Alterations of VEGF Expression and Ovarian Follicles in Doxorubicin Treated Rats: Role of Curcumin Selenium Nanoparticles

Green nanoparticle synthesis has great potential in the biomedical and environmental scientific fields (Jadoun et al., 2021). The green chemistry method promotes the utilization of plants and plant extracts, providing a credible, uncomplicated, non-toxic, cost-effective, stable, and environmentally friendly method (Sood and Khudiar, 2019; Wali and Alqayim, 2019). Selenium is an essential trace mineral for an individual’s body, with properties of both minerals and nonmetallic compounds. Selenium exhibits biological characteristics associated with the immune system, antioxidation, antiviral activity, and anticancer activities (Zhou et al., 2020). Additionally, selenium nanoparticles (SeNPs) have the ability to reduce and alleviate heavy metal toxicity. They offer advantages such as a high absorption rate, increased biological activity, and lower toxicity levels (Zhang et al., 2023).

The SeNPs s demonstrates efficacy in decreasing the rate of cancer cell growth (Geoffrion et al., 2020). Because, the stability of these nanoparticles is enhanced by their unique organic molecule covering, which effectively inhibits any gradual buildup over time (Wali and Alqayim, 2019; Shareef et al., 2024). The black currant selenium nanoparticle works as an antioxidant. It exhibits both prophylactic and therapeutic properties in mitigating the harmful impact of D-galactose (Khudair et al., 2020). Also, Avena sativa seed crude extract protects the heart from DOX-induced toxicity in rats (Salim and Hasan, 2022). Curcumin is a polyphenolic substance that is naturally found in the rhizome of Curcuma longa (Patel et al., 2020). Besides, curcumin demonstrates notable multi-pharmacological capabilities, including anti-tumor and anti-inflammatory activities (Peng et al., 2021). Curcumin phytosome nanoparticles have the capacity to lower inflammation and decrease cholesterol levels, as proven by Jadaan and Khudair, 2023. Additional research has provided further evidence to support the concept that phytochemicals play a vital role in controlling the creation of metal nanoparticles (Al-Kurdy and Khudair, 2020).The anticancer effects of curcumin on ovarian cancer act through various mechanisms, including disrupting cellular interactions essential for the spread and recurrence of ovarian cancer cells, enhancing the production of pro-apoptotic proteins, and regulating the generation of various molecules such as cytokines, transcription factors, enzymes, protein kinases, and growth factors (Bhatia et al., 2021) .These nanoparticles have potential applications in several fields such as medicine and therapy (Jaffar and Hussein, 2015). Doxorubicin, a type of medication used to treat various tumors, has been related to adverse effects and toxicity in crucial organs, particularly cardiotoxicity (Dulf et al., 2023), in addition ovarian toxicity, which is manifested by decreased ovulation and associated with a decrement in ovarian size, caused probably by ovarian injury (Gao et al., 2023).DOX caused damage to actively dividing granulosa cells in the ovary, resulting in the programmed cell death of follicles and ultimately disrupting female ovarian activity and ovulation (Xiao et al., 2017). DOX has harmful effects on ovarian follicles in rodents, such as follicular atresia, excessive activation (Wang et al., 2019), and decreased synthesis of 17beta-estradiol (E2) (Xiao et al., 2017). Therefore, this study was conducted to examine modern approaches for manufacturing and analyzing CurSeNPs and their ability to lower the possible hazards of doxorubicin exposure on several reproductive aspects of the ovaries in healthy adult female rats.

Curcuma longa alcoholic extract was prepared as described by Jayarambabu et al. (2020) using a magnetic stirrer. The rhizomes were dried, pulverized, and mixed with ethanol for six hours at 70-80°C. The alcohol was extracted using a rotary evaporator. Selenium nanoparticles were created by mixing sodium hydrogen selenite with distilled water, subjected to sonication, and stored in a dark place. The alcoholic extract of Curcuma longa was used as an efficient reducing agent to synthesize CurSeNPs) from sodium selenite (0.04M). The extract were then added dropwise to the selenite solution, stirring for four hours. The color changed from lighter orangish-red to deeper orangish-red, and a dark orange-red color was observed. The solution was then spun at 10,000 rpm for 20 minutes, centrifuged at 6,000 rpm for 15 minutes, and iterated three times to eliminate organic impurities. The SeNPs were dried at 45 ºC. (Al-Ebadi et al., 2021).

