
==== Front
Redox Biol
Redox Biol
Redox Biology
2213-2317
Elsevier

S2213-2317(24)00293-3
10.1016/j.redox.2024.103315
103315
Research Paper
Copper overload exacerbates testicular aging mediated by lncRNA:CR43306 deficiency through ferroptosis in Drosophila
Huang Qiuru a1
Li Jiaxin a1
Qi Yujuan b1
He Xuxin a1
Shen Cong c1
Wang Chenyu a
Wang Xinda a
Xia Qiushi a
Zhang Yi a
Pan Ziyue a
Hu Qingqing a
Cao Ziyu a
Liu Yiheng a
Huang Jingqi a
Han Guoqing a
Zheng Ying d
Zheng Bo bozheng@njmu.edu.cn
c⁎⁎
Zeng Xuhui zengxuhui@ntu.edu.cn
a⁎⁎⁎
Bi Xiaolin bixl@ntu.edu.cn
e⁎⁎⁎⁎
Yu Jun yujun9117@126.com
a⁎
a Institute of Reproductive Medicine, School of Medicine, Nantong University, Nantong, 226001, China
b Clinical Center of Reproductive Medicine, Xuzhou Central Hospital, Xuzhou Clinical School of Xuzhou Medical University, Xuzhou, 221000, China
c State Key Laboratory of Reproductive Medicine and Offspring Health, Center for Reproduction and Genetics, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School of Nanjing Medical University, Suzhou, 215002, China
d Department of Histology and Embryology, School of Medicine, Yangzhou University, Yangzhou, 225009, China
e School of Medicine, Nantong University, Nantong, 226001, China
⁎ Corresponding authors. yujun9117@126.com
⁎⁎ Corresponding author. bozheng@njmu.edu.cn
⁎⁎⁎ Corresponding authors. zengxuhui@ntu.edu.cn
⁎⁎⁎⁎ Corresponding author. bixl@ntu.edu.cn
1 These authors contributed equally to this work.

15 8 2024
10 2024
15 8 2024
76 10331517 7 2024
15 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Testicular aging manifests as impaired spermatogenesis and morphological alterations in Drosophila. Nonetheless, the comprehensive molecular regulatory framework remains largely undisclosed. This investigation illustrates the impact of copper overload on testicular aging and underscores the interplay between copper overload and lncRNA. Copper overload triggers Cuproptosis through the mitochondrial TCA cycle, facilitating intracellular interactions with Ferroptosis, thereby governing testicular aging. Dysfunction of lncRNA:CR43306 also contributes to testicular aging in Drosophila, emphasizing the significance of lncRNA:CR43306 as a novel aging-associated lncRNA. Moreover, copper overload exacerbates spermatid differentiation defects mediated by lncRNA:CR43306 deficiency through oxidative stress, copper, and iron transport. Therapeutically, Ferrostatin-1 and Resveratrol emerge as potential remedies for addressing testicular aging. This study offers perspectives on the regulatory mechanisms involving copper overload and lncRNA:CR43306 deficiency in the context of testicular aging.

Graphical abstract

Image 1

Highlights

• Copper overload induced Cuproptosis to trigger crosstalk with Ferroptosis for testicular aging.

• LncRNA:CR43306 was recognized as a novel lncRNA associated with aging.

• Synergistic effects of copper overload and lncRNA:CR43306 deficiency intensified testicular aging and male infertility.

• Ferrostatin-1 and resveratrol were identified as pivotal compounds for alleviating testicular aging.

Keywords

Copper overload
Testicular aging
lncRNA:CR43306
Ferroptosis
Cuproptosis
==== Body
pmc1 Introduction

The gradual decline in testicular function with advancing age among men has spurred research interest in testicular aging [1]. Prior investigations have indicated a negative association between aging and parameters such as sperm concentration, motility, normal morphology, and reproductive outcomes [[2], [3], [4]]. While extended observations have revealed structural changes in the testes during aging in Drosophila, the specific features of aging have not been comprehensively delineated. Notably, the synergistic effect between environmental exposure and genetic factors in testicular aging remains unexplored.

Copper serves as a vital trace element in numerous processes such as the detoxification of reactive oxygen species (ROS) and the uptake of iron in eukaryotic organisms [5,6]. The recommended daily intake of copper for humans should not exceed 1.5 mg, as 0.8 mg is deemed sufficient to regulate and uphold copper levels within the body [5]. Cellular copper homeostasis is intricately governed by a network of copper-dependent proteins to avert the repercussions of copper overload and deficiency [7]. Excessive copper consumption leads to bodily accumulation, instigating cytotoxicity through the buildup of ROS, oxidative stress, and mitochondrial dysfunction [8]. A recent study has unveiled that intracellular copper accumulation initiates a novel form of regulated cell death by directing copper to the mitochondria, termed Cuproptosis, which triggers the aggregation of mitochondrial lipoylated proteins and the destabilization of Fe–S cluster proteins [9].

Long noncoding RNAs (lncRNAs) are typically delineated as a subset of noncoding RNAs exceeding 200 nucleotides in length, discerned through genome-wide transcriptome analyses [10,11]. In contrast to messenger RNA (mRNA), lncRNAs are characterized by diminished conservation, reduced expression levels, and heightened variability in expression across different tissues [12,13]. Wen et al. identified a substantial cohort of lncRNAs expressed specifically in the testis of Drosophila [14]. Our investigation further demonstrated that the deletion of lncRNA:CR43306 led to a partial decline in male fertility and influenced the process of testicular aging in Drosophila. Additionally, Zhang et al. constructed a prognostic signature of Cuproptosis-associated lncRNAs for disease prognosis prediction [15], unveiling pivotal roles in the interplay between Cuproptosis and lncRNAs.

Numerous aging biomarkers have been identified in relation to testicular aging [16]. However, the impact of copper overload on testicular aging and the synergistic effects between copper overload and lncRNA signatures have yet to be investigated. This study employs in vivo and in vitro assays to elucidate the cellular repercussions of testicular aging, particularly focusing on the synergistic effects of aging markers influenced by copper overload and the deficiency of lncRNA:CR43306 in the Drosophila testis.

2 Materials and methods

2.1 Fly strains

The w1118 line was used as control group and lncRNA:CR43306 KO was a gift from Prof. Xiaolin Bi (Nantong University) and Prof. Guanjun Gao (ShanghaiTech University). All flies for in vivo experiments were raised at 25 °C and in a relative humidity of 40 %–60 %.

