THE EFFECTS OF POMEGRANATE SEED OIL ON CHRONIC LIVER DAMAGE INDUCED CARBON TETRACHLORIDE IN RATS AND DETERMINATION OF HEPATIC APOPTOSIS AND CASPASE ACTIVITY

Abstract

In this study, the protective effect, and biochemical values of pomegranate seed oil (PSO) on liver tissue lesions in rats with a chronic liver injury caused by carbon tetrachloride(CCl4)were investigated.Thirty-two rats were divided into 4 groups as 8 rats in each group. The first group was determined as control. The second group received PSO gavage at 2 ml/kg daily for 12 weeks. In the third group, 0.2 ml/kg two doses of CCl4 were injected intraperitoneally at 3-day intervals in each week. The fourth group received PSO at 2 ml/kg daily by gavage for 12 weeks, while 2 doses CCl4 at 3 days intervals in each week. While normal liver tissue structure was observed in the control groups; Macro and microvesicular fat vacuoles were less common in CCl4+PSO groups hepatocytes than CCl4 group. In PSO applications, histopathological changes were partially decreased compared to the CCl4 group, while caspase 3 and 9 activity decreased to a limited number. Serum ALT activity and liver MDA levels were significantly increased in the chronic CCl4 treated groups, while PSO applications were not significantly affected. In the experimental groups treated with chronic CCl4 with PSO, no significant effect was observed on biochemical data and histopathological lesions on liver damage.

Keywords

• Histopathology
• Immunohistochemistry
• Carbon tetrachloride
• Pomegranate seed oil
• Rat

RATLARDA KARBON TETRAKLORÜR İLE OLUŞTURULAN KRONİK KARACİĞER HASARI ÜZERİNE NAR ÇEKİRDEĞİ YAĞININ ETKİSİ VE KASPAZ AKTİVİTESİ İLE HEPATİK APOPTOZİSİN BELİRLENMESİ

Abstract

Bu çalışmada, karbon tetraklorür (CCl4) ile kronik karaciğer hasarı oluşturulan ratlarda nar çekirdeği yağının (NÇY) karaciğer dokusunda oluşabilecek lezyonlar üzerine koruyucu etkisi ve biyokimyasal değerleri araştırılmıştır.Çalışmada 32 adet, her grupta 8 rat olmak üzere 4 gruba ayrıldı. İlk grup kontrol olarak belirlendi. İkinci gruba 12 hafta her gün 2 ml/kg dozunda NÇY gavaj ile verildi. Üçüncü gruba dilüe edilen 0.2 ml/kg iki doz CCl4 her hafta 3 gün ara ile intraperitoneal olarak enjekte edildi. Dördüncü gruba 2 ml/kg dozunda NÇY 12 hafta her gün, gavaj ile verilirken her hafta 3 gün ara ile 2 doz CCl4 uygulandı.Kontrol gruplarında normal karaciğer doku yapısı gözlenirken; CCl4+NÇY grubu hepatositlerde CCl4 grubuna göre daha az yoğunlukta makro ve mikroveziküler yağ vakuolleri görüldü. NÇY uygulamalarında histopatolojik değişiklikler kısmen CCl4 grubuna göre kısmen azalırken, kaspaz 3 ve 9 aktivitesi de sınırlı sayıda azaldı. Kronik olarak CCl4 uygulanan gruplarda serum ALT aktivitesi ve karaciğer MDA düzeyleri önemli düzeyde artış gösterirken, NÇY uygulamaları istatistiki önemde etkilenmedi.Kronik CCl4 ile NÇY verilen deney gruplarında karaciğer hasarı üzerinde biyokimyasal veriler ve histopatolojik lezyonlar üzerine iyileştirici yönden anlamlı bir etkisi gözlenmedi.

Keywords

• Histopatoloji
• İmmunhistokimya
• Karbon tetraklorür
• Nar çekirdeği yağı
• Rat

References

1. Referans1 Recknagel RO, Glende EA JR, Dolak JA, Waller RL. Mechanisms of carbon tetrachloride toxicity. Pharmacol Therapeut 1989; 43: 139-154.
2. Referans2 Sun F, Hamagawa E, Tsutsui C, et al. Evaluation of oxidative stress during apoptosis and necrosis caused by carbon tetrachloride in rat liver. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 2001; 1535(2): 186-191.
3. Referans3 Weber LW, Boll M, Stampfl A. Hepatotoxicity and mechanism of action of haloalkanes: Carbon tetrachloride as a toxicological model. Crc Cr Rev Toxicol 2003; 33: 105-136.
4. Referans4 Shyur LF, Huang CC, Lo CP, et al. Hepatoprotective phytocompounds from Cryptomeria japonica are potent modulators of inflammatory mediators. Phytochemistry 2008; 69(6): 1348-1358.
5. Referans5 Moure A, Cruz JM, Franco D, et al. Natural antioxidants from residual sources. Food Chemistry, 2001; 72(2): 145-171.
6. Referans6 Naczk M, Shahidi F. Extraction and analysis of phenolics in food. Journal of chromatography A 2004; 1054(1): 95-111.
7. Referans7 Namiki M. Antioxidants/antimutagens in food. Critical Reviews in Food Science & Nutrition 1990; 29(4): 273-300.
8. Referans8 Schinella G, Mosca S, Cienfuegos-Jovellanos E, et al. Antioxidant properties of polyphenol-rich cocoa products industrially processed. Food Research International 2010; 43(6): 1614-1623.