Characterization of CurSeNPs was performed by: Ultraviolet– visible spectroscopy (Metertech SP-8001 Taiwan) as described by Soleimani et al. (2018); X-ray diffraction (Shemadzu-6000 Japan)as describe by Fouda et al. (2022), Fourier-transform infrared spectroscopy (Shimadzu8400s, Japan), this method is likely comes from the research by Al-Kurdy and Khudair, (2020) Zeta potential and Field emission scanning electron microscope (FE-SEM) ( SEM-Tescan Vega III, Czech) and atomic force microscopy(AFM) (TOSHIBA/Japan).

The current study was conducted at an animal house at the College of Veterinary Medicine / University of Baghdad. The study’s use of animals got ethical approval by the local committee of animal care and use (P.G. 2641 date 29-11-2023). Thirty-two adult healthy female wistar rats were utilized in this investigation. After the acclimatization phase, they were randomly and evenly divided into four experimental groups. These groups were subjected to the given treatment for a duration of two weeks: Rats in control group (C) were orally delivered distilled water, Group G1: Rats in the this group had injected intraperitoneal of doxorubicin (STADA /Germany) at a dose of 4.40mg/kg B.W, G2 group, Rats administered CurSeNPs at a dose of 10.47 μg/kg B.W, while both of doxorubicin and CurSeNPs at the same doses were given to G3 group. Ovarian tissue specimens were taken to measure the gene expression VGEF mRNA, and the RNA extraction kit from Genaid Korea was used to extract total RNA from the ovarian tissue. The measurement of gene expression was performed utilizing the Forward primer (5’-GCACCCATGGCAGAAGG-3’) and Reverse primer (5’-CTCGATTGGATGGCAGTAGCT-3’) specific to the VEGF gene (Pang et al., 2020). The AddScript RT-qPCR Syber master kit from AddBio, Korea was utilized for this study. The RT-qPCR findings were assessed according to the guidelines given by Schmittgen and Livak (2008). Besides, another part of the specimens was fixed in a 10% formal saline solution to preserve their structure; then the fixed tissues were embedded in paraffin wax. Thin slices, 5 micrometers thick, were cut from the paraffin blocks and the sections were A staining with hematoxylin and eosin (H and E) for pathohistological examination under a microscope according to Suvarna et al. (2018) furthermore, histomorphometric analysis of ovarian tissues were conducting (Suad et al., 2018).

The data were analyzed using SPSS version 26, and the outcomes are presented as means ± Standard deviation (SD). One-way analysis of variance (ANOVA) followed by post hoc test to compare between groups P value < 0.05 pointed a statistical significance (Baarda and van Dijkum, 2019). A post hoc multiple comparison test was employed to compare the treatment groups with the control group. An analysis of variance (ANOVA) was conducted, followed by a post hoc multiple comparison test. A probability value significance criterion of 0.05 was utilized.

Figure 1 illustrates the gradual change in colour of the reaction mixture throughout the biosynthesis process. The formation of CurSeNPs was demonstrated by the gradual change in colour from brilliant orange to a darker orange-red shade after 40 minutes, indicating enhanced stability. The optical absorbance of curcuma longa extract and synthesized CurSeNPs was measured the change in the color using UV-Vis spectroscopy demonstrating that theabsorption peak between 427-214 nm confirm the presence of CurSeNPs (Figure 2). Results of FTIR of alcoholic extract of Curcuma longa and CurSeNPs revealed distinct bands at different peaks (Figure 3). The XRD test provides precise data on the size of nanomaterials and the crystal type present in a substance (Figure 4).

