Dose-dependent therapeutic effects of liposomal nano-curcumin on ovarian toxicity induced by chronic environmental diclofenac exposure via Bcl-2 upregulation and IL-6 modulation in female Wistar rats
Article information
Abstract
An emerging but poorly understood risk to female reproductive health is the pervasiveness of non-steroidal anti-inflammatory drugs (NSAIDs) as environmental contaminants, especially the pseudo-persistent medication diclofenac. This study examined the effectiveness of liposomal nano-curcumin (NC), a bioactive nanocarrier with strong anti-inflammatory and antioxidant qualities, in treating (therapeutic model) ovarian toxicity in adult female Wistar rats brought on by long-term exposure to an environmental concentration of diclofenac (609.12 μg/L in drinking water). Based on an average water intake of 12.5 mL/100g body weight, this exposure translates to a systemic dose of approximately 76.14 μg/kg/day. Following the cessation of a 100-day exposure period, rats were treated with NC (5 and 20 mg/kg, IP) for 21 days. Thirty animals were divided into six groups (n=5) at random: diclofenac (DIC), NC5 (5 mg/kg), NC20 (20 mg/kg), control, DIC+NC5, and DIC+NC20. In addition to causing significant ovarian damage, such as follicular atresia, stromal hyperplasia, and a decreased healthy/atretic follicle ratio, diclofenac exposure also markedly increased relative ovarian weight. These effects were accompanied by downregulation of the anti-apoptotic protein Bcl2. Only a partial histological recovery was obtained with low-dose NC (5 mg/kg). Compared to animals exposed to diclofenac, high-dose NC (20 mg/kg) showed remarkable restorative efficacy, notably increasing primary and developing follicle populations, decreasing atresia, restoring ovarian cytoarchitecture, and significantly upregulating Bcl-2 expression while modulating IL-6 levels. These results offer the first proof that liposomal nano-curcumin, by means of coordinated modulation of follicular survival pathways and inflammatory mediators, represents a promising therapeutic approach for reducing reproductive toxicity caused by environmental pharmaceutical contamination.
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used worldwide for their analgesic, antipyretic, and anti-inflammatory properties, However, their extensive consumption and incomplete metabolic degradation have led to their frequent detection in surface and groundwater, including drinking water sources [1], and are therefore classified as pseudo-persistent pharmaceuticals posing substantial risks to both wildlife and human health [2].
Diclofenac sodium 2-[2-[(2,6-chlorophenyl) amino] phenyl] acetic acid is among the most commonly identified NSAIDs in aquatic systems, with environmentally relevant concentrations ranging from nanograms to micrograms per liter [3]. Due to its physicochemical stability and persistence, several toxicological studies have reported its adverse effect, including oxidative stress, inflammatory disturbances, genotoxicity, and tissue injury in various aquatic and terrestrial organisms [4]. Mechanistically, diclofenac acts primarily through cyclooxygenase enzymes (COX-1 and COX-2) inhibition, thereby reducing the synthesis of prostaglandin E2 (PGE2), a key inflammatory mediator that contribute to an inflammatory response [5]. In the female reproductive system, a finely regulated balance between pro-inflammatory and anti-inflammatory responses is considered essential for normal ovarian function, Ovulation itself is regarded as a controlled inflammatory-like process, with COX-2 derived prostaglandin contributing to a crucial role in follicular rupture, luteinization and vascular permeability [6]. While, disruption of prostaglandin signaling can affect granulosa cell viability, impair cell–cell communication within the follicle, and direct certain follicles toward apoptotic pathways [7].
Given these concerns, there is rising interest in natural compounds with protective properties against environmental and pharmaceutical-induced ovarian injury. Liposomal nano-curcumin, the nanostructured formulation of curcumin, has recently gained notable attention in biomedical and toxicological research, due to its improved bioavailability, enhanced cellular uptake, and significant antioxidant efficacy [8]. Curcumin or diferuloylmethane [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione], a naturally occurring hydrophobic polyphenolic compound derived from the perennial herb, Curcuma longa [9]. Exhibiting a wide therapeutic profile, including anti-inflammatory, anti-oxidant, anti-apoptotic and estrogenic activities [10]. It has been thoroughly investigated for its clinical potential in several disorders, such as cancer, asthma, fibrosis, arthritis, digestive and liver abnormalities, diabetes, and infertility [11].
