The Intra Gastric Effects of Grape Juice on Histological Structure of Liver Tissue in Castrated Treated Mice

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Homady MH*1, ALquraishi LO2, Tanya S Salih1 and Juma ASM1 Research Article 1College of Science, Department of Biomedical Sciences, Cihan University-Erbil, Kurdistan Volume 4 Issue 1 Region, Iraq Received Date: July 27, 2020 2College of Dentistry, Babylon University, Iraq Published Date: August 11, 2020 DOI: 10.23880/jhua-16000147 *Corresponding author: Homady MH, College of Science, Department of Biomedical Sciences, Cihan University-Erbil, Kurdistan Region, Iraq, Tel: 009647517118678; Email: merzahh@ yahoo.com

Abstract

The effect of ingestion of freshly prepared ethanoic extract of grape juice was investigated on the histology of liver tissue on both castrated and castrated treated with 10 µl/g of grape juice. The histological sections of liver tissue from castrated

group showed ballooning degeneration, macro and micro vesicular steatosis, lobular inflammation, hydropic degeneration, administration of 10µl/g grape juice for 6 weeks to castrated subjects was able to restore the histological structure of liver cellular inflammatory of lymphocytes and acidophil bodies (Councilman Body) as compared with the control group. The orally tissue to normal structure as compared with the control group. These results suggest that the fresh extract of grape juice can exert hepatoprotective effects of castration.

Keywords: Liver; Histology; Castration; Steatosis; Grape Juice

Introduction hyperglycaemia, hypertriglyceridemia, hypercholerteremia

The liver tissue is made up of the largely predominant risk factors of cardiovascular disease, stroke, and diabetes [8].and high LDL (low density lipoprotein) that are predictive

endothelialparenchymatous cells [1-3]. cells (hepatocytes),Nonalcoholic fatty as wellliver disease as Ito (perisinusoidal cells), Kupffer cells and sinusoidal world. It is present in 30% of the general adult population. insulinTestosterone resistance and deficiency accumulation has beenintrahepatic associated lipids, withthus (NAFLD) is the most common chronic liver disease in the theincreased lower testosterone accumulation levels of Visceral are associated Adipose with Tissues nonalcoholic (VAT), abnormalities that are observable in liver histological slides, In reality, NAFLD comprise as a spectrum of hepatic to eliminate breeding and reduce aggressive behavior [11,12]fatty liver Hypogonadism disease [9,10]. after Farm castration animals have caused been abdominal castrated from a simple intrahepatic accumulation of fat (steatosis or obesity, impaired fasting glucose, excess accumulation of Simplenonalcoholic steatosis fatty rarelyliver, NAFL) progresses to various to degreesadvanced of necroticdisease, liver triglyceride, and thigh muscle loss. Castration also whereas,inflammation in approximately (NASH nonalcoholic 20% of steatohepatitis)patients with NASH, [4-7]. induced increase of visceral fat mass and decreased thigh muscle mass in mice [13]. Castration promotes progression

