Bindarit ameliorates doxorubicin-induced ovarian damage by suppressing CCL8-dependent macrophage infiltration and NF-κB-mediated inflammation

Bindarit ameliorates doxorubicin-induced ovarian damage by suppressing CCL8-dependent macrophage infiltration and NF-κB-mediated inflammation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bindarit ameliorates doxorubicin-induced ovarian damage by suppressing CCL8-dependent macrophage infiltration and NF-κB-mediated inflammation Xiang Ma, chenzi Feng, Nan Lu, Yi Qian, Mingyu Yang, Xin Ning, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8939659/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Doxorubicin (DOX), a widely used chemotherapeutic agent, induces severe ovarian damage resulting in premature ovarian insufficiency and reduced fertility. In the present study, through transcriptome sequencing and immune cell infiltration analysis, we demonstrated that DOX significantly upregulates ovarian CCL8 expression, thereby activating the CCL8/CCR5 axis to promote the recruitment and polarization of proinflammatory M1 macrophages. Mechanistically, CCL8 exacerbates local inflammation through NF-κB signaling (evidenced by p65 phosphorylation and IκBα degradation), initiating granulosa cell apoptosis and follicular atresia. The CCL8-specific inhibitor Bindarit effectively mitigated this pathological cascade by (1) reducing macrophage infiltration by 90.9% ( P < 0.01), (2) inhibiting NF-κB activation (99.3% decrease in p65 nuclear translocation, P < 0.001), and (3) downregulating proinflammatory cytokines (reduction in TNF-α/IL-1β expression: 80–90%, P < 0.001). Notably, Bindarit treatment restored 54.5% of fertility capacity ( P < 0.01) in DOX-treated mice (which showed 84.1% fertility loss). These findings illuminate the crucial role of the CCL8/NF-κB axis in DOX-induced ovarian toxicity and propose a novel therapeutic strategy for fertility preservation. Bindarit could serve as a promising CCL8-targeted agent with substantial clinical potential. Biological sciences/Cell biology/Cell signalling Health sciences/Medical research Chemotherapy-induced ovarian damage CCL8/CCR5 axis M1 macrophages NF-κB signaling fertility preservation Bindarit Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The ovary is the central organ of the female reproductive system, performing two critical functions: reproduction and endocrine. In the reproduction function, the ovary regulates folliculogenesis and ovulation, which are crucial for female fertility. In the endocrine function, the ovary secretes sex hormones—including estrogens, progesterone, and small amounts of androgens—that regulate the hypothalamic-pituitary-ovarian axis to maintain menstrual cyclicity, promote maturation of the reproductive organs, and modulate systemic functions such as bone metabolism and immune homeostasis. With advancements in cancer therapeutics, patient survival rates have improved substantially. Data from the National Cancer Center of China indicate that the 5-year survival rate increased from 40.5% in 2015 to 43.7% in 2022 [ 1 , 2 ] . However, with the increase in the number of cancer survivors, concerns regarding treatment-related toxicities have also increased. Recent statistics indicate a 38% reduction in pregnancy rates among female cancer survivors compared to their expected rate prior to cancer diagnosis [ 3 ] . Doxorubicin (DOX), an anthracycline chemotherapeutic, is widely used to treat leukemias, lymphomas, sarcomas, and breast/ovarian cancers because of its potent antitumor activity [ 4 ] . The primary mechanism of action of DOX involves intercalation into the DNA strand to inhibit polymerase activity, thereby blocking nucleic acid synthesis. DOX also generates superoxide radicals that disrupt the cellular membrane of tumor cells. Although DOX is effective against malignancies, its clinical utility is limited by cardiotoxicity mediated by opening of the mitochondrial permeability transition pore, accumulation of reactive oxygen species, and activation of the cGAS-STING pathway [ 5 ] . Notably, DOX also exhibits significant ovarian toxicity [ 6 ] . Clinical studies report an increased incidence of amenorrhea post-DOX chemotherapy, reflecting follicular depletion and hypoestrogenism [ 7 ] . In in vitro experiments, DOX induces DNA double-strand breaks in granulosa cells and oocytes, leading to cell apoptosis [ 8 , 9 ] . These findings highlight DOX-induced ovarian failure as a critical clinical challenge, necessitating mechanistic insights and protective strategies. Current approaches, such as GnRH agonists, natural compounds (e.g., resveratrol and gallic acid), and antioxidants, have limitations. GnRH agonists exhibit inconsistent efficacy, while phytochemicals show poor bioavailability and lack standardized protocols [ 10 – 12 ] . Therefore, it is imperative to identify novel interventions to preserve ovarian function during chemotherapy. Emerging evidence implicates inflammatory dysregulation in DOX-induced ovarian damage. DOX triggers infiltration of monocytes and induces sustained inflammation through the production of proinflammatory cytokines (IL-1, IL-6, and TNF-α) and matrix metalloproteinase-mediated extracellular matrix (ECM) degradation. Chemokine receptor signaling promotes the recruitment of monocytes to inflamed tissues [ 13 , 14 ] . Among the chemokines, C-C motif chemokine 8 (CCL8/MCP-2), structurally homologous to CCL2/MCP-1, remains understudied in ovarian pathology [ 15 – 17 ] . Bindarit [2-(1-benzyl-1H-indazol-3-yl)methoxy)-2-methylpropanoic acid], a selective inhibitor of MCP-1/2/3, inhibits monocyte/macrophage recruitment without causing immunosuppression [ 18 , 20 ] . Preclinical studies have demonstrated the efficacy of Bindarit in nephritis, arthritis, and pancreatitis models. Clinical Phase II trials have established the favorable safety profile of Bindarit. Notably, the perivascular administration of Bindarit reduces venous fibrosis and stenosis following angioplasty by suppressing macrophage-mediated inflammation. These characteristics suggest the potential utility of Bindarit as a promising candidate for mitigating ovarian inflammation [ 21 ] . Despite advances in research, the precise mechanisms linking DOX to ovarian inflammatory cascades remain elusive, and effective therapeutic interventions are not yet available. Here, we established a murine model of DOX-induced ovarian injury to investigate the role of CCL8 and evaluate the therapeutic efficacy of bindarit. The results may contribute to developing new approaches for preserving ovarian function during chemotherapy, thereby enhancing the quality of life of female cancer survivors. Materials and Methods Mice Female ICR mice and male mice aged 7–8 weeks were purchased from and maintained in the animal facility at Nanjing Medical University. The mice were kept under a 12/12-h light/dark cycle at 22°C with free access to food and water. All animal experimental protocols were approved by the Committee on the Ethics of Animal Experiments at Nanjing Medical University (Approval/accreditation number: IACUC-2408025). All animal experiments adhered to the ARRIVE guidelines. Animal experiments A total of 60 female ICR mice at 7 weeks of age were used in this study. They were randomly assigned to three groups, with 20 mice per group. The control group (Ctrl group) was intraperitoneally administered phosphate-buffered saline. The DOX group received a single intraperitoneal injection of doxorubicin hydrochloride (10 mg/kg). The DOX + Bin group was intraperitoneally administered doxorubicin hydrochloride (10 mg/kg) following an intracapsular injection of Bindarit (4 mg/kg) into the ovaries. Ovarian tissues were collected at 24 and 48 h after injection for fixation, embedding, sectioning, protein extraction, and RNA extraction. Immunohistochemistry Ovaries were collected and fixed in 10% buffered formalin for paraffin embedding and sectioning. To detect the expression of Cleaved Caspase-3(Cell Signaling Technology [CST], Beverly, USA, Cat. no. catalog no.9661, 1: 400) and gamma H2A.X (Abcam, Cambridge, UK, Cat. no. Ab26350, 1: 400)5-µm sections were deparaffinized, rehydrated, and endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide in methanol for 15 min. The sections were then boiled in 0.01 M citrate buffer for antigen retrieval. After blocking with goat serum (ZSGB-Bio, China) for 1 h, primary antibodies against cleaved-caspase-3 and γ-H2AX were incubated overnight at 4°C. Diaminobenzidine (DAB) reagent was used for coloration on the following day. Non-immune immunoglobulin G (IgG) was applied as a negative control. Immunofluorescence assay For ovarian tissues, immunofluorescence assay was performed using antibodies for DDX4 (Abcam, Cambridge, UK, Cat. no. Ab13840, 1:400), PCNA (Cell Signaling Technology, Beverly, USA, Cat. no. 13110, 1:16,000), CCL8 (ABclonal, Wuhan, China, Cat. no. A6977, 1:100), IBA-1 (MedChemExpress, Monmouth Junction, USA, Cat. no. HY-P80501, 1:100), CD68 (Proteintech, Wuhan, China, Cat. no. 28058-1-AP, 1:500), CD206 (Proteintech, Wuhan, China, Cat. no. 18704-1-AP, 1:800), iNOS (Proteintech, Wuhan, China, Cat. no. 18985-1-AP, 1:500), and phospho-NF-κB p65 (Cell Signaling Technology, Beverly, USA, Cat. no. 3033, 1:3000). Following overnight incubation at 4°C, the primary antibodies were removed, and the sections were incubated with the corresponding secondary antibodies at room temperature for 1 h: Alexa Fluor 488-conjugated donkey anti-rabbit (Invitrogen, USA, Cat. No. A21206; 1:500), Alexa Fluor 594-conjugated donkey anti-mouse (Invitrogen, USA, Cat. No. A21203, 1:500), and Alexa Fluor 488-conjugated goat anti-mouse (Invitrogen, USA, Cat. No. A21202, 1:500). The nuclei were then stained with 0.01 mg/mL Hoechst 33342 (Invitrogen, USA, Cat. No. H1339) for 20 min, and the sections were observed under a confocal laser scanning microscope (Zeiss, Germany, Model No. LSM900). TUNEL assay TUNEL assay was performed on 5-µm-thick ovarian tissue sections by using the TUNEL Apoptosis Detection Kit (Alexa Fluor 640) (Yeasen Biotech, Shanghai, China, Cat. No 40308) in accordance with the manufacturer’s instructions. RNA isolation, reverse transcription, and reverse transcription- quantitative polymerase chain reaction (RT-qPCR) Total RNA from ovarian tissues was isolated using TRIzol reagent (Invitrogen, USA) in accordance with the manufacturer’s instructions. RNA concentration in ovarian tissues was measured using a spectrophotometer (NanoDrop 2000c, Thermo Scientific, Waltham, USA). Next, 500 ng of RNA per sample was reverse transcribed to cDNA by using the FastQuant RT Kit (Tianyuan Biotechnology, China). The obtained cDNA was then subjected to real-time PCR on an ABI Step One Plus platform (Thermo Scientific, Waltham, USA) by using a SYBR Green Master Mix (Applied Biological Materials, Canada), with actin amplification as an internal control. The specificity of the PCR products was assessed by melting curve analysis, and the amplicon size was determined by 2% agarose gel electrophoresis. RNA sequencing (RNA-seq) analysis A total of 3 µg RNA per sample was used as the input material for RNA sample preparation. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) in accordance with the manufacturer’s recommendations, with index codes added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was performed using divalent cations under elevated temperature in the NEBNext First Strand Synthesis Reaction Buffer (5×). First-strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA was subsequently synthesized using DNA polymerase I and RNase H. The remaining overhangs were converted into blunt ends through exonuclease/polymerase activity. After adenylation of the 3ʹ-ends of DNA fragments, NEBNext Adaptor with a hairpin loop structure was ligated for hybridization. To select cDNA fragments of preferentially 250 ~ 300 bp in length, the library fragments were purified with the AMPure XP System (Beckman Coulter, Beverly, USA). Next, 3 µL of USER ™ Enzyme (NEB, USA) was applied to size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. PCR was subsequently performed with Phusion ™ High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) primer. Finally, the PCR products were subjected to purification (AMPure XP system) and library quality assessment on the Agilent Bioanalyzer 2100 system. Bindarit administration The dosage of Bindarit (4 mg/kg) was determined based on prior preclinical studies employing systemic administration (e.g., 10 mg/kg via oral gavage) which demonstrated efficacy in inflammatory disease models [ 22 ] . Given that our study utilized a novel, targeted intracapsular injection method which ensures direct ovarian delivery and likely higher local drug concentration, a moderately lower dose of 4 mg/kg was selected to achieve therapeutic efficacy while minimizing potential systemic exposure. Bindarit was dissolved in DMSO and further diluted in phosphate-buffered saline (PBS) to the final working concentration. The injection volume was standardized at 10 µL per ovary. Surgical procedure for intracapsular ovarian injection Mice were anesthetized with avertin (240 mg/kg, i.p.). After shaving and disinfecting the dorsal skin near the kidney region, a small incision was made to expose the ovarian fat pad. The ovary was carefully externalized, and a 1 mL syringe needle was inserted into the ovarian capsule. Bindarit solution (10 µL per ovary) was slowly injected into the intracapsular space under a stereomicroscope to ensure proper distribution without leakage. The ovary was then returned to the abdominal cavity, and the incision was sutured layer by layer. Mice were monitored until fully recovered. CCL8 siRNA transfection in KGN cells. To investigate the role of CCL8 in DOX-induced inflammation, human granulosa cell line KGN was transfected with CCL8-specific siRNA (GenePharma,Shanghai GenePharma Co.,Ltd). After 48 h, cells were treated with 