2.2 In vivo exposure strategy

For flies subjected to CuCl2 exposure, copper (II) chloride hydrate (CuCl2, 99.999 % purity, #010698, Alfa-Aesar) was diluted in 0.9 % normal saline and subsequently incorporated into standard corn syrup food to establish a 10 mM CuCl2 medium for the flies. In the case of flies exposed to Ferrostatin-1 + CuCl2 and Resveratrol + CuCl2, Ferrostatin-1 (Fer-1; 10 mM, HY-100579, MCE) or Resveratrol (Res; 10 mM, HY-16561, MCE) was co-administered with CuCl2. Consequently, mediums containing CuCl2 (10 mM) + Ferrostatin-1 (10 μM) and CuCl2 (10 mM) + Resveratrol (10 μM) were prepared for the flies. The flies were exposed to these various mediums for a duration of 30 days.

2.3 Cell culture and treatment

The Drosophila Genomics Resource Center provided the Drosophila Schneider 2 (S2) cells, which were then maintained at 28 °C in Schneider's Drosophila medium (#21720024, Gibco) supplemented with 10 % heatinactivated fetal bovine serum (FBS) (#04-001-1ACS, Bioind). S2 cells were divided every 3–4 days with the supplemented medium at a ratio of 1:3. CuCl2 (final concentrations: 0, 100 and 200 μM), Ferrostatin-1 (final concentration: 0, 5, 10 μM), Resveratrol (final concentration: 0, 5, 10 μM), Ferric ammonium citrate (FAC) (F5879, Sigma-Aldrich; final concentrations: 0, 100 and 200 μM), FeSO4 (T20691, Shanghai Yuanye Biotechnology; final concentrations: 0, 100 and 200 μM) were added into the medium for the treatment of 48h, and S2 cells were collected for subsequent analysis.

2.4 Cell death assays

According to the manufacturer's guidelines, one-step TdT-mediated dUTP Nick-End Labeling (TUNEL) Apoptosis Assay Kit (#C1090, Beyotime) was employed to identify cell death. S2 cells were fixed for 20 min in 4 % paraformaldehyde (PFA) and then three times in 1 × phosphate buffered saline (PBS). 45 L of fluorescent labeling solution and 5 L of TdT enzyme were combined in the dark. After exposing to the mixture for 1 h at 28 °C in the dark and washing three times in 1 × PBS, S2 cells were stained with Hoechst33342 (1.0 mg/mL, #C0031, Solarbio).

2.5 Copper ion assays

Copper and iron ions within S2 cells were assessed using the copper content assay kit (BC5755, Solarbio), the iron content assay kit (BC5315, Solarbio) and the ferrous ion content assay kit (BC5415, Solarbio). A total of 1 × 106 S2 cells were seeded in a 6-well plate. Following a 48-h incubation period, S2 cells from distinct groups were harvested and lysed utilizing an ultrasonic homogenizer. Subsequently, the supernatants were collected after centrifugation at 10,000 g for 10 min at 4 °C. According to the manufacturer's guidelines, the resulting supernatants and detection reagents were transferred to 96-well plates and mixed thoroughly. The absorbance of different experiments (copper: λ580nm; iron: λ510nm; ferrous: λ593nm) were measured using FlexStation 3 Multi-Mode Microplate Reader. All experiments were repeated three times, and the data were standardized based on the cell counts.

2.6 TCA cycle related assays

Metabolite levels associated with TCA cycle were determined using citric acid (CA; BC2155, Solarbio), pyruvate (PA; BC2205, Solarbio), α-ketoglutarate dehydrogenase (α-KGDH; BC0715, Solarbio), and mitochondrial isocitrate dehydrogenase (ICDHm; BC2165, Solarbio). A total of 1 × 106 S2 cells were seeded in a 6-well plate. Subsequent to copper treatment for 48 h, S2 cells were promptly harvested, homogenized and centrifugated at respective speeds and temperatures. Following the instructions provided by the manufacturer, the resulting supernatants and detection reagents were transferred to a 96-well plate for the respective assays. The absorbance of different experiments (CA: λ545nm; PA: λ520nm; α-KGDH: λ340nm; ICDHm: λ505nm) were measured using FlexStation 3 Multi-Mode Microplate Reader. All experiments were repeated three times, and the data were standardized based on the cell counts.

2.7 Oxidative stress assays

Oxidative stress was evaluated using lipid peroxidation (LPO; BC5245, Solarbio), Glutathione (GSH; BC1175, Solarbio) assays, 4-Hydroxynonenal ELISA kit (4-HNE; E-EL-0128, Elabscience) and GSH and GSSG assay kit (S0053, Beyotime). 1 × 106 S2 cells were inoculated into a 6-well culture plate. After treatment in each groups for 48 h, S2 cells were homogenized. As per the manufacturer's guidelines, the supernatants of the cells were collected. The quantification of LPO, GSH and GSH:GSSG ratio were subsequently assayed in 96-well plates, while 4-HNE was performed in pre-coated micro ELISA plate. The absorbance of different experiments (LPO: λ532nm; GSH: λ412nm; GSH:GSSG ratio: λ412nm; 4-HNE: λ450nm) were measured using FlexStation 3 Multi-Mode Microplate Reader. All experiments were repeated three times, and the data were standardized based on the cell counts.

2.8 Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted using TRIzol Reagent (15596026, Invitrogen, Waltham, MA, USA) in accordance with the manufacturer's procedure. Reverse transcription was performed and first strand cDNA was generated using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (6210A, Takara). The fluorescent reporter dye employed in the qPCR process was TB Green Premix Ex TaqII (RR820, Takara), and the LightCycler® System (Roche) was utilized. Primer sequences used in this study were shown in Table S4.