At the molecular level, curcumin regulate multiple intracellular signaling pathways via the modulation of transcription factors, cytokines, and enzymes including protein kinases, histone-modifying enzymes, thereby exerting a comprehensive modulatory action associated with several chronic diseases [12]. Recent finding suggest that curcumin exerts protective effects against various female reproductive system dysfunctions, including PCOS, endometriosis and ovarian damage, largely attributed to its anti-inflammatory and redox-balancing ability [13], and estrogenic properties [14]. However, its protective potential against environmental relevant dose diclofenac-induced ovarian injury has not been fully investigated.
Therefore, the present study was designed to evaluate the post-exposure therapeutic potential of liposomal nano-curcumin against ovarian toxicity induced by environmentally relevant doses of diclofenac in adult female Wistar rats. The investigation focuses on ovarian histopathological alterations and follicular dynamic, highlighting the modulation of Bcl-2 expression as a pivotal marker of cellular survival and interleukin-6 (IL-6) as an indicator of ovarian inflammation.
Graphical abstract illustrating the experimental design and main finding of the study.
Materials and Methods
Chemicals and Reagents
Diclofenac Sodium (Diclamid 75 mg/3 mL ampoule; Btch No. (12/004 B04/433) was purchased from a local pharmacy and manufactured by FRATER-RAZES Laboratory (Algeria). Nano-curcumin (14 mg/ 3 ml) was obtained from a commercial supplier via an online platform (u-Bay).
Experimental Animals
Thirty adult female Westar rats (180–200 g) were procured from the Pasteur Institute of Algeria. Animals presenting regular estrus cycles were selected and housed in climate–controlled environments, with a temperature set at 24±2°C and humidity levels maintained between 55± 5%, following a 12–hour light/dark cycle to ensure consistent environmental conditions. Food and water were provided ad libitum. All animal procedures were conducted following the 1986 law and Directive 2010/63/EU on the protection of animals used for scientific purpose and were approved by the Institutional Animal Ethics Committee (CCE) at the Biotechnology Research Center (CRBT), Constantine, Algeria, (Decision Number: 27-BS-05/2024).
Dose Determination and Administration
The diclofenac concentration (609.12 µg/L) was selected based on reports of environmental residues in industrial effluents and polluted water bodies [15]. Given an average daily water consumption of 25 mL for a 200g rat, this concentration represents a chronic 'micro-dose', corresponding to an Estimated Daily Intake (EDI) of approximately 76.14 µg/kg/day. This model was specifically chosen to simulate realistic, long-term environmental exposure rather than acute pharmacological overdose. To maintain chemical stability, water bottles were shielded from light and solutions were renewed every 24 hours.
The commercially prepared liposomal nano-curcumin (14 mg/3 ml) was administered via intraperitoneal (IP) route. To ensure the doses of 5 and 20 mg/kg, the injection volumes were individually adjusted for each animal based on its body weight. This route was chosen to achieve maximal dosing precision and to bypass the variables associated with gastrointestinal absorption and first-pass metabolism, which often limit curcumin’s bioavailability. While liposomal carriers are designed to improve oral uptake, IP delivery in this study allowed for a clearer assessment of the nano-formulation’s systemic efficacy at low doses (5−20 mg/kg), which proved significantly more potent than the high doses (>100 mg/kg) typically required for free curcumin.
Experimental Design and Treatment Exposure
After two week acclimatization period, animals were randomly assigned into six experimental groups (n=5 per group):
Control Group (n=5): Served as control (-) and rats were received drinking water.
NC5 Group (n=5): Served as Nano-curcumin control (+), rats were received liposomal nano-curcumin intraperitoneally at a low dose of 5mg/kg body weight for 21 consecutive days.