100 µM DOX for 24 h. Knockdown efficiency was validated by RT-qPCR. Immunoblotting assay Proteins were extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China, Cat. No. P0013B) containing protease inhibitor cocktails (Amresco, Solon, Ohio, USA, Cat. No. M221). A total of 10–30 µg proteins from each sample were loaded and separated by electrophoresis (Mini-PROTEAN® Tetra Cell, Bio-Rad, USA, Cat. No. 165–8000). The separated proteins were electroblotted (Mini Trans-Blot Electrophoretic Transfer Cell, Bio-Rad, Cat. No. 170–3930) onto polyvinylidene fluoride membranes (Thermo Fisher, USA, Cat. No. 88250), and the membranes were then blocked with 5% skimmed milk-TBST (Tris-buffered saline (TBS) containing 0.1% Tween 20) for 30 min; subsequently, the membranes were incubated overnight at 4°C with primary antibodies. Following washing with TBST (5 mL) for three times, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies to detect proteins through enhanced chemiluminescence (ECL Prime Western blotting detection reagent, GE Healthcare, Washington, NY, USA, Cat. No. RPN2232) on the Tanon 5200 analysis system. Statistical analysis Statistical analyses between groups were performed using an unpaired two-tailed Student’s t test or one-way analysis of variance with Tukey’s multiple comparison test. Error bars represent the standard error of the mean value. A P-value of < 0.05 was considered statistically significant. All statistical analyses were performed using Prism software (GraphPad Software, San Diego, CA, USA). Results DOX induces severe acute and chronic ovarian injury The chemotherapeutic agent DOX, although a crucial oncological drug, exerts substantial ovarian toxicity that warrants detailed investigation. In this study, we established a murine model of DOX-induced ovarian injury to comprehensively characterize the effects of DOX on ovarian histoarchitecture, fibrotic progression, and primordial follicle pool. By conducting integrated histomorphometric analysis, fibrosis marker assessment, and DNA damage evaluation, we elucidated the direct cytotoxic effects of DOX on ovarian tissues, thereby providing foundational evidence for subsequent mechanistic studies. In healthy, sexually mature mice, the ovarian surface is covered with an intact epithelial layer. The ovarian parenchyma shows a typical cortical-medullary structure; the cortical region contains abundant follicles at various developmental stages, including primordial, primary, and secondary follicles, with relatively few atretic follicles. The medullary region is less prominent and has a loosely organized structure. Hematoxylin and eosin (H&E) staining of ovarian sections obtained at time points following DOX treatment (Fig. 1A) revealed significant structural damage in DOX-treated ovaries, characterized by markedly increased atretic follicles within the cortex, cortical thinning with loosened tissue organization, significantly reduced number of growing follicles, medullary expansion, thickened follicular membrane, and decline in ovarian volume. Masson’s trichrome staining of paraffin-embedded ovarian sections revealed substantial pathological alterations in DOX-treated groups (Fig. 1B). Histological examination showed significant disruption of normal ovarian architecture in DOX-treated mice: the originally intact blue-stained reticular framework displayed fragmentation, with only a few follicle structures retaining intact zona pellucida. Notably, DOX administration induced extensive collagen deposition around follicular membranes, accompanied by abnormal invasion of luteal membrane granulosa cells into surrounding tissues. These pathological changes resulted in markedly increased fibrosis in both luteal and interstitial regions. These findings suggest that DOX may adversely affect ovarian function by disrupting collagen metabolism and compromising the structural integrity of ovarian tissues. To replicate the microenvironment of early ovarian development, neonatal mouse ovaries at postnatal day 3.5 were subjected to 3D ovarian culture (3D-Ovary Culture) for toxicity assessment. After 24-h culture under various treatment conditions, immunofluorescence assay revealed that compared to the normal Ctrl group, ovaries treated with doxorubicin hydrochloride (100 nM DOX) showed substantial accumulation of TUNEL-positive cells within primordial follicles (Fig. 1C-D), indicating DOX-induced premature apoptotic activation in primordial follicles. We developed a mouse model of ovarian injury through a single intraperitoneal injection of doxorubicin hydrochloride (10 mg/kg DOX); ovarian tissues were collected at 24 h post-injection for immunohistochemical analysis of paraffin-embedded tissues. The results showed a marked increase in cleaved caspase-3-positive cells in DOX-treated ovarian tissues compared to that in the Ctrl group (Fig. 1E, DAB staining). Additionally, significant accumulation of γ-H2A.X-positive signals was detected (Fig. 1F, DAB staining), indicating the occurrence of DNA double-strand breaks [ 22 ] . These observations confirmed that DOX rapidly activates ovarian cell apoptosis pathways and induces follicular DNA damage within 24 h, providing direct experimental evidence for elucidating the reproductive toxicity mechanisms of DOX. To assess the chronic effects of doxorubicin hydrochloride on ovarian tissues, we measured the ovarian coefficient (ovarian weight/body weight) at multiple time points (24, 36, 48, and 96 h, and 14 and 21 d) following a single intraperitoneal injection of 10 mg/kg doxorubicin hydrochloride. The results revealed a significant reduction in ovarian coefficient at all time points compared to that in the Ctrl group (Fig. 1H, P < 0.05). Concurrent quantitative analysis of follicles at different developmental stages (Fig. 1I) demonstrated a time-dependent decrease in the total follicle count in the DOX-treated group, with the degree of reduction showing a negative correlation with time post-injection. At 21 days post-DOX administration, female ICR mice from the model group were paired 1:1 with healthy male ICR mice for 19–21 days. Reproductive outcomes revealed that the DOX-treated group produced significantly fewer offspring (4.6 ± 1.52) compared to the Ctrl group (15.4 ± 1.10) (Fig. 1J-K, P < 0.001, unpaired t-test). These findings demonstrate that DOX exposure significantly impairs reproductive capacity in female mice. Integrated transcriptome and CIBERSORT analysis reveals the CCL8/immune cell regulatory network in DOX-induced ovarian toxicity To elucidate the mechanisms underlying DOX-induced ovarian toxicity, we conducted RNA-seq analysis on bilateral ovarian tissues from Ctrl mice (n = 5) and DOX-treated (n = 5) mice (Fig. 2A-B; sample inclusion criteria shown in Fig. S1 ). The sequencing analysis identified 350 differentially expressed genes (DEGs) in the DOX group (|logFC| > 1, P < 0.05). Gene Ontology (GO) enrichment analysis revealed the following significant terms: Extracellular space (GO:0005615, false discovery rate (FDR) = 0.0001021) and Extracellular region (GO:0005576, FDR = 0.0001584) (Fig. 2C); this finding suggests that DOX disrupts ovarian tissue architecture and ECM homeostasis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (Fig. 2D) showed that the DEGs were primarily enriched in the following pathways: Cytokine-cytokine receptor interaction (mmu04060, P = 0.001854) and Chemokine signaling pathway (mmu04062, P = 0.008657). Notably, CCL8 (log2FC = 2.53, P = 1.81E-06) was significantly upregulated in both pathways, suggesting its potential role in DOX-induced ovarian injury through cytokine network regulation and chemokine-mediated signaling. This observation provided important insights into subsequent mechanistic investigations. Immunofluorescence assay confirmed elevated CCL8 expression in DOX-treated ovaries (Fig. 2E-F). RT-qPCR further verified the sequencing results (Fig. 2G-H), with CCL8 showing the most prominent upregulation in DOX mice. According to CIBERSORT-based immune infiltration analysis and visualization by ggplot2, M1 macrophages, activated NK cells, and Th1 cells were in the immunoactive state in the DOX group; however, these cells were in the immunosuppressive state in the Ctrl group (Fig. 2I-K). These results indicate that DOX treatment restructures the immune microenvironment by modifying cellular composition and function, thereby activating immune responses. Collectively, these findings establish a theoretical foundation for further investigation of immunoregulatory mechanisms and optimization of immunotherapeutic strategies. CCL8/CCR5-mediated M1 macrophage activation promotes doxorubicin-induced ovarian inflammation and apoptosis DOX treatment induces proinflammatory monocyte infiltration that promotes M1 macrophage polarization in ovarian tissues, exacerbating inflammatory responses through the increased secretion of IL-1β and TNF-α, subsequently resulting in tissue damage. Immunofluorescence assay revealed substantial accumulation of IBA-1⁺ macrophages in the follicular, luteal, and stromal regions of DOX-treated ovaries (Fig. 3A-B), exhibiting distinct spatial colocalization with TUNEL⁺ apoptotic cells (Fig. 3C-D); this finding suggests active macrophage participation in apoptotic clearance. RT-qPCR revealed significant upregulation of proinflammatory cytokines (IL-1α, IL-1β, and TNF-α) in the DOX group (Fig. 3E, P < 0.01). A detailed immunophenotyping analysis showed a significant shift toward M1-dominant polarization, characterized by an increased number of CD68⁺ M1 macrophages (Fig. 3F-G), an enhanced expression of M1 markers (CD40, CD86, and TLR4; Fig. 3J, P < 0.05), and a stable level of M2 macrophage markers (CD36, Arg1, and IL-10) (Fig. 3H-I, K). Bioinformatics analysis based on the KEGG pathway enrichment analysis and protein-protein interaction network identified CCL8-CCR5 as the primary interaction pair, showing a strong correlation at the mRNA level (Fig. 3L-M). These observations support a mechanistic model wherein DOX upregulates CCL8 expression to stimulate CCR5-mediated macrophage recruitment and M1 polarization, resulting in ovarian inflammatory injury and functional deterioration. To further elucidate the key role of CCL8 in mediating ovarian inflammatory responses, in vitro experiments were conducted using the human granulosa cell line KGN. KGN cells were divided into three groups: a blank control group (no treatment), a DOX-treated group (100 µM doxorubicin for 24 hours), and a CCL8-siRNA + DOX group (transfected with CCL8-specific siRNA for 48 hours followed by the same dose of doxorubicin). By targeting CCL8 with small interfering RNA (siRNA), RT-qPCR results (Fig. 4A) showed that CCL8 expression in the DOX-treated group increased to 2.897 ± 0.512 (vs. Control, P < 0.001), while in the CCL8-siRNA group, expression decreased to 0.399 ± 0.073, achieving a knockdown efficiency of 66.1% (P = 0.003). RT-qPCR analysis further demonstrated that, compared to the DOX-treated group, knockdown of CCL8 significantly downregulated the mRNA levels of the pro-inflammatory cytokines IL-1α and TNF-α (Fig. 4B, p < 0.05). Concurrently, the mRNA expression of M1 macrophage activation markers iNOS, CD86, and CD40 was also markedly reduced (Fig. 4C, p < 0.01), indicating that CCL8 knockdown effectively suppresses inflammatory responses and pro-inflammatory polarization of macrophages. In summary, CCL8 serves as a key chemokine mediating macrophage recruitment to ovarian tissue. Specific inhibition of CCL8 effectively blocks the activation and accumulation of inflammatory macrophages, thereby ameliorating doxorubicin-induced ovarian injury. Bindarit ameliorates DOX-induced ovarian damage and fertility decline by simultaneously suppressing inflammation and apoptosis To examine the protective effects of Bindarit against DOX-induced ovarian inflammation, we established an in vivo intervention model. The results demonstrated that while DOX treatment significantly reduced the ovarian coefficient in mice, immediate subcapsular ovarian administration of Bindarit (4 mg/kg) post-DOX modeling effectively restored this parameter (Fig. 5A). The Bindarit-only group exhibited no abnormalities, confirming its safety profile. Histological analyses revealed that Bindarit treatment markedly ameliorated DOX-induced structural damage (H&E staining, Fig. 5B) and disruption of collagen metabolism (Masson’s trichrome staining, Fig. 5C) in ovarian tissues. Immunofluorescence assay demonstrated that Bindarit significantly inhibited DOX-induced abnormal apoptosis of granulosa cells (Fig. 5D-G), indicating its potential anti-apoptotic mechanism. Functional assessments revealed that while DOX administration substantially decreased litter size compared to that in the Ctrl group, Bindarit when administered alone partially reversed this fertility impairment (Fig. 5H-I), without affecting reproductive function. These findings demonstrate that Bindarit restores DOX-induced fertility dysfunction through three mechanisms: reduction of the ovarian inflammatory response, maintenance of tissue integrity, and inhibition of apoptotic pathways. Bindarit attenuates ovarian inflammatory injury by suppressing CCL8-dependent macrophage infiltration To validate the pivotal role of Bindarit in influencing CCL8-mediated macrophage recruitment to granulosa cells, we examined the effects of Bindarit on macrophage infiltration and activation. Immunofluorescence assay demonstrated that Bindarit treatment significantly reduced the number of IBA-1⁺ macrophages and CD68⁺ phagocytic activity in ovarian tissues (Fig. 6A-D), indicating effective suppression of macrophage accumulation. Ovarian sections from Bindarit-treated mice exhibited markedly decreased CCL8 immunoreactivity concurrent with reduced infiltration of iNOS⁺ macrophages (Fig. 6E-F, P < 0.01). RT-qPCR analysis confirmed that Bindarit reduced mRNA expression of proinflammatory cytokines (IL-1α, IL-1β, and TNF-α; Fig. 