Degradation reshapes the toxic identity of polylactic acid microplastics through MSR1-dependent immune decoding in mouse kidney

Abstract

Background

Biodegradable plastics are increasingly being used as a sustainable alternative, but their degradation in biological environments may produce transformation products with unexpected toxicological characteristics. These products generate a distribution pattern in the kidneys that differs from other organs, with the accumulation of low-molecular-weight polylactic acid microplastics (PLA MPs) being far higher than that of high-molecular-weight tissues.

Methods

PLA is used as a representative bioplastic. We used polymeric and oligomeric MPs to simulate their original and partially degraded states. Mice were exposed to these MPs through repeated oral administration under controlled experimental exposure conditions for 28 consecutive days to study the accumulation and inflammatory damage caused by PLA oligomer and polymer MPs in the kidneys. In combination with in vitro transcriptomic analysis, we explored the potential mechanisms by which oligomers drive nephrotoxicity.

Results

Exposure to PLA oligomer MPs results in significantly higher accumulation in the kidneys compared to PLA polymer MPs, and triggers more severe inflammatory damage. The mechanism is that renal macrophages preferentially phagocytose PLA oligomer MPs and decode them through macrophage scavenger receptor 1 (MSR1), activating phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling and inducing chemokine ligand 2 (CCL2)-dependent macrophage recruitment, thereby establishing a self-amplifying inflammatory loop. Inhibiting MSR1 or PI3K/AKT effectively reduces oligomer-driven cytokine production, macrophage infiltration, and renal injury, narrowing the toxicity gap between oligomeric and polymeric PLA MPs in the kidneys.

Conclusions

These findings reveal that biodegradation can heighten the inflammatory potential of MPs, and that distinct polymerization states of the same material elicit different immune interpretations. Our work provides mechanistic clarity on how degradability reshapes microplastic toxicity, underscoring the need to incorporate degradation-state profiling into the safety assessment of biodegradable MPs.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12989-026-00690-9.

Background

Polylactic acid (PLA) is one of the most widely used biodegradable plastics in medical, food-packaging, and consumer-product applications [1, 2]. Although considered biocompatible and approved for clinical use [3, 4], PLA-based materials inevitably fragment during manufacturing, use, and environmental degradation, generating substantial quantities of microplastics (MPs) that enter biological and ecological environments [5–7], PLA MPs have now been detected in human placenta and feces, indicating widespread human exposure and raising concerns regarding their potential health effects [8], Notably, unlike conventional non-degradable MPs such as polystyrene or polyethylene, PLA MPs undergo continuous depolymerization in biological systems, generating lower–molecular-weight oligomers with distinct physicochemical and biological behaviors [9]. Multiple studies, including our own, have reported that these oligomeric particles elicit stronger intestinal and neurotoxic effects than their polymeric precursors [9, 10], suggesting that degradation does more than simply alter particle size and instead generates particulate states with altered toxic potential. However, whether these degradation-dependent changes trigger distinct toxicological mechanisms in organs beyond the gut and brain remains poorly understood.

The kidney, as a central filtration and excretory organ, has been reported to continuously exposed to circulating particulates and has been reported to be susceptible to particle retention and injury [11–14]. This vulnerability may be especially relevant for biodegradable particles, whose physicochemical properties continue to evolve after exposure. Our previous work revealed a distinct biodistribution pattern of PLA MPs in vivo: among all examined organs, the kidney was the only tissue in which low–molecular-weight PLA MPs accumulated at substantially higher levels than their high–molecular-weight counterparts [9]. This molecular-weight–dependent enrichment suggests that PLA degradation fundamentally alters particle-organ interactions and may confer a renal tropism not observed for conventional MPs. Yet, the biological processes that drive this selective renal accumulation, as well as the resulting nephrotoxic effects and mechanisms, remain largely unknown.

