Impact of Vitamin B17 Against Growth of Colitis Bearing Mice Induced Variations in Colon Structure, AFP, CEA and PCNA Immunoreactivity

Corresponding Author: Ehab Tousson Department of Zoology, Faculty of Science, Tanta University, Egypt Email: toussonehab@yahoo.com ehabtousson@science.tanta.edu.eg Abstract: Colitis alludes to inflammation of the internal coating of the colon that accompanied clinically through abdominal pains, diarrhea, weight loss, nausea. Current study aimed to study the impact of vitamin B17 against growth of colitis bearing mice induced variations in colon structure, AFP, CEA and PCNA immune reactivity. Forty female Swiss albino mice separated into 4 groups (Control, vitamin B17, colitis and post treated colitis with vitamin B17 groups). Our results revealed significant increase in serum AFP, CEA, colon injury and PCNA expressions. In contrast; post treated colitis with vitamin B17 improved the colon functions and structure, inhibit the proliferations, AFP and CEA. If established in human beings, these outcomes could recommend that; vitamin B17 can be utilized as an adjuvant treatment with chemotherapy drugs.


Introduction
The colon is a significant piece of the gastrointestinal lot. Its capacities incorporate re-take-up of liquids and electrolytes just as capacity and end of rest results of food. Colitis suggests the irritation in the inward covering of the colon (Ali et al., 2018). There are different purposes behind colitis including defilement brought about by an infection or a parasite, Crohn illness, absence of blood stream and radiation colitis and injuries (Ohlsson, 2015;Saad et al., 2019). Colitis influences the distal piece of the colon and makes non-transmural irritation, huge rot and ulceration in the layers of mucosa and submucosa (Randhawa et al., 2014). These sicknesses are characterized clinically through provocative confusion in gastrointestinal lot went with looseness of the bowels, torments in midsection, weight decrease, nausea and obsessive changes like a deficiency of mucosal dysfunctions, aggravation and cell invasion (Cengiz et al., 2017). Acceptance of colitis may be because of the openness to various incendiary specialists and because of creation of free extremists (Hanauer, 2006;Saad et al., 2019). Furthermore, free extremists lessen cell reinforcement specialists and along these lines increment the seriousness of the illness (Radko and Cybulski, 2007).
So far there are three distinguished models used to initiate test colitis in rodents and mice (Antoniou et al., 2016). These models incorporate dextran sulfate sodium, oxalone and acidic corrosive (Tahan et al., 2011;Ali et al., 2018). The utilization of correlative and elective medication has consistently expanded over the previous many years (Salama et al., 2014;Tousson et al., 2018;Abd Eldaim et al., 2019a). In these examinations, a few phytochemicals were exhibited to be protected and conceivably valuable remedially (Abd Eldaim et al., 2019b;Tousson et al., 2019;Altwaijry et al., 2020Altwaijry et al., , 2021. Phytochemicals are normal composites that have exercises against oxidation, irritation and malignant growth or tumors which might be a fundamental for the treatment of a few disastrous sicknesses (Salama et al., 2015;Elmasry et al., 2018;Alotaibi et al., 2020;.Vitamin B17 is called as apricot kernels or amygdalin extracted from the kernels of apricots by a biochemist named Ernst T Krebs Jr. and it is one of numerous nitrilosides which are normal cyanide containing substances (Bolarinwa et al., 2014;Elmasry et al., 2018). Vitamin B17 is a kind of sugar happening normally in plants and it is a cyanogenic diglucoside found basically in fruit kernels such as apricot, peach, cashews and macadamias (Santos Pimenta et al., 2014). Amygdalin are natural compounds that has been utilized as conventional Chinese medication for the treatment of numerous illnesses and it has numerous pharmacological properties include antioxidant, anti-inflammatory, antitussive, anti-asthmatic, activities (Zhou et al., 2012;Mutar et al., 2020;Tousson et al., 2020). A few examinations revealed that amygdalin can be utilized to lessen the development of prostate, renal and lung cell carcinoma (Qian et al., 2015;Juengel et al., 2016). Therefore; this study was designed the impact of vitamin B17 against growth of colitis induced variations in colon structure, AFP, CEA and PCNA-immunoreactivity.

Chemical and Reagent Vitamin B17
Vitamin B17 was purchased from the Amazon for Natural Oils Company.