6G) and M1 macrophage markers (CD40, CD86, and TLR4; Fig. 6H), suggesting inhibition of proinflammatory macrophage polarization. Notably, Bindarit significantly decreased CCL8 expression at both protein (immunofluorescence assay) and mRNA levels (Fig. 6E-G), with CCL8 suppression showing a strong correlation with reduced macrophage infiltration and enhanced granulosa cell apoptosis (Fig. 6I-M). These findings establish CCL8 as the primary chemokine that regulates macrophage recruitment to ovarian tissues, while demonstrating that Bindarit’s targeted inhibition of CCL8 prevents inflammatory macrophage activation and accumulation, thus mitigating DOX-induced ovarian injury. Bindarit mitigates doxorubicin-induced ovarian toxicity by targeting the NF-κB/p65-IκBα axis Given the established role of CCL8 in promoting NF-κB activation via macrophage-derived cytokines, we next investigated whether Bindarit’s protective effects involve suppression of the CCL8/NF-κB axis. Our data demonstrate that DOX-induced CCL8 upregulation precedes NF-κB activation, as evidenced by increased p65 phosphorylation and IκBα degradation. Bindarit treatment significantly reduced CCL8 expression, which in turn attenuated NF-κB signaling, indicating a causal link between CCL8 suppression and NF-κB inactivation. To investigate the critical role of NF-κB signaling in DOX-induced ovarian injury, we examined the subcellular localization of phosphorylated p65 (p-p65) by immunofluorescence assay. The analysis revealed significantly elevated p-p65 expression in both nuclear and cytoplasmic compartments of DOX-treated ovarian cells, while Bindarit treatment markedly reduced the number of p-p65-positive cells (Fig. 7A-B). Western blotting assay demonstrated that DOX exposure decreased the cytoplasmic level of inactive p65 while increasing the levels of phosphorylated p65 (p-p65) and phosphorylated IκB-α, indicating sustained NF-κB pathway activation. Conversely, Bindarit administration effectively reversed these alterations (Fig. 7C). Further analyses revealed that DOX treatment downregulated the expression of prosurvival p-AKT and upregulated the apoptosis marker cleaved-PARP1; both these effects were significantly ameliorated by Bindarit intervention (Fig. 7D). Notably, the expression of iNOS—a downstream NF-κB target and M1 macrophage marker—was elevated in the DOX group but suppressed by Bindarit treatment (Fig. 7E-F). In summary, Bindarit alleviates inflammatory responses, suppresses macrophage M1 polarization, and ultimately mitigates DOX-induced apoptosis and functional impairment in ovarian granulosa cells by inhibiting the activation of the NF-κB pathway—specifically through reducing p65 phosphorylation and nuclear translocation, while promoting IκB-α-mediated negative feedback regulation. These findings not only underscore the central role of the NF-κB pathway in ovarian inflammatory injury but also provide a mechanistic basis for the clinical application of Bindarit. Discussion Chronic inflammation is the primary driver of chemotherapy-induced ovarian damage (CIOD). The present study is the first to demonstrate that Bindarit significantly mitigates the ovarian toxicity of DOX by targeting the CCL8-mediated immune regulatory network. Bindarit improves the ovarian inflammatory microenvironment and granulosa cell survival by inhibiting CCL8-dependent macrophage recruitment and M1 macrophage polarization, thereby suppressing the excessive activation of the NF-κB/p65 pathway. Previous studies have shown a significant decline in follicular reserve among chemotherapy patients, although the underlying mechanism remains unclear [ 23 ] . The present study simulated clinical chemotherapy scenarios by using animal models and found that DOX-induced sustained overexpression of CCL8 promotes inflammatory monocyte infiltration. The GO enrichment analysis and the KEGG pathway enrichment analysis confirmed that these changes are closely associated with CCR5-mediated immune cell migration pathways. Notably, unlike the self-limiting nature of physiological inflammation, CCL8/NF-κB signaling in the DOX group exhibited persistent activation and showed a strong correlation with irreversible ovarian tissue damage. These findings elucidate the central role of the CCL8-CCR5 axis in CIOD and provide a theoretical foundation for the clinical application of Bindarit. Bindarit, a broad-spectrum chemokine inhibitor, shows protective effects in various inflammatory diseases by inhibiting CCL2-mediated monocyte infiltration [ 24 ] . This investigation represents the first application of Bindarit in CIOD. Ovarian granulosa cell apoptosis and follicular atresia constitute the primary pathological features of DOX-induced ovarian toxicity. This study demonstrated that Bindarit treatment substantially reduces DOX-triggered granulosa cell apoptosis (as evidenced by downregulated expression of cleaved-PARP1) and enhances primordial follicle survival. The maintenance of follicular microenvironment homeostasis is essential for female reproductive function, and controlling excessive inflammatory responses may be crucial for ovarian reserve protection. The analysis of local ovarian inflammatory status revealed that Bindarit-treated groups showed markedly reduced levels of proinflammatory cytokines (IL-1β and TNF-α). Notably, chemotherapy-induced TNF-α elevation promotes granulosa cell apoptosis through NF-κB signaling activation, while clinical studies indicate that anti-TNF-α therapy improves ovarian function in chemotherapy patients. Therefore, one of the main mechanisms through which Bindarit mitigates DOX-induced ovarian toxicity may involve disruption of this vicious cycle by reducing TNF-α levels. Moreover, current evidence suggests that proinflammatory cytokines accelerate follicular atresia by activating the NF-κB pathway within the follicular microenvironment [ 25 ] . The findings of the present study confirm this hypothesis: Bindarit preserves ovarian structure and function by inhibiting CCL8-dependent NF-κB activation, evidenced by a decrease in p-p65 nuclear translocation and suppression of IκB-α degradation. Macrophage polarization status plays a dual role in the progression and restoration of CIOD. Previous research indicates that the dynamic balance between proinflammatory (M1) and anti-inflammatory (M2) macrophages determines the extent of ovarian tissue damage [ 26 ] . The present study revealed that DOX treatment significantly upregulated M1 macrophage markers (iNOS, CD86, IL-1β, and TNF-α) and suppressed M2 markers (CD36, Arg1, and IL-10) in the ovarian microenvironment, and this imbalance showed a positive correlation with follicular atresia severity (Fig. 3). Notably, Bindarit treatment effectively downregulated M1-associated gene expression (P < 0.01), suggesting its capacity to reprogram macrophage phenotype. In vitro experiments further demonstrated that ovarian macrophages in the Bindarit treatment group secreted 60–70% lower levels of TNF-α, IL-1α, and IL-1β compared to the macrophages in the DOX group (P < 0.001); this finding was consistent with previous reports that M2 macrophages inhibit ovarian fibrosis through IL-10 secretion. The present research demonstrates that Bindarit significantly ameliorated DOX-induced ovarian damage by targeting the CCL8/NF-κB axis through three primary mechanisms: inhibition of CCL8-dependent macrophage (particularly M1-type) recruitment to ovarian tissues, suppression of NF-κB overactivation, and reduction of proinflammatory cytokine levels (IL-1β, TNF-α, etc.). Notably, Bindarit effectively increased follicular reserve and subsequent embryonic developmental potential by reducing apoptosis (evidenced by decreased cleaved-PARP1 expression), ultimately improving reproductive outcomes. Although previous studies have reported conflicting results regarding CCL2/CCL7-mediated regulation of macrophage polarization with M1/M2 phenotypic discrepancies, the present research revealed the distinct role of CCL8 in DOX-induced ovarian toxicity—specifically driving M1 polarization and establishing a positive feedback loop with the NF-κB pathway. Notably, the inhibitory effect of Bindarit on CCL8 exceeds that of other broad-spectrum chemokine inhibitors, emphasizing its clinical potential for targeted therapy. Further investigations should address the following questions: (1) whether CCL8 indirectly modulates ovarian inflammation by regulating T-cell subsets (e.g., Th17/Treg balance) and (2) how CCL8 secreted by follicular granulosa cells forms paracrine circuits with macrophages. Future studies should focus on developing ovarian-targeted delivery systems for Bindarit (e.g., exosome-based carriers) and evaluating its synergistic effects with existing ovarian protectants (e.g., GnRH agonists). The proposed “CCL8-targeting strategy” presents a novel therapeutic approach for preserving ovarian function in chemotherapy patients, particularly in prepubertal cancer survivors. Declarations Funding This work was supported by the National Science Foundation of China | Key Programme (2024YFC2706704); Natural Science Research of Jiangsu Higher Education Institutions of China (22KJB320004); Special Project for High-quality Development of Maternal and Child Health of Jiangsu Provincial Hospital (Grant No. [GZL2501]); Cell Therapy Foundation (Grant No. [303100784AA25]) Contributions CF: conception and design, collecting and assembling data, analyzing and interpreting data, writing manuscripts; NL,YQ: collecting and assembling data, data analysis, and interpretation, writing manuscripts; MY,XN,QC: collecting and assembling data, data analysis; FW,YC,QC: conception and design, experimental technical support; JL, XM: conception and design, administrative support, financial support, manuscript writing and final approval of the manuscript. All authors reviewed the manuscript. Corresponding author Correspondence to Jing Li and Xiang Ma Ethical approval and consent to participate All animal protocols were approved by the Committee on the Ethics of Nanjing Medical University. [Approval/accreditation number: IACUC-2408025] Consent for publication All authors have approved the manuscript for submission. Competing interests The authors declare no conflict of interest. Clinical trial number: not applicable. Data availability All data generated or analyzed during this study are included in this published article. References Zeng H, Zheng R, Sun K, et al. Cancer survival statistics in China 2019–2021: a multicenter, population-based study. J Natl Cancer Cent. 2024;4(3):203-13. doi: 10.1016/j.jncc.2024.06.005. Zeng H, Chen W, Zheng R, et al. Changing cancer survival in China during 2003–15: a pooled analysis of 17 population-based cancer registries. Lancet Glob Health. 2018;6(5):e555-67. doi: 10.1016/S2214-109X(18)30127-X. Anderson RA, Brewster DH, Wood R, et al. The impact of cancer on subsequent chance of pregnancy: a population-based analysis. Hum Reprod. 2018;33(7):1281-90. doi: 10.1093/humrep/dey216. D’Angelo NA, Noronha MA, Câmara MCC, et al. Doxorubicin nano formulations on therapy against cancer: An overview from the last 10 years. Biomater Adv. 2022;133:112623. doi: 10.1016/j.msec.2021.112623. Zhang S, Liu X, Bawa-Khalfe T, et al. Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat Med. 2012;18(11):1639-42. doi: 10.1038/nm.2919. Morgan S, Anderson RA, Gourley C, et al. How do chemotherapeutic agents damage the ovary? Hum Reprod Update. 2012;18(5):525-35. doi: 10.1093/humupd/dms022. Aziz AUR, Yu X, Jiang Q, et al. Doxorubicin-induced toxicity to 3D-cultured rat ovarian follicles on a microfluidic chip. Toxicol In Vitro. 2020;62:104677. doi: 10.1016/j.tiv.2019.104677. Ben-Aharon I, Bar-Joseph H, Tzarafaty G, et al. Doxorubicin-induced ovarian toxicity. Reprod Biol Endocrinol. 2010;8:20. doi: 10.1186/1477-7827-8-20. Jurisicova A, Lee HJ, D’Estaing SG, et al. Molecular requirements for doxorubicin-mediated death in murine oocytes. Cell Death Differ. 2006;13(9):1466-74. doi: 10.1038/sj.cdd.4401819. Blumenfeld Z, von Wolff M. GnRH-analogues and oral contraceptives for fertility preservation in women during chemotherapy. Hum Reprod Update. 2008;14(6):543-52. doi: 10.1093/humupd/dmn022. Bildik G, Akin N, Senbabaoglu F, et al. GnRH agonist leuprolide acetate does not confer any protection against ovarian damage induced by chemotherapy and radiation in vitro. Hum Reprod. 2015;30(12):2912-25. doi: 10.1093/humrep/dev257. (注：原DOI指向该页码) Turner NH, Partridge A, Sanna G, et al. Utility of gonadotropin-releasing hormone agonists for fertility preservation in young breast cancer patients: the benefit remains uncertain. Ann Oncol. 2013;24(9):2224-35. doi: 10.1093/annonc/mdt196. Charo IF, Ransohoff RM. The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med. 2006;354(6):610-21. doi: 10.1056/NEJMra052723. Zernecke A, Shagdarsuren E, Weber C. Chemokines in atherosclerosis: an update. Arterioscler Thromb Vasc Biol. 2008;28(11):1897-908. doi: 10.1161/ATVBAHA.107.161174. Van Damme J, Proost P, Put W, et al. Induction of monocyte chemotactic proteins MCP-1 and MCP-2 in human fibroblasts and leukocytes by cytokines and cytokine inducers. Chemical synthesis of MCP-2 and development of a specific RIA. J Immunol. 1994;152(11):5495-502. Van Coillie E, Proost P, Van Aelst I, et al. Functional comparison of two human monocyte chemotactic protein-2 isoforms, role of the amino-terminal pyroglutamic acid and processing by CD26/dipeptidyl peptidase IV. Biochemistry. 1998;37(36):12672-80. doi: 10.1021/bi980497d. Van Damme J, Proost P, Lenaerts JP, et al. Structural and functional identification of two human, tumor-derived monocyte chemotactic proteins (MCP-2 and MCP-3) belonging to the chemokine family. J Exp Med. 1992;176(1):59-65. doi: 10.1084/jem.176.1.59. Bhatia M, Ramnath RD, Chevali L, et al. Treatment with bindarit, a blocker of MCP-1 synthesis, protects mice against acute pancreatitis. Am J Physiol Gastrointest Liver Physiol. 