¹ Department of Physiology, Faculty of Basic Medical Sciences, College of Health Sciences, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria;

² Department of Physiology, Faculty of Basic Medical Sciences, College of Medical Sciences, Edo State University Uzuairue, Nigeria

Corresponding Author: Serah Funke Ige, PhD, Senior Lecturer and the Former Head, Department of Physiology, Faculty of Basic Medical Sciences, College of Health Sciences, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria. E-mail: sfige@lautech.edu.ng.

Running title: Hematological and Immunological Characterizations of Alcoholic Colitis

	ABSTRACT
	INTRODUCTION
	MATERIALS AND METHODS
	RESULTS
	DISCUSSION
	CONCLUSION
	ACKNOWLEDGEMENT
	REFERENCES

	ABSTRACT

BACKGROUND: Studies have documented the contributions of alcohol to ulcerative colitis (UC) development. The present study sought to determine the specific pattern of hematological and immunological indices in experimental ulcerative colitis rats exposed to different alcohol intervention schedules. METHODS: 28 female Wistar rats weighing between 150-200 g were randomly divided into control, non-alcohol colitis (NAC), low-frequency alcohol colitis (LAC) and high-frequency alcohol colitis (HAC) groups. Experimental UC was induced through a single intra-colic instillation of 1 ml/100g of 7% acetic acid and rats were administered 20% v/v of ethanol ad libitum. RESULTS: When compared with NAC group, LAC elicited a significant increase (P<0.05) in white blood cells (WBC), neutrophils, eosinophils, basophils, and myeloperoxidase while monocytes, lymphocytes, platelet, superoxide dismutase (SOD), catalase and glutathione decreased. Also, HAC caused an increase in myeloperoxidase and a decrease in Mean Corpuscular Volume (MCV), neutrophils, platelet, SOD, catalase, glutathione and tumor necrosis factor (α-TNF) when compared with NAC group. On the other hand, LAC raised WBC, neutrophils, eosinophils, basophils, platelet, MCV and α-TNF but decreased lymphocyte and monocyte when compared with HAC group. Histological analyses also revealed disruption of normal colonic integrity in NAC, LAC, and HAC. CONCLUSION: Low-frequency alcohol intake was relatively more granulocytic, inflammatory, thrombocytic, macrocytic and agranulopenic in colitis female rats than high-frequency alcohol intake.

	INTRODUCTION

   Alcohol, an organic compound and a nutritional agent, is an active integral component of beverages, most especially bear, wine and liquor (1). It is produced through fermentation of foodstuffs. In scientific consensus, alcohol is known as a psychoactive substance and one of the commonest recreational agents in the world (1). In 2010, the total alcohol consumption per capital for male and female stood at 19.4 liters and 7.0 liters worldwide (2). In 2016, the average alcohol global consumption was 6.4 liters per person older than 15 years (2). In Nigeria, alcohol consumption per capita in 2010 was estimated at 9.1%, the highest in West Africa (2).

   Alcohol consumption has been widely documented as an important risk factor for mortality and morbidity (1). Excess alcohol consumption has been reported to result in euphoria, decreased social inhibition, tachycardia, high blood pressure, vomiting, nausea, decreased depth of perception, anterograde amnesia, muscle incoordination (3, 4), hypoventilation, transient erectile dysfunction and hyporeflexia, culminating in pulmonary aspiration (5). These effects were reported to be mediated courtesy of the allosteric modulatory effect of alcohol on Gamma Amino Butyric Acid (GABA) receptors, inhibition of N-Methyl D Aspartate receptor and induction of serotonin and dopamine secretions (1, 6). Furthermore, other most extensively documented effects of excess alcohol consumption include decreased attentional control, slurred speed, impaired motor planning, urinary incontinence, increased risk of atrial fibrillation (7), hypertriglyceridemia, hypertension, alcoholic liver diseases, cancer, nervous dysfunction (5, 6, 8), stroke, heart failure (9, 10) and inflammatory bowel diseases, usually in combination with other factors.

   Ulcerative colitis, an inflammatory bowel disease, is a chronic disorder that affects the lining of the large intestine (11-13). It affects females and males in the same proportions at the rate of 5 to 500 per 100,000 people (14, 15). Although, there is no actual figure of ulcerative colitis prevalence in Nigeria and West Africa at large, the disease remains a relatively uncommon diagnosis (16). In addition to inflammation and ulceration, disruption of oxidant/antioxidant ratio, modulation of normal leucocyte, erythrocyte and thrombocyte counts, anemia, pain and weight loss have been reported to characterize the chronic inflammatory condition (17).