manifestationit progresses of to metabolic fibrosis and syndrome, cirrhosis which and is potentially a cluster toof complexhepatocellular conditions, carcinoma. including NAFLD central is also obesity, deemed hypertension, to be hepatic to steatohepatitis through activation of the ER (endoplasmic reticulum) stress pathway and enhancement of macro The Intra Gastric Effects of Grape Juice on Histological Structure of Liver Tissue in Castrated Treated J Human Anat Mice 2 Journal of Human Anatomy vesicular droplet. Testosterone suppresses ER stress, inhibits given daily as orally administered for six weeks. Animals the formation of macro vesicular lipid droplets, promotes lipid export, and ameliorates steatohepatitis induced by ketamine and Xylazine as anesthetic drugs to anesthetize the castration [14] Low serum testosterone levels have a higher mice.were sacrificed at the end of the experiments, with using risk of developing hepatic steatosis [15]. Histological sections were prepared according to the procedure described by Chong WC [25] as following: fruits globally. It possesses a wide range of pharmacological activitiesVitis viniferadue to its (Grape) rich polyphenol is one of theingredients most consumed most of hours. which have been demonstrated to have therapeutic or health a) WashingFixation: samplesand dehydration: were fixed samplesin formalin were 10% washed for 24-48 by promoting properties [16]. As the largest group of grape b) series of increasing ethanol concentrations 50%, 70%, to have biological properties, including but not limited to 80%,water 90%, to prevent 95%, forover 1-2 fixing. hours And than transferred 100% for overnight.to graded polyphenols, flavonoids are the main candidates considered Clearing: using xylene for twice at half – one hour. antiviral, cardioprotective, and hepatoprotective [17]. Embedding: the samples were embedded by using antioxidant, anti-inflammatory, anti-cancer antimicrobial, c) ° in the thermal console for apoptotic effects of cytokines, chemotherapeutic agents, and d) 1-2 hour. gammaMoreover, radiation Flavonoids [18]. The also present have shownstudy was to aimed influence to study the solubleAfter Embedding paraffin wax tissues, in 60C samples transferred to blocks, the effects of both castration and manipulation of fresh grape let go in room temperature to acquire solidly by cryo juice on structure of liver tissue. e) console. Sectioning: histological sections were prepared by using Materials and Methods f) ° Then,auto-microtome dry by slide at dryer. thickness of (5µm), cut by tapes transferred to water bath in temperature (55-50C ). Swiss albino male mice weighting between (14-17) g., Sectionsg) are stained by using Acid Eosin Hematoxylin stain Universityand aged (3weeks) of Kufa. wereAnimals used were in the kept present in ventilated study, the cages, mice [25] as following: were obtained from the Animal House, Faculty of Science/ A. was used balanced, rodent food pellet and water were in the oven at 58-65 C° temperature for a half hour and providedwith a temperature ad libitum of [19]. (25±2Cº) All experimental at 12:12 h light,protocols dark usingcycle Paraffin wax was removed from glass slides by placing B. Section are dried from xylene then rehydration by according to guidelines for the care and use of laboratory passingplaced in through xylene (twice) a series for ofa half alcohol hour tofrom remove descending all wax. animalslive animals in biomedical were first research reviewed, [20]. approved and accepted

A total number of 45 Swiss albino mice were used in the C. concentrations for (2-3 minutes), then placed in tap D. water for (5 minutes). castration and treatment was started at the age of 21 days E. Sections are duckedplaced in in Hematoxylin acid – alcohol for for (10-15) little seconds minutes. to forpresent 6 weeks study. as: Animals were divided into 3 groups (N=15), preventSections over are washed the stain. by tap water (5) minutes. Group 1: Intact male mice received tap-water as control. Sections are washed by tap water for 5 minutes until Group 11: Castrated male mice received tap-water as return the blue color. G.F. Group 111: Castrated male mice treated daily with 10µl/g. H. Sections are washed by tap water. grape(positive juice, group). the surgical castration method was done I. DehydrationSections are placedfrom glass in Eosin slides stain by passingfor (10-30) through seconds. series of ascending concentrations of ethanol alcohol for 2-3 Plant material extract: Vitis vinifera minutes for each concentrate. according to Al-Fatlawi AA [21]. J. Sections are placed in xylene for half hour. blundered by using a commercialBlack grape blender ( without separating) obtained K. from local market (Baghdad, Iraq) 100g of grape was Then, cover specimen by cover slide with Histofluid the seeds, and then it was filtered to remove the residue. (DPX).Results accordingThe resulting to Al-Ahmadi extract (10 AA mls) [22]. was stored in the refrigerator Doseat 4˚C, Selection: and used A after previous one hour.study The documented extract was that prepared 10µl/g/ The histological sections of liver tissue from intact male protocol, 10µl /g/day are used in the present study and was can identify the boundaries of the liver lobule by locating day of grape juice extract was effective dose [23,24]. For this mice (Figure 1) showed a normal histological structure that

Homady MH, et al. The Intra Gastric Effects of Grape Juice on Histological Structure of Copyright© Homady MH, et al. Liver Tissue in Castrated Treated Mice. J Human Anat 2020, 4(1): 000147. 3 Journal of Human Anatomy

arethe centralcuboidal vein in shapeat its center,and are and arranged portal triadsin plates, (portal one vein, cell thick,hepatic usually, artery andseparated bile duct) by at sinusoids its periphery. contain The hepatocytessome blood cells (Figure 2).