2005;288(6):G1259-65. doi: 10.1152/ajpgi.00435.2004. Mirolo M, Fabbri M, Sironi M, et al. Impact of the anti-inflammatory agent bindarit on the chemokinome: selective inhibition of the monocyte chemotactic proteins. Eur Cytokine Netw. 2008;19(3):119-22. doi: 10.1684/ecn.2008.0133. Perico N, Benigni A, Remuzzi G. Present and future drug treatments for chronic kidney diseases: evolving targets in renoprotection. Nat Rev Drug Discov. 2008;7(11):936-53. doi: 10.1038/nrd2685. Singh AK, Kilari S, Cai C, et al. Bindarit encapsulated nanoparticles prevent venous neointimal hyperplasia and restenosis in a murine angioplasty model. Transl Res. 2022;248:68-86. doi: 10.1016/j.trsl.2022.06.002. Zoja C, Abbate M, Corna D, et al. Bindarit retards renal disease and prolongs survival in murine lupus autoimmune disease. Kidney Int. 1998;53(3):726-34. doi: 10.1046/j.1523-1755.1998.00791.x. Roti Roti EC, Leisman SK, Abbott DH, et al. Acute doxorubicin insult in the mouse ovary is cell- and follicle-type dependent. PLoS One. 2012;7(8):e42293. doi: 10.1371/journal.pone.0042293. Mora E, Guglielmotti A, Biondi G, et al. Bindarit: An anti-inflammatory small molecule that modulates the NF-κB pathway. Cell Cycle. 2012;11(1):159-69. doi: 10.4161/cc.11.1.18559. Fabbri R, Macciocca M, Vicenti R, et al. Epigallocatechin-3-gallate inhibits doxorubicin-induced inflammation on human ovarian tissue. Biosci Rep. 2019;39(5):BSR20181424. doi: 10.1042/BSR20181424. Lech M, Gröbmayr R, Ryu M, et al. Macrophage phenotype controls long-term AKI outcomes—kidney regeneration versus atrophy. J Am Soc Nephrol. 2014;25(2):292-304. doi: 10.1681/ASN.2013020152. Additional Declarations (Not answered) Supplementary Files OriginalimagesforFigure7.pdf Original images for Figure 7 Supportinginformation.tif Table S1 Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 31 Mar, 2026 Review # 2 received at journal 31 Mar, 2026 Review # 1 received at journal 19 Mar, 2026 Reviewer # 2 agreed at journal 16 Mar, 2026 Reviewer # 1 agreed at journal 15 Mar, 2026 Reviewers invited by journal 03 Mar, 2026 Submission checks completed at journal 23 Feb, 2026 Editor assigned by journal 22 Feb, 2026 First submitted to journal 22 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8939659","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":599914497,"identity":"92c8c3c3-9310-4d29-8a03-cdeb73ece205","order_by":0,"name":"Xiang Ma","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYNACAyBmb2x88IE0LTyHmw1nkGaTRHqbNAcxCuVnZCd+5imwy5OPfNggzcBgJ6fbQMhJZ85ulpxhkFxseDuxwbiAIdnY7AAhLey9GyQ+GDAnbpyd2JA8g+FA4jZCWuSbeTf/SDCoT9w482DDYR5itDAc790GtOVw4nwJxsZmorQA/bLNcobB8cQNPInNjEBPEfaL/Izczbd5/lQnzm8//vzHhwo7OYJaENaBVRoQqxxsXQMpqkfBKBgFo2BEAQDXFUYuhhlr5QAAAABJRU5ErkJggg==","orcid":"","institution":"Jiangsu Province Hospital","correspondingAuthor":true,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Ma","suffix":""},{"id":599914498,"identity":"f7e00411-834c-4af4-ac37-145144380e97","order_by":1,"name":"chenzi Feng","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"chenzi","middleName":"","lastName":"Feng","suffix":""},{"id":599914499,"identity":"7ffcfb80-82e6-4b48-bb41-1b71edd4c3bb","order_by":2,"name":"Nan Lu","email":"","orcid":"","institution":"The First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Nan","middleName":"","lastName":"Lu","suffix":""},{"id":599914500,"identity":"464cf3d2-e4db-42d8-b03c-5f4be09f7bbf","order_by":3,"name":"Yi Qian","email":"","orcid":"","institution":"the First Affiliated Hospital of Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Qian","suffix":""},{"id":599914501,"identity":"8202d15a-4e6b-4414-98b6-6ba8a304c99e","order_by":4,"name":"Mingyu Yang","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Mingyu","middleName":"","lastName":"Yang","suffix":""},{"id":599914502,"identity":"1594e3f6-5f21-46b1-9389-7a2cddc958c5","order_by":5,"name":"Xin Ning","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Ning","suffix":""},{"id":599914503,"identity":"4ede6ab4-7de2-4062-8704-0b0470ce6afa","order_by":6,"name":"Qi Chen","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Chen","suffix":""},{"id":599914504,"identity":"e8e86554-43ff-4c38-b4ab-a3d485b326a6","order_by":7,"name":"HaoFeng Wang","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"HaoFeng","middleName":"","lastName":"Wang","suffix":""},{"id":599914505,"identity":"098386a9-272c-433b-9414-6810575de8ec","order_by":8,"name":"YaTing Chen","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"YaTing","middleName":"","lastName":"Chen","suffix":""},{"id":599914506,"identity":"d8794cb5-6ec7-4a7d-bdaf-374aba779953","order_by":9,"name":"Qi Chen","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Chen","suffix":""},{"id":599914507,"identity":"e7d76529-27c9-4065-a9e1-3282e8cdd71d","order_by":10,"name":"Jing Li","email":"","orcid":"https://orcid.org/0000-0001-8692-4981","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2026-02-22 14:15:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8939659/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8939659/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104113555,"identity":"6ce735ce-ebdd-4c02-aed5-f3fd45163d26","added_by":"auto","created_at":"2026-03-07 03:34:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":10213557,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e H\u0026amp;E-stained pathological sections of mouse ovarian tissues at different time points after a single intraperitoneal injection of doxorubicin (10 mg/kg). Scale bar = 200 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e Representative Masson-stained images of mouse ovaries at different time intervals post single doxorubicin injection (10 mg/kg). Collagen fibers and zona pellucida appear blue, while other components stain red. Scale bar = 200 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eTUNEL and DDX4 co-staining showing apoptosis in cultured ovaries after 24 hours. Germ cells display green fluorescence, apoptotic signals show red fluorescence, and nuclei are counterstained blue with DAPI. Scale bar = 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003e\u0026nbsp;Quantitative analysis of DDX4 and TUNEL immunofluorescence intensity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u003c/strong\u003eImmunohistochemical staining of Cleaved-caspase-3 in mouse ovarian tissues collected 24 hours after doxorubicin injection (10 mg/kg). Scale bar = 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003eγ-H2A.X immunohistochemistry detecting DNA damage in primordial follicles 24 hours post doxorubicin administration in vivo. Scale bar = 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.\u003c/strong\u003e Quantitative analysis of Cleaved-caspase-3 and γ-H2A.X positive signals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH.\u003c/strong\u003e Ovarian coefficient (ovary weight/body weight ratio) at different time points after single doxorubicin injection (10 mg/kg). Data presented as mean ± SD (n=6). *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI. \u003c/strong\u003eDynamic quantification of follicular counts (primordial, primary, secondary, and atretic follicles) in ovarian sections at different post-treatment intervals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ. \u003c/strong\u003eRepresentative photographs of female mice and their offspring in control versus doxorubicin-treated groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK. \u003c/strong\u003eStatistical analysis of litter sizes between groups (n=6).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8939659/v1/10bf3b9e6ee9c95c21a9df37.png"},{"id":104113550,"identity":"6aa68577-b435-4d6e-9f36-8de94a112c45","added_by":"auto","created_at":"2026-03-07 03:34:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4413260,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eDifferentially expressed genes (DEGs) between control and doxorubicin (DOX)-treated mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eVolcano plot illustrating DEGs in mouse ovaries 24 hours after DOX treatment (10 mg/kg) compared to controls (|logFC| \u0026gt; 1, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eTop 30 enriched GO terms of DEGs (DOX vs. control). Green: Biological Process (BP); orange: Cellular Component (CC); purple: Molecular Function (MF).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eSignificantly enriched signaling pathways of DEGs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u003c/strong\u003eImmunofluorescence staining of CCL8 (red) in DOX-treated mouse ovaries. Scale bar = 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003eQuantitative analysis of CCL8-positive signals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG.\u003c/strong\u003eRNA-seq analysis revealed significantly upregulated CCL8 expression in DOX-treated ovaries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH.\u003c/strong\u003e qPCR validation confirmed elevated CCL8 mRNA levels in DOX-treated ovaries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI.\u003c/strong\u003e Inferred composition of 25 immune cell subsets in control and DOX-treated groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ. \u003c/strong\u003eDifferentially expressed immune-related genes between control and DOX-treated groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK. \u003c/strong\u003eCIBERSORT analysis indicated a significant increase in monocyte infiltration after DOX treatment.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8939659/v1/2f9f5a461e889e94a0e5c09e.png"},{"id":104113551,"identity":"6d3796b5-8ea3-41de-85e7-bcf6476da494","added_by":"auto","created_at":"2026-03-07 03:34:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6192929,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eImmunofluorescence labeling of IBA-1⁺ cells (red) in mouse ovaries after doxorubicin treatment. Scale bar = 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eQuantitative analysis of IBA-1 fluorescence intensity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eCo-localization of TUNEL⁺ apoptotic cells (green) and IBA-1⁺ macrophages (red) in doxorubicin-treated ovaries. Scale bar = 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eStatistical analysis of TUNEL and IBA-1 fluorescence signals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u003c/strong\u003eExpression levels of classical inflammatory cytokines in doxorubicin-treated vs control ovaries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF.\u003c/strong\u003e Immunofluorescence staining of CD68⁺IBA-1⁺ macrophages (red). Scale bar = 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG. \u003c/strong\u003eQuantitative analysis of CD68 and IBA-1 co-expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH.\u003c/strong\u003e Immunofluorescence staining of CD206⁺IBA-1⁺ macrophages (red). Scale bar = 50 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI. \u003c/strong\u003eQuantitative analysis of CD206 and IBA-1 co-expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ. \u003c/strong\u003emRNA expression levels of M1 macrophage markers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK. \u003c/strong\u003emRNA expression levels of M2 macrophage markers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL. \u003c/strong\u003eProtein-protein interaction network showing the most significant interaction between CCL8 and its receptor CCR5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM. \u003c/strong\u003emRNA expression analysis of CCL8 and CCR5.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8939659/v1/f0fd2efa7749daf40b84069c.png"},{"id":104403346,"identity":"4d5a46f1-07f8-4955-9def-f8a574a2a3c2","added_by":"auto","created_at":"2026-03-11 12:18:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":666368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003emRNA expression levels of CCL8 in KGN cells after small interfering RNA (siRNA)-mediated knockdown (n = 3 biological replicates).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003emRNA levels of pro-inflammatory cytokines (IL-1α, TNF-α) in CCL8-knockdown KGN cells (siCCL8) compared to negative control (siNC) (n = 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003emRNA expression of M1 macrophage activation markers (iNOS, CD86, CD40) in siCCL8-treated KGN cells (n = 3).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8939659/v1/1538e1603aec80d0dc712ff2.png"},{"id":104403278,"identity":"82068409-ecb6-4ab3-8724-9342c78f1e2a","added_by":"auto","created_at":"2026-03-11 12:17:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7158194,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eComparison of ovarian coefficients among the control group, doxorubicin (DOX)-treated group, DOX-treated + Bindarit subcapsular injection group, and Bindarit-only subcapsular injection group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eH\u0026amp;E staining of paraffin-embedded ovarian sections from each group. Scale bar = 200 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eMasson staining of paraffin-embedded ovarian sections from each group. Scale bar = 200 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eImmunofluorescence staining of γ-H2A.X⁺ cells (red) in ovarian tissues from control and DOX-treated mice (n = 5 animals per group). Nuclei were counterstained with DAPI (blue). Scale bar = 50 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u003c/strong\u003eQuantitative analysis of γ-H2A.X⁺ cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003eRepresentative immunofluorescence images showing TUNEL⁺ (red) and DDX4⁺ (green) germ cells in neonatal mouse ovaries cultured in 3D conditions (n = 5 replicates per group). Nuclei were stained with DAPI (blue). Scale bar = 50 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG. \u003c/strong\u003eStatistical analysis of TUNEL⁺ and DDX4⁺ cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH. \u003c/strong\u003eRepresentative images of offspring from each group (n = 5 animals per group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI. \u003c/strong\u003eStatistical analysis of litter sizes. Error bars represent SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001 (unpaired two-tailed Student’s t-test).