   As far as ulcerative colitis is concerned, heavy alcohol consumption has been reported to exaggerate symptoms (18-20). Swanson et al., (21) showed that Inflammatory Bowel Disease (IBD) patients who consumed red wine demonstrated increased long-term risk of a relapse or inflammation. Mechanisms underlying alcohol-induced ulcerative colitis might include release of pro-inflammatory cytokines such as alpha tumor necrosis factor and interleukin-I, alteration of colonic microbial composition, reduction in the number of beneficial microorganisms, increase in the population of latently harmful microorganisms, modulation of immune strength and disruption of mucosal barrier (22, 23).

   Conversely, studies have also indicated the beneficial effect of alcohol consumption in ulcerative colitis patients. According to the report of Boyko et al, increase in alcohol consumption was found to be associated with in reduction in risk of ulcerative colitis. Frequent consumption of alcohol was also shown to reduce ulcerative colitis risk when compared with less frequent consumption (24) but empirical data are insufficient. Also, light alcohol consumption has been speculated to exert a protective effect against the development of ulcerative colitis (25). Despite the plethora information on the implications of alcohol in ulcerative colitis development, the specific pattern of hematological and immunological indices associated with ulcerative colitis patients who are on different alcohol consumption frequencies was not well understood. Therefore, the present study aimed at determining the specific pattern of hematological and immunological indices in experimental ulcerative colitis rats exposed to different alcohol intervention schedules.

	MATERIALS AND METHODS

   Animal Care and Management

   28 female Wistar rats weighing between 150-200 g were used for this research work. They were obtained from the Animal House, Department of Physiology, Ladoke Akintola University of Technology, Ogbomoso, Oyo State. They were kept in a plastic cage (37 cm × 27 cm) (26-28) and lined with wood shavings. The animals were maintained in 12-hour light/ 12-hour dark cycle at room temperature. All rats were provided with standard rats’ chow through feeding troughs and water adlibitum.

   Ethical Certification

   The study was conducted in line with the guidelines of National Institute of Health (NIH) for the use of laboratory rats and LAUTECH ethical codes for animal research.

   Experimental Design

   The animals were randomly grouped into five groups containing seven animals each;

   Group 1: received 1ml/100g body weight of distilled water and was designated as control group (CTRL).

   Group II: received a single instillation of 1 ml/100g of 7% acetic acid (pr) to induce colitis and 0ml of alcohol for 21 days and was designated as non-alcohol colitis group (NAC).

   Group III: received a single instillation of 1 ml/100g of 7% acetic acid (pr) to induce colitis. 20% v/v ethanol was administered for seven days and was designated as low frequency alcohol colitis group (LAC).

   Group IV: received a single instillation of 1 ml/100g of 7% acetic acid (pr) to induce colitis and 20% v/v ethanol for twenty-one days and was designated as high frequency alcohol colitis group (HAC).

   20% v/v of ethanol (adlibitum) was administered during the day-time. The duration of the study was 21 days.

   Preparation of Ethanol

   Ethanol used was commercially procured. 20 millimeters of ethanol was added to 80 millimeters of distilled water to form 20% v/v ethanol. It was freshly prepared.

   Induction of Colitis

   Preparation of 7% acetic acid. The 7% acetic acid was prepared by adding the acid to water method. 93 mls of distilled water was measured with the measuring cylinder, 7% of 100% acetic acid was added to the water. Distilled water was gradually added to the mixture till it gets to 100ml and the mixture was then thoroughly stirred.

   Induction of colitis. The rats were fasted prior to induction of colitis and the rats were weighed before induction. Rectal flushing was done to remove fecal remnant. Intrarectal administration of 1 ml /100 g body weight of 7% acetic acid was used in the induction of colitis with the aid of rectal cannula in Trendelenburg position. To prevent the acid reflux, the rats were pressed rectally and held upside down for about 30 seconds. Their daily weight after induction were recorded and rectal temperature were measured. The animals were given access to water and normal feed after induction.