Sections of liver tissue from castrated male mice (Figures peripherally3 & 4) appeared located ballooning nuclei. degenerationMoreover, castration of hepatocyte also and lipid accumulation (Macro vesicular steatosis), with hydropic degeneration and Micro vesicular steatosis with resulted in cellular inflammation (Figure 5) as well as the sections also showed acidophilic bodies (Councilman Figure 3: Cross histological section of liver tissue from cellular inflammatory of lymphocytes (Figure 6). Mean time and lipid accumulation in hepatocytes. Macro vesicular liverBody) tissue as seen to inthe (Figure normal 7). structure Treatment as of compared castrated withsubjects the castrated male mic showing Ballooning Degeneration (BD) controlwith 10µl/g group. grape juice (Figure 8) was able to restore the steatosis (MA) (X100).

Figure 1: Cross histological section of liver tissue from Figure 4: Cross histological section of liver tissue from intact male mice showing the normal structure of castrated male mice showing lipid accumulation Macro

hepatocytes (H), central vein (CV) and sinusoid(S), (X100). vesicular steatosis (MA) (X400).

Figure 2: Cross histological section of liver tissue from intact male mice showing Normal histological structure Figure 5: Cross histological section of the liver tissue from

central vein (CV), sinusoid (S), Ito cells (I) and Kupffer cells castrated male mice showing lobular inflammation (LO), (K) (X400) (X400).

Discussion The histological sections of liver tissue from castrated male mice showed ballooning degeneration in hepatocytes

and lipid accumulation (macro vesicular steatosis) lobular inflammation, Hydropic Councilman degeneration, micro vesicular mightsteatosis be withdue inflammatoryto the molecular cellular mechanisms of lymphocyte by which and apoptosis in hepatocytes ( Body). These changes

adiposetestosterone tissue deficiencyincreased hepatic is involved lipogenesis, in the in pathogenesiscreased hepatic of NAFLD.fatty acid Several B-oxidation, pathways and decreased have been export proposed: of lipids increased from the liver caused Obesity, hyperglycemia, and insulin resistance often increased lipogenesis and hepatic steatosis. Another Figure 6: Cross histological section of the liver tissue from reason may be attributed to increase the reactive oxygen

to induced cells injuries in the liver. Minehira and Gual [26] castrated male mice showing Hydropic degeneration (HD), noticedspecies (ROS) that disturbancespathway, in which in fatty was stimulatedacid oxidation by castration lead to Micro vesicular steatosis (MI) with inflammatory cellular excess lipid storage in the liver. Moreover, this observation of lymphocyte (IN), (X400) demonstrated that there are many genes involved in fatty

acid oxidation were regulated in the liver of castrated animals, and these effects were prevented by testosterone replacement like peroxisome proliferator-activated receptor Saez Lopez, et al. [27] found that the levels of PPARD, mRNA (PPARD) regulates lipid oxidation processes. castrated animals and treatment with testosterone was ableand proteinto restore were the significantlyexpression of reduced PPARD. inThis the provides livers ofa novel view of PPARD function in the modulation of hepatic lipid metabolism.

Figure 7: Cross histological section of liver tissue from in liver tissue In the present study, the increased inflammatory cells castrated male mice showing Councilman B and apoptosis consideredplay critical “two-hit” roles in theory the pathological of NAFLD ody (CO), progression proposes that [28]. inflammation, Harada, et oxidative al. [29] stress, who

Kupffer cell (K), Ito cell (I), (H&E, X 400) demonstrated that low testosterone levels increase the risk progressionfor cardiovascular of NAFLD disease in men and lead to shorter life spans. Androgen deprivation via castration caused obesity, impaired fasting glucose, excess hepatic triglyceride accumulation, thigh muscle weight loss, gene expression

response genes were activated in the livers of castrated mice.profiling Zhang indicated N, et al. that [30] several concluded immune that castration and inflammatory promote progression to steatohepatitis through activation of endoplasmic reticulum stress pathway and enhancement micro vesicular droplet steatosis. Cia Z, et al. [31] noticed

the pro-inflammatory of chemokines contributes to Figure 8: Cross histological section of liver tissue from the mechanism of inflammatory recruitment in hepatic castrated group treated with 10µl/g grape juice showing steatosis induced by testosterone deficiency.