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8939659/v1/38a60d5e8933020bf2b6e712.png"},{"id":104113558,"identity":"6f6dc8f4-c008-4291-ba6d-bd6b2a5b6bf2","added_by":"auto","created_at":"2026-03-07 03:34:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9535671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eImmunofluorescence staining of IBA-1⁺ cells (red) in ovarian tissues from control, doxorubicin (DOX)-treated, and DOX+Bindarit-treated mice (n = 5 animals per group). Nuclei were counterstained with DAPI (blue). Scale bar = 50 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eQuantitative analysis of IBA-1⁺ cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eImmunostaining of IBA-1⁺ macrophage infiltration and CD68⁺ phagocytic activity in ovarian tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eStatistical analysis of IBA-1 and CD68 co-expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e Immunostaining of CCL8 and iNOS⁺ macrophage infiltration in ovarian tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF. \u003c/strong\u003eQuantitative analysis of CCL8 and iNOS⁺ cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG. \u003c/strong\u003emRNA expression levels of pro-inflammatory cytokines (IL-1α, IL-1β, TNF-α).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH. \u003c/strong\u003emRNA expression of M1 macrophage markers (CD40, CD86, TLR4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI. \u003c/strong\u003eAnalysis of CCL8 and its receptor CCR5 mRNA expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ. \u003c/strong\u003eImmunostaining of CCL8 in ovarian tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eK. \u003c/strong\u003eQuantitative analysis of CCL8⁺ cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eL. \u003c/strong\u003eCo-localization of TUNEL⁺ apoptotic cells (green) and IBA-1⁺ macrophages (red) in ovarian tissues. Scale bar = 50 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM. \u003c/strong\u003eStatistical analysis of TUNEL and IBA-1 co-localization.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8939659/v1/21260051cbb7e4a9d0afb494.png"},{"id":104403498,"identity":"958125e6-6a7d-4945-b225-167e5fca714e","added_by":"auto","created_at":"2026-03-11 12:18:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4002659,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eImmunofluorescence staining of phosphorylated p65 (p-p65, red) in ovarian tissues. Nuclei were counterstained with DAPI (blue). Scale bar = 50 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eQuantitative analysis of p-p65⁺ cells (n = 5 fields/section, 3 mice/group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003eWestern blot analysis of total p65, phosphorylated p65 (p-p65), and phosphorylated IκB-α (p-IκBα) protein levels in ovarian lysates. β-actin served as a loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD. \u003c/strong\u003eProtein levels of pro-survival signaling marker (AKT，p-AKT) detected by Western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE. \u003c/strong\u003eProtein expression of iNOS (an NF-κB downstream target and M1 macrophage marker) and apoptosis marker Cleaved-PARP1 in ovarian tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e. Protein quantification analysis of Figures\u003cstrong\u003e C-E\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8939659/v1/8000cb3bcaf631bc034c010b.png"},{"id":104409252,"identity":"29d90d96-eab1-4593-bd26-44220dde2b6c","added_by":"auto","created_at":"2026-03-11 12:44:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":39824723,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8939659/v1/a916a1ca-ee4d-4e3f-9999-94c69584238e.pdf"},{"id":104403022,"identity":"53e031b5-fbc2-4f29-a184-e97227bb8686","added_by":"auto","created_at":"2026-03-11 12:17:12","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":196677,"visible":true,"origin":"","legend":"Original images for Figure 7","description":"","filename":"OriginalimagesforFigure7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8939659/v1/7a79bf191200820c64cf9346.pdf"},{"id":104403543,"identity":"91c8d29e-afc8-4f9a-8b61-f68fdac1c3a3","added_by":"auto","created_at":"2026-03-11 12:18:32","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":507460,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"Supportinginformation.tif","url":"https://assets-eu.researchsquare.com/files/rs-8939659/v1/d4899b3e9992a50999e1147e.tif"}],"financialInterests":"(Not answered)","formattedTitle":"Bindarit ameliorates doxorubicin-induced ovarian damage by suppressing CCL8-dependent macrophage infiltration and NF-κB-mediated inflammation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe ovary is the central organ of the female reproductive system, performing two critical functions: reproduction and endocrine. In the reproduction function, the ovary regulates folliculogenesis and ovulation, which are crucial for female fertility. In the endocrine function, the ovary secretes sex hormones\u0026mdash;including estrogens, progesterone, and small amounts of androgens\u0026mdash;that regulate the hypothalamic-pituitary-ovarian axis to maintain menstrual cyclicity, promote maturation of the reproductive organs, and modulate systemic functions such as bone metabolism and immune homeostasis.\u003c/p\u003e \u003cp\u003eWith advancements in cancer therapeutics, patient survival rates have improved substantially. Data from the National Cancer Center of China indicate that the 5-year survival rate increased from 40.5% in 2015 to 43.7% in 2022\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. However, with the increase in the number of cancer survivors, concerns regarding treatment-related toxicities have also increased. Recent statistics indicate a 38% reduction in pregnancy rates among female cancer survivors compared to their expected rate prior to cancer diagnosis\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDoxorubicin (DOX), an anthracycline chemotherapeutic, is widely used to treat leukemias, lymphomas, sarcomas, and breast/ovarian cancers because of its potent antitumor activity\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. The primary mechanism of action of DOX involves intercalation into the DNA strand to inhibit polymerase activity, thereby blocking nucleic acid synthesis. DOX also generates superoxide radicals that disrupt the cellular membrane of tumor cells. Although DOX is effective against malignancies, its clinical utility is limited by cardiotoxicity mediated by opening of the mitochondrial permeability transition pore, accumulation of reactive oxygen species, and activation of the cGAS-STING pathway\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Notably, DOX also exhibits significant ovarian toxicity\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Clinical studies report an increased incidence of amenorrhea post-DOX chemotherapy, reflecting follicular depletion and hypoestrogenism\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003ein vitro\u003c/em\u003e experiments, DOX induces DNA double-strand breaks in granulosa cells and oocytes, leading to cell apoptosis\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. These findings highlight DOX-induced ovarian failure as a critical clinical challenge, necessitating mechanistic insights and protective strategies.\u003c/p\u003e \u003cp\u003eCurrent approaches, such as GnRH agonists, natural compounds (e.g., resveratrol and gallic acid), and antioxidants, have limitations. GnRH agonists exhibit inconsistent efficacy, while phytochemicals show poor bioavailability and lack standardized protocols\u003csup\u003e[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Therefore, it is imperative to identify novel interventions to preserve ovarian function during chemotherapy.\u003c/p\u003e \u003cp\u003eEmerging evidence implicates inflammatory dysregulation in DOX-induced ovarian damage. DOX triggers infiltration of monocytes and induces sustained inflammation through the production of proinflammatory cytokines (IL-1, IL-6, and TNF-α) and matrix metalloproteinase-mediated extracellular matrix (ECM) degradation. Chemokine receptor signaling promotes the recruitment of monocytes to inflamed tissues\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Among the chemokines, C-C motif chemokine 8 (CCL8/MCP-2), structurally homologous to CCL2/MCP-1, remains understudied in ovarian pathology\u003csup\u003e[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBindarit [2-(1-benzyl-1H-indazol-3-yl)methoxy)-2-methylpropanoic acid], a selective inhibitor of MCP-1/2/3, inhibits monocyte/macrophage recruitment without causing immunosuppression\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Preclinical studies have demonstrated the efficacy of Bindarit in nephritis, arthritis, and pancreatitis models. Clinical Phase II trials have established the favorable safety profile of Bindarit. Notably, the perivascular administration of Bindarit reduces venous fibrosis and stenosis following angioplasty by suppressing macrophage-mediated inflammation. These characteristics suggest the potential utility of Bindarit as a promising candidate for mitigating ovarian inflammation\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite advances in research, the precise mechanisms linking DOX to ovarian inflammatory cascades remain elusive, and effective therapeutic interventions are not yet available. Here, we established a murine model of DOX-induced ovarian injury to investigate the role of CCL8 and evaluate the therapeutic efficacy of bindarit. The results may contribute to developing new approaches for preserving ovarian function during chemotherapy, thereby enhancing the quality of life of female cancer survivors.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003eFemale ICR mice and male mice aged 7\u0026ndash;8 weeks were purchased from and maintained in the animal facility at Nanjing Medical University. The mice were kept under a 12/12-h light/dark cycle at 22\u0026deg;C with free access to food and water. All animal experimental protocols were approved by the Committee on the Ethics of Animal Experiments at Nanjing Medical University (Approval/accreditation number: IACUC-2408025). All animal experiments adhered to the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal experiments\u003c/h3\u003e\n\u003cp\u003eA total of 60 female ICR mice at 7 weeks of age were used in this study. They were randomly assigned to three groups, with 20 mice per group. The control group (Ctrl group) was intraperitoneally administered phosphate-buffered saline. The DOX group received a single intraperitoneal injection of doxorubicin hydrochloride (10 mg/kg). The DOX\u0026thinsp;+\u0026thinsp;Bin group was intraperitoneally administered doxorubicin hydrochloride (10 mg/kg) following an intracapsular injection of Bindarit (4 mg/kg) into the ovaries. Ovarian tissues were collected at 24 and 48 h after injection for fixation, embedding, sectioning, protein extraction, and RNA extraction.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eOvaries were collected and fixed in 10% buffered formalin for paraffin embedding and sectioning. To detect the expression of Cleaved Caspase-3(Cell Signaling Technology [CST], Beverly, USA, Cat. no. catalog no.9661, 1: 400) and gamma H2A.X (Abcam, Cambridge, UK, Cat. no. Ab26350, 1: 400)5-\u0026micro;m sections were deparaffinized, rehydrated, and endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide in methanol for 15 min. The sections were then boiled in 0.01 M citrate buffer for antigen retrieval. After blocking with goat serum (ZSGB-Bio, China) for 1 h, primary antibodies against cleaved-caspase-3 and γ-H2AX were incubated overnight at 4\u0026deg;C. Diaminobenzidine (DAB) reagent was used for coloration on the following day. Non-immune immunoglobulin G (IgG) was applied as a negative control.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence assay\u003c/h3\u003e\n\u003cp\u003eFor ovarian tissues, immunofluorescence assay was performed using antibodies for DDX4 (Abcam, Cambridge, UK, Cat. no. Ab13840, 1:400), PCNA (Cell Signaling Technology, Beverly, USA, Cat. no. 13110, 1:16,000), CCL8 (ABclonal, Wuhan, China, Cat. no. A6977, 1:100), IBA-1 (MedChemExpress, Monmouth Junction, USA, Cat. no. HY-P80501, 1:100), CD68 (Proteintech, Wuhan, China, Cat. no. 28058-1-AP, 1:500), CD206 (Proteintech, Wuhan, China, Cat. no. 18704-1-AP, 1:800), iNOS (Proteintech, Wuhan, China, Cat. no. 18985-1-AP, 1:500), and phospho-NF-κB p65 (Cell Signaling Technology, Beverly, USA, Cat. no. 3033, 1:3000). Following overnight incubation at 4\u0026deg;C, the primary antibodies were removed, and the sections were incubated with the corresponding secondary antibodies at room temperature for 1 h: Alexa Fluor 488-conjugated donkey anti-rabbit (Invitrogen, USA, Cat. No. A21206; 1:500), Alexa Fluor 594-conjugated donkey anti-mouse (Invitrogen, USA, Cat. No. A21203, 1:500), and Alexa Fluor 488-conjugated goat anti-mouse (Invitrogen, USA, Cat. No. A21202, 1:500). The nuclei were then stained with 0.01 mg/mL Hoechst 33342 (Invitrogen, USA, Cat. No. H1339) for 20 min, and the sections were observed under a confocal laser scanning microscope (Zeiss, Germany, Model No. LSM900).