2.9 Immunostaining and antibodies

After dissecting the fly testes in 1 × phosphate-buffered saline (PBS), they were fixed in 4 % PFA for 30 min, rinsed three times with 0.3 % PBS-Triton X-100 (PBST), and blocked for 30 min in 5 % bovine serum albumin (BSA). Primary antibodies were applied to the tests and incubated for 1 h at room temperature. Testes were then rinsed 3 times in 0.3 % PBST and left to incubate for 1 h at room temperature in the dark with secondary antibodies. Following three additional rounds of washing with 0.3 % PBST, testes were stained for 5 min with Hoechst33342 (Solarbio, 1.0 mg/ml) before being mounted. The following primary antibodies were used: rabbit anti-Vasa (1:2000, a gift from Chao Tong's lab), mouse anti-FasIII (DSHB, 1:50), mouse anti-Orb (DSHB, 1:50), rabbit anti-β-Gal (#A11132, invitrogen, 1:2000). Secondary antibodies conjugated with Cy3, or A647 (Jackson ImmunoResearch Laboratories) were diluted at 1:400.

2.10 RNA isolation and bulk RNA sequencing

Total RNA was extracted utilizing TRIzol (15596026, Invitrogen, Waltham, MA, USA) for subsequent analysis. RNA integrity was assessed employing an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and validated through RNase-free agarose gel electrophoresis. Post RNA isolation, mRNA was enriched utilizing Oligo (dT) beads. The enriched mRNA was then fragmented into short sequences and reverse transcribed into cDNA utilizing the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB #7530, New England Biolabs, Ipswich, MA, USA). The purified double-stranded cDNA fragments underwent end repair, addition of an adenine base, and ligation to Illumina sequencing adapters. The ligation product was purified using AMPure XP Beads (1.0X). Ligated fragments were size-selected via agarose gel electrophoresis and amplified through PCR. The resulting cDNA library was subjected to sequencing using the Illumina Novaseq6000 platform by Gene Denovo Biotechnology Co (Guangzhou, China).

For the acquisition of high-quality, pristine reads, reads underwent additional filtration using fastp (version 0.18.0) [17]. A Drosophila melanogaster reference genome index was established, and paired-end immaculate reads were aligned to the reference genome utilizing HISAT2.2.4 with the parameter "-rna-strandness RF" and default configurations [18]. Transcript abundances were quantified using Fragments Per Kilobase per Million mapped reads (FPKM) values employing the RSEM tool [19]. RNA differential expression analysis was executed utilizing the DESeq2 software [20] between two distinct groups. Genes meeting the criteria of a false discovery rate (FDR) below 0.05 and an absolute fold change (FC) of ≥2 were recognized as DEGs. Enrichment analyses, employing Sankey views, were utilized to explore pivotal functional occurrences linked with DEGs based on subset databases like GO, STRING clusters, KEGG pathways, Reactome pathways, Pfam domains, and InterPro domains, with a statistical significance threshold of FDR below 0.05 [21].

2.11 Statistical analysis

All findings are depicted as mean ± standard error of the mean (SEM). Statistical analysis was carried out using GraphPad Prism software Version 6.01 (GraphPad Inc., La Jolla, CA, USA). For parametric analysis, student's t-test was employed for comparisons between two groups. One-way and two-way ANOVA was utilized for comparisons among multiple groups with Dunnett's or Turkey's multiple comparisons test. For non-parametric analysis, the Chi-square (Fisher's exact) test was applied to assess male fertility rate comparisons. Significance levels were denoted as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

3 Results

3.1 Copper overload induces cell demise through cuproptosis and ferroptosis

To explore the cellular effects of copper overload, we exposed S2 cells to CuCl2. Initially, we observed a significant increase in copper levels (Fig. 1A) and a rise in TUNEL-positive cells (Fig. 1B and C) following copper overload. Studies have indicated that copper-triggered cell demise is modulated through the direct interaction of copper with lipoylated components of the tricarboxylic acid (TCA) cycle [9]. Subsequently, we investigated the impact of copper overload on the TCA cycle, revealing elevated levels of citric acid (CA) and pyruvic acid (PA), along with heightened activities of isocitrate dehydrogenase (ICDH) and alpha-ketoglutarate dehydrogenase (α-KGDH) (Fig. 1D–H), mirroring the process of Cuproptosis. Additionally, qRT-PCR was conducted to evaluate the relative expression levels of genes associated with Cuproptosis, unveiling the up-regulations of the Cuproptosis regulatory gene Ferredoxin 1 (Fdx1) and lipoylation-related genes, e.g. Lipoyltransferase 1 (Lipt1), Dihydrolipoamide S-acetyltransferase (Dlat, also known as ‘muc’), Pyruvate dehydrogenase E1 alpha subunit 1 (Pdha), and Pyruvate dehydrogenase E1 beta subunit (Pdhb), following copper overload (Fig. S1A and B). Unexpectedly, we observed an increase in iron accumulation (Fig. 1I), followed by elevated levels of LPO and 4-HNE (Fig. 1J and K), and a reduction in GSH/GSSG ratio (Fig. 1L) in S2 cells as a result of copper overload. These changes potentially indicated that copper overload induced Ferroptosis, which was an iron-dependent regulated cell death process [22,23]. In conclusion, our results demonstrate that excessive copper induces Cuproptosis, thereby further promoting regulated cell death through Ferroptosis.Fig. 1 Excessive copper accumulation triggers cellular demise via cuproptosis and ferroptosis. (A) The content of copper ions in S2 cells after CuCl2 treatments. (B) The percentage of TUNEL-positive cells. Control, n = 10; 100 μM CuCl2 group, n = 10; 200 μM CuCl2 group, n = 10. (C) TUNEL staining for control, 100 μM CuCl2 and 200 μM CuCl2 groups in S2 cells. DNA was stained with Hoechst 33342 (blue). (D) Diagram illustrating the TCA Cycle. (E) PA level in S2 cells after CuCl2 treatments. (F) CA level in S2 cells after CuCl2 treatments. (G) ICDH activity in S2 cells after CuCl2 treatments. (H) α-KGDH activity in S2 cells after CuCl2 treatments. (I) The content of iron ions in S2 cells after CuCl2 treatments. (J) LPO level in S2 cells after CuCl2 treatments. (K) 4-HNE level in S2 cells after CuCl2 treatments. (L) GSH/GSSG ratio in S2 cells after CuCl2 treatments. All values are presented as mean ± SEM. Asterisks represent a significant difference compared to control group. *P < 0.05, **P < 0.01, ***P < 0.001, Scale bar: 50 μm.