NC20 Group (n=5): Served as Nano-curcumin control (+), rats were received liposomal nano-curcumin intraperitoneally at a high dose of 20 mg/kg body weight for 21 consecutive days.
DIC Group (n= 5): Rats were exposed to Diclofenac Sodium via drinking water for 100 days at an environmental relevant micro-dose (609.12 µg.L-1).
DIC+NC5 Group (n=5): Rats were received environmental relevant dose of Diclofenac Sodium in drinking water for 100 days, followed by intraperitoneal administration of liposomal nano-curcumin at a low dose of 5mg/kg body weight for 21 days.
DIC+NC20 Group (n=5) rats were received environmental relevant dose of Diclofenac Sodium in drinking water for 100 days, followed by intraperitoneal administration of liposomal nano-curcumin at a low dose of 20mg/kg body weight for 21 days.
Sample Collection
Following 100 days of treatment and overnight fast, all rats were weighted and euthanized during the estrus phase. Blood samples were collected and centrifuged at 3000 rpm for 15 minutes at 4 °C to obtain serum that was kept at -20 °C for IL-6 analysis. Ovaries were immediately excised, cleaned of surrounding fat tissue, and fixed in 10% neutral buffered formalin solution for histological and immunohistochemical analyses.
Body weight Gain and relative ovarian weights
weight was checked daily during the experimental period, Body weight gain was calculated based on the body weights measured at the beginning and end of the experiment [16]. The weight of ovary was taken after sacrifice in order to estimate the ROW [17].
Histopathological Examination and Follicular Count
Fixed ovaries were dehydrated through graded of ethanol series, cleared in xylene, and embedded in paraffin. Serial sections of 5-µm-thick were prepared and stained with Hematoxylin and Eosin (H&E). Sections were examined using a light microscope (Optika Srl, DM–25, Italy) at different magnifications. Histopathological alteration, including stromal edema, vascular congestion, and inflammatory cell infiltration, were semi-quantitatively scored as: none (0), mild (1), moderate (2) and severe (3) [18]. Follicle counting was performed in every tenth section (5 section per ovary), including primordial, growing (primary, secondary and antral) and atretic follicles (Atr F). Only follicles with a visible oocyte nucleus were counted to avoid duplication. Atretic follicles were identified based on granulosa cell pyknosis, detachment of granulosa layers, disrupted oocyte-granulosa cell communication, oocyte degeneration and cellular debris within the antral cavity [19]. Follicle counts were conducted using a semi-automated morphometric analysis method based on machine learning tool (Trainable Weka Segmentation, WEKA (version 3.7.6). All evaluations were performed independently by two blinded pathologists.
Serum IL-6 Levels Measurement
Serum level of interleukin 6 (IL-6) was quantified using IL-6 ELISA kit (Springfield, NJ 07081 USA) according to the manufacturer’s instructions.
Immunohistochemical Analysis of Bcl-2
Paraffin-embedded ovarian sections (4-µm) were mounted on positively charged slides. Sections were deparaffinized, rehydrated, and subjected to antigen retrieval using Tris/EDTA buffer, pH 9 (EnVision FLEX Target Retrieval Solution. K8004 -Dako). Slides were then incubated overnight at 4 °C with primary anti-Bcl-2 antibody, followed by incubation in the anti-mouse EnVision FLEX, High pH (Link) kit (Dako; K8000) to assess the immune complexes. Finally, the sections were counterstained with Mayer’s hematoxylin, dehydrated through a graded ethanol series, cleared in xylene, and coverslipped using a mounting medium. Slides were examined (5–10 fields; 4 rats/group), and the positive brown immunoreaction was assessed under a light microscope (Optika Srl, DM–25, Italy). Image analysis was performed using computer-assisted Fiji software, which converts brown staining into pixel-intensity values. The immunostaining (IS) grade was classified as follows: 0 (Negative ; 181-255 Pixel), 1 (Weak ; 121- 180 Pixel), 2 (Moderate ; 61 – 120 Pixel), 3 (Strong ; 0 – 60 Pixel) [20].