9. Referans9 Freeman BA, Crapo JD. Biology of disease: free radicals and tissue injury. Laboratory investigation; a journal of technical methods and pathology 1982; 47(5): 412.
10. Referans10 Radi, R. Oxygen radicals, nitric oxide, and peroxynitrite: Redox pathways in molecular medicine. Proceedings of the National Academy of Sciences 2018; 115(23): 5839-5848.
11. Referans11 Manual of Histologic Staining Methods; of the Armed Forces Institute of Pathology, Luna LG (Edt), New York Blakiston Division, McGraw-Hill, 1968, 34-75.
12. Referans12 Lv P, Luo HS, Zhou XP et al. Thalidomide prevents rat liver cirrhosis via inhibition of oxidative stress. Pathol Res Pract 2006; 202: 777-788.
13. Referans13 Yehia HM, Al Olayan EM, Elkhadragy MF. Hepatoprotective role of the pomegranate (Punica granatum) juice on carbon tetrachloride induced oxidative stress in rats. Life Sci J 2013; 10: 1534-1544.
14. Referans14 Tasci I, Mas N, Mas MR, Tuncer M, Comert B. Ultrastructural changes in hepatocytes after taurine treatment in CCl4 induced liver injury. World J Gastroenterol 2008; 14: 4897-4902.
15. Referans15 Ochenashko OV, Volkova NA, Mazur SP,et al. Cryopreserved fetal liver cell transplants support the chronic failing liver in rats with CCl4-induced cirrhosis. Cell transplantation 2006; 15(1), 23-33.
16. Referans16 Gil MI, Tomás-Barberán FA, Hess-Pierce B, Holcroft DM, Kader AA. Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. Journal of Agricultural and Food chemistry 2000; 48(10): 4581-4589.
17. Referans17 Ignarro LJ, Byrns RE, Sumi D, de Nigris F, Napoli C. Pomegranate juice protects nitric oxide against oxidative destruction and enhances the biological actions of nitric oxide. Nitric oxide 2006; 15(2): 93-102.
18. Referans18 Lansky EP, Newman RA. Punica granatum (pomegranate) and its potential for prevention and treatment of inflammation and cancer. Journal of ethnopharmacology 2007; 109(2): 177-206.
19. Referans 19 Gram Yaman D, Atasever A, Eren M. Effect of pomegranate (Punica granatum) seed oil on carbon tetrachloride-induced acute and chronic hepatotoxicity in rats. Pharmacognosy Research 2018; 10(2): 124-129.
20. Referans20 Ali EH, Al-Okaily BN. Role of Pomegranate Seed Oil (PSO) Against Hepatotoxicity Induced by Sodium Fluoride in Adult Female Rats. Journal of Kerbala for Agricultural Sciences 2017; 10(3): 28-43.‎
21. Referans21 El-Khadragy MF. Hepatoprotective role of the pomegranate (Punica granatum) juice on carbon tetrachloride-induced oxidative stress in rats. Afr J Biol Sci 2011; 7: 135-49.
22. Referans22 Aram G, Potter JJ, Liu X, Torbenson MS, Mezey E. Lack of inducible nitric oxide synthase leads to increased hepatic apoptosis and decreased fibrosis in mice after chronic carbon tetrachloride administration. Hepatology 2008; 47(6), 2051-2058.
23. Referans23 Chan CC, Lee KC, Huang YH, et al. Regulation by resveratrol of the cellular factors mediating liver damage and regeneration after acute toxic liver injury. Journal of gastroenterology and hepatology 2014; 29(3): 603-613.
24. Referans24 Leu JI, Crissey MAS, Taub R. Massive hepatic apoptosis associated with TGF-β1 activation after Fas ligand treatment of IGF binding protein-1–deficient mice. The Journal of clinical investigation 2003; 111(1): 129-139.
25. Referans25 Lu B, Xu Y, Xu L, et al. Mechanism investigation of dioscin against CCl4-induced acute liver damage in mice. Environmental toxicology and pharmacology 2012; 34(2): 127-135.
26. Referans26 Xie J, Liu J, Chen TM, et al. Dihydromyricetin alleviates carbon tetrachloride-induced acute liver injury via JNK-dependent mechanism in mice. World Journal of Gastroenterology: WJG 2015; 21(18): 5473-5481.
27. Referans27 Parajuli DR, Park EJ, Che XH, et al. PF2401-SF, standardized fraction of Salvia miltiorrhiza, induces apoptosis of activated hepatic stellate cells in vitro and in vivo. Molecules 2013; 18(2): 2122-2134.
28. Referans28 Tao LL, Cheng YY, Ding D, et al. C/EBP-α ameliorates CCl4-induced liver fibrosis in mice through promoting apoptosis of hepatic stellate cells with little apoptotic effect on hepatocytes in vitro and in vivo. Apoptosis 2012; 17(5): 492-502.
