Baicalin targets YTHDC2 and alleviates male reproductive toxicity caused by co-exposure to nanoplastics and manganese through m6A-dependent pathway

Abstract

Nanoplastics (NPs) pollution has become a pressing global environmental issue. NPs possess the ability to adsorb heavy metals, thereby acting as vectors that facilitate the entry of these toxic substances into living organisms. However, the synergistic toxic effects of NPs and heavy metals, particularly with regard to male reproductive health, remain poorly understood. This study establishes in vivo and in vitro models to assess the effects of single and co-exposure to polystyrene nanoplastics (PS-NPs, 0.1 μm) and manganese (Mn) on male reproductive function. Our results reveal that co-exposure leads to a synergistic toxic effect, aggravating testicular damage, sperm abnormalities, and hormone disruption. Mechanistically, PS-NPs and Mn collaboratively suppress the RNA-binding protein YTHDC2, which in turn impairs Mdm2 transcription and translation in an m⁶A-dependent manner. This disruption results in cell cycle arrest via the Mdm2-p53 pathway, ultimately hindering spermatogenesis. Notably, baicalin, a natural compound, effectively targets YTHDC2 and mitigates the reproductive toxicity induced by co-exposure. These findings provide the novel evidence of the synergistic reproductive toxicity of PS-NPs and Mn in male mammals, offering new insights into their combined toxic effects and highlighting the potential of baicalin as a therapeutic intervention.

Graphical Abstract:

Supplementary Information: The online version contains supplementary material available at 10.1186/s12951-025-03535-3.

Keywords: Baicalin; Male reproductive toxicity; Manganese; Mdm2-p53 Pathway; Polystyrene nanoplastics; m6A.

Fig. 1

Characterization of PS-NPs/PS-MPs and PS-NPs + Mn/PS-MPs+ Mn. (a and b) SEM image of PS-NPs/PS-MPs and PS-NPs + Mn/PS-MPs + Mn. (c) The particles size of PS-NPs + Mn/PS-MPs + Mn (DLS). (d) Zeta Potential of PS-NPs + Mn (0.1 μm). (e and f). The entry of fluorescent microplastics with particle sizes of 0.1 μm, 1 μm, and 5 μm into spermatogenic cells. e: GC-1 spg. f: GC-2 spg. The magnification was set to ×200. (g) Adsorption of heavy metal Mn by microplastics with particle sizes of 0.1 μm, 1 μm, and 5 μm. (h) Elemental distribution mapping of C, Mn, and O in PS-NPs and PS-NPs + Mn (scale bar, 100 nm)

Fig. 2

Co-exposure to PS-NPs + Mn causes spermatogenesis blockage and spermatogenic cell abnormalities in mice. (a) Schematic of PS-NPs and PS-NPs + Mn exposure. (b-c) Typical images of testicular ultrasound (b). Testicular volume statistics (c). (d) The extent of Mn accumulation in the testis. (e) Sperm counts. (f) Serum T levels. (g) The malformation rate of sperm. (h) Representative images of sperm. (i) Testicular images with PAS staining. The magnification was set to ×200, scale bar = 50 μm. (j) Number of DDX4-positive cells in testicular tissue. (k) Number of SYCP3-positive cells in testicular tissue. (l) The levels of spermatogenic cell marker (DDX4), spermatocyte marker (SYCP3) in testicular tissues were measured by immunofluorescence. The magnification was set to ×400, scale bar = 25 μm

Fig. 3

Co-exposure to PS-NPs + Mn leads to down-regulation of the binding protein YTHDC2. (a) Schematic of PS-NPs + Mn treated GC-1 spg cells. (b) GO enrichment of differential genes after Co-exposure to PS-NPs and Mn (Molecular Function). (c) Scatterplot of differential genes (Ythdc2, Igf2bp1, Igf2bp2, and Igf2bp3) in the “N6-methyladenosine-containing RNA binding” pathway. (d) Mouse germ cell lineage atlas. (e) YTHDC2 expression in germ cell lineage. (f) YTHDC2 expression levels in germ cell lines at different stages. (g) YTHDC2 expression in normal and NOA. (h) Relative mRNA level of YTHDC2 in GC-1 spg. (i) Relative mRNA level of YTHDC2 in testis. (j) Relative protein level of YTHDC2 in GC-1 spg. (k) Relative protein level of YTHDC2 in testis. (l and n) The levels of YTHDC2 in GC-1 was measured by immunofluorescence. The magnification was set to ×200, scale bar = 50 μm. (m and o) The levels of YTHDC2 in testicular tissues was measured by immunofluorescence. The magnification was set to ×200, scale bar = 50 μm