Statistical Analysis
Statistical analyses were performed using Graph Pad Prism 6 (ver. 5.02, GraphPad Software, Inc., CA, USA). Initially, data were tested for normality and homogeneity (Shapiro-Wilk test), then one-way ANOVA variance analysis followed by Dunnet post hoc multiple comparison test were applied. Data are expressed as mean ± SEM. A value of P <0.05 was considered statistically significant.
Results and Discussion
Body Weight Gain and Relative Ovarian Weight
As shown in Figure 2, non-significant change in the body weight gain was observed between among the experimental groups (p > 0.05). However, there was a significant increase in the ROW in diclofenac-treated group respecting the control group (p = 0.0198).While, non-significant change was observed in DIC+NC (5mg/kg) and DIC+NC (20mg/kg) compared to the diclofenac-treated group (p>0.05).
Body weight gain and relative ovarian weight changes in animals of different experimental groups. BWG: body weight gain; OI: ovarian index (relative ovarian weight). Values are expressed as the mean ± SEM deviation; ANOVA. * Symbol indicates significant difference compared to DIC group (P < 0.05).
Histological Examination and Follicular Count
Exposure to environmental relevant doses of diclofenac induced marked ovarian injury including, stromal edema, vascular congestion and neutrophil cell infiltration. As shown in (Figure 3, Table 1), significant elevation in histopathological lesion scores was prominently observed in the Diclofenac-treated group compared to controls group (p < 0.05). Interestingly, nano-curcumin dose dependently restored these scores, with a significant greater improvement observed in the high-dose nano-curcumin group (p < 0.05) compared to Diclofenac- treated group. Quantitatively, as shown in Figure 4, a significantly greater reduction in the number of primordial, growing, healthy follicles, and in the healthy/atretic ratio (p < 0.0001), accompanied by a significant increase in the number of atretic follicular (p < 0.0001), was observed in Diclofenac-treated group compared to the control group. Consistently, high-dose nano-curcumin (20 mg/kg) exhibited a pronounced restorative action of ovarian follicular dynamics (p < 0.05). No significant improvement was identified in the number of primordial follicle and in the healthy/Atretic ratio compared to the Diclofenac-treated group (p > 0.05). While, significant rise in the number of growing and healthy follicle was observed in DIC+ NC (20mg/kg) group compared to the Diclofenac-treated group (p < 0.01). With a concomitant decline in the atretic follicle proportion compared with Diclofenac treated group (p = 0.0013). Regarding the low dose Nano-curcumin (5mg/kg), no significant changes were observed in the number of primordial, growing, healthy, atretic follicle and in the healthy/Atretic ratio compared to the diclofenac-treated group (p > 0.05).
Photomicrograph of female Wistar rat ovarian tissue stained with H&E (x10, x40). (A,B,C) Diclofenac-treated group exhibits significant histopathological alterations characterized by granulosa cells vacuolation (circle), hemorrhage (yellow arrow heads), congestion (black arrow heads), inflammatory cell infiltration (red arrow heads). Note the marked increase in atretic follicle (AF, AtrF) and the significant thinnig of ovarian cortex (black double-headed arow), reflecting severe follicular depeletion and structural damage. (D) control group shows a normal ovarian architecture with follicls at deferent stages of maturation. (E): DIC+NC (20mg/kg) group demonstrated a remarkable restoration of ovarian medulla and stroma with the presence of developing follicles at different stage and corpus luteum (CL) and some vascular edema (green arrow heads). Abbreviations: PF: primordial; Prf: primary follicle; SF: secondary follicle; AF: atretic follicle; AtrF: atretic follicle; GC: granulosa cell; IG: interstitial glands; CL: corpus luteum; BV: blood vessels. (Scale bar = 400 µm).
Semi-quantitative scoring of ovarian histopathological changes.
