دراسة نسجية فسلجية ووراثية لتأثير المستخلص المائي لثمار نبات توت العليق Rubus ulmifolius في الاجهاد التاكسدي المستحث بعقار السيكلوسبورين في ذكور الجرذان البيض

Histological, physiological and genetic study of the effect of the aqueous extract of the fruits of the raspberry plant Rubus ulmifolius on oxidative stress induced by cyclosporine in male albino rats

The current study aimed to investigate the protective effect of the aqueous extract of Rubus ulmifolius fruit in male albino rats treated with cyclosporine (CsA) by studying some histological, physiological, and genetic changes. The study revealed the chemical content of the aqueous extract of raspberry fruit and characterized the compounds using Gas Chromatography Maas Spectrometry (GC-MS). Nine chemical compounds were identified: 12,9-Octadecadienoic acid / Hexadecanoic acid, cyclohexyl ester / Methyl trans-9-octadecenoate / 2-Chloroethyl linoleate / Dimethylcyclohexene 4,4 / Ethylimidazole 2- / Pentanedinitrile, 2-methyl / -Dioxolane, 2-pheny 1,3- / 2-Amino-2-oxoethyl acetate
The study was conducted in the holy Karbala Governorate during the period extending from mid-October 2024 to the end of February 2025. The study was implemented in the animal house of the College of Pharmacy / University of Karbala and in the laboratories of the College of Education for Pure Sciences / Department of Biology.
Forty-eight adult male rats, weighing between (230-250 g) and aged between (12-14) weeks, were divided into two experiments. The first experiment was to test the effective dose of the aqueous extract of Rubus ulmifolius plant. It included determining the most effective concentration of the aqueous extract of Rubus ulmifolius plant among three ascending concentrations (200, 300, 400 mg/kg). Its protective role against oxidative stress caused by treatment with cyclosporine was tested for 30 days. 24 male white rats were used and divided systematically into four groups, with (6) animals for each group. The first group was the negative control group, which received normal water, and the first prevention group was dosed orally with the aqueous extract of Rubus ulmifolius plant at a concentration of (200 mg/kg) of body weight, 4 hours before being dosed with cyclosporine at a concentration of (0.2) mg/kg of The second prevention group received an oral dose of 300 mg/kg of raspberry aqueous extract, 4 hours before receiving 0.2 mg/kg of cyclosporine. The third prevention group received an oral dose of 400 mg/kg of Rubus ulmifolius aqueous extract, 4 hours before receiving 0.2 mg/kg of cyclosporine. 24 hours after the last dose, blood samples were drawn by cardiac puncture and left for 30 minutes to clot. They were then centrifuged (3,000 rpm for 15 minutes) to separate the serum, which was used to estimate superoxide dismutase (SOD) activity, which has been used as an indicator of the extract’s effectiveness against the oxidative stress caused by cyclosporine treatment. The second experiment used (24) white male rats, which were randomly distributed into four groups (6 rats per group). The first was left untreated and was considered the negative control group (G1). The second group (G2) was dosed every other day with cyclosporine at a concentration of (0.2) mg/kg of body weight for a period of (30) days and was considered the positive control group. The third group (G3) was dosed daily with the aqueous extract of Rubus ulmifolius fruit at a concentration of (400) mg/kg of body weight for a period of (30) days. As for the fourth group (G4), it was dosed daily with the aqueous extract of Rubus ulmifolius fruit at a concentration of (400 mg/kg) of body weight, 4 hours before being orally dosed with cyclosporine at a concentration of (0.2 mg/kg) every other day for a period of 30 days. Blood samples were collected for the above groups one month after dosing. Histological sections of the liver, kidneys, and testes were taken to study the histological changes, which included: measuring the diameters of the hepatocytes, sinusoids, and central vein diameters, as well as measuring the diameters of the glomerulus, proximal convoluted tubule, and distal convoluted tubule. Blood serum was taken to measure the following biochemical parameters: levels of liver enzymes Aspartate transaminase (AST), Alanine transaminase (ALT), Alkaline phosphatas, and estimating the concentration rate of total protein, albumin, globulin, urea, creatinine, Malondialdehyde (MDA), and glutathione. Glutathione (GSH), superoxide dismutase (SOD) activity, catalase (CAT) concentration, and serum levels of testicular hormones including testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured. A comet assay, also called the liver comet test, was also performed to measure the rate of DNA damage.