Acetic Acid
Acetic acid was bought from Sigma Company and reconstituted according to the manufacture description, then diluted with saline solution to prepare 2% concentration.

Animals
Forty mice (female; 22±2 g) were acquired from the animal community colony Egypt Vaccine Company, Egypt. The animals were randomized and housed under ambient room-temperature at 22 -25°C and relative humidity conditions a 12: 12 h light/dark cycle, a commercial diet and water were provided ad libitum for two weeks.

Induction of Ulcerative Colitis
Mice (n = 20) were received100 µL of 2% acetic acid in combination with 5 µL of olive oil (acetic acid/olive oil mix) using a gavage Intra-Rectally (IR) injection day by day for one week (Ali et al., 2018). Immediately after administration, the mice were hold horizontally for 2 min to prevent fluid leakage. Acetic acid was then retained in the colon for 30 seconds after which the fluid was withdrawn (Ali et al., 2018).

Experimental Design and Animal Groups
The mice were equally divided into four groups (10 mice in each): G 1: Control group in which mice were received Intra-Rectal (IR) injection only 5 µL of olive oil. G 2: The mice in this group were received orally Vitamin B17 (175 mg/Kg body weight/day), for 2 weeks according to Mutar et al. (2020). G 3: Colitis group with induced mice ulcerative colitis, mice were administrated with intra-rectal injection of 100 µL of 2% acetic acid in combination with 5 µL of olive oil for one week (Ali et al., 2018).
G 4: Post treated group, induced mice ulcerative colitis for one week and then treated with Vitamin B17 for another 2 weeks

Sample Collection
At the end of experiment, overnight fasted mice were euthanized, blood samples were collected from eye, centrifugation and saved in clean plug vial at -20°C until measure. Sera were used for estimation of AFP and CEA. Necropsy was conducted, colon were removed, cleaned in saline solution and fixed in a 10 per cent neutral buffer formalin buffer for up to 48 h for histopathology and immunohistochemistry studied.

Histopathological Examination
Colon samples were immediately taken after decapitation, washed with 0.9 saline solutions and fixed with 10% neutral buffered formalin. Following the protocol of Tousson (2016), fixed colon from each groups were stained by standard haematoxylin and eosin counterstain techniques.

Immunohistochemical Investigations
Mice colon sections were utilized to stain with PCNA following the protocol of Tousson et al. (2011;.

Statistical Analysis
The investigation of results was finished utilizing SPSS programming adaptation 16. Information were introduced as the mean ± SEM and the foundation for measurable hugeness was set at p<0.05.

Toxicity and Clinical Symptoms
The dose of vitamin B17 didn't inductee any reactions for the test creatures. Anal injection of mice with 2% acetic acid induced colitis with diarrhea and rectal bleeding.

Changes in Serum Carcinoembryonic Antigen (CEA)
In the current study; Fig. (1) revealed a significant elevation in CEA levels in colitis group as compared to control and vitamin B17 groups. In contrast; treatment of colitis with vitamin B17 revealed significant decrease in the CEA levels when compared with colitis group (Fig. 1).

Changes in Serum Alpha-Fetoprotein (AFP)
In the current study; Fig. (1) revealed a significant elevation in AFP levels in colitis group as compared to control and vitamin B17 groups. In contrast; treatment of colitis with vitamin B17 revealed significant decrease in the AFP levels when compared with colitis group (Fig. 1).

Histopathological investigations
Colon sections in control mice and in mice treated with vitamin B17 groups revealed well preserved mucosal integrity with well -arranged finger like villi and their epithelial linings with a few inflammatory cells were seen to infiltrate into the villous stroma and sub mucosa (Fig. 2a and 2b).
Colon sections in colitis group as the colonic mucosa appeared atrophy, degenerated, ulcerated with shortening villus with crypt damage, depletion in goblet cell numbers and moderate marked leukocyte infiltration in the villi lamina and in sub mucosal layer (Fig. 2c). Colon sections in treated colitis with vitamin B17 revealed moderate degree of improvement as mild atrophy in villi with moderate crypt hyperplasia, mild increase in goblet cells and moderate decrease in the leukocyte infiltration (Fig. 2d).