Fig. 1

3.2 Copper overload facilitated testicular aging in Drosophila

Based on our extensive longitudinal experimental observations, Drosophila testes exhibit a series of morphological alterations throughout the aging progression. Subsequently, we delved into the testicular repercussions to elucidate the correlation between copper overload and testicular aging. Following CuCl2 administration, a notable reduction in the apical width of the testes was observed (Fig. 2A and B). Subsequently, we utilized Vasa and FasIII staining to delineate the localization of germline stem cells (GSCs) and noted a significant decrease in the GSC population post-copper overload (Fig. 2C and D). Remarkably, an augmented presence of TUNEL-positive cells was identified at the apex of the testes post-copper overload (Fig. 2E and F), potentially influencing spermatogenesis. Furthermore, evaluation of spermiogenesis at the post-meiotic phase unveiled a substantial decline in both the number of elongated spermatid clusters and Orb protein-labeled spermatids in the CuCl2-treated group compared to the control group (Fig. 2G–J). These findings collectively underscore the profound impact of copper overload on spermatogenesis. Additionally, our investigations revealed that the reproductive toxicity induced by copper overload resulted in a diminished male fertility rate (Fig. S2A) and expedited the process of testicular aging through intensifying the beta Galactosidase (β-Gal) signaling (Fig. 2K and L). Our data strongly suggest that copper overload can directly modulate male fertility by regulating testicular aging.Fig. 2 Excessive copper levels accelerated testicular aging in Drosophila. (A) DNA (grey) stainings in control and CuCl2 (10 mM) treated testes at 30-Days. (B) The apical width of Drosophila testes in control (n = 24) and CuCl2 (10 mM) treatment (n = 24) groups at 30-Days. (C) Immunostainings of Vasa (red) and FasIII (green) in control and CuCl2 (10 mM) treated testes at 30-Days. (D) The number of GSC in control (n = 10) and CuCl2 (10 mM) treated (n = 10) testes at 30-Days. (E) TUNEL (red) stainings in control and CuCl2 (10 mM) treated testes at 30-Days. (F) The number of TUNEL positive cells in control (n = 9) and CuCl2 (10 mM) treated (n = 10) testes at 30-Days. (G) Visualizations for elongated spermatid clusters at the tails of testes in control and CuCl2 (10 mM) treatment groups at 30-Days. (H) The number of elongated spermatid clusters in control (n = 13) and CuCl2 (10 mM) treatment (n = 13) groups at 30-Days. (I) Immunostainings of Orb (green) to label elongated spermatids in control and CuCl2 (10 mM) treated testes at 30-Days. (J) The number of Orb positive cells in control (n = 8) and CuCl2 (10 mM) treated (n = 11) testes at 30-Days. (K) Immunostainings of β-Gal (green) in control and CuCl2 (10 mM) treated testes at 30-Days. (L) The integrated density of β-Gal in control (n = 8) and CuCl2 (10 mM) treated (n = 8) testes at 30-Days. DNA was stained with Hoechst33342 (blue). All values are presented as mean ± SEM. Asterisks represent a significant difference compared to control group. *P < 0.05, **P < 0.01, ***P < 0.001, Scale bar: 50 μm.

Fig. 2

3.3 Copper overload mediated transcriptional regulation network in Drosophila testes

We proceeded to conduct RNA sequencing (RNA-seq) analysis on control and CuCl2-treated testes to delve deeper into the regulatory framework of copper overload in Drosophila. Within the transcriptional profiles, we identified 602 differentially expressed genes (DEGs), comprising 409 up-regulated and 193 down-regulated DEGs (Fig. 3A and Table S1). In the context of processes linked to Ferroptosis and Cuproptosis, we observed a notable enrichment of oxidative stress, copper transport, and iron transport, with the majority of related DEGs exhibiting up-regulation post-copper overload (Fig. 3B and C). Subsequent qRT-PCR analysis was conducted to validate the increased expression of pro-oxidative markers, e.g., CG10337, CG14907, Activity-regulated cytoskeleton associated protein 1 (Arc1), and Activity-regulated cytoskeleton associated protein 2 (Arc2); copper transport elements, e.g., Copper transporter 1B (Ctr1B), Metallothionein A (MtnA), and Metallothionein D (MtnD); and iron transport factors, such as Transferrin 1 (Tsf1); in CuCl2-treated testes compared to control testes (Fig. 3D–F).Fig. 3 Transcriptional regulation network in Drosophila testes influenced by copper overload. (A) Statistics of DEGs for the comparison between control and CuCl2 (10 mM) treatment groups. (B) Sankey view of representative DEGs and biological events for oxidative stress, copper ion transport and iron ion transport. (C) Heatmap view of representative DEGs for oxidative stress, copper ion transport and iron ion transport. (D) Relative mRNA of CG10337, CG14907, Arc1 and Arc2 in control and CuCl2 (10 mM) treated testes at 30-Days. (E) Relative mRNA of Ctr1B, MtnA and MtnD in control and CuCl2 (10 mM) treated testes at 30-Days. (F) Relative mRNA of Tsf1 in control and CuCl2 (10 mM) treated testes at 30-Days. All values are presented as mean ± SEM. Asterisks represent a significant difference compared to control group. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3

3.4 lncRNA:CR43306 plays pivotal roles in governing testicular aging through the modulation of oxidative stress