Given that immune cells govern particle recognition, uptake, and inflammatory activation, we hypothesized that PLA biodegradation reshapes the biological identity and toxic potential of derived MPs by altering their immune decoding within the kidney. To address this hypothesis, we established a molecular-weight-stratified framework comparing intact 40 kDa polymers with 2 kDa oligomeric fragments that mimic physiologically relevant degradation states. Using this framework, we investigated the immune mechanisms by which PLA degradation state influences renal pathogenicity, focusing on macrophage-associated recognition of oligomeric particles, including the potential involvement of macrophage scavenger receptor 1 (MSR1), and downstream phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling. This study provides a mechanistic basis for understanding how polymer degradation products, rather than the parent material alone, acquire heightened renal toxicity.

Methods

PLA MPs

The red-fluorescent PLA oligomer and polymer MPs were obtained from Shanghai Sur-Release Biotech Inc. (Shanghai, China). Morphological characterization of the PLA oligomer and polymer MPs was carried out using scanning electron microscope (SEM) (Zeiss Supra55, Carl Zeiss AG, Germany). The particle size distribution and zeta potential of these suspensions were determined by dynamic light scattering (DLS) and Zetasizer nano ZS (Malvern Panalytical GmbH, Kassel, Germany). Both particle types exhibited > 99% purity and high physicochemical stability, as confirmed in our previous study [9].

Mouse husbandry

Male C57BL/6J mice aged 6–8 weeks were housed in a temperature-controlled facility (23–25 °C) under a 12 h light/dark cycle (lights on 08:00–20:00) with 50–60% humidity. Food and water were provided ad libitum. After a 7-day acclimation period, randomization was used to allocate animals to control and treatment groups. The number of animals is determined based on the minimum number required to observe statistically and biologically meaningful effects, thereby reducing the use of animals. Animals were first stratified by initial body weight to reduce intergroup variation, then assigned to groups using a computer-generated random number sequence produced with the Excel RAND() function, with sequential allocation based on the sorted random values. During group allocation, the investigator responsible for randomization was aware of group assignments in order to implement the allocation sequence. During the conduct of the experiment, animal caretakers and technicians performing interventions were unaware of group allocation. Animal cage location was not controlled, as all cages were housed in a single rack with uniform environmental conditions during the study.

28-Day repeated dose oral toxicity experiment in mice

A 28-day oral gavage study was performed using C57BL/6J mice. For biodistribution analyses, nine mice were randomized into three groups (n = 3 per group): control, PLA oligomer MPs (25 mg/kg), and PLA polymer MPs (25 mg/kg). For renal injury assessment, fifteen mice were randomized into five groups (n = 3 per group): control; PLA oligomer MPs (2.5 or 25 mg/kg); and PLA polymer MPs (2.5 or 25 mg/kg). Body weight was recorded every 7 days, and gavage volumes were adjusted weekly. The 2.5 mg/kg/day dose was selected as a low-dose condition approximating reported estimates of total environmental MP exposure, whereas 25 mg/kg/day was used as a high-dose condition to improve detection of renal accumulation and early toxicological responses [15]. All administrations were performed between 08:00 and 09:00. The order of treatments and measurements was randomized daily.

Macrophage-specific clearance experiment

Macrophages were selectively depleted using clodronate-containing liposomes following established protocols [16]. Six experimental groups were included (n = 5 per group): control liposomes; clodronate liposomes; PLA oligomer MPs + control liposomes; PLA oligomer MPs + clodronate liposomes; PLA polymer MPs + control liposomes; and PLA polymer MPs + clodronate liposomes. On day 1, mice received an orbital vein injection of either control liposomes or clodronate liposomes (200 µL per mouse; Yensen, Shanghai, China) according to group assignment. The exposure dose of PLA MPs was 25 mg/kg, administered once daily by oral gavage for 7 consecutive days. Mice were euthanized by cervical dislocation on day 7. All administrations were performed between 08:00 and 09:00. The order of treatments and measurements was randomized daily.