   Blood and Tissue Investigations. The animals were sacrificed by cervical dislocation after 21 days and blood and tissue samples were recovered. Blood samples were collected by cardiac punctures into EDTA bottles and mixed thoroughly with the anticoagulant. After hematological analyses were done, the blood was centrifuged and plasma was recovered into plain bottles. Colonic tissues (distal colons) were exercised using dissecting kits, washed in normal saline and transfer into phosphate buffer solution and 10% buffered formalin for biochemical, histological and immunohistochemical analyses.

   Determination of biological parameters

   White blood cells (WBC) and Platelet determinations

   White blood cell count

   White blood cell count was determined using

   blood autoanalyzer.

   Differential white blood cell count

   Differential white blood cell count was

   determined using blood autoanalyzer.

   Platelet count

   Whole blood was diluted with a 1% ammonium oxalate solution. The isotonic balance of the diluent was such that all erythrocytes were lysed while the leukocytes, platelets, and reticulocytes remain intact.

   Red blood cell (RBC) determinations

   Hematocrit assay

   It was based on the application of centrifugal force to recover blood cells from anticoagulated blood in a tube.

   Hemoglobin assay

   Hemoglobin concentration was measured using Sahli’s method.

   RBC indices

   MCV, MCH and MCHC was determined using blood autoanalyzer.

   Determination of biochemical parameters

   Myeloperoxidase assay. Myeloperoxidase was analyzed using ELISA.

   Alpha tumor necrosis factor assay. Tumor necrotic factor was done using ELISA.

   Glutathione assay. Concentration of GSH in the homogenate was measured spectrophotometrically according to Ellman’s method (29). Briefly, aliquots of tissue homogenate were mixed with distilled water and 50% trichloroacetic acid in glass tubes and centrifuged at 2,000 × g for 15 min at 4 °C. Supernatants were mixed with 0.4 mol Tris buffer (pH 8.9) and 0.01 mol 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB) was added. Following agitation of the reaction mixture, absorbance was measured at 412 nm within 5 min of the addition of DTNB against a blank sample that contained no homogenate. Absorbance values were extrapolated from a glutathione standard curve and provided as GSH (µM/g tissue).

   Catalase assay.Determination of CAT activity: CAT activity was determined according to the method outlined by (30). The principle of the assay is based on the determination of the rate constant (k, s-1) or H₂O₂ decomposition rate at 240 nm. Results are provided as k/g protein.

   Superoxide dismutase assay.Total SOD activity was determined according to the method used by Sun et al., (31), which is based upon the principle of the inhibition of Nitro Blue-Tetrazolium (NBT) reduction by the xanthine oxidase system as a superoxide generator. One unit of SOD was defined as the enzyme amount required to induce a 50% reduction in the NBT reduction rate. SOD activity was calculated as U/mg protein.

   Histological studies

   A histological study was carried out as described by Ogihara and Okabe (32).

   Statistical analysis

   All data are expressed as mean ± standard error of the mean (SEM) using SPSS 21. Statistically significant differences were accepted at p<0.05. Least Significance Difference (LSD) test was used to identify the significance of pair wise comparison of mean values among the groups.

	RESULTS

   Effect of alcohol consumption pattern on inflammatory markers in ulcerative colitis rats

   Non-alcohol colitis, low frequency alcohol colitis and high frequency alcohol colitis groups showed significantly increased colonic myeloperoxidase when compared with control respectively. Low frequency alcohol colitis and high frequency alcohol colitis groups showed significantly increased colonic myeloperoxidase when compared with non-alcohol colitis group respectively (Figure 1A).

   Non-alcohol colitis and low frequency alcohol colitis groups showed significantly increased colonic alpha-tumor necrosis factor when compared with control respectively. High frequency alcohol colitis group showed significantly increased colonic alpha-tumor necrosis factor when compared with non-alcohol colitis group. Low frequency alcohol colitis group significantly increased colonic alpha-tumor necrosis factor when compared with high frequency alcohol colitis group respectively (Figure 1B).

   Effect of alcohol consumption pattern on antioxidant markers in ulcerative colitis rats

   Non-alcohol colitis, low frequency alcohol colitis and high frequency alcohol colitis groups showed significantly decreased colonic catalase when compared with control respectively. Low frequency alcohol colitis and high frequency alcohol colitis groups showed significantly decreased colonic catalase when compared with non-alcohol colitis group respectively (Figure 2A).