Histomorphometric analysis of primordial (PF), growing GF), healthy (HF) and atretic (AtrF) follicle number; HF/AnF Ratio: ratio of healthy to abnormal follicle. The symbols represent statistical significance: * P < 0.05, ** P < 0.01, and *** P < 0.001, compared to the diclofenac treated group.
Pro-inflammatory marker (Serum IL-6)
As shown in Figure 5. No significant difference was observed in the diclofenac-treated group compared to the control(-) group (p > 0.05). Post-treatment of Nano-curcumin resulted in a significant dose-dependent modulation of serum IL-6 levels in the DIC+NC (5mg/kg) and DIC+NC (20mg/kg) group (p = 0.0189; p = 0.0123, respectively) compared to the diclofenac-treated group.
Serum levels of IL-6 analyzed by ELISA method. Data are stated as mean ± SEM deviation, *: the difference was significant compared to the diclofenac treated group. The symbols represent statistical significance: *P < 0.05 and ** P < 0.01. ELISA: enzyme–linked immunosorbent assay.
Anti-apoptotic pathways-Bcl-2
The percent area analysis of ovarian tissue Bcl-2 immunoreaction expression, revealed a significant reduction of Bcl-2 expression in Diclofenac-treated group compared to the control (p < 0.05). While, significant upregulation was observed in DIC+NC (20mg/kg) compared to diclofenac-treated group (p = 0.0480) (Figure 6).
Bcl-2 expression in ovaries of control and treatment groups; (A) Control group and (B) control(+) NC (20mg/kg) group show intense and widespread cytoplasmic Bcl-2 expression (brown staining) within granulosa cells (GC) and ovarian stroma. Red arrowheads: indicate strong immunopositive reactivity, reflecting a robust anti-apoptotic status in both follicular and stromal compartments, (C) Diclofenac-treated group exhibits a marked reduction in Bcl-2 immunoreactivity. Green arrowheads: point to immunonegative cells (predominantly blue hematoxylin staining) within the stroma (cell debris and nuclear pyknosis) and degenerating primordial follicles (PF), indicating a localized pro-apoptotic effect of diclofenac, (D) DIC+NC (20mg/kg) demonstrates a significant restoration of protein expression. Red arrowheads: highlight the reappearance of strong positive cytoplasmic immunostaining in granulosa cells and stromal tissue, indicating the therapeutic efficacy of nano-curcumin against diclofenac-induced ovarian damage. Abbreviations: PF: primordial folicle; SF: secondary follicle; Atrf: atretic follicle; GC: granulosa cell; TI: theca interna; BV: blood vessels. Scale bars: 400µm (A,B,D); 200µm (C, x40).
Discussion
Chronic exposure to emerging environmental contaminants, including non-steroidal anti-inflammatory drugs such as diclofenac, has been increasingly recognized as a potential reproductive toxicant, even at environmentally relevant doses. The present study assessed ovarian effect of realistic relevant diclofenac exposure and evaluated the restorative potential of Nano-curcumin dose-dependent.
Histopathological assessment revealed pronounced ovarian injury in the diclofenac-treated group, characterized by stromal edema, vascular congestion and neutrophil cell infiltration. Lesion scoring demonstrated a significant increase in tissue damage compared with the control group (p < 0.05). These morphological lesions likely contributed to the observed increase in the relative ovarian index in diclofenac-treated group (p < 0.05 vs. control). Quantitatively, a higher significant depletion in the number of primordial, growing and healthy follicle was observed relative to control (p < 0.0001). This was accompanied by a substantial increase in the number of atretic follicle (p < 0.0001 vs. control), leading to a dramatic decline in the healthy to abnormal follicle ratio (p < 0.0001 vs. control). This findings indicate that diclofenac not only reduces the total follicle pool but also actively accelerates follicular atresia, reflecting an impaired ovarian reserve, follicular degeneration and early signs of stromal atrophy, thus highlighting its potential to induce reproductive toxicity even at environmentally relevant doses. Our results are consistent with previous studies reporting significant alterations in follicular dynamics in rats following exposure to low dose (0.2 mg/kg) of diclofenac [21]. Similar findings were describing with those of E Kia et al, who reported that diclofenac-induced several degenerative follicles at different stages of follicular maturation and interstitial stromal hyperplasia in rats receiving 1.7 mg/kg [22].