\u003c/p\u003e\n\u003ch3\u003eTUNEL assay\u003c/h3\u003e\n\u003cp\u003eTUNEL assay was performed on 5-\u0026micro;m-thick ovarian tissue sections by using the TUNEL Apoptosis Detection Kit (Alexa Fluor 640) (Yeasen Biotech, Shanghai, China, Cat. No 40308) in accordance with the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation, reverse transcription, and reverse transcription- quantitative polymerase chain reaction (RT-qPCR)\u003c/h2\u003e \u003cp\u003eTotal RNA from ovarian tissues was isolated using TRIzol reagent (Invitrogen, USA) in accordance with the manufacturer\u0026rsquo;s instructions. RNA concentration in ovarian tissues was measured using a spectrophotometer (NanoDrop 2000c, Thermo Scientific, Waltham, USA). Next, 500 ng of RNA per sample was reverse transcribed to cDNA by using the FastQuant RT Kit (Tianyuan Biotechnology, China). The obtained cDNA was then subjected to real-time PCR on an ABI Step One Plus platform (Thermo Scientific, Waltham, USA) by using a SYBR Green Master Mix (Applied Biological Materials, Canada), with actin amplification as an internal control. The specificity of the PCR products was assessed by melting curve analysis, and the amplicon size was determined by 2% agarose gel electrophoresis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRNA sequencing (RNA-seq) analysis\u003c/h3\u003e\n\u003cp\u003eA total of 3 \u0026micro;g RNA per sample was used as the input material for RNA sample preparation. Sequencing libraries were generated using the NEBNext\u0026reg; Ultra\u0026trade; RNA Library Prep Kit for Illumina\u0026reg; (NEB, USA) in accordance with the manufacturer\u0026rsquo;s recommendations, with index codes added to attribute sequences to each sample.\u003c/p\u003e \u003cp\u003eBriefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was performed using divalent cations under elevated temperature in the NEBNext First Strand Synthesis Reaction Buffer (5\u0026times;). First-strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA was subsequently synthesized using DNA polymerase I and RNase H. The remaining overhangs were converted into blunt ends through exonuclease/polymerase activity. After adenylation of the 3ʹ-ends of DNA fragments, NEBNext Adaptor with a hairpin loop structure was ligated for hybridization. To select cDNA fragments of preferentially 250\u0026thinsp;~\u0026thinsp;300 bp in length, the library fragments were purified with the AMPure XP System (Beckman Coulter, Beverly, USA). Next, 3 \u0026micro;L of USER\u003csup\u003e\u0026trade;\u003c/sup\u003e Enzyme (NEB, USA) was applied to size-selected, adaptor-ligated cDNA at 37\u0026deg;C for 15 min followed by 5 min at 95\u0026deg;C before PCR. PCR was subsequently performed with Phusion\u003csup\u003e\u0026trade;\u003c/sup\u003e High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) primer. Finally, the PCR products were subjected to purification (AMPure XP system) and library quality assessment on the Agilent Bioanalyzer 2100 system.\u003c/p\u003e \u003cp\u003eBindarit administration\u003c/p\u003e \u003cp\u003eThe dosage of Bindarit (4 mg/kg) was determined based on prior preclinical studies employing systemic administration (e.g., 10 mg/kg via oral gavage) which demonstrated efficacy in inflammatory disease models \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Given that our study utilized a novel, targeted intracapsular injection method which ensures direct ovarian delivery and likely higher local drug concentration, a moderately lower dose of 4 mg/kg was selected to achieve therapeutic efficacy while minimizing potential systemic exposure. Bindarit was dissolved in DMSO and further diluted in phosphate-buffered saline (PBS) to the final working concentration. The injection volume was standardized at 10 \u0026micro;L per ovary.\u003c/p\u003e \u003cp\u003eSurgical procedure for intracapsular ovarian injection\u003c/p\u003e \u003cp\u003eMice were anesthetized with avertin (240 mg/kg, i.p.). After shaving and disinfecting the dorsal skin near the kidney region, a small incision was made to expose the ovarian fat pad. The ovary was carefully externalized, and a 1 mL syringe needle was inserted into the ovarian capsule. Bindarit solution (10 \u0026micro;L per ovary) was slowly injected into the intracapsular space under a stereomicroscope to ensure proper distribution without leakage. The ovary was then returned to the abdominal cavity, and the incision was sutured layer by layer. Mice were monitored until fully recovered. CCL8 siRNA transfection in KGN cells.\u003c/p\u003e \u003cp\u003eTo investigate the role of CCL8 in DOX-induced inflammation, human granulosa cell line KGN was transfected with CCL8-specific siRNA (GenePharma,Shanghai GenePharma Co.,Ltd). After 48 h, cells were treated with 100 \u0026micro;M DOX for 24 h. Knockdown efficiency was validated by RT-qPCR.\u003c/p\u003e\n\u003ch3\u003eImmunoblotting assay\u003c/h3\u003e\n\u003cp\u003eProteins were extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China, Cat. No. P0013B) containing protease inhibitor cocktails (Amresco, Solon, Ohio, USA, Cat. No. M221). A total of 10\u0026ndash;30 \u0026micro;g proteins from each sample were loaded and separated by electrophoresis (Mini-PROTEAN\u0026reg; Tetra Cell, Bio-Rad, USA, Cat. No. 165\u0026ndash;8000). The separated proteins were electroblotted (Mini Trans-Blot Electrophoretic Transfer Cell, Bio-Rad, Cat. No. 170\u0026ndash;3930) onto polyvinylidene fluoride membranes (Thermo Fisher, USA, Cat. No. 88250), and the membranes were then blocked with 5% skimmed milk-TBST (Tris-buffered saline (TBS) containing 0.1% Tween 20) for 30 min; subsequently, the membranes were incubated overnight at 4\u0026deg;C with primary antibodies. Following washing with TBST (5 mL) for three times, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies to detect proteins through enhanced chemiluminescence (ECL Prime Western blotting detection reagent, GE Healthcare, Washington, NY, USA, Cat. No. RPN2232) on the Tanon 5200 analysis system.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses between groups were performed using an unpaired two-tailed Student\u0026rsquo;s t test or one-way analysis of variance with Tukey\u0026rsquo;s multiple comparison test. Error bars represent the standard error of the mean value. A P-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical analyses were performed using Prism software (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDOX induces severe acute and chronic ovarian injury\u003c/h2\u003e \u003cp\u003eThe chemotherapeutic agent DOX, although a crucial oncological drug, exerts substantial ovarian toxicity that warrants detailed investigation. In this study, we established a murine model of DOX-induced ovarian injury to comprehensively characterize the effects of DOX on ovarian histoarchitecture, fibrotic progression, and primordial follicle pool. By conducting integrated histomorphometric analysis, fibrosis marker assessment, and DNA damage evaluation, we elucidated the direct cytotoxic effects of DOX on ovarian tissues, thereby providing foundational evidence for subsequent mechanistic studies.\u003c/p\u003e \u003cp\u003eIn healthy, sexually mature mice, the ovarian surface is covered with an intact epithelial layer. The ovarian parenchyma shows a typical cortical-medullary structure; the cortical region contains abundant follicles at various developmental stages, including primordial, primary, and secondary follicles, with relatively few atretic follicles. The medullary region is less prominent and has a loosely organized structure.\u003c/p\u003e \u003cp\u003eHematoxylin and eosin (H\u0026amp;E) staining of ovarian sections obtained at time points following DOX treatment (Fig.\u0026nbsp;1A) revealed significant structural damage in DOX-treated ovaries, characterized by markedly increased atretic follicles within the cortex, cortical thinning with loosened tissue organization, significantly reduced number of growing follicles, medullary expansion, thickened follicular membrane, and decline in ovarian volume.\u003c/p\u003e \u003cp\u003eMasson\u0026rsquo;s trichrome staining of paraffin-embedded ovarian sections revealed substantial pathological alterations in DOX-treated groups (Fig.\u0026nbsp;1B). Histological examination showed significant disruption of normal ovarian architecture in DOX-treated mice: the originally intact blue-stained reticular framework displayed fragmentation, with only a few follicle structures retaining intact zona pellucida. Notably, DOX administration induced extensive collagen deposition around follicular membranes, accompanied by abnormal invasion of luteal membrane granulosa cells into surrounding tissues. These pathological changes resulted in markedly increased fibrosis in both luteal and interstitial regions.\u003c/p\u003e \u003cp\u003eThese findings suggest that DOX may adversely affect ovarian function by disrupting collagen metabolism and compromising the structural integrity of ovarian tissues.\u003c/p\u003e \u003cp\u003eTo replicate the microenvironment of early ovarian development, neonatal mouse ovaries at postnatal day 3.5 were subjected to 3D ovarian culture (3D-Ovary Culture) for toxicity assessment. After 24-h culture under various treatment conditions, immunofluorescence assay revealed that compared to the normal Ctrl group, ovaries treated with doxorubicin hydrochloride (100 nM DOX) showed substantial accumulation of TUNEL-positive cells within primordial follicles (Fig.\u0026nbsp;1C-D), indicating DOX-induced premature apoptotic activation in primordial follicles.\u003c/p\u003e \u003cp\u003eWe developed a mouse model of ovarian injury through a single intraperitoneal injection of doxorubicin hydrochloride (10 mg/kg DOX); ovarian tissues were collected at 24 h post-injection for immunohistochemical analysis of paraffin-embedded tissues. The results showed a marked increase in cleaved caspase-3-positive cells in DOX-treated ovarian tissues compared to that in the Ctrl group (Fig.\u0026nbsp;1E, DAB staining). Additionally, significant accumulation of γ-H2A.X-positive signals was detected (Fig.\u0026nbsp;1F, DAB staining), indicating the occurrence of DNA double-strand breaks\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. These observations confirmed that DOX rapidly activates ovarian cell apoptosis pathways and induces follicular DNA damage within 24 h, providing direct experimental evidence for elucidating the reproductive toxicity mechanisms of DOX.\u003c/p\u003e \u003cp\u003eTo assess the chronic effects of doxorubicin hydrochloride on ovarian tissues, we measured the ovarian coefficient (ovarian weight/body weight) at multiple time points (24, 36, 48, and 96 h, and 14 and 21 d) following a single intraperitoneal injection of 10 mg/kg doxorubicin hydrochloride. The results revealed a significant reduction in ovarian coefficient at all time points compared to that in the Ctrl group (Fig.\u0026nbsp;1H, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eConcurrent quantitative analysis of follicles at different developmental stages (Fig.\u0026nbsp;1I) demonstrated a time-dependent decrease in the total follicle count in the DOX-treated group, with the degree of reduction showing a negative correlation with time post-injection.\u003c/p\u003e \u003cp\u003eAt 21 days post-DOX administration, female ICR mice from the model group were paired 1:1 with healthy male ICR mice for 19\u0026ndash;21 days. Reproductive outcomes revealed that the DOX-treated group produced significantly fewer offspring (4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52) compared to the Ctrl group (15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10) (Fig.\u0026nbsp;1J-K, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, unpaired t-test). These findings demonstrate that DOX exposure significantly impairs reproductive capacity in female mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIntegrated transcriptome and CIBERSORT analysis reveals the CCL8/immune cell regulatory network in DOX-induced ovarian toxicity\u003c/h2\u003e \u003cp\u003eTo elucidate the mechanisms underlying DOX-induced ovarian toxicity, we conducted RNA-seq analysis on bilateral ovarian tissues from Ctrl mice (n\u0026thinsp;=\u0026thinsp;5) and DOX-treated (n\u0026thinsp;=\u0026thinsp;5) mice (Fig.\u0026nbsp;2A-B; sample inclusion criteria shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The sequencing analysis identified 350 differentially expressed genes (DEGs) in the DOX group (|logFC| \u0026gt; 1, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Gene Ontology (GO) enrichment analysis revealed the following significant terms: Extracellular space (GO:0005615, false discovery rate (FDR)\u0026thinsp;=\u0026thinsp;0.0001021) and Extracellular region (GO:0005576, FDR\u0026thinsp;=\u0026thinsp;0.0001584) (Fig.\u0026nbsp;2C); this finding suggests that DOX disrupts ovarian tissue architecture and ECM homeostasis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (Fig.\u0026nbsp;2D) showed that the DEGs were primarily enriched in the following pathways: Cytokine-cytokine receptor interaction (mmu04060, P\u0026thinsp;=\u0026thinsp;0.001854) and Chemokine signaling pathway (mmu04062, P\u0026thinsp;=\u0026thinsp;0.008657). Notably, CCL8 (log2FC\u0026thinsp;=\u0026thinsp;2.53, P\u0026thinsp;=\u0026thinsp;1.81E-06) was significantly upregulated in both pathways, suggesting its potential role in DOX-induced ovarian injury through cytokine network regulation and chemokine-mediated signaling. This observation provided important insights into subsequent mechanistic investigations.\u003c/p\u003e \u003cp\u003eImmunofluorescence assay confirmed elevated CCL8 expression in DOX-treated ovaries (Fig.\u0026nbsp;2E-F). RT-qPCR further verified the sequencing results (Fig.\u0026nbsp;2G-H), with CCL8 showing the most prominent upregulation in DOX mice.\u003c/p\u003e \u003cp\u003eAccording to CIBERSORT-based immune infiltration analysis and visualization by ggplot2, M1 macrophages, activated NK cells, and Th1 cells were in the immunoactive state in the DOX group; however, these cells were in the immunosuppressive state in the Ctrl group (Fig.\u0026nbsp;2I-K).