الفهرس 
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Abstract
Cancer is a very complicated difficulty affecting people around the world. There are many experimental and clinical trails of scientists to find an acceptable strategy to combat tumors. The most common primary malignancy of the liver tumors is hepatocellular carcinoma (HCC). The liver plays many vitalmetabolic functions as expulsion of waste metabolites, detoxification, blood coagulation, homeostatic activities, storage of vitamins and excretion of bile and hormones withother significant functions that make it exposed to injury and development of tumors.
In view of this fact, this study is designed to assess the preventive effects of pomegranate(Punicagranatum) aril juice, aqueous extract of seeds, aqueous extract of husks and their mixture on the hepatic injury and hepatocarcinogenesis-induced by 7,12-dimethylbenz(a)anthracene (DMBA) and carbon tetrachloride (CCl4) administration as well as, on cardiorenal toxicity in male Wistar rats.
For this target, sixty male Wistar rats were used in the experiment and were alocated into six groups as follow:
1-Normal group:
Animals of this group were given orally 10 ml/kg b.w. mineral oil for two days and distilled water by oral gavages 10 ml/kg b.w. every day for sixteen weeks and injected saline subcutaneously at dose of 3 ml/kg b.w. (weekly for thirteen weeks). The distilled water and mineral oil were vehicles in which Punica granatuimwas infused and DMBA was dissolved respectively while saline was given as isotonic solution equal to the volume of CCl4solution.
2- DMBA/CCl4 control group:
Animals of this group were injected with DMBA at dose 50 mg/kg b.w. in 10 ml mineral oil divided on two days as hepatocarcinogenisesinitiator. After three weeks CCl4 has been injected subcutaneously at dose level of 3 ml/kg b.w./week for thirteen weeks in the thoracic area till the end of the experiment as a promotor. Distilled water was orally and daily given also at10 ml/kg b.w.during the experimental period as a vehicle of P. granatum infusions.
3- DMBA/CCl4 and PJ group:
Rats of this group were injected as the second group and administered fresh crude P. granatumaril juice (PJ) at a dose level of 10 ml/kg b.w. daily for the sixteen weeks of the experiment. After the third week, CCl4 injection started at dose 3 ml/kg b.w./week for thirteen weeks after the third week of DMBA injection.
4-DMBA/CCl4 and PSE group:
Rats of this group were given DMBA and CCl4 solutions at the same doses and were orally administered P. granatum seed extract (PSE) at dose 400 mg/kg b.w./day for sixteen weeks.
5-DMBA/CCl4 and PHE group:
This group was administered the same doses of DMBA and CCl4 for the same duration and route of the DMBA/CCl4 control group. Furthermore, this group was orally treated with P. granatum husk extract (PHE) at dose 400 mg/kg b.w./day for sixteen weeks.
6-DMBA/CCl4 and PM group:
This group was treated with the same solutions of DMBA and CCl4 exactly as in DMBA/CCl4 control group and treated also with P. granatum mixture of extracts and juice (PM) at a dose level of 10 ml/kg b.w./day for sixteen weeks.
Data of the present study revealed a significant elevation in serum ALT, AST, ALP andγGTenzymesactivites as well as, total bilirubin and globulin levels in DMBA/CCl4-administeredcontrol groupwhile albumin and total protein levels and albumin/globulin were significantly reduced as compared with normal group. In addition, oxidative stress parameters as malondialdehyde (MDA) and nitric oxide (NO) levels showed an increased production in the liver tissue.
Conversely, antioxidants as GPx, GST and SOD activities as well as level of GSH andTAC were decreased in DMBA/CCl4-administered control group. The mRNA expressions of P53, Bcl-2and IL4 in liver tissue were decreased while expressions of COX-2, TNF-α and NF-κB were increased in DMBA/CCl4-administered groups. These results are supported by the histopathological examination which established a precancerous lesions andmassive destruction in the liver tissue represented as oval cells of proliferated fibrous connective tissue, inflammatory cells infiltration, necrosis of hepatic cells, dilated hyperemic sinusoids, karyomegally, and fatty change of hepatocytes.