Post-treatment with law dose nano-curcumin (5mg/kg) revealed noticeable, though partial, improvement of ovarian histoarchitecture with mild reduction of stromal congestion, where, a significantly greater improvement was observed in the high-dose DIC+NC20 group compared to the diclofenac-treated group (p < 0.05). Consistently, significant rise in the number of growing and healthy follicle was observed in DIC+NC (20mg/kg) group compared to the diclofenac-treated group (p < 0.01). With a concomitant decline in the atretic follicle proportion compared with diclofenac treated group (p = 0.0013), confirming the restorative dose dependent effect of liposomal nano-curcumin on ovarian tissue and follicle dynamic. Our findings are in line with those of previous studies which have widely reported the protective and therapeutic effects of nano-curcumin against ovarian damage caused by environmental agents. In irradiated mice, curcumin attenuated follicular atresia and promoted granulosa cell proliferation, indicating potent anti-apoptotic and cytoprotective effects [23]. However, further study revealed that nano-curcumin administration in PCOS models, resulted in a pronounced restoration of ovarian histoarchitecture, reflected by a reduced number of cystic and atretic antral follicles and an increased numbers of corpora lutea indicating enhanced follicular development and luteinization [14]. Similar findings were observed in endometriosis mice following administration of 5 and 10 mg/kg nano-curcumin, while higher significant enhancement of folliculogenesis was recorded specifically at 10 mg/kg nano-curcumin dose [24].
The mammalian ovary is a highly dynamic organ within the female reproductive system, where follicular growth carefully regulates the selection and fate of developing follicles, maintaining cellular homeostasis is therefore essential for proper oocyte development [25]. Accordingly, maintaining a delicate equilibrium between pro-inflammatory and anti-inflammatory mediators within the ovarian follicle is critical for normal follicular growth and ovulation [26]. However, at the molecular level, diclofenac recognized for its pro-oxidative properties, disrupted the pro-inflammatory response by modulating oxidative status, enhancing lipid peroxidation and impairing prostaglandin synthesis in both plasma and tissues [27]. Hence, deregulated inflammatory resulting from the over activity of immune cells, accompanied by oxidative stress, are considered to be the crucial factors in ovarian tissue damage [28]. Mechanistically, the variations in protein expression changed the permeability of mitochondrial membrane and viability causing the discharge of cytochrome C into the cytosol, which results in the stimulation of the adaptor molecule apoptotic protease activating factor 1 (Apaf1), and producing the apoptosome complex. Thus, mitochondrial facilitated apoptosis comprises induction of Bax and suppression of Bcl2 [29]. In the present study, while chronic diclofenac exposure did not significantly alter systemic IL-6 levels compared to the control (p > 0.05), it led to significant downregulation Bcl-2 expression (p < 0.05 vs. control) in ovarian tissue. This suggests a localized tissue-specific toxicity where follicular degeneration occurs through direct apoptotic pathways independent of a generalized inflammatory response. This process is primarily driven by the fact that diclofenac inhibits cyclooxygenase (COX) enzymes, Thereby reducing prostaglandin synthesis, which is vital for granulosa cell survival and follicular maturation [30]. this internal disruption contributes to follicular degeneration which in turn triggers intra-ovarian NF-kB signaling that promotes the pro-oxidative cascades [27]. Consequently, persistent micro-environmental inflammation driven by these molecular changes, further disrupts follicular development through intricate paracrine interactions, ultimately contributing follicular depletion and impaired infertility [31].