\u003c/p\u003e \u003cp\u003eThese results indicate that DOX treatment restructures the immune microenvironment by modifying cellular composition and function, thereby activating immune responses. Collectively, these findings establish a theoretical foundation for further investigation of immunoregulatory mechanisms and optimization of immunotherapeutic strategies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCCL8/CCR5-mediated M1 macrophage activation promotes doxorubicin-induced ovarian inflammation and apoptosis\u003c/h2\u003e \u003cp\u003eDOX treatment induces proinflammatory monocyte infiltration that promotes M1 macrophage polarization in ovarian tissues, exacerbating inflammatory responses through the increased secretion of IL-1β and TNF-α, subsequently resulting in tissue damage. Immunofluorescence assay revealed substantial accumulation of IBA-1⁺ macrophages in the follicular, luteal, and stromal regions of DOX-treated ovaries (Fig.\u0026nbsp;3A-B), exhibiting distinct spatial colocalization with TUNEL⁺ apoptotic cells (Fig.\u0026nbsp;3C-D); this finding suggests active macrophage participation in apoptotic clearance. RT-qPCR revealed significant upregulation of proinflammatory cytokines (IL-1α, IL-1β, and TNF-α) in the DOX group (Fig.\u0026nbsp;3E, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). A detailed immunophenotyping analysis showed a significant shift toward M1-dominant polarization, characterized by an increased number of CD68⁺ M1 macrophages (Fig.\u0026nbsp;3F-G), an enhanced expression of M1 markers (CD40, CD86, and TLR4; Fig.\u0026nbsp;3J, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and a stable level of M2 macrophage markers (CD36, Arg1, and IL-10) (Fig.\u0026nbsp;3H-I, K). Bioinformatics analysis based on the KEGG pathway enrichment analysis and protein-protein interaction network identified CCL8-CCR5 as the primary interaction pair, showing a strong correlation at the mRNA level (Fig.\u0026nbsp;3L-M). These observations support a mechanistic model wherein DOX upregulates CCL8 expression to stimulate CCR5-mediated macrophage recruitment and M1 polarization, resulting in ovarian inflammatory injury and functional deterioration.\u003c/p\u003e \u003cp\u003eTo further elucidate the key role of CCL8 in mediating ovarian inflammatory responses, in vitro experiments were conducted using the human granulosa cell line KGN. KGN cells were divided into three groups: a blank control group (no treatment), a DOX-treated group (100 \u0026micro;M doxorubicin for 24 hours), and a CCL8-siRNA\u0026thinsp;+\u0026thinsp;DOX group (transfected with CCL8-specific siRNA for 48 hours followed by the same dose of doxorubicin). By targeting CCL8 with small interfering RNA (siRNA), RT-qPCR results (Fig.\u0026nbsp;4A) showed that CCL8 expression in the DOX-treated group increased to 2.897\u0026thinsp;\u0026plusmn;\u0026thinsp;0.512 (vs. Control, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while in the CCL8-siRNA group, expression decreased to 0.399\u0026thinsp;\u0026plusmn;\u0026thinsp;0.073, achieving a knockdown efficiency of 66.1% (P\u0026thinsp;=\u0026thinsp;0.003). RT-qPCR analysis further demonstrated that, compared to the DOX-treated group, knockdown of CCL8 significantly downregulated the mRNA levels of the pro-inflammatory cytokines IL-1α and TNF-α (Fig.\u0026nbsp;4B, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Concurrently, the mRNA expression of M1 macrophage activation markers iNOS, CD86, and CD40 was also markedly reduced (Fig.\u0026nbsp;4C, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), indicating that CCL8 knockdown effectively suppresses inflammatory responses and pro-inflammatory polarization of macrophages. In summary, CCL8 serves as a key chemokine mediating macrophage recruitment to ovarian tissue. Specific inhibition of CCL8 effectively blocks the activation and accumulation of inflammatory macrophages, thereby ameliorating doxorubicin-induced ovarian injury.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBindarit ameliorates DOX-induced ovarian damage and fertility decline by simultaneously suppressing inflammation and apoptosis\u003c/h2\u003e \u003cp\u003eTo examine the protective effects of Bindarit against DOX-induced ovarian inflammation, we established an \u003cem\u003ein vivo\u003c/em\u003e intervention model. The results demonstrated that while DOX treatment significantly reduced the ovarian coefficient in mice, immediate subcapsular ovarian administration of Bindarit (4 mg/kg) post-DOX modeling effectively restored this parameter (Fig.\u0026nbsp;5A). The Bindarit-only group exhibited no abnormalities, confirming its safety profile. Histological analyses revealed that Bindarit treatment markedly ameliorated DOX-induced structural damage (H\u0026amp;E staining, Fig.\u0026nbsp;5B) and disruption of collagen metabolism (Masson\u0026rsquo;s trichrome staining, Fig.\u0026nbsp;5C) in ovarian tissues. Immunofluorescence assay demonstrated that Bindarit significantly inhibited DOX-induced abnormal apoptosis of granulosa cells (Fig.\u0026nbsp;5D-G), indicating its potential anti-apoptotic mechanism. Functional assessments revealed that while DOX administration substantially decreased litter size compared to that in the Ctrl group, Bindarit when administered alone partially reversed this fertility impairment (Fig.\u0026nbsp;5H-I), without affecting reproductive function. These findings demonstrate that Bindarit restores DOX-induced fertility dysfunction through three mechanisms: reduction of the ovarian inflammatory response, maintenance of tissue integrity, and inhibition of apoptotic pathways.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBindarit attenuates ovarian inflammatory injury by suppressing CCL8-dependent macrophage infiltration\u003c/h2\u003e \u003cp\u003eTo validate the pivotal role of Bindarit in influencing CCL8-mediated macrophage recruitment to granulosa cells, we examined the effects of Bindarit on macrophage infiltration and activation. Immunofluorescence assay demonstrated that Bindarit treatment significantly reduced the number of IBA-1⁺ macrophages and CD68⁺ phagocytic activity in ovarian tissues (Fig.\u0026nbsp;6A-D), indicating effective suppression of macrophage accumulation. Ovarian sections from Bindarit-treated mice exhibited markedly decreased CCL8 immunoreactivity concurrent with reduced infiltration of iNOS⁺ macrophages (Fig.\u0026nbsp;6E-F, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). RT-qPCR analysis confirmed that Bindarit reduced mRNA expression of proinflammatory cytokines (IL-1α, IL-1β, and TNF-α; Fig.\u0026nbsp;6G) and M1 macrophage markers (CD40, CD86, and TLR4; Fig.\u0026nbsp;6H), suggesting inhibition of proinflammatory macrophage polarization. Notably, Bindarit significantly decreased CCL8 expression at both protein (immunofluorescence assay) and mRNA levels (Fig.\u0026nbsp;6E-G), with CCL8 suppression showing a strong correlation with reduced macrophage infiltration and enhanced granulosa cell apoptosis (Fig.\u0026nbsp;6I-M). These findings establish CCL8 as the primary chemokine that regulates macrophage recruitment to ovarian tissues, while demonstrating that Bindarit\u0026rsquo;s targeted inhibition of CCL8 prevents inflammatory macrophage activation and accumulation, thus mitigating DOX-induced ovarian injury.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBindarit mitigates doxorubicin-induced ovarian toxicity by targeting the NF-κB/p65-IκBα axis\u003c/h2\u003e \u003cp\u003eGiven the established role of CCL8 in promoting NF-κB activation via macrophage-derived cytokines, we next investigated whether Bindarit\u0026rsquo;s protective effects involve suppression of the CCL8/NF-κB axis. Our data demonstrate that DOX-induced CCL8 upregulation precedes NF-κB activation, as evidenced by increased p65 phosphorylation and IκBα degradation. Bindarit treatment significantly reduced CCL8 expression, which in turn attenuated NF-κB signaling, indicating a causal link between CCL8 suppression and NF-κB inactivation.\u003c/p\u003e \u003cp\u003eTo investigate the critical role of NF-κB signaling in DOX-induced ovarian injury, we examined the subcellular localization of phosphorylated p65 (p-p65) by immunofluorescence assay. The analysis revealed significantly elevated p-p65 expression in both nuclear and cytoplasmic compartments of DOX-treated ovarian cells, while Bindarit treatment markedly reduced the number of p-p65-positive cells (Fig.\u0026nbsp;7A-B). Western blotting assay demonstrated that DOX exposure decreased the cytoplasmic level of inactive p65 while increasing the levels of phosphorylated p65 (p-p65) and phosphorylated IκB-α, indicating sustained NF-κB pathway activation. Conversely, Bindarit administration effectively reversed these alterations (Fig.\u0026nbsp;7C).\u003c/p\u003e \u003cp\u003eFurther analyses revealed that DOX treatment downregulated the expression of prosurvival p-AKT and upregulated the apoptosis marker cleaved-PARP1; both these effects were significantly ameliorated by Bindarit intervention (Fig.\u0026nbsp;7D). Notably, the expression of iNOS\u0026mdash;a downstream NF-κB target and M1 macrophage marker\u0026mdash;was elevated in the DOX group but suppressed by Bindarit treatment (Fig.\u0026nbsp;7E-F).\u003c/p\u003e \u003cp\u003eIn summary, Bindarit alleviates inflammatory responses, suppresses macrophage M1 polarization, and ultimately mitigates DOX-induced apoptosis and functional impairment in ovarian granulosa cells by inhibiting the activation of the NF-κB pathway\u0026mdash;specifically through reducing p65 phosphorylation and nuclear translocation, while promoting IκB-α-mediated negative feedback regulation. These findings not only underscore the central role of the NF-κB pathway in ovarian inflammatory injury but also provide a mechanistic basis for the clinical application of Bindarit.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eChronic inflammation is the primary driver of chemotherapy-induced ovarian damage (CIOD). The present study is the first to demonstrate that Bindarit significantly mitigates the ovarian toxicity of DOX by targeting the CCL8-mediated immune regulatory network. Bindarit improves the ovarian inflammatory microenvironment and granulosa cell survival by inhibiting CCL8-dependent macrophage recruitment and M1 macrophage polarization, thereby suppressing the excessive activation of the NF-κB/p65 pathway. Previous studies have shown a significant decline in follicular reserve among chemotherapy patients, although the underlying mechanism remains unclear\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. The present study simulated clinical chemotherapy scenarios by using animal models and found that DOX-induced sustained overexpression of CCL8 promotes inflammatory monocyte infiltration. The GO enrichment analysis and the KEGG pathway enrichment analysis confirmed that these changes are closely associated with CCR5-mediated immune cell migration pathways. Notably, unlike the self-limiting nature of physiological inflammation, CCL8/NF-κB signaling in the DOX group exhibited persistent activation and showed a strong correlation with irreversible ovarian tissue damage. These findings elucidate the central role of the CCL8-CCR5 axis in CIOD and provide a theoretical foundation for the clinical application of Bindarit.\u003c/p\u003e \u003cp\u003eBindarit, a broad-spectrum chemokine inhibitor, shows protective effects in various inflammatory diseases by inhibiting CCL2-mediated monocyte infiltration\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. This investigation represents the first application of Bindarit in CIOD. Ovarian granulosa cell apoptosis and follicular atresia constitute the primary pathological features of DOX-induced ovarian toxicity. This study demonstrated that Bindarit treatment substantially reduces DOX-triggered granulosa cell apoptosis (as evidenced by downregulated expression of cleaved-PARP1) and enhances primordial follicle survival. The maintenance of follicular microenvironment homeostasis is essential for female reproductive function, and controlling excessive inflammatory responses may be crucial for ovarian reserve protection. The analysis of local ovarian inflammatory status revealed that Bindarit-treated groups showed markedly reduced levels of proinflammatory cytokines (IL-1β and TNF-α). Notably, chemotherapy-induced TNF-α elevation promotes granulosa cell apoptosis through NF-κB signaling activation, while clinical studies indicate that anti-TNF-α therapy improves ovarian function in chemotherapy patients. Therefore, one of the main mechanisms through which Bindarit mitigates DOX-induced ovarian toxicity may involve disruption of this vicious cycle by reducing TNF-α levels. Moreover, current evidence suggests that proinflammatory cytokines accelerate follicular atresia by activating the NF-κB pathway within the follicular microenvironment\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. The findings of the present study confirm this hypothesis: Bindarit preserves ovarian structure and function by inhibiting CCL8-dependent NF-κB activation, evidenced by a decrease in p-p65 nuclear translocation and suppression of IκB-α degradation.\u003c/p\u003e \u003cp\u003eMacrophage polarization status plays a dual role in the progression and restoration of CIOD. Previous research indicates that the dynamic balance between proinflammatory (M1) and anti-inflammatory (M2) macrophages determines the extent of ovarian tissue damage\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. The present study revealed that DOX treatment significantly upregulated M1 macrophage markers (iNOS, CD86, IL-1β, and TNF-α) and suppressed M2 markers (CD36, Arg1, and IL-10) in the ovarian microenvironment, and this imbalance showed a positive correlation with follicular atresia severity (Fig.