29. Referans29 Domitrović R, Škoda M, Marchesi VV, et al. Rosmarinic acid ameliorates acute liver damage and fibrogenesis in carbon tetrachloride-intoxicated mice. Food and chemical toxicology 2013; 51: 370-378.
30. Referans30 Ibrahim MAR, Okail HAM, Emam NMM. Ameliorative Effects of Pomegranate Peel Extract on Hepatotoxicity Induced by Carbon Tetrachloride in Mice.International Journal of Research Studies in Biosciences (IJRSB) 2016; 4(10): 23-31.
31. Referans31 Çayır K, Karadeniz A, Şimşek N, et al. Pomegranate seed extract attenuates chemotherapy-induced acute nephrotoxicity and hepatotoxicity in rats. Journal of medicinal food 2011; 14(10): 1254-1262.
32. Referans32 Shaban NZ, El-Kersh MA, Bader-Eldin MM, Kato SA, Hamoda AF. Effect of Punica granatum (pomegranate) juice extract on healthy liver and hepatotoxicity induced by diethylnitrosamine and phenobarbital in male rats. J Med Food 2014; 17: 339-349.
33. Referans33 Choudhury S, Ghosh S, Mukherjee S, et al. Pomegranate protects against arsenic-induced p53-dependent ROS-mediated inflammation and apoptosis in liver cells. The Journal of nutritional biochemistry 2016; 38: 25-40.
34. Referans34 Tan H, He Q, Li R, Lei F, Lei X. Trillin reduces liver chronic inflammation and fibrosis in carbon tetrachloride (CCl4) induced liver injury in mice. Immunological investigations 2016; 45(5): 371-382.
35. Referans35 Yim YK, Lee H, Hong KE, et al. Hepatoprotective effect of manual acupuncture at acupoint GB34 against CCl4-induced chronic liver damage in rats. World journal of gastroenterology: WJG 2006; 12(14): 2245.
36. Referans36 Ahmed SK, Mohammed SA, Khalaf G, Fikry H. Role of bone marrow mesenchymal stem cells in the treatment of CCl4 induced liver fibrosis in albino rats: a histological and immunohistochemical study. International journal of stem cells 2014; 7(2): 87-97.
37. Referans37 Achliya GS, Wadodkar SG, Dorle AK. Evaluation of hepatoprotective effect of Amalkadi ghrita against carbon tetrachloride induced hepatic damage in rats. J Ethnopharmacol 2004; 90: 229-232.
38. Referans38 Zeashan H, Amresh G, Singh S, Rao CV. Hepatoprotective activity of Amaranthus spinosus in experimental animals. Food and Chemical Toxicology 2008; 46: 3417-3421.
39. Referans39 Gnanaprakash K, Madhusudhana CC, Ramkanth S et al. Aqueous extract of Flacourtia indica prevents carbon tetrachloride induced hepatotoxicity in rat. Int J Biol Sci 2010; 6: 51-55.
40. Referans40 Abdelaziz DH, Ali SA. The protective effect of Phoenix dactylifera L. seeds against CCl4-induced hepatotoxicity in rats. Journal of ethnopharmacology 2014; 155: 736-743.
41. Referans41 Botsoglou NA, Taitzoglou IA, Botsoglou E et al. Effect of long-term dietary administration of oregano and rosemary on the antioxidant status of rat serum, liver, kidney and heart after carbon tetrachloride-induced oxidative stress. J Sci Food Agric 2009; 89: 1397-1406.
42. Referans42 Ahsan R, Islam KM, Musaddik A, Haque E. Hepatoprotective activity of methanol extract of some medicinal plants against carbon tetrachloride induced hepatotoxicity in Albino rats. Global J Pharm 2009; 3: 116-122.
43. Referans43 Palaniswamy R, Raghunathan PP. Protective effect of Bacopa monnieri leaf extract against oxidative stress induced hepatotoxicity in rats. Int J Pharm Pharmac Sci 2013; 5: 555-558.
44. Referans44 Melo ILP, Silva AMO, Carvalho EBT, et al. Effect of Pomegranate (Punica granatum L.) Seed Oil on Markers of Oxidative Stress Induced by Carbon Tetra-chloride in Wistar Rats. Int J Food Sci Nutr Diet 2015; 5, 1-8.
45. Referans45 Boroushaki MT, Mollazadeh H, Rajabian A, et al. Protective effect of pomegranate seed oil against mercuric chloride-induced nephrotoxicity in rat. Renal failure 2014; 36(10), 1581-1586.
46. Referans46 Ali NA, Saeed SZ. Pomegranate seed oil ameliorates paracetamol induced hepato-and nephro-toxicity in rats. Middle East Journal of Internal Medicine 2013; 6(2): 1-9.
47. Referans47 Abozid MM, Farid HE. The anti-fatty liver effects of guava leaves and pomegranate peel extracts on ethanol-exposed rats. J Biol Chem Environ Sci 2013; 8(3): 83-104.