Given these deleterious effects of diclofenac, the therapeutic potential of medicinal plants rich in bioactive phyto-constituents such as curcumin, play a crucial role in maintaining female reproductive health by modulating endocrine balance, enhancing intercellular communication, stabilization of membrane integrity, and exerting anti-inflammatory effects that support normal ovarian physiology [32]. In the present study, the intraperitoneal administration of liposomal nano-curcumin served as a key factor in achieving the observed histological recovery of the ovarian tissue. This approach ensured optimized bioavailability of nano curcumin within the plasma and ovarian tissues compared to its free form [33]. Mechanistically, curcumin suppresses the nuclear transcription factor NF-kB activation [26], leading to downregulation of pro-inflammatory cytokines such as IL-1, IL-6, and TNF-α, as well as decreased COX-2 expression, thereby mitigating cytokine-mediated disruption of follicular development [24]. In oocytes, the PI3K/AKT pathway plays a crucial role in follicular dynamics by regulating the activity of the transcription factor FOXO3. When PI3K/AKT signaling is activated, FOXO3 is phosphorylated, leading to altered transcriptional control of genes involved in cell survival and apoptosis. Activation of AKT also modulates the balance between Bax and Bcl-2 proteins, thereby enhancing mitochondrial oxidative stress, increasing reactive oxygen species production, and inducing cytochrome c release, and caspase-3 activation, ultimately resulting in oocyte and granulosa cell apoptosis [34]. Furthermore, it was reported that curcumin exerts a protective effect on the ovarian reserve by modulating the PTEN-AKT-FOXO3a pathway [35]. In accordance with our study, this mechanism may underlie the significant upregulation of Bcl-2 expression in the DIC+NC (20mg/kg) (p = 0.0480 vs. Dic, Figure 4), the modulation of pro-apoptotic IL-6 levels (p = 0.0123 vs. Dic group) , amelioration of follicular dynamics and reduction in atretic follicles observed in post-nano-curcumin treated group (p < 0.05), supporting its potent anti-inflammatory activity, thereby, preventing granulosa cell apoptosis and promoting follicular survival in a dose-dependent manner [36].
Conclusions
In vivo, this study offers strong evidence that long-term exposure to environmentally relevant levels of diclofenac sodium causes significant ovarian toxicity in adult Wistar rats. This toxicity manifests as follicular atresia, stromal disruption, and downregulation of the anti-apoptotic protein Bcl-2. These results highlight the real risks to reproductive health that pseudo-persistent pharmaceutical pollutants pose, going beyond ecological disruption to include direct endocrine and gonadal impairment in mammals. Crucially, our research shows that ovarian damage brought on by diclofenac is not irreversible. Liposomal nano-curcumin, especially at 20 mg/kg, demonstrated strong, multifaceted restorative effects as a post-exposure therapeutic intervention. In addition to effectively restoring ovarian histoarchitecture, high-dose NC treatment also significantly increased Bcl-2 expression, which countered the apoptotic cascade that diclofenac had started, and rebalanced follicular dynamics by increasing the pool of healthy growing follicles while suppressing atresia. The dual anti-apoptotic and anti-inflammatory mechanisms that underlie nano-curcumin's therapeutic efficacy are further supported by the concurrent modulation of serum IL-6 levels. The observed dose-dependent response emphasizes how important the liposomal formulation's increased bioavailability is to obtaining the best possible ovarian tissue penetration and therapeutic result. This work positions nano-curcumin as a potential therapeutic agent for reducing acquired ovarian damage after exposure to environmental toxicants, going beyond its traditional use as a preventative antioxidant. Our findings provide a promising approach to address the often-overlooked effects of pharmaceutical pollution on female fertility by bringing phytochemical-based nanotherapeutics into the field of environmental reproductive toxicology. To demonstrate clinical relevance for populations at risk of environmental pharmaceutical exposure, more research examining chronic safety, translational potential, and molecular pathways is necessary.
Notes
Acknowledgement
Laboratory of LBMBPC, CRBT.
Conflict of interest
All authors report there are no competing interests to declare.
CRediT author statement
AC: Investigation, Data curation, Writing- Original draft preparation; MY: Supervision, Writing- Reviewing and Editing, Resources; SB: Conceptualization, Resources, Methodology, Data curation, Visualization; AO: Investigation; SMB: Investigation; KY: Investigation.