Previously, Wen et al. developed a three-component Cas9 microinjection system based on CRISPR/Cas9-catalyzed homology-directed repair, elucidating the pivotal roles of 33 out of 105 testis-specific lncRNAs, including lncRNA:CR43306, in the regulation of spermiogenesis in Drosophila [14,24]. It is noteworthy that we observed a significant reduction in the expression level of lncRNA:CR43306 during the aging process in Drosophila testes (Fig. 4A). Here, we systematically examined the function of lncRNA:CR43306 in Drosophila testes.Fig. 4 lncRNA:CR43306 deficiency led to testicular aging by modulating oxidative stress. (A) Relative mRNA levels of lncRNA:CR43306 in 2-Days and 30-Days testes. (B) DNA (grey) stainings in control and lncRNA:CR43306 KO testes at 30-Days. (C) The apical width of Drosophila testes in control (n = 24) and lncRNA:CR43306 KO (n = 21) groups at 30-Days. (D) Immunostainings of Vasa (red) and FasIII (green) in control and lncRNA:CR43306 KO testes at 30-Days. (E) The number of GSC in control (n = 9) and lncRNA:CR43306 KO (n = 11) testes at 30-Days. (F) TUNEL (red) stainings in control and lncRNA:CR43306 KO testes at 30-Days. (G) The number of TUNEL positive cells in control (n = 10) and lncRNA:CR43306 KO (n = 10) testes at 30-Days. (H) Visualizations for elongated spermatid clusters at the tails of testes in control and lncRNA:CR43306 KO groups at 30-Days. (I) The number of elongated spermatid clusters in control (n = 13) and lncRNA:CR43306 KO (n = 11) groups at 30-Days. (J) The number of Orb positive cells in control (n = 8) and lncRNA:CR43306 KO (n = 10) testes at 30-Days. (K) Immunostainings of Orb (green) to label elongated spermatids in control and lncRNA:CR43306 KO testes at 30-Days. (L) Immunostainings of β-Gal (green) in control and lncRNA:CR43306 KO testes at 30-Days. (M) The integrated density of β-Gal in control (n = 9) and 30-Days lncRNA:CR43306 KO (n = 8) testes at 30-Days. (N) Statistics of DEGs between control and lncRNA:CR43306 KO testes at 30-Days. (O) Heatmap view of representative DEGs for oxidative stress. DNA was stained with Hoechst33342 (blue). All values are presented as mean ± SEM. Asterisks represent a significant difference compared to control group. *P < 0.05, **P < 0.01, ***P < 0.001, Scale bar: 50 μm.

Fig. 4

Similarly, the apical width of the testes exhibited a decrease in lncRNA:CR43306 knockout (KO) testes compared to control testes (Fig. 4B and C). Loss of lncRNA:CR43306 led to a reduction in the number of GSCs and induced cell death at the apex of the testes (Fig. 4D–G). Towards the tail of the testes, a notable decrease in the number of elongated spermatid clusters and Orb-positive spermatids was observed in lncRNA:CR43306 KO testes relative to control testes (Fig. 4H–K). Furthermore, lncRNA:CR43306 deficiency resulted in a diminished male fertility rate and triggered β-Gal signals (Fig. 4L–M, and Fig. S2B). These findings collectively highlight the indispensable role of lncRNA:CR43306 in the process of testicular aging. Subsequent RNA-seq analysis in control and lncRNA:CR43306 KO testes unveiled a series of gene sets crucial for the oxidative stress process (Fig. 4N and O). Notably, qRT-PCR validation indicated an upregulation in the expression levels of CG10337, CG14907, Arc1, and Arc2 following lncRNA:CR43306 deficiency in the testes (Fig. S3A and B), emphasizing the pivotal involvement of pro-oxidative markers in lncRNA:CR43306-mediated testicular aging.

3.5 Synergistic effects for copper overload and lncRNA:CR43306 deficiency mediated testicular aging

Following a 2-day exposure to CuCl2, the expression level of lncRNA:CR43306 underwent down-regulation, a trend that intensified after a 30-day CuCl2 treatment, emphasizing the critical role of synergistic interactions between copper overload and lncRNA:CR43306 deficiency in testicular aging (Fig. 5A and B). Remarkably, male fertility was entirely compromised due to the synergistic effects induced by copper overload and lncRNA:CR43306 deficiency (Fig. 5C).Fig. 5 Cumulative effects between copper overload and lncRNA:CR43306 deficiency for testicular aging. (A) Relative mRNA levels of lncRNA:CR43306 in control and CuCl2-treated (10 mM) testes at 2-Days. (B) Relative mRNA levels of lncRNA:CR43306 in control and CuCl2 (10 mM) treated testes at 30-Days. (C) Male fertility rate for CuCl2-treated (n = 20) and CuCl2 + lncRNA:CR43306 KO (n = 20) flies at 30-Days. (D) DNA (grey) stainings in CuCl2 (10 mM) treated and CuCl2 + lncRNA:CR43306 KO testes at 30-Days. (E) The apical width of Drosophila testes in CuCl2 (10 mM) treated (n = 21) and CuCl2 + lncRNA:CR43306 KO (n = 18) groups at 30-Days. (F) Immunostainings of Vasa (red) and FasIII (green) in CuCl2 (10 mM) treated and CuCl2 + lncRNA:CR43306 KO testes at 30-Days. (G) The number of GSC in CuCl2 (10 mM) treated (n = 11) and CuCl2 + lncRNA:CR43306 KO (n = 11) testes at 30-Days. (H) TUNEL (red) stainings in CuCl2 (10 mM) treated and CuCl2 + lncRNA:CR43306 KO testes at 30-Days. (I) The number of TUNEL positive cells in CuCl2 (10 mM) treated (n = 9) and CuCl2 + lncRNA:CR43306 KO (n = 13) testes at 30-Days. (J) Visualizations for elongated spermatid clusters at the tails of testes in CuCl2 (10 mM) treated and CuCl2 + lncRNA:CR43306 KO groups at 30-Days. (K) The number of elongated spermatid clusters in CuCl2 (10 mM) treated (n = 10) and CuCl2 + lncRNA:CR43306 KO (n = 8) groups at 30-Days. (L) Immunostainings of Orb (green) to label elongated spermatids in CuCl2 (10 mM) treated and CuCl2 + lncRNA:CR43306 KO testes at 30-Days. (M) The number of Orb positive cells in CuCl2 (10 mM) treated (n = 11) and CuCl2 + lncRNA:CR43306 KO (n = 9) testes at 30-Days. (N) The integrated density of β-Gal in CuCl2 (10 mM) treated (n = 7) and CuCl2 + lncRNA:CR43306 KO (n = 10) testes at 30-Days. (O) Immunostainings of β-Gal (green) in CuCl2 (10 mM) treated and CuCl2 + lncRNA:CR43306 KO testes at 30-Days. DNA was stained with Hoechst33342 (blue). All values are presented as mean ± SEM. Asterisks represent a significant difference compared to CuCl2-treated group. *P < 0.05, **P < 0.01, ***P < 0.001, Scale bar: 50 μm.