\u0026nbsp;3). Notably, Bindarit treatment effectively downregulated M1-associated gene expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), suggesting its capacity to reprogram macrophage phenotype. \u003cem\u003eIn vitro\u003c/em\u003e experiments further demonstrated that ovarian macrophages in the Bindarit treatment group secreted 60\u0026ndash;70% lower levels of TNF-α, IL-1α, and IL-1β compared to the macrophages in the DOX group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001); this finding was consistent with previous reports that M2 macrophages inhibit ovarian fibrosis through IL-10 secretion.\u003c/p\u003e \u003cp\u003eThe present research demonstrates that Bindarit significantly ameliorated DOX-induced ovarian damage by targeting the CCL8/NF-κB axis through three primary mechanisms: inhibition of CCL8-dependent macrophage (particularly M1-type) recruitment to ovarian tissues, suppression of NF-κB overactivation, and reduction of proinflammatory cytokine levels (IL-1β, TNF-α, etc.). Notably, Bindarit effectively increased follicular reserve and subsequent embryonic developmental potential by reducing apoptosis (evidenced by decreased cleaved-PARP1 expression), ultimately improving reproductive outcomes.\u003c/p\u003e \u003cp\u003eAlthough previous studies have reported conflicting results regarding CCL2/CCL7-mediated regulation of macrophage polarization with M1/M2 phenotypic discrepancies, the present research revealed the distinct role of CCL8 in DOX-induced ovarian toxicity\u0026mdash;specifically driving M1 polarization and establishing a positive feedback loop with the NF-κB pathway. Notably, the inhibitory effect of Bindarit on CCL8 exceeds that of other broad-spectrum chemokine inhibitors, emphasizing its clinical potential for targeted therapy.\u003c/p\u003e \u003cp\u003eFurther investigations should address the following questions: (1) whether CCL8 indirectly modulates ovarian inflammation by regulating T-cell subsets (e.g., Th17/Treg balance) and (2) how CCL8 secreted by follicular granulosa cells forms paracrine circuits with macrophages. Future studies should focus on developing ovarian-targeted delivery systems for Bindarit (e.g., exosome-based carriers) and evaluating its synergistic effects with existing ovarian protectants (e.g., GnRH agonists).\u003c/p\u003e \u003cp\u003eThe proposed \u0026ldquo;CCL8-targeting strategy\u0026rdquo; presents a novel therapeutic approach for preserving ovarian function in chemotherapy patients, particularly in prepubertal cancer survivors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Science Foundation of China | Key Programme (2024YFC2706704); Natural Science Research of Jiangsu Higher Education Institutions of China (22KJB320004); Special Project for High-quality Development of Maternal and Child Health of Jiangsu Provincial Hospital (Grant No. [GZL2501]); Cell Therapy Foundation (Grant No. [303100784AA25])\u003c/p\u003e\n\u003cp\u003eContributions\u003c/p\u003e\n\u003cp\u003eCF: conception and design, collecting and assembling data, analyzing and interpreting data, writing manuscripts; NL,YQ: collecting and assembling data, data analysis, and interpretation, writing manuscripts; MY,XN,QC: collecting and assembling data, data analysis; FW,YC,QC: conception and design, experimental technical support; JL, XM: conception and design, administrative support, financial support, manuscript writing and final approval of the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003eCorresponding author\u003c/p\u003e\n\u003cp\u003eCorrespondence to Jing Li and Xiang Ma\u003c/p\u003e\n\u003cp\u003eEthical approval and consent to participate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll animal protocols were approved by the Committee on the Ethics of Nanjing Medical University. [Approval/accreditation number: IACUC-2408025]\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent for publication\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors have approved the manuscript for submission.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting interests\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003eData availability\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e Zeng H, Zheng R, Sun K, et al. Cancer survival statistics in China 2019\u0026ndash;2021: a multicenter, population-based study. J Natl Cancer Cent. 2024;4(3):203-13. doi: 10.1016/j.jncc.2024.06.005.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Zeng H, Chen W, Zheng R, et al. Changing cancer survival in China during 2003\u0026ndash;15: a pooled analysis of 17 population-based cancer registries. Lancet Glob Health. 2018;6(5):e555-67. doi: 10.1016/S2214-109X(18)30127-X.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Anderson RA, Brewster DH, Wood R, et al. The impact of cancer on subsequent chance of pregnancy: a population-based analysis. Hum Reprod. 2018;33(7):1281-90. doi: 10.1093/humrep/dey216.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;D\u0026rsquo;Angelo NA, Noronha MA, C\u0026acirc;mara MCC, et al. Doxorubicin nano formulations on therapy against cancer: An overview from the last 10 years. Biomater Adv. 2022;133:112623. doi: 10.1016/j.msec.2021.112623.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Zhang S, Liu X, Bawa-Khalfe T, et al. Identification of the molecular basis of doxorubicin-induced cardiotoxicity. Nat Med. 2012;18(11):1639-42. doi: 10.1038/nm.2919.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Morgan S, Anderson RA, Gourley C, et al. How do chemotherapeutic agents damage the ovary? Hum Reprod Update. 2012;18(5):525-35. doi: 10.1093/humupd/dms022.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Aziz AUR, Yu X, Jiang Q, et al. Doxorubicin-induced toxicity to 3D-cultured rat ovarian follicles on a microfluidic chip. Toxicol In Vitro. 2020;62:104677. doi: 10.1016/j.tiv.2019.104677.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Ben-Aharon I, Bar-Joseph H, Tzarafaty G, et al. Doxorubicin-induced ovarian toxicity. Reprod Biol Endocrinol. 2010;8:20. doi: 10.1186/1477-7827-8-20.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Jurisicova A, Lee HJ, D\u0026rsquo;Estaing SG, et al. Molecular requirements for doxorubicin-mediated death in murine oocytes. Cell Death Differ. 2006;13(9):1466-74. doi: 10.1038/sj.cdd.4401819.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Blumenfeld Z, von Wolff M. GnRH-analogues and oral contraceptives for fertility preservation in women during chemotherapy. Hum Reprod Update. 2008;14(6):543-52. doi: 10.1093/humupd/dmn022.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Bildik G, Akin N, Senbabaoglu F, et al. GnRH agonist leuprolide acetate does not confer any protection against ovarian damage induced by chemotherapy and radiation in vitro. Hum Reprod. 2015;30(12):2912-25. doi: 10.1093/humrep/dev257. (注：原DOI指向该页码)\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Turner NH, Partridge A, Sanna G, et al. Utility of gonadotropin-releasing hormone agonists for fertility preservation in young breast cancer patients: the benefit remains uncertain. Ann Oncol. 2013;24(9):2224-35. doi: 10.1093/annonc/mdt196.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Charo IF, Ransohoff RM. The many roles of chemokines and chemokine receptors in inflammation. N Engl J Med. 2006;354(6):610-21. doi: 10.1056/NEJMra052723.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Zernecke A, Shagdarsuren E, Weber C. Chemokines in atherosclerosis: an update. Arterioscler Thromb Vasc Biol. 2008;28(11):1897-908. doi: 10.1161/ATVBAHA.107.161174.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Van Damme J, Proost P, Put W, et al. Induction of monocyte chemotactic proteins MCP-1 and MCP-2 in human fibroblasts and leukocytes by cytokines and cytokine inducers. Chemical synthesis of MCP-2 and development of a specific RIA. J Immunol. 1994;152(11):5495-502.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Van Coillie E, Proost P, Van Aelst I, et al. Functional comparison of two human monocyte chemotactic protein-2 isoforms, role of the amino-terminal pyroglutamic acid and processing by CD26/dipeptidyl peptidase IV. Biochemistry. 1998;37(36):12672-80. doi: 10.1021/bi980497d.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Van Damme J, Proost P, Lenaerts JP, et al. Structural and functional identification of two human, tumor-derived monocyte chemotactic proteins (MCP-2 and MCP-3) belonging to the chemokine family. J Exp Med. 1992;176(1):59-65. doi: 10.1084/jem.176.1.59.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Bhatia M, Ramnath RD, Chevali L, et al. Treatment with bindarit, a blocker of MCP-1 synthesis, protects mice against acute pancreatitis. Am J Physiol Gastrointest Liver Physiol. 2005;288(6):G1259-65. doi: 10.1152/ajpgi.00435.2004.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Mirolo M, Fabbri M, Sironi M, et al. Impact of the anti-inflammatory agent bindarit on the chemokinome: selective inhibition of the monocyte chemotactic proteins. Eur Cytokine Netw. 2008;19(3):119-22. doi: 10.1684/ecn.2008.0133.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Perico N, Benigni A, Remuzzi G. Present and future drug treatments for chronic kidney diseases: evolving targets in renoprotection. Nat Rev Drug Discov. 2008;7(11):936-53. doi: 10.1038/nrd2685.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Singh AK, Kilari S, Cai C, et al. Bindarit encapsulated nanoparticles prevent venous neointimal hyperplasia and restenosis in a murine angioplasty model. Transl Res. 2022;248:68-86. doi: 10.1016/j.trsl.2022.06.002.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Zoja C, Abbate M, Corna D, et al. Bindarit retards renal disease and prolongs survival in murine lupus autoimmune disease. Kidney Int. 1998;53(3):726-34. doi: 10.1046/j.1523-1755.1998.00791.x.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Roti Roti EC, Leisman SK, Abbott DH, et al. Acute doxorubicin insult in the mouse ovary is cell- and follicle-type dependent. PLoS One. 2012;7(8):e42293. doi: 10.1371/journal.pone.0042293.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Mora E, Guglielmotti A, Biondi G, et al. Bindarit: An anti-inflammatory small molecule that modulates the NF-\u0026kappa;B pathway. Cell Cycle. 2012;11(1):159-69. doi: 10.4161/cc.11.1.18559.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Fabbri R, Macciocca M, Vicenti R, et al. Epigallocatechin-3-gallate inhibits doxorubicin-induced inflammation on human ovarian tissue. Biosci Rep. 2019;39(5):BSR20181424. doi: 10.1042/BSR20181424.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Lech M, Gr\u0026ouml;bmayr R, Ryu M, et al. Macrophage phenotype controls long-term AKI outcomes\u0026mdash;kidney regeneration versus atrophy. J Am Soc Nephrol. 2014;25(2):292-304. doi: 10.1681/ASN.2013020152.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chemotherapy-induced ovarian damage, CCL8/CCR5 axis, M1 macrophages, NF-κB signaling, fertility preservation, Bindarit","lastPublishedDoi":"10.21203/rs.3.rs-8939659/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8939659/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDoxorubicin (DOX), a widely used chemotherapeutic agent, induces severe ovarian damage resulting in premature ovarian insufficiency and reduced fertility. In the present study, through transcriptome sequencing and immune cell infiltration analysis, we demonstrated that DOX significantly upregulates ovarian CCL8 expression, thereby activating the CCL8/CCR5 axis to promote the recruitment and polarization of proinflammatory M1 macrophages. Mechanistically, CCL8 exacerbates local inflammation through NF-κB signaling (evidenced by p65 phosphorylation and IκBα degradation), initiating granulosa cell apoptosis and follicular atresia. The CCL8-specific inhibitor Bindarit effectively mitigated this pathological cascade by (1) reducing macrophage infiltration by 90.9% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), (2) inhibiting NF-κB activation (99.3% decrease in p65 nuclear translocation, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and (3) downregulating proinflammatory cytokines (reduction in TNF-α/IL-1β expression: 80\u0026ndash;90%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Notably, Bindarit treatment restored 54.5% of fertility capacity (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in DOX-treated mice (which showed 84.1% fertility loss). These findings illuminate the crucial role of the CCL8/NF-κB axis in DOX-induced ovarian toxicity and propose a novel therapeutic strategy for fertility preservation. Bindarit could serve as a promising CCL8-targeted agent with substantial clinical potential.\u003c/p\u003e","manuscriptTitle":"Bindarit ameliorates doxorubicin-induced ovarian damage by suppressing CCL8-dependent macrophage infiltration and NF-κB-mediated inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-07 03:34:53","doi":"10.21203/rs.3.rs-8939659/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-03-31T13:06:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-31T04:26:39+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-20T03:30:54+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-03-16T06:16:58+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-03-15T10:34:33+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-03-03T10:44:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-23T14:51:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-22T14:13:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2026-02-22T14:13:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"138053e7-3220-45a8-9edf-1f60c012496f","owner":[],"postedDate":"March 7th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":63838896,"name":"Biological sciences/Cell biology/Cell signalling"},{"id":63838897,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-03-31T13:16:08+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-07 03:34:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8939659","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8939659","identity":"rs-8939659","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}