A zeta potential value exceeding 30 mV indicates a high level of stability (Figure 5). The pattern of FE-SEM revealed the existence of spherical, amorphous structures with diameters ranging from 21 to 37 nm (Figure 6). The atomic force microscope displays a two-dimensional view of CurSeNPs (Figure 7A). The present results demonstrated that mRNA analysis of rats’ ovarian tissues (Figures 8 and 9). A notable (P<0.05) upregulation of the VEGF gene expression in group G1 compared to the other experimental groups was observed. While Following CurSeNPs administration with or without doxorubicin, rats exhibited a significant (P<0.05) reduction in VEGF expression compared to the G1 group (Figure 10). Table 1 showed a significant (p < 0.05) reduction in the number of follicles in doxorubicin treated group compared to the control group. In addition, rats that received CurSeNPs with or without doxorubicin in groups G2 and G3 showed a significant (p< 0.05) increase in the number of ovarian follicles compared to group G1. Also, (Table 2) also demonstrates the impact of injecting doxorubicin and orally intubating CurSeNPs on the diameter of ovarian follicles. The results revealed a significant (p <0.05) reduction in the diameter of all follicles in group G2 (treated with doxorubicin) compared to the control group, as well as groups G2 and G3. In addition, rats in groups G2 and G3, showed a significant (p < 0.05) increase in diameter compared to the G1 group. The ovaries of the control group show normal different type of follicles involved graafian follicles, germinal epithelium, theca layer, granulosa cell layer, oocyte, antrum and oogonium (Figure 11). Compared to the control group (Figure 11) group exhibited clear congestion of Graafian follicles with sloughing and degenerative lesion in the granulosa cell layer), The other section shows a clear necrotic lesion in the griffin follicle with lysis of oocyte, associated with clear damage and necrosis of epithelia of oogonium (Figures 12 and 13). While histological sections of group G2 (Figure 14 and 15) show graffian follicles, germinal epithelium, theca layer, Oocyte, granulosa cell layer, antrum, and Oogonium without damag. Whereas a clear blood vessels congestion of ovarian veins with infiltration of inflammatory cells in the ovarian tissue were observed in group G3 (Figure 16).

The curcumin extract and sodium selenite react to affect the mixture’s color. To create environmentally safe CurSeNPs,curcumin served as a reducing and stabilizing agent. Previous investigation have used a variety of green plants to generate the unique reddish-orange color of CurSeNPs nanoparticles (Sabea and Al-Qaiym, 2023).The color transition from yellow to reddish-orange upon heating serves as an indication of an increase in particle size (Figure 2A) demonstrated that the peak at 427 nm was impeded in the solution of Curcuma longa. In contrast, the solution of Cur-Se-NPs (Figure 2C) exhibited a shift from 427 to 214 nm, indicating that curcumin, the primary constituent of turmeric (Curcuma longa), was present (Soleimani et al., 2018).

The FT-IR spectra exhibited prominent absorption bands at (3755–3429 cm1) and indicating the presence of hydroxyl (-OH) or amine groups (-NH) (3402 cm1) in the Curcuma longa extract. Alternatively, CH stretch alkynes are indicated by the absorption peak at (2924, 2926 and 2854 cm−1).

The plant extract included carbon dioxide (0 = C = 0) as indicated by the peak at wavenumbers (2385 and 2370 cm−1). Around 1734, the absorption peaks. The carboxyl groups are represented by 06 cm-1. The C=C bond is associated with the absorption peaks at 1639 cm-1.

The N-O bond is associated with the absorption peak at 1573 cm-1, whereas the peak represents the C-H bond at 1462.04 cm-1. Concerning the S = O, the absorption peaks may be seen around 1390.68 cm-1. The stretching of the C-N bonds in the amines is shown by the absorption peaks at 1269.16 cm-1 and 1163.80, 1124.50, 1116.78, 1068.56, 1041.56 and 1037.56 cm-1. The stretching of C-X bonds in alkyl halides results in bands at 825.53, 840.96, and675.90 cm−1, whereas the bending of C-N-C bonds in amines generates the weak bands at 592.15 and 484.13 cm−1. Based on these results, it appears that there are a number of biomolecules with functional groups that might help stabilize and reduce the SeNPs. Figure 5 shows the results of Zeta potential investigation carried out on CurSeNPs, which indicates a negative surface charge range from -120 to +60, with a specific value of -36.08 mV, this is due to the polyphenolic and flavonoid parts of the reducing agent in the plant extract, which show electrostatic forces in the nanoparticles made from green materials. All particles in the synthesized solution possess a negative zeta potential value, contributing to their high stability and preventing aggregation and making them easier for cell absorption and maintaining their nanostructure integrity (Nasrollahzadeh and Sajadi, 2016). The FE-SEM pictures, seen at various magnifications, revealed the existence of spherical, amorphous structures with diameters ranging from 21 to 37 nm, as depicted in (Figure 6). The atomic force microscope displays a two-dimensional view of CurSeNPs (Figure 7A)