Fig. 4

YTHDC2 in the testis resists spermatogenesis disorders caused by PS-NPs + Mn exposure. (a) Schematic of ADV-YTHDC2 over-expression in testis. (b) Typical images of testicular ultrasound. (c) Testicular volume statistics. (d) Sperm counts. (e) Sperm motility. (f) The malformation rate of sperm. (g) Serum T levels. (h) Histological score. (i) Representative images of sperm. (j) Testicular images with HE staining. The magnification was set to ×200, scale bar = 50 μm. (k) Testicular images with PAS staining. The magnification was set to ×200, scale bar = 50 μm

Fig. 5

YTHDC2 inhibiton mediates PS-NPs+Mn-induced down-regulation of the Mdm2-p53 pathway. (a) RNA-seq: GO analysis of DEGs with |log2FC|>1 and p < 0.05, comparing the YTHDC2-vector+PS-NPs+Mn group to the YTHDC2-OE+PS-NPs+Mn group, as determined by RNA-seq with a |log2FC|>1 and p < 0.05, (b) KEGG pathway enrichment analysis of DEGs with |log2FC|>1 and p < 0.05, comparing the YTHDC2-vector+PS-NPs+Mn group to the YTHDC2-OE+PS-NPs+Mn group, represented as a bubble chart. (c and d) cell-cycle phase. (e) RIP-seq: KEGG enrichment bubble chart of YTHDC2 binding gene. (f) Overlap of RNA-seq genes, Ythdc2 RIP-seq gene, and genes containing m6Am6A. (g) RIP assays were performed with the YTHDC2 antibody, followed by qPCR analysis. (h) Relative mRNA level of Mdm2 in GC-1 spg. (i) Relative protein level of Mdm2 in GC-1 spg. (j) Docking model of YTHDC2 (white) with MDM2 (blue). (k) Co-localization of YTHDC2 and MDM2 in GC-1 Spg. The magnification was set to ×200, scale bar = 50 μm. (l) Act-D analysis of stability of Mdm2 mRNA. (m) CHX analysis of the stability of Mdm2 protein. (n) Relative protein levels of p53, Ccnd1 and p21 in GC-1 spg

Fig. 6

YTHDC2 regulates Mdm2 in an m⁶A-dependent manner. Methylation modification sites of Mdm2 were predicted using the m⁶A site prediction tool. (b) The MeRIP quantitative PCR verified the presence of m⁶A modification at the P1-3 sites. (c) The m⁶A site of Mdm2 mRNA. (d) Western blot results showing the relative protein levels of Mdm2 in Mettl3 knocked down GC-1 Spg. (e) The relative protein level of Mdm2 in GC-1 Spg treated with 3DAA and MA2. (f) The relative protein level of Mdm2 in YTHDC2-overexpressing GC-1 Spg treated with or without 3-DAA. (g) The molecular mechanism of YTHDC2 regulating cell cycle

Fig. 7

YTHDC2 regulation of Mdm2-p53 pathway alterations involved in PS-NPs + Mn co-exposure-induced cell cycle arrest in spermatogenic cells. (a) Relative mRNA level of p53 in GC-1 spg. (b) Relative protein level of p53 in GC-1 spg. (c) Relative mRNA level of p21 in GC-1 spg. (d) Relative protein level of p21 in GC-1 spg. (e) Relative mRNA level of Ccnd1 in GC-1 spg. (f) Relative protein level of Ccnd1 in GC-1 spg. (g and h) cell-cycle phase

Fig. 8

Baicalin alleviates PS-NPs + Mn-induced male reproductive impairment by targeting YTHDC2. (a) Molecular Docking diagram of Baicalin, C3G, Rutin, Curcumin, and YTHDC2. (b-g) Representative western blot (b) and quantification of YTHDC2 (c), p53 (d), MDM2 (e), CCND1 (f), and p21 (g) in control and PS-NPs + Mn-exposed cells treated with natural compounds (Baicalin, C3G, Rutin, Curcumin). (h) Typical images of testicular ultrasound (i) Testicular volume statistics. (j) Sperm counts. (k) Sperm motility. (l) Representative images of sperm. (m) The malformation rate of sperm. (n) Serum T levels. (o) Histological score. (p) Testicular images with H&E staining. The magnification was set to ×400, scale bar = 25 μm. (q) Testicular images with PAS staining. The magnification was set to ×400, scale bar = 25 μm