The results also showed that the treatment ofDMBA/CCl4-administered rats with P. granatum juice and aqueous extracts of the seeds and husks and their mixture restored the normal activity of ALT, AST and γGT enzymes as well as the values of total bilirubin, total protein, albumin, globulinand A/G. Furthermore,P. granatumaril juice and aqueous extracts of seeds and husks and their mixtureincreased the activities of antioxidant enzymes as GPx, GST and SOD, increased the tissue content of GSH and TAC and decreased oxidative stress parameters MDA and NO.
Additionally, the altered mRNA expressions of detected genes in liver tissue were remarkably improved;the elevated COX-2, TNF-α and NF-κB were decreased and the lowerd P53, Bcl-2and IL4expressions increased as a result of treatment with P. granatumaril juice, aqueous extract of seeds, aqueous extract of husks and their mixture.Regulation of genes expression revealed the antiapoptotic, anti-inflammatory and anticarcinogenesis effects of P. granatumjuice and extracts. These results were confirmed with the improvement of the histopathologicalalterations which were reversed inDMBA/CCl4-administered groups due to treatments reflecting the therapeutic efficacy of P.granatum.
Represented data also showed that DMBA/CCl4 induced considerable nephrotoxicity which was remarked by increased serum urea, uric acid, creatinine and K concentration and decreased Na ions. As well, oxidative stress markers as MDA and NO in the kidney were increased inDMBA/CCl4-administered control group. On the other hand, the activity of antioxidant enzymes as GPx, GST and SOD were decreased and the tissue content of GSH and TAC were diminished.The biochemical alterations come as a result of DMBA/CCl4 metabolites that inducedtissue damage. This damage of the kidney tissue demonstrated as glomerulitis and focal interstitial nephritis, inflammatory cells infiltration, thickening of parietal layer of Bowman’s capsule and necrobiotic changes.
Pomegranate aril juice and aqueous extracts of seeds and husks and their mixture treatment induced remarkabledecrease and normalization in the kidney functions parameters in serum including urea, uric acid, creatinine and K concentration while Na level wasincreased. Moreover, antioxidant enzymes GPx, GST and SOD activities and levels of GSH and TAC were increased in kidney of P. granatum treated groups. On the contrary, oxidative stress markers as MDA and NO were significantly decreased in the kidney of DMBA/CCl4-administered animals treated with P. granatumaril juice and seeds and husks aqueous extracts and their mixture. The improvement of biochemical andantioxidants status reflected recovery of kidney to almost normal histological architecture and integrity. Whoever, there were slight alterations still present in the kidney tissue of the treated groups represented by some congestion of glomerular tuft and necrobiotic changes of epithelial lining renal tubules.The normalization of kidney function parameters and improvement of kidney tissue architecture revealed the antioxidant property and therapeutic effects of P. granatumariljuice,seed and husk aqueous extracts, and their mixture.
The obtained results also showed DMBA/CCl4-induced cardiotoxicity which was remarkedby increase in serum parameters related toheart function CK, CK-MB, AST and LDH enzyme activities of the control group as compared with normal group. Histopathological examination of heart sections from DMBA/CCl4-administered control group showed deleterious effects of DMBA/CCl4 which were illustrated as multifocal myocarditis and inflammatory cell infiltration as well as congestion of myocardial blood vessels.
The treatment of DMBA/CCl4-administered groups with P.granatumaril juice, seeds aqueous extract, husks aqueous extract and their mixture recovered the heart functions and decreased serum activities of CK, CK-MB, AST and LDH enzymes as compared with DMBA/CCl4-administered control group. These results come in the same line with the histopathological results which cleared enhancement of heart tissue as a result of P. granatumtreatment ofDMBA/CCl4-administered animals.
Overall, it can be concluded that, P. granatumaril juice, seed aqueous extract, husk aqueous extract and their mixture havetherapeutic valuesand chemoprevention of DMBA/CCl4-induced hepatic carcinogenesis. This hypothesis was confirmed in terms of normalization of biochemical markers, oxidative stress, genes expression and tissue improvement. Thus, the P. granatumaril juice, seed and husk aqueous extracts and their mixture successfully counteract DMBA and CCl4-induced liver deleterious changes and precancerous lesions. Based on the obtained result, it can be concluded that pomegranate has antioxidant properties and synergistic effects of its bioactive compounds that can prevent DMBA/CCl4-induced liver, kidney and heart damage.