الفهرس 
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Abstract
Aging is marked by a progressive disturbance in the cellular functions and integrity resulting in increased vulnerability to certain age-related diseases. The percentage of older people (≥ 60 years old) is expected to increase from 12% in 2015 to 22% by 2050. In addition, the number of the oldest-old people (≥80 years old) is expected to increase by 3 folds at the same time period. The increase in the aging population needs to live in a healthy status without any diseases. The present study was designed to focus on the antiaging mechanisms of both Astragalus membranaceus telomerase activator (TA-65) and Punica granatum (pomegranate) supplements against aging-associated male infertility, kidney dysfunction, and hepatic apoptosis. In addition, their effects were studied on the expression of cyp3a1 gene as a marker for potential drug interactions.
To fulfill the aim, thirty old Wistar rats (10-12 months) as well as ten young adult rats (2 months) were divided into four groups; young control, aged control, aged rats treated orally with Ta-65 (500mg/ kg/ day) for two consecutive months, aged rats treated orally with pomegranate (250mg/ kg/ day) for two consecutive months.
The weights of rats were recorded weekly throughout the experimental period. At the end of the experiment, serum samples, liver, kidneys, spleen, and testes were collected for analyses. The relative organs weights were calculated. Histological abnormalities were observed in testis by light microscope and liver by light and electron microscope. Kidney function tests (creatinine, uric acid, sodium, potassium), male fertility hormones (testosterone, LH, and FSH), and liver function tests (ALT, total protein, albumin, total globulins) and insulin like growth factor-1 (IGF-1) were assayed in serum. In addition, oxidative stress markers (malondialdehyde “MDA”, glutathione S-transeferase “GST”, and protein carbonyl), the relative expression of hepatic telomerase reverse transcriptase (Tert), thioredoxin reductase 1 (TrxR1), cytochrome 3a1 genes, and the protein expression of p53 were quantified. The hepatic active cleaved Caspase-3 activity was determined in the cytosolic supernatant of the liver homogenate.
Normal aging significantly reduced the body weight and the relative weights of liver, kidney, and testes without affecting the relative spleen weight. These significant declines were not ameliorated after the administration of Ta-65 or pomegranate supplements to the aged rats.
Normal aging significantly elevated the levels of creatinine and uric acid in serum compared to the young rats, while serum levels of sodium and potassium were significantly decreased. In aged rats administered with both Ta-65 and pomegranate, serum levels of creatinine, uric acid, sodium, and potassium were sustained to the young levels.
Aging decreased male fertility by significant reductions in sperms count, sperms motility percentage, in association with significant elevations in the percentages of sperms abnormality and immotility, compared to the young rats. Abnormal sperms were observed in aged rats (tailess, headless, or sperms with U-shaped tail). Treatment of aged rats with Ta-65 improved male fertility by increasing both sperm count and motility percentage as well as decreasing the percentage of abnormalities in morphology, compared to aged rats. Similarly, oral administration of pomegranate improved the percentage of both sperms abnormalities and motility, compared with aged control rats, but this improvement in sperm abnormalities remained significantly different from that of young rats.
Significant reductions in the serum levels of testosterone, FSH, and LH of aged rats were reported. Administration of Ta-65 or pomegranate to aged rats significantly improved serum levels of testosterone, FSH, and LH compared to the aged group, but the levels were still significantly lower than the normal young levels.
Microscopical examination of cross section of testes revealed that normal aging caused deleterious histological abnormalities in testes in the form of progressive testicular degeneration and absence of spermatogenesis as well as loose connective tissue and Leyding cell aggregation in the interstitial compartment. Treatment with Ta-65 and pomegranate improved the histological structure of testes with normal spermatogenesis, well preserved Sertoli cells and well delineated tubular basement membrane. The interstitium between tubules and Leydig cells was also intact.
Normal aging did not cause any significant change in serum albumin level or ALT activity in serum, but significantly declined the serum IGF-1 level compared to the young rats. However, aging significantly increased the levels of total protein and globulins with a significant reduction in A/G ratio compared to the young rats. Administrations of Ta-65 and pomegranate to aged rats normalized the levels of total protein and globulins in serum as well as A/G ratio. Ta-65 treatment of aged rats alleviated the serum IGF-1 level. Administration of pomegranate to aged rats improved the serum level of IGF-1 compared to the aged rats, but was still lower than the young level.
Pronounced elevations in the hepatic contents of MDA and serum protein carbonyl were reported in the aged rats compared to young rats, along with a significant decrease in the hepatic GST activity. In aged+Ta-65 and aged+pomegranate groups, hepatic MDA and protein carbonyl contents as well as GST activity returned to the young control level.
The relative expression of hepatic Cyp3a1, Tert, and TrxR1 genes was significantly down-regulated in aged rats compared to the young expression level. Ta-65 and pomegranate administrations to aged rats significantly up-regulated the relative expression of these genes compared to aged rats.
normal aging increased the hepatocyte apoptosis by up regulating the protein expression of p53 and increasing the hepatic active cleaved Caspase 3 activity. Treatments of aged–induced apoptosis either by Ta-65 or pomegranate supplements declined the expression of p53 protein near to the normal young expression level and reduced hepatic Caspase 3 activity compared to aged rats.
Liver sections from aged rats illustrated the presence of hepatocytes with moderate intra-lobular inflammatory infiltration. Karyomegaly and binucleation were seen in some hepatocytes. In addition, many hepatocytes had dilated rough endoplasmic reticulum, many lysosomes and many fat droplets. Furthermore, hepatocytes showed mild steatosis. Moreover, many apoptotic hepatocytes characterized by the presence of shrunken nuclei were seen, this was also supported by results of the immunohistochemistry.
Treatment of aged rats with Ta-65 or pomegranate for 2 consecutive months improved the histological findings of liver in which normal hepatocytes were radiating from the central vein and arranged in single cell cords. No signs of steatosis or apoptosis were observed in hepatocytes. Liver central vein, portal vein, portal tract and bile duct were with average size. The space of Disce had normal hepatic stellate cells without any signs of collagen strands. Few lysosomes were seen without any signs of apoptosis or necrosis, this was also supported by the results of immunohistochemistry.