MSR1 inhibitor and PI3K/AKT signaling pathway intervention

A 28-day repeated oral gavage study was conducted using 36 C57BL/6J mice to investigate the biological distribution and kidney damage of PLA oligomer MPs in mice treated with Fucoidan (MSR1 inhibitor) (Shanghai Macklin Biochemical Co., Ltd, Shanghai, China) and LY294002 (PI3K/AKT signaling pathway inhibitor) (MCE, Shanghai, China). Both inhibitors were administered via intraperitoneal injection, with doses selected on the basis of prior literature [17, 18]. The mice were randomly divided into six groups (n = 6 per group): control group, PLA oligomer MPs group (25 mg/kg), Fucoidan group (100 µg/mouse, administered every two days), Fucoidan + PLA oligomer MPs group, LY294002 group (50 mg/kg, administered three times per week), and LY294002 + PLA oligomer MPs group. After 28 days, the animals were euthanized by cervical dislocation.

Sample collection

At the end of each experiment, mice were anesthetized by intraperitoneal injection of 3% pentobarbital (150 mg/kg body weight) and perfused with cold physiological saline. Euthanasia was performed by cervical dislocation. Kidneys were collected on ice, snap-frozen in liquid nitrogen, and stored at -80 °C for molecular analyses. For histological assessment, perfused kidneys were pre-filled with 4% paraformaldehyde (PFA), excised intact, fixed in 4% PFA for 24 h, and processed for paraffin embedding.

Hematoxylin-eosin (HE) staining

For histopathological evaluation, paraffin-embedded kidneys were sectioned and stained with HE. After deparaffinization and rehydration, standard HE procedures were performed to visualize renal architecture and pathological lesions. Stained sections were examined by light microscopy. Glomerular injury was defined, relative to the control group, by blurred glomerular boundaries, overall enlargement or shrinkage of the glomerulus, and inflammatory cell infiltration. For each HE stained section, 100 glomeruli were evaluated, and the glomerular injury rate was calculated as the percentage of damaged glomeruli.

Immunofluorescence staining

Paraffin-embedded kidney sections were used for immunofluorescence analysis. After deparaffinization and rehydration, antigen retrieval was performed using proteinase K (Servicebio). Sections were then incubated with 3% hydrogen peroxide (Sinopharm, Beijing, China) and subsequently blocked with 3% bovine serum albumin (Servicebio) for 30 min. Primary antibodies against CD68 (Servicebio, Wuhan, China), CD8 (Servicebio, Wuhan, China), IL-1β (Servicebio, Wuhan, China), and CCL2 (Affinity, China) were applied and incubated overnight at 4 °C. After three washes with phosphate-buffered saline (PBS, pH 7.4), fluorescent secondary antibodies were added and incubated at room temperature. Nuclei were counterstained with DAPI (Servicebio, Wuhan, China). Slides were mounted and scanned using a Pannoramic MIDI digital slide scanner, and images were analyzed with Pannoramic Viewer. Fluorescence intensities of CD68, CD8, IL-1β, and CCL2 were quantified as the number of positive cells normalized to DAPI. At least five fields were analyzed per slide. The complete list of the antibodies is shown in Supplementary Table S1.

Cell culture

The mouse macrophage cell line RAW264.7 was obtained from the American Type Culture Collection. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 mg/mL streptomycin, and 100 IU /mL penicillin (Gibco). Cells were cultured at 37 °C in a humidified incubator with 5% CO₂ and passaged at 75–85% confluence. For treatments, RAW264.7 cells were seeded into 6-well plates at 3 × 10⁵ cells per well and incubated overnight, followed by exposure to PLA oligomer MPs (10–40 µg/mL) or PLA polymer MPs (10–40 µg/mL) for 24 h. Control wells received no MPs.