On the other hand, tumor necrosis factor (TNF) may normal histological structure (X400). have a pathogenic role during NAFLD development, Homady MH, et al. The Intra Gastric Effects of Grape Juice on Histological Structure of Copyright© Homady MH, et al. Liver Tissue in Castrated Treated Mice. J Human Anat 2020, 4(1): 000147. 5 Journal of Human Anatomy

2. with hepatocellular injury. Testosterone has been suggestedspecifically to by act modulating directly on chronic immune lobular cells byinflammation repressing Springer,Kaplan JB, Cham Kalra pp: A, 3-11. Biggins SW (2017) Liver Anatomy oncogenic and Function. In Radiation Therapy for Liver Tumors transcription factors 3. thattranscription increased factorsoxidative (such stress as may FOS, induce JUN hepatocyte Liver. Annual review of immunology 36: 247-277. apoptosis, resulting). in Furman more severe D, et liver al. [32] injury concluded hepatic Kubes P, Jenne C (2018) Immune Responses in the 4. apoptosis was important to note that several genes liver disease–current status and future directions. Journal associated with apoptosis, including B-cell leukemia/ Demir M, Lang S, Steffen HM (2015) Nonalcoholic fatty BCL2 CASP2 BAX 5. of digestive diseases 16(10): 541-557. lymphoma-2 ( ), caspase-2 ( ), and bcl-2. The pathogenesis of nonalcoholic fatty liver disease: associated-x ( ), were up regulated in the livers of. interplayYu J, Marsh between S, Hu diet, J, Fenggut microbiota, W, Wu C (2016)and genetic The castrated pigs in response to testosterone deficiency background. Gastroenterology research and practice. presentHistological findings aresections in aggrement of liver with tissue the findings from castrated [33-35] group treated with grape juice showed the protective 6. effect of this extract on the liver tissue by returning it to its connections with obesity, fatty liver, and cirrhosis. Annual normal structure. These results may be due to the role of reviewMarengo of A,medicine Rosso C,67: Bugianesi 103-117. E (2016) Liver cancer: have been reported to multiple biological effects, including 7. antioxidantpoly phenolic activity, and flavonoids anti-diabetic compounds and hepatoprotective. were commonly steatohepatitis pathogenesis: sublethal hepatocyte Ibrahim SH, Hirsova P, Gores GJ (2018) Non-alcoholic radicals, including the superoxide and hydroxyl radicals. The 972. Flavonoids act as anti-oxidants by neutralizing oxidizing free injury as a driver of liver inflammation. Gut 67(5): 963- 8. properties of flavonoids also allow them to act as reducing have been recognized as excellent scavengers of superoxide, Trauner M (2018) New Insights into the Pathogenesis agents and presence of flavonoids and phenolic compounds hydroxyl ion and peroxyl radicals there by inhibiting lipid of Non-Alcoholic Fatty Liver Disease. Annual Review of peroxidation. 9. Pathology: Mechanisms of Disease 13(1).

Polyphenols are reported to exert hepatoprotective Kelly DM, Jones TH (2015) Testosterone and effects through increasing fatty acid oxidation in the liver 10. obesity.Jaruvongvanich Obesity V,Reviews Sanguankeo 16(7): A, 581-606. Riangwiwat T, Upala S and improving insulin sensitivity. The preventive effect of grape juice against hepatic steatosis may be predominantly nonalcoholic fatty liver disease: a systematic review and due to proanthocyanidins. meta-analysis.(2017) Testosterone, Annals sex of hepatology hormone-binding 16(3): 382-394. globulin and who stated positive effects of several classes of antioxidants,These findings such are as polyphenols within the 11. agreement of Ferramosca A [36] ofMoya intramuscular D, González injectionsLA, Janzen of E, ketoprofen Caulkett NA, on Fireheller behavioral E, reversion(resveratrol, of fatty quercetin, liver, and in coumestrol, some cases as anthocyanins, an indirect andet al. physiological (2014) Effects indicators of castration of pain method in beef and cattle. frequency Journal interactionepigallocatechin with mitochondrial gallate and curcumin), metabolism. carotenoids on the 12. of animal science 92(4): 1686-1697. Conclusion in Bucks and Cattle Calves (Doctoral dissertation, Nanaji Sawhney P (2016) Burdizzo Versus Pinhole Castration Therefore, in the present study we can conclude that ‏ Grape juice appears to act as a protective agent against the 13. DeshmukhHarada N, HanaokaVeterinary R, Science Hanada University K, Izawa T,Jabalpur). Inui H, et al. effects of whole castration.

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