PCNA Immunoreactivity in mice colon
Negative reactions for PCNA were observed in the mucosal layers of colon in control and in treated mice with vitamin B7 (Fig. 3a and 3b). In contrast strong positive reactions for PCNA were observed in colitis group (Fig. 3c). On the other hand; colon sections in treated colitis with vitamin B17 revealed mild positive reactions for PCNA (Fig. 3d).

Discussion
Currently we designed the impact of vitamin B17 against growth of colitis induced variations in colon structure, AFP, CEA and PCNA immunoreactivity. Current results showed that; anal injection of 2% acetic acid and Olive oil induced colitis with diarrhea and visible rectal bleeding. Our results agree with Ali et al. (2018) who find that; 2% acetic acid in the presence of olive oil induced colitis in albino CD1 mice.
CEA is overexpressed by an assortment of malignancies and is usually connected with colorectal disease, it can likewise be raised in different malignancies, for example, liver, pancreas, stomach and breast (Bel Hadj Hmida et al., 2001;Perkins et al., 2003). Current study revealed that a significant elevation in CEA levels in colitis group as compared to control and vitamin B17 group. In contrast; treatment of colitis with vitamin B17 revealed significant depletion in the CEA levels when compared with colitis group. Current results agree with Kamada et al. (2013) who find an increase in the levels of CEA in colitis. Current results also agree with Rocklin et al. (1991) who find that; a significant elevation in CEA and in liver function in colorectal carcinoma. Also; the current results are in agreement with Umesalma and Sudhandiran, (2010) who find that; serum CEA levels were elevated with DMH-induced colon cancer in rats.
AFP is a tumor-related fetal glycoprotein containing 3-4% sugar moieties and is a plasma protein delivered by the early stage yolk sac and the fetal liver. AFP levels in serum, amniotic liquid and pee works as a screening test for innate inabilities, chromosomal variations from the norm, just as some other grown-up happening tumors and pathologies (Shumway et al., 1996). Current investigation; uncovered a critical increment in the degrees of AFP in colitis bunch when contrasted with control and nutrient B17 gatherings while treatment of colitis with nutrient B17 uncovered huge diminishing in the AFP when contrasted with colitis gathering. Our outcomes concur with Thompson et al. (1974) who revealed that; an expansion in the degrees of AFP in ulcerative colitis and territorial enteritis. Additionally Linson and Hanauer (2019) considers the potential and significant job of AFP in incendiary gut infection.
Many abnormalities were reported in mouse colon after 2% acetic acid induced colitis as a marked atrophy, degenerated, ulcerated with shortening villus with crypt damage, depletion in goblet cell numbers and moderate marked leukocyte infiltration in the villi lamina and in sub mucosal layer. Colitis was experimentally induced by acetic acid anal injection and it is similar to human ulcerative colitis in inflammation and ulcers of the colon and rectum. These results agree with Ali et al. (2018) who find that; 1, 2 and 4% acetic acid induced colitis with many changes in mucosa and in sub mucosa layers in CD1 albino mice. PCNA is one antigen used to calculate a proliferation index and is a non-histone protein detected 1st in the mid-G1 phase of the cell cycle, reaching a maximum in S and declining in G2 . Current results revealed that; colitis induced an increase in PCNA immunoreactivity as compared to control group and treatments of colitis with vitamin B17 inhibit this PCNA elevation. Our results agree with Sipos et al. (2005) who reported that; an increased in proliferation and apoptosis expression in inflammatory bowel diseases. Also; current results agree with Sjöqvist et al. (1999) who reported increased expression of proliferative Ki-67 nuclear antigen is correlated with dysplastic colorectal epithelium in ulcerative colitis. Vitamin B17 extract have the ability to improve the colon functions and structure, inhibit the proliferations, AFP and CEA. If established in human beings, these outcomes could recommend th at; vitamin B17 can be utilized as an adjuvant treatment with chemotherapy drugs in the treatments of different types of cancer and tumors.

Author's Contributions
This work was carried out in collaboration among all authors. 'Gihan Abd Elsamie, Sabah El-Banna, Ehab Tousson and Shatha Felemban designed the study, performed the statistical analysis and wrote the protocol, Mohanad Hussein wrote the first draft of the manuscript, managed the analyses of the study and managed the literature searches. 'All authors read and approved the final manuscript

Ethics
This article is original and contains unpublished material. The corresponding author confirms that have read and approved the manuscript and no ethical issues involved.