دراســة منــاعية و نســجية لإناث الجرذان المصــابات بتكيـس المبايض المتعدد المســتحث وتقييم تأثير المعاملة للمســتخلص النــانوي لنبات الهليون Asparagus officinalis

Immunological and Histological study of female rats with induced Polycystic Ovary Syndrome and Evaluation of the treatment effect of Asparagus officinalis Nano-Extract

The present study aimed to prepare and characterize the green Nanocomposite from fresh asparagus stems Asparagus officinalis (Asp.), improve its biological activity and evaluate its biological efficacy before and after loading it with Metformin (Met.) in female Albino Rats induced with Polycystic Ovary Syndrome (PCOS). This study was conducted in the laboratories of Department Biology / College of Education for Pure Sciences / University of Kerbala, during the period from 10/8/2023 to 2/20/2024. 40 white rats were used, including five males for pregnancy only in the last axis of the experiment and 35 female rats were divided into seven groups, each group included five females. The first group (G1) was the control group, dosed with Physiological Saline solution, while the second group (G2) was the induction group, dosed orally with Letrozole dissolved in 1% Carboxymethylcellulose (CMC), whose concentration was prepared according to the average weights of the females for a period of 28 days. The third group (G3) was dosed orally with Metformin. At a concentration of 500 mg/60 kg of body weight, and the fourth group (G4) was orally dosed with the aqueous extract of fresh tender asparagus stems Asp. At a concentration of 300 mg/kg of body weight, the fifth group (G5) was orally dosed with the nano-asparagus complex Asp.NPs at a concentration of 300 mg/kg of body weight, the sixth group (G6) was orally dosed with the aqueous extract of fresh tender asparagus stems loaded with metformin Asp.+Met. At a concentration of (300) mg/kg of body weight, the seventh group (G7) was orally dosed with the nano-asparagus complex loaded with metformin Asp.NPs+Met. At a concentration of (300) mg/kg of body weight.
Polycystic Ovary Syndrome was induced in all groups except the control group, after which the second group was left as the positive control group and the remaining groups were treated, where the animals in all the treated groups were dosed once a day for 28 days, after which two of each group were sacrificed and three females remained to complete the recovery process and pregnancy until they were sacrificed at the end of the experiment.
In this study, Nano-Asparagus complex and fresh soft asparagus stem extract were prepared and characterized before and after loading with Metformin. Nano-Asparagus complex was prepared by sieving 20% of Asparagus stem extract on 80% of silver nitrate AgNO3 solution and loaded with the drug after which it was diagnosed.
Fourier Transform Infrared Technique (FT-IR) spectroscopy showed the appearance of new diffraction levels and a clear shift in the waves of the hybrid nanocomposites and the aqueous extract of asparagus stems, which clearly indicates the success of the loading process. Atomic Force Microscope (AFM) images also showed changes in the surface of Nanocomposite loaded with the treatment and the aqueous extract of Asparagus plant loaded with the treatment, and that these changes are within the nanoscale sizes and dimensions that are all consistent with the results of the infrared spectrum (FT-IR). Scanning Electron Microscopy (SEM) images also showed the homogeneity of the distribution of the particles and most of the shapes of the particles are spherical, with a clear difference between the images of the nanocomposite and the aqueous extract before and after loading them with metformin, and it also showed that the average particle sizes are within the specified range for Hybrid Nanocomposites.
The results of the current study on Physiological Axis indicated changes in the studied hormones, as the results of the PCOS induction group in female white rats (G2) indicated a significant decrease (P˂0.05) in the level of both Progesterone hormone (Prog) and Estradiol hormone (E2), and a significant increase (P˂0.05) in the level of Testosterone hormone (Testo) compared with the negative control group (G1), while in the treatment groups the results showed a significant increase (P˂0.05) in the level of (Prog) hormone and a significant decrease in the level of (E2) hormone in (G3) compared with (G2), as well as indications of a significant increase (P˂0.05) in the level of Testosterone hormone (Testo) and a significant decrease in the level of (E2, Prog) hormones in (G4) compared with (G2), while it indicated a significant decrease (P˂0.05) in the level of (E2 and Testo) hormones and a significant increase (P˂0.05) in the level of (Prog) hormone in (G5) compared to (G2), and a significant decrease (P˂0.05) in the level of (Testo) hormone and a significant increase (P˂0.05) in the level of (Prog) hormone in (G6) compared to (G2), and it also showed a significant decrease (P˂0.05) in the level of (E2 and Testo) hormones and a significant increase (P˂0.05) in the level of (Prog) hormone in (G7) compared to (G2).