siRNA transfection

To silence gene expression, RAW264.7 macrophages were transfected with 50 nM siRNA using Tsingke Transfection Reagent (Tsingke Biological Technology, Beijing, China) according to the manufacturer’s protocol. Transfections were performed in serum-free medium for 6 h, after which the medium was replaced with complete DMEM and cells were exposed to PLA oligomer MPs. Cells were harvested 24 h after transfection for further analysis. The following siRNA sequences were used: mouse si-Cd36-1 sense, 5′-GGAUCUGAAAUCGACCUUA-3′, mouse si-Cd36-1 antisense, 5′-UAAGGUCGAUUUCAGAUCC-3′; mouse si-Cd36-2 sense, 5′-GCACCACUGUGUACAGACA-3′; mouse si-Cd36-2 antisense, 5′-UGUCUGUACACAGUGGUGC-3′; mouse si-Msr1-1 sense, 5′-GAAUGUCAGAGUCCGUGAA-3′, mouse si-Msr1-1 antisense, 5′-UUCACGGACUCUGACAUUC-3′; mouse si-Msr1-2 sense, 5′-CGACCUUAUAGACACGGAA-3′; mouse si-Msr1-2 antisense, 5′-UUCCGUGUCUAUAAGGUCG-3′.

Transwell migration assay

The transwell migration assay was used to evaluate macrophage recruitment. RAW264.7 macrophages (1 × 10⁵ cells) were seeded into the upper chamber of a transwell insert (Corning, New York, USA), and 3 × 10⁵ RAW264.7 macrophages were seeded into the lower chamber. Cells were allowed to attach for 24 h in DMEM. Only the lower-chamber cells were treated with PLA oligomer MPs for 24 h, with selected groups co-treated with the CCR2 inhibitor INCB3284 (MCE, Shanghai, China) during the same period (n = 3 per group). The upper-chamber cells received no treatment. After 24 h, the cells that migrated to the lower surface of the membrane were fixed, stained with crystal violet (Beyotime, Guangzhou, China) [19].

PLA MPs uptake and accumulation assessment

Red-fluorescent PLA MPs were detected at an excitation wavelength of 530 nm and an emission wavelength of 580 nm. Untreated control samples were imaged under identical acquisition settings to define the autofluorescence baseline. For in vitro uptake assays, RAW264.7 macrophages were exposed to red-fluorescent PLA oligomer or polymer MPs for 24 h, washed with PBS three times, fixed, stained with phalloidin and DAPI (Beyotime), and imaged using a fluorescence microscope (Nikon Eclipse Ti/C2, Japan). To assess intracellular localization, Z-stack imaging was performed in at least five randomly selected fields per slide, with a scanning depth of 10 μm and an interval of 0.25 μm. For each cell, the first and last optical sections containing detectable cell-body signal were defined as the upper and lower boundaries of the cell. The Z-position of each particle was assigned to the optical section showing its maximum fluorescence intensity. A particle was classified as intracellular only when its intensity maximum occurred within the cell-body Z-range and its centroid was located inside the cell boundary region in the corresponding XY optical section. Image analysis was performed in a blinded manner by two independent observers. For kidney tissues, PLA MP fluorescence and CD68 immunofluorescence were acquired using identical fluorescence-channel and exposure settings across all groups. Untreated control kidney sections were imaged under the same acquisition conditions to define the tissue autofluorescence baseline. PLA MP–CD68 co-localization was quantified using ImageJ with automatic thresholding for background subtraction. At least five non-overlapping fields were randomly selected from each section, samples were blindly coded before analysis, and co-localization was quantified based on the overlap between PLA-MP fluorescence and CD68 immunofluorescence.

RNA isolation and quantitative polymerase chain reaction (qPCR) analysis

Total RNA was extracted from homogenized animal tissues using Trizol reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions. cDNA synthesis was performed using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme). qPCR was conducted with SYBR^® Green Premix Pro Taq HS (Accurate Biotechnology) on an ABI QuantStudio 6 Flex system (Applied Biosystems, USA). β-actin served as the internal reference gene. Relative mRNA expression was calculated using the 2^⁻ΔΔCt method, with the control group as the calibrator. qPCR cycling conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Primer sequences are listed in Supplementary Table S2.