   Non-alcohol colitis, late alcohol colitis and high frequency alcohol colitis groups showed significantly decreased colonic superoxide dismutase when compared with control respectively. Late alcohol colitis and high frequency alcohol colitis groups significantly decreased colonic superoxide dismutase when compared with non-alcohol colitis group respectively (Figure 2B).

   Non-alcohol colitis, low frequency alcohol colitis and high frequency alcohol colitis groups showed significantly decreased colonic glutathione when compared to control respectively. Low frequency alcohol colitis and high frequency alcohol colitis groups showed significantly decreased colonic when compared with non-alcohol colitis respectively (Figure 2C).

   Effect of alcohol consumption pattern on White Blood Cells in ulcerative colitis rats

   Low frequency alcohol colitis and high frequency alcohol colitis groups showed significantly increased neutrophil and basophil levels when compared with control respectively. Low frequency alcohol colitis group showed increased absolute white blood cell count, neutrophils, eosinophils, basophils, neutrophil: lymphocyte ratio and lymphocyte: monocyte ratio when compared with non-alcohol colitis and early alcohol colitis groups respectively (Table 1).

   Effect of alcohol consumption pattern on Red Blood Cells in ulcerative colitis rats

   Non-alcohol colitis, low frequency alcohol colitis and high frequency alcohol colitis groups exhibited significantly low packed cell volume, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and hemoglobin when compared with control respectively. Late alcohol colitis group showed a significant increase in mean corpuscular volume when compared with high frequency alcohol colitis group (Table 2).

   Effect of alcohol consumption pattern on platelet count in ulcerative colitis rats

   Non-alcohol colitis, low frequency alcohol colitis and high frequency alcohol colitis groups showed significantly increased platelet when compared with control respectively. Low frequency alcohol colitis and high frequency alcohol colitis groups showed significantly decreased platelet count when compared with non-alcohol colitis group respectively. Low frequency alcohol colitis group showed significantly increased platelet count when compared with high frequency alcohol colitis group (Figure 3).

   Effect of alcohol consumption pattern on colonic histology in ulcerative colitis rats

   Control group showed normal colonic layer. Non-alcohol colitis, high frequency alcohol colitis and late colitis groups showed poorly preserved colonic layers (Figure 4).

 

View larger version : [in a new window] Figure 1. a, Effect of alcohol consumption pattern on myeloperoxidase in ulcerative colitis rats. Non-alcohol colitis (NAC), low-frequency alcohol colitis (LAC) and high-frequency alcohol colitis (HAC); b, Effect of alcohol consumption pattern on alpha-tumor necrosis factor in ulcerative colitis rats. Non-alcohol colitis (NAC), low-frequency alcohol colitis (LAC) and high-frequency alcohol colitis (HAC). abcrepresent significant difference from CTRL, NAC and HAC respectively.

 

View larger version : [in a new window] Figure 2. a, Effect of alcohol consumption pattern on catalase in ulcerative colitis rats. Non-alcohol colitis (NAC), low-frequency alcohol colitis (LAC) and high-frequency alcohol colitis (HAC); b, Effect of alcohol consumption pattern on superoxide dismutase in ulcerative colitis rats. Non-alcohol colitis (NAC), low-frequency alcohol colitis (LAC) and high-frequency alcohol colitis (HAC); c, Effect of alcohol consumption pattern on glutathione in ulcerative colitis rats. Non-alcohol colitis (NAC), low-frequency alcohol colitis (LAC) and high-frequency alcohol colitis (HAC). abrepresent significant difference from CTRL and NAC respectively.

 

View larger version : [in a new window] Figure 3. Effect of alcohol consumption pattern on platelet count in ulcerative colitis rats. Non-alcohol intervention colitis (NAC), low-frequency alcohol colitis (LAC) and high-frequency alcohol colitis (HAC). abc represent significant difference from CTRL, NAC and HAC respectively.

 

View larger version : [in a new window] Figure 4. Colonic histology stained by H&E showing the effect of alcohol consumption pattern on experimental ulcerative colitis rats. Scale bar: 40 μm. Control group (a), non- alcohol colitis group (b), low-frequency alcohol colitis group (c), high-frequency alcohol ulcerative colitis group (d).   Blue star represents poorly preserved colonic layers.

	DISCUSSION

   Compromise in hematological and immunological indices is a feature of impaired health status and disease conditions representing increased predisposition to opportunistic infection, high likelihood of relapse and fatalities. Studies have indicated higher incidence rate of ulcerative colitis in alcoholics and alcohol addicts (33). The present study sought to determine the specific pattern of hematological and immunological indices in experimental ulcerative colitis rats exposed to different alcohol consumption frequencies.