Table 1: Effect of CurSeNPs, doxorubicin and both on number of primary and secondary, Graafian follicle in Ovarian tissue after 14 days.

*Values are expresses as mean ± SD; n:5; C: Control group; G1: Injected with i/p doxorubicin 4.40 mg /kg. b. w.; G2: Administrated with CurSeNPs 10.47 μg / kg bw. Orally; G3: Administrated with CurSeNPs 10.47 μg / kg bw. orally and with doxorubicin 4.40 mg! kg b. w. i/p.; *Different small letters mean significantly differences between groups at P<0.05.

Table 2: Effect of CurSeNPs, doxorubicin and both on diameter mm of primary and secondary, Graafian follicle in Ovarian tissue after 14 days.

*Values are expresses as mean ± SD; n: 5; C: Control group; G1: Injected with i/p doxorubicin 4.40 mg /kg. b. w.; G2: Administrated with CurSeNPs 10.47 μg / kg bw. Orally; G3: Administrated with CurSeNPs 10.47 μg / kg bw. orally and with doxorubicin 4.40 mg! kg b. w. i/p.; *Different small letters mean significantly differences between groups at P<0.05.

which appear as clusters of spherical shapes in either isolated or aggregated forms. While, the (Figure 7B) displays a three-dimensional image of CurSeNPs, which exhibits a population of uniform particles with a smooth surface. In addition, (Figures 7C and D) demonstrated that the diameter of the synthesized curSeNPs varied between 3 and 43 nm, with a height of around 46.1n–48. The current results revealed that mRNA analysis of rats’ ovarian tissues indicated upregulation of the VEGF gene expression in group G1 compared to the other experimental groups in agreement with study of Torres-Ruiz et al. (2023), who confirmed the upregulation of main lymphangiogenic receptor characterized with higher mRNA and protein levels of VEGFR3 after a short treatment with doxorubicin in cellular models when compared to the parental cell line. In addition, hypoxia triggers the activation of hypoxia-inducible factor 1α (HIF-1α), which manages the transcription of key molecules such as VEGF and sirtuin-1 (SIRT-1) (Pinto et al., 2024). As well as that acute or chronic hypoxia caused an increase in VEGF expression and decreased the pro-angiogenic potential factors in liver cancer cells, associated with a notable activation of the ROS/NF-κB/hypoxia-inducible factor-1α/VEGF signaling pathway (Refaie et al., 2022). The downregulation of VEGFR3receptor leads to a reduction of cancer in vivo models through reduced migration and the proliferation capacities of cancer cells (Walsh et al., 2022).