Bulk RNA sequencing

Total RNA extracted from RAW264.7 macrophages (seeded in 6-well plates; n = 3 per group) was further purified using the RNAClean XP Kit (Beckman Coulter, CA, USA) and the RNase-Free DNase Set (QIAGEN, Germany). Strand-specific libraries were prepared using the VAHTS Universal V6 RNA-seq Library Preparation Kit for Illumina (Vazyme, Nanjing, China) following the manufacturer’s instructions. Sequencing reads were quantified using StringTie (v1.3.0) [20]. Gene expression levels were normalized using the M-value pruning average method [21], and converted to fragments per kilobase of transcript per million mapped reads (FPKM). Differential gene expression analysis was performed using the edgeR package (v3.32.1) in R, with P-values adjusted for multiple testing to control the false discovery rate [22].

Protein extraction and western blotting

For protein extraction, cultured cells (n = 3 per group) were washed with cold PBS and lysed in ice-cold RIPA buffer (Beyotime, Guangzhou, China) supplemented with protease and phosphatase inhibitors (1:100; Keygen, Nanjing, China). Lysates were incubated on ice for 30 min and centrifuged at 13,000 × g for 30 min at 4 °C, and the supernatants were collected. Equal amounts of protein were separated by 8% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA, USA). Membranes were blocked in 5% skimmed milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature and incubated overnight at 4 °C with primary antibodies against β-actin (Sigma-Aldrich, Germany), MSR1 (Abclonal, Wuhan, China), CD36 (Abclonal, Wuhan, China), phosphorylated AKT (P-AKT), and total AKT (Proteintech, Wuhan, China). After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies (Proteintech, Wuhan, China) for 1 h at room temperature. Protein bands were visualized using ECL reagent (Millipore, Billerica, MA, USA) and imaged with a Tanon-5200 chemiluminescence system (Tanon, Shanghai, China). Band intensity was quantified using ImageJ. Protein expression levels were normalized to β-ACTIN for total protein and to total AKT for phosphorylated AKT. The complete list of the antibodies is shown in Supplementary Table S3.

SDS-PAGE

After incubation of PLA oligomer and polymer MPs with 10% FBS‑DMEM for 24 h, the bound proteins were eluted, denatured by boiling, separated by SDS‑PAGE, and stained with Coomassie Blue.

Enzyme-linked immunosorbent assay (ELISA)

Cell culture supernatants were centrifuged at 3,000 rpm for 20 min at 4 °C to remove precipitates. The clarified supernatants were analyzed using CCL2 and CCL5 ELISA kits (MEIMIAN, Guangzhou, China) according to the manufacturer’s instructions. Each sample was tested in triplicate. Absorbance was measured at 450 nm using a microplate reader, and cytokine concentrations were calculated from standard curves generated for each assay.

Statistical analysis

The data are presented as mean ± standard deviation (SD) unless otherwise specified. Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) and SPSS 22.0 (IBM, Armonk, NY, USA). The hypotheses of statistical tests are evaluated as follows. The normality of continuous outcomes is assessed using the Shapiro–Wilk test. Homogeneity of variance between groups is tested using Levene’s test. Comparisons between two groups were conducted using two-tailed unpaired Student’s t-tests. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. When normality and homogeneity of variance are not met, Mann–Whitney U test or the Kruskal–Wallis test was used. A two-tailed P-value < 0.05 was considered statistically significant. Data analysis was conducted by a statistician who was initially blinded to group identities.