Fig. 5

To gain deeper insights into the synergistic effects between copper overload and lncRNA:CR43306 deficiency in mediating testicular aging, we conducted an analysis of the testicular phenotype in CuCl2-treated testes and CuCl2 + lncRNA:CR43306 KO testes. In comparison to the CuCl2 treatment group, the apical width of the testes exhibited a significant decrease in CuCl2 + lncRNA:CR43306 KO testes (Fig. 5D and E). Notably, in testes subjected to CuCl2 treatment, the deficiency of lncRNA:CR43306 further exacerbated the reduction in GSC quantity and intensified the presence of TUNEL-positive cells at the apex of the testes (Fig. 5F–I). Additionally, we made a surprising observation that the elongated spermatid cluster was nearly completely absent, and Orb protein-labeled spermatids showed no signal in CuCl2 + lncRNA:CR43306 KO testes (Fig. 5J–M). Furthermore, lncRNA:CR43306 deficiency exacerbated the β-Gal signals in testes treated with CuCl2 (Fig. 5N and O). Moreover, Lam has been recognized as a crucial marker for evaluating aging in Drosophila [25]. Notably, in our research, both copper overload and lncRNA:CR43306 KO resulted in decreased expression of Lam, and its expression level exhibited a further decline in CuCl2 + lncRNA:CR43306 KO testes (Fig. S4A).

Through the examination of transcriptomic data, we noted that lncRNA:CR43306 deficiency could modulate the expression of DEGs associated with iron transport, oxidative stress, and processes related to elongated spermatids in CuCl2-treated testes (Fig. 6A–C). Specifically, DEGs related to elongated spermatids, such as sungrazer (sunz) and CG31244, exhibited decreased expression in CuCl2-treated and lncRNA:CR43306 KO testes compared to control testes, with their expression levels further declining in CuCl2 + lncRNA:CR43306 KO testes (Fig. 6D and E). Notably, qRT-PCR analysis also validated the up-regulation of the expression levels of Tsf1, a DEG involved in iron uptake and transport, following lncRNA:CR43306 deficiency in CuCl2-treated testes (Fig. 6F). Additional genes with known functions in Drosophila iron absorption were also assessed by qRT-PCR, including Ferritin 1 heavy chain homologue (Fer1HCH), Ferritin 2 light chain homologue (Fer2LCH), mitoferrin (mfrn), Multicopper oxidase 1 (MCO1), Multicopper oxidase 3 (MCO3) [26,27]. Consistent with Tsf1, the expression levels of additional iron metabolism genes (Fer1HCH, Fer2LCH, mfrn, MCO1, MCO3) were enhanced by CuCl2 treatment and lncRNA:CR43306 KO, with further exacerbation observed in CuCl2 + lncRNA:CR43306 KO testes (Fig. S4B). Furthermore, copper overload can also further increase the expression levels of copper transporter genes (Ctr1B, MtnA and MtnD) in the context of lncRNA:CR43306 KO testes (Fig. S4C). In our subsequent experiments, we treated S2 cells with FAC and FeSO4 separately to explore the impact of iron on lncRNA:CR43306. Our results revealed a significant accumulation of ferrous ion content level and a notable decrease in the expression level of lncRNA:CR43306 following the treatment with FAC and FeSO4 (Fig. S5). Collectively, these findings underscore the significant roles played by the combined effects of copper overload and lncRNA:CR43306 deficiency in regulating copper and iron transports and contributing to testicular aging.Fig. 6 Transcriptional regulation network influenced by copper overload and lncRNA CR43306 deficiency. (A) Statistics of DEGs for the comparison between CuCl2 (10 mM) treatment and CuCl2 + lncRNA:CR43306 KO groups. (B) Heatmap view of representative DEGs for iron ion transport, elongated spermatids and oxidative stress. (C) Heatmap view of representative DEGs for iron ion transport, elongated spermatids and oxidative stress. (D) Relative mRNA levels of sunz in control, CuCl2 (10 mM) treated, lncRNA:CR43306 KO, and CuCl2 + lncRNA:CR43306 KO testes at 30-Days. (E) Relative mRNA levels of CG31244 in control, CuCl2 (10 mM) treated, lncRNA:CR43306 KO, and CuCl2 + lncRNA:CR43306 KO testes at 30-Days. (F) Relative mRNA levels of Tsf1 in CuCl2 (10 mM) treated and CuCl2 + lncRNA:CR43306 KO testes at 30-Days. All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6

3.6 Ferrostatin-1 and resveratrol ameliorates copper overload mediated cell death and testicular aging

To further emphasize the counteractive effects facilitated by copper overload, we administered dietary Ferrostatin-1 and Resveratrol to investigate the mitigation of cell death and testicular aging. S2 cells were treated with copper overload in combination with Ferrostatin-1 for 48 h, revealing that Ferrostatin-1 effectively restored the copper-induced iron content (Fig. 7A). Additionally, Ferrostatin-1 significantly improved LPO and GSH levels in CuCl2-treated S2 cells (Fig. 7B and C).Fig. 7 Ferrostatin-1 and resveratrol alleviate cell death and mitigate testicular aging induced by copper overload. (A) The content of iron ions in S2 cells after CuCl2 (200 μM) and CuCl2 (200 μM) + Ferrostatin-1 (0, 5, 10 μM) treatments. (B) LPO level in S2 cells after CuCl2 (200 μM) and CuCl2 (200 μM) + Ferrostatin-1 (0, 5, 10 μM) treatments. (C) GSH level in S2 cells after CuCl2 (200 μM) and CuCl2 (200 μM) + Ferrostatin-1 (0, 5, 10 μM) treatments. (D) The content of iron ions in S2 cells after CuCl2 (200 μM) and CuCl2 (200 μM) + Resveratrol (0, 5, 10 μM) treatments. (E) LPO level in S2 cells after CuCl2 (200 μM) and CuCl2 (200 μM) + Resveratrol (0, 5, 10 μM) treatments. (F) GSH level in S2 cells after CuCl2 (200 μM) and CuCl2 (200 μM) + Resveratrol (0, 5, 10 μM) treatments. (G) Male fertility rate of CuCl2 + lncRNA:CR43306 KO (n = 20), Ferrostatin-1 + CuCl2 + lncRNA:CR43306 KO (n = 20), and Resveratrol + CuCl2 + lncRNA:CR43306 KO (n = 20) flies at 30-Days. (H) DNA (grey) stainings in CuCl2 + lncRNA:CR43306 KO, Ferrostatin-1 + CuCl2 + lncRNA:CR43306 KO, and Resveratrol + CuCl2 + lncRNA:CR43306 KO testes at 30-Days. (I) The apical width of Drosophila testes in CuCl2 + lncRNA:CR43306 KO (n = 20), Ferrostatin-1 + CuCl2 + lncRNA:CR43306 KO (n = 13), and Resveratrol + CuCl2 + lncRNA:CR43306 KO (n = 20) testes at 30-Days. (J) Immunostainings of Orb (green) to label elongated spermatids in CuCl2 + lncRNA:CR43306 KO, Ferrostatin-1 + CuCl2 + lncRNA:CR43306 KO, and Resveratrol + CuCl2 + lncRNA:CR43306 KO testes at 30-Days. (K) The number of Orb positive cells in CuCl2 + lncRNA:CR43306 KO (n = 20), Ferrostatin-1 + CuCl2 + lncRNA:CR43306 KO (n = 11), and Resveratrol + CuCl2 + lncRNA:CR43306 KO (n = 10) flies at 30-Days. DNA was stained with Hoechst33342 (blue). All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Scale bar: 50 μm.