Dosing experimental animals with cyclosporine (CsA) resulted in histological changes in the liver, represented by congestion and dilation of the central vein, severe irregularity of the hepatic cords, congestion and dilation of the portal vein, severe infiltration of inflammatory cells with dilation of the hepatic sinusoids, degeneration and necrosis of hepatocytes. A significant increase (P<0.05) was observed in the average diameters of the hepatocytes, sinusoids, and central vein compared to the negative control group. The histological changes of the kidney were represented by atrophy of the glomerulus, increased Bowman’s space, and destruction of the walls of the urinary tubules, with congestion of the blood vessels between the renal tubules, sloughing of the lining epithelium, infiltration of inflammatory cells, hemorrhage between the renal tubules, degeneration of the urinary tubule cells, and necrosis of the tubular lining, with a significant decrease (P<0.05) in the average diameters of the glomerulus, the proximal convoluted tubule, and the distal convoluted tubule. As for the histological changes specific to the testis, they were represented by the presence of interstitial spaces between the seminiferous tubules, and there were few sperm in the tubule cavities, with a reduction and disintegration of the interstitial tissue, a decrease in the Leydig cells, a decrease in the size of the germinal epithelial cell layer, degeneration of the germinal layer, and disintegration of its cells, compared to the negative control group (G1). A significant increase (P<0.05) was observed in the average levels of liver enzymes (AST, ALT, ALP), urea, creatinine, and MDA, while a significant decrease (P<0.05) was observed in the average levels of total protein, albumin, globulin, GSH, SOD, CAT, and testosterone (LH) (FSH). Oral administration of the aqueous extract of Rubus ulmifolius to rats in the G3 did not show any histological changes in the liver, kidney, and testicular tissues compared to the negative control group (G1). There were no significant differences in the average diameters of both the liver and kidneys in the G3 compared to the negative control group. A significant increase (P < 0.05) was observed in the level of total protein, globulin, albumin, GSH, SOD, and testosterone (LH) (FSH) for the G3 compared with the positive control group (G2). A significant decrease (P < 0.05) occurred in the level of enzymes (ALP, AST, ALT), urea and creatinine levels, and in the level of MDA in the G3 compared with G2.
In the fourth group (G4), the tissue was closer to normal compared to the negative control group (G1). As for the diameter rate, a significant decrease (P<0.05) was observed in both groups for the diameter rate of each of the hepatocytes, central vein, and sinusoids compared to the positive control group (G2). As for the kidney tissue, the histological changes in the fourth group (G4) were represented by no clear damage to the glomerulus, and the tissue was closer to normal. There was the presence of Bowman’s capsule and Bowman’s space with the normal structure of the distal and proximal urinary tubules compared to the positive control group (G2). There was a significant increase (P<0.05) in the diameter rate of each of the glomerulus, the proximal convoluted tubule, and the distal convoluted tubule compared to the positive control group (G2), respectively. As for the histological changes in the testicular tissue in the G4, the tissue was observed to be closer to normal, as sperm-generating cells appeared, and their cavities were filled with sperm, with the presence of Leydig cells in the interstitial tissue and an increase in the number of cell layers. The germinal epithelium that forms sperm. The results of the current study also showed that the group treated with aqueous extract of Rubus ulmifolius at a concentration of (400) mg/kg with cyclosporine showed a significant decrease (P<0.05) in the levels of liver enzymes (ALT, AST, ALP), urea, creatinine and MDA and a significant increase (P<0.05) in the levels of total protein, globulin, albumin, SOD, GSH, CAT, and the levels of testosterone ,LH and FSH compared to the positive control group (G2). Regarding the genetic aspect of liver tissue, a significant increase (P<0.05) was observed in the rate of DNA molecule fragmentation in the second group (G2) dosed with (0.2 ) mg/kg cyclosporine compared to the negative control group (G1).
While there was a significant decrease (P<0.05) in the percentage of DNA fragmentation in the third group (G3), which was treated with the aqueous extract of mulberry fruit, and also in the G4, which was treated with the aqueous extract of Rubus ulmifolius four hours before cyclosporine, compared to the positive control group (G2). The current study concludes the effectiveness of the aqueous extract of Rubus ulmifolius at a concentration of 400 mg/kg in inhibiting free radical activity and reducing cyclosporine-induced oxidative stress in liver, kidney and testicular tissue, as well as some functional and genetic parameters in male albino rats.