   Comparative evaluation of histological results of colitis rats and colitis rats that received high frequency and low frequency of alcohol administrations revealed structural changes in colonic integrity from normal. With respect to colitis rats, remission and natural healing tendency reportedly exhibited by some sufferers of ulcerative colitis (34) could not be replicated in the present study. One of the reasons for the difference might be our methodology; we did not place the rats on special chow like fat-free diet, maize free diet, sugar-, meat-, and sulphite-free diet and fiber-depleted meal, all of which have been reported to mitigate colitis severity in animal and human studies (35, 36).

   In the present study, depression in glutathione, superoxide dismutase and catalase observed in acetic acid-induced colitis rats parleyed with the findings of previous studies (16, 37, 38). The role of oxidative stress in the progression of diseases cannot be underestimated (13, 16, 37-41). We observed that colitis rats that were on low frequency and high frequency of alcohol consumptions exhibited lower antioxidant profile when compared with colitis rats respectively. This finding was significant as it portrayed the immunologic risk extent posed by exposures to alcohol in ulcerative colitis subjects.

   In addition to the reported colitis-induced depression in antioxidant markers, our study revealed an elevation in myeloperoxidase profile in colitis rats. Increase in myeloperoxidase, an enzyme produced courtesy of degranulation of neutrophilic polymorphonuclear leucocytes at the infection site (42, 43), symbolized increased peroxidation of halides and pseudohalides and formation of hypohalous acids which are toxic to microorganisms. Our study indicated that colitis rats that received high frequency and low frequency of alcohol administration showed higher myeloperoxidase level when compared to colitis rats respectively.

   Colitis rats that received high frequency and low frequency of alcohol administrations exhibited heightened alpha necrosis factor when compared with colitis rats respectively. A rise in alpha necrosis factor occurred in colitis rats given low frequency of alcohol administration when compared with colitis rats that received high frequency of alcohol administration. Although, we are aware that at least a study has reported that cessation and infrequent of alcohol consumption increased the risk of inflammatory bowel disease (23, 24), the rats used in the present study have never been previously exposed to alcohol. This implied that low frequency of alcohol intervention exacerbated inflammatory response in ulcerative colitis subjects who have not been previously exposed to alcohol. The finding also indicated the relative immunologic risk level posed by low frequency exposures to alcohol in ulcerative colitis subjects.

   When compared with colitis rats, the present study showed an evidence of granulocytosis in colitis rats that received high frequency and low frequency of alcohol administrations respectively. For example, basophilic, eosinophilic and neutrophilic leucocytes were significantly raised in colitis rats that received high frequency and low frequency of alcohol administrations when compared with colitis rats respectively. Neutrophils are a group of white blood cells known to be activated through formyl peptide receptors (44). There was an increase in neutrophil count in colitis rats given low frequency of alcohol when compared with colitis rats that received high frequency of alcohol administration. In popular consensus, neutrophilia occurs in acute infection and inflammation. Besides neutrophils, increase in basophils signified chronic inflammation and infestation. In inflammatory Bowel Disease, a study has shown that the survival and attraction of basophil was mediated by activator effector T cells-induced increase in interleukin-3 (45). Eosinophilia characterizing active and inactive cases of UC (46) has also been reported to be due to eotaxin, a chemoattractant (47, 48).

   Although ulcerative colitis did not significantly affect neutrophil count, there were significant changes in agranulocytes. For instance, the significant increase in monocytes in colitis rats indicated chronic inflammatory response (49, 50). Works have shown that intestinal inflammation increases monocyte recruitment through CCR2. In the study, the low lymphocyte count observed in colitis rats concurred with previous findings (51, 52). Lymphocyte count significant reduced in colitis rats that received low frequency of alcohol administration when compared with colitis rats. There was also a reduction in monocyte count in colitis rats given low frequency of alcohol administration when compared with colitis rats that received high frequency of alcohol administration. Lymphocyte count reduced in colitis rats when compared with the control group. Lymphocyte count was significantly reduced in colitis rats that received low frequency of alcohol administration when compared with colitis rats. There was also a reduction in lymphocyte count in colitis rats given low frequency of alcohol administration when compared with colitis rats that received high frequency of alcohol administration. The finding also indicated the relative immunosuppressive effect associated with low frequency exposure to alcohol in ulcerative colitis subjects.