Fig. 7

Resveratrol, a polyphenolic phytoalexin, is recognized for its diverse pharmacological advantages, encompassing anti-inflammatory and antioxidant attributes [28,29]. Studies have indicated that Resveratrol provided tissue protection by mitigating Ferroptosis [30]. Likewise, in S2 cells, Resveratrol rescued CuCl2-induced Ferroptosis, resulting in the reinstatement of iron content, LPO, and GSH levels (Fig. 7D–F).

Through in vivo experiments, we demonstrated that male fertility could be partially restored by Ferrostatin-1 and Resveratrol in CuCl2 + lncRNA:CR43306 KO flies (Fig. 7G). In order to elucidate the mechanisms behind the recovery of male fertility, we further examined the testicular phenotype in Drosophila. Ferrostatin-1 and Resveratrol widened the apical width of the testes in both copper overload and lncRNA:CR43306 deficiency conditions (Fig. 7H and I). Given the absence of spermatids in CuCl2 + lncRNA:CR43306 KO testes, Orb-labeled spermatids showed significant improvement in Ferrostatin-1 + CuCl2 + lncRNA:CR43306 KO and Resveratrol + CuCl2 + lncRNA:CR43306 KO testes (Fig. 7J and K). Our findings suggest that the inhibition of Ferroptosis can rescue testicular aging mediated by copper overload and lncRNA:CR43306 deficiency.

4 Discussion

Aging impacts spermatogenesis, sperm function, and the spermatogenic microenvironment, leading to decreased male fertility [31]. Although a recent review has preliminarily described the aging characteristics of mammalian testes [16], these characteristics still lack systematic evaluation and quantification. In this study, we systematically analyzed the aging characteristics of several significant phenotypes during testicular aging in Drosophila. Our findings reveal that both copper overload and lncRNA:CR43306 play pivotal roles during aging process, collectively accelerating testicular aging in Drosophila.

Emerging evidence suggests that lncRNAs play roles in modulating critical molecular and biological events associated with the aging process, including the regulation of telomere length, chromatin modulation, genomic imprinting, proteostasis, cell growth and differentiation, cell proliferation, and cellular aging [32,33]. Consequently, the dysregulated expression of lncRNAs may contribute to a range of age-related pathologies and disorders [33]. While certain lncRNAs, such as lncRNA:dntRL (CR45923), have been identified as potential target genes regulated by Rpd3 and Loco for longevity, the identification of lncRNAs related to testicular aging in Drosophila remains limited [34]. Furthermore, the specific role and mechanism of lncRNA:CR43306 in testicular aging have yet to be fully elucidated. Interestingly, our study revealed that copper overload led to a down-regulation of lncRNA:CR43306 expression, identifying it as a novel lncRNA associated with aging.

Copper, as an essential cofactor, plays a crucial role in maintaining cellular homeostasis and is vital for a range of physiological functions [7,35]. Disruption of intracellular copper levels can lead to oxidative stress and cytotoxicity [36]. From prokaryotes to eukaryotes, the regulation of copper homeostasis primarily involves preventing the excessive accumulation of copper ions in cells, which poses a threat to cell survival [35]. In mammals, seven pro-Cuproptosis genes, e.g. ferredoxin 1 (FDX1), lipoic acid synthetase (LIAS), lipoyltransferase 1 (LIPT1), dihydrolipoamide dehydrogenase (DLD), dihydrolipoamide S-acetyltransferase (DLAT), pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1), and pyruvate dehydrogenase E1 subunit beta (PDHB); three anti-Cuproptosis genes, e.g. metal regulatory transcription factor 1 (MTF1), glutaminase (GLS), and cyclin dependent kinase inhibitor 2A (CDKN2A); and two crucial copper transporters, e.g. ATPase copper transporting beta (ATP7B) and solute carrier family 31 member 1 (SLC31A1); were identified associated with Cuproptosis [37,38]. Copper deficiency can disrupt the physiological functions of iron, impacting superoxide dismutase 1 (SOD1) activity, thereby impairing ROS detoxification and shortening cellular lifespan [39]. Additionally, research has shown that duodenal cytochrome b (DCYTB) protein, acting as a reductase for both iron and copper, is rapidly induced in response to iron and oxygen deficiency, enhancing cellular copper uptake [40]. Multicopper ferroxidases, with copper as a prosthetic factor, such as ceruloplasmin (Cp) and hephaestin (Heph), play a crucial role as a vital connection between copper and iron in ensuring the balance of iron homeostasis [26,41]. MCO1 and MCO3 represent multicopper ferroxidases in Drosophila linked to iron metabolism [26]. Interestingly, MCO1 and MCO3 displayed up-regulations in response to copper overload and lncRNA:CR43306 deficiency. Furthermore, our findings confirmed that both copper overload and lncRNA:CR43306 deficiency led to the up-regulation of iron metabolism genes (Fer1HCH, Fer2LCH, mfrn) and copper transport factors (Ctr1B, MtnA and MtnD), with a more pronounced effect on iron metabolism. These results suggest that testicular aging is accelerated by copper overload and lncRNA:CR43306 deficiency through their impact on copper and iron metabolism, potentially exacerbated by synergistic effects.