Rats received CurSeNPs with or without doxorubicin rats exhibited a reduction in VEGF expression compared to doxorubicin-treated rats. This result was in accordance with other studies (Elneklawi et al., 2023). The chemical structure of curcumin as a phytochemical compound has been (Giordano and Tommonaro, 2019) safe and effective, and the role of curcumin as an anticancer had been widely proven. A recent study by Liu et al. (2023), demonstrated that curcumin decreases VEGF, activity and expression of matrix metalloproteinases (MMPs), urokinase plasminogen activator (uPA), and modulating E-Cadherin (E-Cad) leading to inhibit the metastasis in case of ovarian cancer by the effect of bisdemethoxycurcumin (BDMC) (Liu et al., 2023). Besides, strawberry extract loaded within niosomes showed alleviating VEGF gene expression by improving XBP1/mRNA-21/mRNA-330 signaling pathways in rats’ uterus injury induced by cisplatin (Elneklawi et al., 2023). Also, the effect of curcumin on VEGF expression may be via the inhibition of the NF-κB pathway and activation of the sirtuin-1 molecule (Ungurianu et al., 2022), in addition, it regulate of NF-kB and cyclooxygenase-2 (COX-2) (Hutomo et al., 2017). The anticancer effects of SeNPs and their mechanisms were well evaluated by Menon et al. (2018), who showed that cancer cells have an acidic pH that leads to a peroxidative and triggers an increased production of free radicals, causing leakage of mitochondrial proteins and stress of the endoplasmic reticulum resulting to triggers of apoptosis, through activation of many molecular signaling pathways for oncogenic, including MAPK/Erk, PI3K/Akt/mTOR, NFκB, Wnt/β-catenin, and apoptosis pathways, While SeNPs cause modulating and/or diminished VEGF, PI3K/Akt/mTOR, MAPK/Erk, and Wnt/β-catenin pathways by decreased cellular proliferation associated with impeding the enhancement of growth-promoting signaling in the cancer cell microenvironment. Recently, Khaledizade et al. (2024), showed that the expression levels of VEGF,, BCL2, MMP2, MMP9, CCNE1, CCND1, and ERBB2 were downregulated after SeNPs treatment in MCF-7 breast cancer cell line (Khaledizade et al., 2024). The histopathological results in group G1 were in agreement with Samare-Najaf et al. 2020, who showed that the administration of doxorubicin causes the development of ascites, ovarian hemorrhage, reduced number of ovarian follicles, and tissue atrophy. The study has demonstrated the impact of chemotherapy drugs on the vascular system of the ovaries with follicular loss (Regulska et al., 2019) is by indirectly impairing the stroma and the vascular system (Soleimani et al., 2011). Also, apoptosis in ovarian cells can lead to significant follicular atresia or regression and is regarded as one of the primary mechanisms responsible for ovarian aging (Yang et al., 2019). Besides, oxidative stress can trigger programmed cell death in ovarian germ cells through multiple mechanisms, such as external or endogenous pathways (Jalouli et al., 2022). Apoptosis of granulosa cells causes a lack of nutrients and triggers metabolic dysfunction in the ovarian microenvironment (Palmerini et al., 2016) apoptosis increases the concentration of cell-free DNA in the fluid surrounding the ovarian follicles, hence increasing the deterioration in ovarian function. The present investigation showed a positive effect of CurSeNPs and demonstrated improvement in the ovarian function of rats by enhancing histological modifications in the ovaries caused by modulation of histomorphometric analysis and histopathological changes caused by doxorubicin (group 3). This was evidenced by an improvement in folliculogenesis and a decrease in the number of atretic follicles. These findings are consistent with previous research that showed selenium supplementation can protect rat ovaries from the effects of arsenic (Alhealy et al., 2021). Current results show that polyphenols found in foods and spices, such as curcumin, gallate, epicatechin, and resveratrol, have strong anti-tumor solid effects and provide protection to the reproductive system in cancer patients (Carvalho et al., 2017).

In total, based on our knowledge, CurSeNPs can be produced biologically by employing Curcuma longa extract to create nanoparticles naturally. This is a simple, low-cost method that can be applied in biomedicine and has the potential to prevent or treat ovarian doxorubicin toxicity in adult female rats. This is the first study to confirm the protective effects of CuSeNPs against doxorubicin on VEGF gene expression and histological changes in the ovarian tissues of healthy female rats. The roles of CurSeNPs in regulating oestrogen receptors (ESR1 and ESR2) in both female and male rats subjected to anti-tumor medicines, as well as in mitigating the negative effects of experimentally-induced polycystic ovary and solid tumour in female rats.

Authors of this study would like to thank College of Veterinary Medicine, University of Baghdad-Iraq for their support.

The novelty of the current study was to illustrate the role of Curcumin selenium nanoparticles in decreasing the adverse effects of anticancer drugs on female reproductive system by modulating folliculogenesis and expression of the VEGE gene of rats ovary.

Baraa Najim was responsible for designing the experiment. Rawya Shakir conducted data analysis and experimental procedures. Besides, Baraa Najim wrote the article, interpretation and also provided conceptual advice.

No conflict of interest are declared by authors for the contents of the manuscript.