   Furthermore, the high neutrophil: lymphocyte ratio observed in colitis rats that were administered low frequency of alcohol administration when compared with high frequency alcohol colitis group has been shown to reflect severity of IBD severity in human studies in combination with erythrocyte sedimentation rate, C-Reactive Protein and lymphocyte: monocyte ratio (51-53). As far as the study was concerned, we noticed that colitis rats that were given high frequency of alcohol administration exhibited higher lymphocyte: monocyte ratio when compared with low frequency alcohol colitis rats. Nevertheless, the study contained more proofs in support of the exacerbating influence of low frequency of alcohol administration in colitis rats.

   Anemia is another symptom of clinical and experimental IBD. In our study, the decline in hematocrit, hemoglobin, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration and increase in mean corpuscular volume that characterized colitis rats that received high frequency and low frequency of alcohol administrations. Studies have linked IBD related anemia to chronic hemorrhage. Evidence from the study showed that it is also possible that diarrhea-induced depletion of vitamins and minerals most especially vitamin B12 and iron participates in colitis induced anemia.

   Acute hemorrhage is known to increase platelet count. In addition to increased platelet level in colitis rats, there was an increase in platelet count of high frequency and low frequency alcohol colitis groups when compared with colitis group respectively. Heavy alcohol has for long time been known to increase platelet count (54, 55). The pathophysiological mechanisms underlying augmentation of platelet concentration in colitis rats that received high frequency and low frequency of alcohol administrations are not well understood.

	CONCLUSION

   The present study highlighted the specific hematological and immunological risks associated with different alcohol consumption frequencies in female subjects with ulcerative colitis. In conclusion, the findings of the study indicated that low frequency alcohol intake was relatively more granulocytic, inflammatory, thrombocytic, macrocytic and agranulopenic in colitis female rats than high frequency alcohol intake.

	ACKNOWLEDGEMENT

   The authors are grateful to the laboratory staff of hematology department, Ladoke Akintola University of Teaching Hospital, Ogbomoso.