The results of the treatment groups compared with Metformin indicated clear changes in the level of hormones, as they indicated a significant increase in the hormone (LH) for all treatment groups compared to (G3), while they indicated a significant increase (P˂0.05) in the level of the hormone (FSH) in (G5) and a significant decrease in (G7, G6), and indicated a significant increase (P˂0.05) in the hormone (E2) in each of (G7, G6, G5, G4), and a significant decrease (P˂0.05) in the level of the hormone (Testo.) in each of (G7, G6, G5, G4), and also showed a significant increase (P˂0.05) in the level of the hormone (Prog.) in each of the groups (G7, G6) and a significant decrease in the group (G5, G4).
The results of the current study of Blood Parameters showed a significant increase (P<0.05) in Blood parameters (White Blood Cell count WBC, Lymphocyte ratio Lym, Granulocyte ratio GRAN, and Red Blood Cell count RBC) in the PCOS induction group (G2) compared with the control group (G1). As for the groups treated with treatments (G7, G6, G5, G4, G3), there was a significant decrease (P<0.05) in all blood parameters (WBC, Lym, GRAN, RBC) compared with the PCOS induction group (G2). While comparing the treatment groups with metformin, we note a significant increase (P<0.05) in the level of (Lym, WBC) in (G7, G4) and a significant increase in (G6, G5), and a significant increase (P<0.05) in the level of (GRAN) in (G7, G6, G4) and a significant increase in (G5), and indicated a significant increase in the level of (RBC) in (G7, G6, G5, G4).
The results of the studied Immune Parameters, namely the cytokines IL-17 and IL-10, indicated a significant increase (P<0.05) in the level of (IL-17, IL-10) in the second group (G2), the PCOS induction group, compared to the control group (G1), while we note that there was a significant increase (P<0.05) in the level of the cytokine IL-10 in all treatment groups (G7, G6, G5, G4, G3) compared to the PCOS induction group (G2), while a significant decrease (P<0.05) in the level of IL-17 was observed in all treatment groups (G3-Met., G4-Asp., Asp.NPs – G5, G6- Asp.+Met., G7- Asp.NPs+Met.) compared to the PCOS induction group (G2).
While comparing the IL-10 level of the treatment groups with Metformin, we noticed a significant decrease (P<0.05) in (G7, G5, G4) and an increase in (G6), and indicated a significant decrease (P<0.05) in the IL-17 level in (G7, G5, G4) and a significant increase in (G6).
The results of Hemotoxylin-Eosin stained Tissue sections of the ovaries of female rats in the control group and the groups in which polycystic ovaries were induced and treated with therapeutic treatments showed that the results were almost similar to those in normal rats, as the induction was successful in all groups and changes appeared in the shape and size of the ovary and the number of cysts, which was large. Then this induction was treated well, as (G3) showed that Metformin drug treated the cysts, but it needed a long period of time, while (G4), (G5) and (G7) the treatment period was only 28 days, which reduced the number of cysts that formed in the ovary, while (G6) took more than 28 days to fully recover.
While the results of Current study showed that the average number of births was 8-9 in the (G7) group treated with Asp.NPs+Met. and a very high recovery rate occurred within a very short period, while the average number of births was 5-6 in the groups (G5,G4) treated with Asp. and Asp.NPs and the recovery rate was high in them and occurred within a short period, while the average number of births was 3-4 in the groups (G6,G3) treated with Meto. and Asp.+Met. The recovery rate was good in the (G6) group and recovery occurred within an average period, while in the (G3) group the recovery period was longer, 28 days and needed a long period to recover.
We conclude from this study that when treated with Nano-Complex loaded with Metformin for 28 days, it gave good results in reducing the level of hormones and raising others, lowering blood parameters, inhibiting inflammation, and significantly reducing the number of cysts in the studied tissue sections or eliminating the presence of these cysts. We also conclude that treatment with aqueous extract of fresh Asparagus stems loaded with Metformin gave good results no less good than the results of the green Nano-Asparagus Complex.