On the contrary, another of our discoveries offers insights indicating that copper overload induces cell death through Cuproptosis, impacting the mitochondrial TCA cycle, a crucial process in testicular aging in Drosophila. We also observed that copper overload-induced Cuproptosis is intricately regulated, with oxidative stress playing hub roles in testicular aging through the Ferroptosis signature. GSH acts as the central node for both Ferroptosis and Cuproptosis, albeit with distinct functions [9,42]. In Ferroptosis, GSH acts as an antioxidant, preventing LPO, whereas in Cuproptosis, it functions as a copper chaperone, binding copper to reduce the aggregation of lipoylated proteins [9,42]. Interestingly, GSH demonstrates inhibitory effects on both Ferroptosis and Cuproptosis, suggesting a coordinated relationship where GSH may play a crucial role in mediating interactions between these pathways [43]. Wang et al. also illustrated a crosstalk between Ferroptosis and Cuproptosis, where inducers of Ferroptosis enhanced Cuproptosis by activating protein lipoylation and suppressing GSH synthesis [44].

LncRNAs exhibit a wide array of biological functions, positioning them as promising contenders for biological markers and therapeutic targets in various diseases. Research has validated the potential of lncRNAs to act as genetic markers and therapeutic targets in numerous conditions, exemplified by lncRNAs such as H19 [45,46], MALAT1 [47], and MAFG-AS1 [48]. Ferrostatin-1, an inhibitor of Ferroptosis, has been demonstrated to effectively reduce ROS [49,50]. Nevertheless, the limited metabolic stability and suboptimal pharmacokinetic profile of Ferrostatin-1 have hindered its progression in drug development [51]. Similarly, Resveratrol exhibits a diverse range of biological functions and properties, including antioxidant, anti-inflammatory, and immunomodulatory effects [52,53]. Recent investigations have highlighted the significant role of Resveratrol in mitigating age-related diseases through the attenuation of oxidative stress, reduction of inflammatory responses, enhancement of mitochondrial function, and regulation of apoptosis [54,55]. Overcoming the challenge of enhancing the bioavailability of Resveratrol in clinical settings remains a critical hurdle [56,57]. We confirmed that Ferrostatin-1 and Resveratrol could alleviate copper overload and lncRNA:CR43306 deficiency-induced testicular aging phenotypes, proposing that targeting both Ferroptosis and Cuproptosis concurrently could represent a promising strategy for treating testicular aging.

Through comprehensive RNA-seq analysis, it was ascertained that an excess of copper led to the activation of genes associated with copper and iron transport. A prior investigation delineated that heightened copper levels in senescent cells coincided with increased SLC31A1/CTR1 expression and reduced levels of copper efflux proteins like ATPase copper transporting alpha (ATP7A) [58]. Notably, copper transporters play a pivotal role during these processes, where ATPase enzymes ATP7A and ATP7B facilitate copper concentration within the trans-Golgi network, and the primary plasma membrane-localized copper transporter SLC31A1/CTR1 imports Cu+ from the extracellular milieu [59]. Cells react to elevated extracellular copper concentrations by up-regulating the expression of Refrex1, a regulator of CTR1-mediated copper accumulation [59]. Furthermore, metallothionein genes are transcriptionally induced by heavy metals through the metal-responsive transcription factor MTF-1. Our investigation revealed that following copper overload in Drosophila testes, MtnA and MtnD were up-regulated, indicating their involvement in the detoxification of metal ions [60]. Tsf1 is an extracellular protein with a strong affinity for iron [61]. Tsf1 is implicated in iron trafficking between the gut and fat body, competing with ferritin for iron in the intestines of Drosophila [62]. Suppression of Tsf1 expression disrupts iron homeostasis in the central nervous system and significantly mitigates the neurodegenerative effects of rotenone exposure in Drosophila [63]. In the context of Drosophila testes, our findings indicated that copper overload elevated Tsf1 expression levels and increased iron content, potentially exacerbating cellular aging and damage through the mechanism of ferroptosis.

In conclusion, our research underscored the potential of copper overload-induced Cuproptosis to trigger intracellular crosstalk with Ferroptosis, both pivotal in the context of testicular aging in Drosophila. We also elucidated the significance of lncRNA:CR43306 as an emerging aging-associated lncRNA. Furthermore, we observed that copper overload exacerbates spermatid differentiation defects mediated by lncRNA:CR43306 deficiency, contributing to testicular aging and male infertility. Notably, our investigation identified Ferrostatin-1 and Resveratrol as key pharmaceutical agents for mitigating testicular aging. This study offers novel insights into the regulatory mechanisms involving copper overload and lncRNA during the process of testicular aging.

Availability of data and materials

The data that support the findings of the current study are available from the corresponding author upon reasonable request.

CRediT authorship contribution statement

Qiuru Huang: Investigation, Methodology, Validation, Writing – original draft. Jiaxin Li: Investigation, Methodology. Yujuan Qi: Funding acquisition, Investigation, Validation, Writing – review & editing. Xuxin He: Investigation. Cong Shen: Investigation. Chenyu Wang: Investigation. Xinda Wang: Investigation. Qiushi Xia: Investigation. Yi Zhang: Investigation. Ziyue Pan: Investigation. Qingqing Hu: Investigation. Ziyu Cao: Investigation. Yiheng Liu: Investigation. Jingqi Huang: Investigation. Guoqing Han: Investigation. Ying Zheng: Investigation, Writing – review & editing. Bo Zheng: Conceptualization, Data curation, Funding acquisition, Investigation, Project administration, Validation, Visualization, Writing – review & editing. Xuhui Zeng: Data curation, Formal analysis, Investigation, Project administration, Supervision, Writing – review & editing. Xiaolin Bi: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Writing – review & editing. Jun Yu: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix ASupplementary data

The following are the Supplementary data to this article:Multimedia component 1

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Acknowledgments

The authors wish to thank all study participants, research staff, and students who assisted with this work. We are grateful to Gene Denovo Biotechnology for RNA-seq in this study. We thank Prof. Chao Tong (Zhejiang University) and Prof. Guanjun Gao (ShanghaiTech University) for sharing fly stocks and reagents. This work was supported partly by 10.13039/501100004608 Natural Science Foundation of Jiangsu Province (BK20221376 ), the 10.13039/501100001809 National Natural Science Foundation of China (82271633 ), Basic Science Research Program of Nantong City (JC12022006 ), the Fund of Xuzhou Science and Technology (KC20092 ), the Open Fund of Jiangsu Key Laboratory for New Drug Research and Clinical Pharmacy, and Xuzhou Pengcheng Talents - Medical young reserve talents (XWRCHT20220011 ).

Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2024.103315.
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