th & 7th days followed by administration of 0.9 % saline solution orally daily for 21 days. Oxazolone group; untreated oxazolone group received 0.9 % saline solution orally daily for 21 days. Mesalazine group; oxazolone group treated with mesalazine (100mg/kg; dissolved in 0.9 saline orally daily for 21 days (Li et al., 2014). Metformin group ; oxazolone group treated with metformin (100 mg/kg; dissolved in 0.9 saline administered orally daily for 21 days. Atorvastatin group ;oxazolone group treated with atorvastatin (20 mg/kg dissolved in 0.9 saline administered orally daily for 21 days (Zhang et al.,2009) . Combination groups; oxazolone group treated with a combination of mesalazine with atorvastatin or metformin with the same dosage regimen Treatments were applied 24-h after colitis induction (Gonzalez-Ramirez et al., 2016) .During the experiment animal body weights, occurrence of diarrhea, and rectal bleeding were recorded twice a week over the experiment. DAI was derived according to the method of (Cooper et al., 1993). Biochemical analysis Twenty four hours after the end of the experiment, rats were anaesthetized by diethyl ether then blood was collected via cardiac puncture into a heparinized syringe. Blood was centrifuged at 3000 rpm for 10 min. Plasma was carefully separated and kept at -20ºC until used for determination of plasma S1P. Then rats were euthanized by cervical dislocation under light ether anesthesia. Colons were excised, washed with ice cold phosphate buffered saline and dried between two filter papers to remove excess water and weighed to measure the colon wet mass .Index of the colon wet mass=colon wet mass (gm) / body weight (kg) (Zhang et al., 2009). Then colons were gently stretched and the distance from the colocecal junction to the end of the distal rectum was recorded (Nagib et al., 2013). Then distal 8 cm of the colons were divided; one part was used for histopathological and the other parts were kept frozen at −80 °C till biochemical analysis and quantitative real time PCR studies.