	REFERENCES

1. Rehm J, Mathers C, Popova S, et al. Global burden of disease and injury and economic costattributable to alcohol use and alcohol-use disorders. Lancet. 2009; 373 (9682): 2223–2233.
2. WHO Global status report on alcohol and health 2014 (PDF). who.int. Archived (PDF) from the original on 2018-02-14. Accessed 20/11/2020
3. Coriale, et al. Fetal Alcohol Spectrum Disorder (FASD): neurobehavioral profile, indications for diagnosis and treatment. Rivista di Psichiatria. 2013; 48 (5): 359–369.
4. Chudley, et al. Fetal alcohol spectrum disorder: Canadian guidelines for diagnosis. CMAJ. 2005; 172 (5 Suppl): S1–S21.
5. Muller D, Koch RD, von Specht H, Völker W, et al. Neurophysiologic findings in chronic alcohol abuse. Psychiatr Neurol Med Psychol (Leipz) (in German). 1985; 37 (3): 129–132.
6. Wood AM, Kaptoge S, Butterworth AS, Willeit P, et al. Risk thresholds for alcohol consumption: combined analysis of individual-participant data for 599 912 current drinkers in 83 prospective studies. The Lancet. 2018; 391 (10129): 1513–1523.
7. Larsson SC, Drca N, Wolk A. Alcohol Consumption and Risk of Atrial Fibrillation. Journal of the American College of Cardiology. 2014; 64 (3): 281–289.
8. Testino G. Alcoholic diseases in hepato-gastroenterology: a point of view. Hepatogastroenterology. 2008; 55 (82–83): 371–377.
9. Awtry EH, Philippides GJ. Alcoholic and cocaine-associated cardiomyopathies. Prog Cardiovasc Dis. 2010; 52 (4): 289–299.
10. Djousse L, Gaziano JM. Alcohol consumption and heart failure: a systematic review. Curr Atheroscler Rep. 2008; 10 (2): 117–120.
11. Danese S, Fiocchi C. Ulcerative colitis. The New England Journal of Medicine. 2011; 365 (18): 1713–1725.
12. Ford AC, Moayyedi P, Hanauer SB. Ulcerative colitis. BMJ. 2013; 346: f432.
13. Adeniyi MJ, Ige SF, Adeyemi WJ, Oni TJ, et al. Changes in Body Temperature and Intestinal Antioxidant enzymes in Healthy and Colitis Male Rats: Roles of Garcinia kola (Bitter Kola). International Journal of Physiology. 2016; 4 (2): 36-41.
14. Molodecky NA, Soon IS, Rabi DM, Ghali WA, et al. Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review. Gastroenterology. 2012;142 (1): 46–54.
15. Oludara Mobolaji, Akinola Rachael, Popoola Abiodun, Makanjuola Samira. Inflammatory Bowel Disease, a Rare or Under Diagnosed Disease in Nigeria. British Journal of Medicine & Medical Research. 2014; 4 (35): 5620-5628.
16. Adeniyi MJ, Ige SF. Therapeutic Effect of Crude Extract of Garcinia kola (Bitter Kola)on Acetic Acid Induced Colitis in Male Rats. International Journal of Healthcare Sciences. 2016; 4 (2): 1141-1149.
17. Ungaro R, Mehandru S, Allen PB, Peyrin-Biroulet L, et al. Ulcerative colitis. Lancet. 2017; 389 (10080): 1756–1770.
18. Jowett SL, Seal CJ, Pearce MS, et al. Influence of dietary factors on the clinical course of ulcerative colitis: a prospective cohort study. Gut. 2004; 53: 1479–1484.
19. Zutshi M, Hull TL, Hammel J. Crohn’s disease: a patient’s perspective. Int J Colorectal Dis. 2007; 22: 1437-1444.
20. Cohen AB, Lee D, Long MD, et al. Dietary patterns and self-reported associations of diet with symptoms of inflammatory bowel disease. Dig Dis Sci. 2013; 58: 1322-1328.
21. Swanson GR, Sedghi S, Farhadi A, Keshavarzian A. Pattern of alcohol consumption and its effect on gastrointestinal symptoms in inflammatory bowel disease. Alcohol. 2010; 44: 223-228.
22. Hirsch S, Cháves G, Gotteland M, Maza PDL, et al. Intestinal permeability in alcoholic patients without liver damage. Rev Med Chil. 1997; 125 (6): 653-658.
23. Khoruts A, Stahnke L, McClain CJ, Logan G, et al. Circulating tumor necrosis factor, interleukin-1 and interleukin-6 concentrations in chronic alcoholic patients. Alcoholism: clinical and experimental research. 1991; 13 (2): 267-276.
24. Nakamura Y, Labarthe DR. A case-control study of ulcerative colitis with relation to smoking habits and alcohol consumption in Japan. Am J Epidemiol. 1994; 140 (10): 902-911.
25. El-Tawil Ahmed Mahmoud. Epidemiology and inflammatory bowel diseases. World J Gastroenterol. 2013; 19 (10): 1505–1507.
26. Adeniyi MJ, Agoreyo FO. Nigeria and the Selenium Micronutrient: A Review. Ann Med Health Sci Res. 2018; 8: 5-11.
27. Adeniyi MJ, Agoreyo FO. Estrous Cycle Ratio as a Reproductive Index in the Rats. Am J Biomed Sci & Res. 2019; 4 (2): 100-103.
28. Adeniyi MJ, Agoreyo FO, Olorunnisola OL, Olaniyan OT, et al. Photo-pollution disrupts reproductive homeostasis in female rats: The duration-dependent role of selenium administrations. Chin J Physiol. 2020; 63: 235-243.
29. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. Journal of Laboratory and Clinical Medicine. 1967; 70 (1): 158-169.
30. Aebi H. Catalase in vitro. Methods Enzymol. 1984; 105: 121-126.
31. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 1988; 34: 497-500.
32. Ogihara Y, Okabe S. Effect and mechanism of sucralfate on healing of acetic acid-induced gastric ulcers in rats. J Physiol Pharmacol. 1993; 44: 109–118.
33. Hsu TY, Shih HM, Wang YC, et al. Effect of alcoholic intoxication on the risk of inflammatory bowel disease: a nationwide retrospective cohort study. Plos One. 2016; 11: e0165411.
34. Rubin DT, Ananthakrishnan AN, Siegel CA, Sauer BG, et al. ACG Clinical Guideline: Ulcerative Colitis in Adults. Am J Gastroenterol. 2019; 114 (3): 384–413.
35. Mascolo N, Izzo A, Autore G, Maiello F, et al. Acetic acid induced colitis in normal and essential fatty acid deficient rats. J Pharmacol Exp Ther. 1999; 272: 469-475.
36. Ige SF, Adeniyi MJ, Olayinka AT, Kehinde IC. Role of dietary maize formulations in the healing of experimental acetic acid induced ulcerative colitis in male rats. Chin J Physiol.  2020; 63 (4): 156-162
37. Ige SF, Adeniyi MJ, Iyalla GO. Allium cepa Mitigates Aluminum Chloride-Induced Hepatotoxicity in Male Wistar Rats. J Biomedical Sci. 2017; 6 (4): 27.
38. Serah Funke Ige, Mayowa Jeremiah Adeniyi, Jelili A. Badmus, Suliyat Aremu. Experimental Acetic Acid-Induced Colitis Impairs Fertility Indices through Disrupted Hormonal Feedback in Male Rats. European Journal of Pharmaceutical and Medical Research. 2018; 5 (9): 414-417.
39. Adeniyi MJ, Agoreyo FO. Duration-related modulation of body temperature rhythm and reproductive cycle in rats by photoperiodic perturbation. DRJHP. 2020; 8 (1): 1-6.
40. Adeniyi MJ, Oni TJ. Water Contamination in Nigeria and Body Defense Issues. Research chronicler. 2016; 3 (3): 11-21.
41. Serah Funke Ige, Mayowa Jeremiah Adeniyi, Ademilua OB, Fatola AO, et al. Allium cepa remediates oxidative stress-mediated hepatic DNA damage in cadmium-exposed rats through enhanced p53 expression and inhibition of bcl2. Int J Biomed Sci. 2020; 16 (2): 11-17.
42. Nicholls SJ, Hazen SL. Myeloperoxidase and cardiovascular disease. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1102–1111.
43. Deimann W. Endogenous peroxidase activity in mononuclear phagocytes. Prog. Histochem. Cytochem. 1984; 15: 1–56.
44. Dorward DA, Lucas CD, Chapman GB, Haslett C, et al. The role of formylated peptides and formyl peptide receptor-1 in governing neutrophil function during acute inflammation. Am. J. Pathol. 2015; 185: 1172–1184.
45. Siracusa MC, Kim BS, Spergel JM, Artis D. Basophils and allergic inflammation. J. Allergy Clin Immunol. 2013; 132: 789-801.
46. Al-Haddad S, Riddell RH. The role of eosinophils in inflammatory bowel disease. Gut. 2005; 54 (12): 072595.
47. Mir A, Minguez M, Tatay J, et al. Elevated serum eotaxin levels in patients with inflammatory bowel disease. Am J Gastroenterol. 2002; 97:1452–1457.
48. Chen W, Paulus B, Shu D, et al. Increased serum levels of eotaxin in patients with inflammatory bowel disease. Scand J Gastroenterol. 2001; 36: 515–520.
49. Mee AS, Berney J, Jewell DP. Monocytosis in IBD: absolute monocyte counts: J Clin Path. 1980. 33 (10): 917-920.
50. Ashraf M. Oleba, Mariam M. Amin, Abdelmoaty AS, et al., Neutrophil/Lymphocyte ratio and lymphocyte/monocyte ratio in ulcerative colitis as non-invasive biomarkers of disease activity and severity. Autoimmune Highlight. 2019; 10 (4): 0114-0118.
51. AlRifal A, Prasad N, Shuttleworth E, MCBurney Hm Pushpakom S, et al. Natural history of Zathioprine-associated lymphopenia in inflammatory bowel disease patients: A prospective observational study. Eur J Gastroent Hepatolol. 2011; 23 (2): 153-158.
52. Bincy P. Abraham, Joseph H. Sellim. Lymphopenia in IBD patients in immunosuppressive medications. Journal of Gastroenterol and Hepatol Research. 2016; 15 (1):1890-1894.
53. Cherfane Cynthia, Gessel Luke, Grillo Dominic, Steven Polyak. Monocytosis and low lymphocyte: monocyte ratio are effective biomarkers. America journal of gastroenterology. 2013; 108: s526-s527.
54. Cowan DH. Effect of alcohol on haemostasis. SeminHematol; 17:137-47. Ballard, Harold S. The Hematological Complications of Alcoholism. Alcohol Health & Research World. 1997; 21: 44.

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