Results

Impact of PLA oligomer and polymer MPs on renal injury in mice

To investigate the renal effects of PLA MPs with different degradation states, we established a 28-day mouse model with oral administration of PLA oligomers and polymers. SEM showed that both types of particles were spherical and ranged from 1 to 5 μm in diameter (Figure S1A). DLS analysis showed that PLA oligomer and polymer MPs had comparable size distributions, low PDI values, and negative zeta potentials, indicating stable dispersion in double-distilled water (Figure S1B). Furthermore, SDS-PAGE analysis showed no obvious difference in protein corona composition between the two particle types (Figure S1C). Kidneys were harvested on day 28 for subsequent analyses (Fig. 1A). Fluorescence imaging showed greater renal accumulation of PLA oligomer MPs than PLA polymer MPs (Fig. 1B and S1D). Histopathological analysis revealed mild renal alterations in the PLA polymer MP group, whereas PLA oligomer MPs produced higher glomerular injury scores (Fig. 1C and F). PLA oligomer MPs also increased blood urea nitrogen (BUN) levels, whereas serum creatinine (CRE) remained unchanged; PLA polymer MPs did not significantly affect either marker (Figure S2A and S2B). Immunofluorescence staining showed increased renal IL-1β expression, particularly in the oligomer group (Fig. 1D and G). To assess whether changes in macrophage or T-cell populations accompanied renal inflammatory alterations, we performed targeted staining for CD68 and CD8. The results showed that Macrophage infiltration was markedly elevated in a pattern similar to that of IL-1β, whereas no notable change was observed in CD8⁺ T-cell abundance (Fig. 1E and H-I). Collectively, these findings indicated that both PLA oligomer and polymer MPs induce renal inflammatory alterations and histopathological injury, with oligomer MPs eliciting a more pronounced, macrophage-driven inflammatory response than their polymeric counterparts.

Fig. 1.

Renal accumulation and inflammatory injury induced by PLA oligomer and polymer MPs. A Schematic diagram of the experimental design; B Representative images of PLA MPs autofluorescence in mouse kidneys; C HE staining of mouse kidneys, with black arrows indicating inflammatory infiltration; D Representative immunofluorescence images showing IL-1β expression in mouse kidneys; E Representative immunofluorescence images of CD68⁺ macrophages and CD8⁺ T lymphocytes in mouse kidneys; F Relative quantitative results of glomerular injury rates; G Relative quantitative results of fluorescence intensity of IL-1β in mouse kidneys; H Relative quantitative results of CD68 positive cells; I Relative quantitative results of CD8 positive cell. n = 3 mice per group. *P < 0.05. HE, hematoxylin-eosin; PLA MPs, polylactic acid microplastics; IL-1β, interleukin 1β

Macrophages mediate renal accumulation and inflammation induced by PLA oligomer MPs

Given that renal accumulation and macrophage-driven inflammation were more pronounced in the oligomer MP groups, we next investigated whether macrophages mediated these effects. In vivo colocalization analysis revealed that over 87% of PLA oligomer MPs colocalized with renal macrophages, while only about 46% of PLA polymer MPs showed such colocalization (Figure S3A and S3B). These results indicate a stronger phagocytic response toward oligomer MPs, which may underlie their greater renal inflammatory injury. To verify this hypothesis, we depleted renal macrophages using clodronate disodium liposomes and then exposed the mice to PLA oligomer or polymer MPs via oral gavage (Fig. 2A). Clodronate treatment markedly reduced macrophage abundance in all groups, confirming successful establishment of the macrophage-depletion model (Fig. 2B and F, second column). After depleting macrophages, renal accumulation of both PLA oligomer and polymer MPs decreased significantly (Fig. 2C and F, third column). Notably, the glomerular injury rate decreased significantly only under oligomer MP exposure (Fig. 2D and F, fourth column), whereas IL-1β expression was reduced under both oligomer and polymer MPs exposure conditions (Fig. 2E and F, fifth column). These findings demonstrated that macrophages play a key role in the renal accumulation and inflammation induced by PLA oligomer and polymer MPs, accounting for the greater nephrotoxic effects of oligomeric compared with polymeric MPs.

Fig. 2.

Effects of macrophage depletion on renal accumulation and inflammation induced by PLA oligomer and polymer MPs. A Schematic diagram of the experimental design for the mouse model with specific renal macrophage depletion; B Number of renal macrophages; C Quantification of PLA MPs engulfed by renal macrophages; D Quantification of glomerular injury rates; E Relative quantitative results of fluorescence intensity of IL-1β in mouse kidneys; F Representative images of CD68⁺ immunofluorescence, PLA autofluorescence (orange arrows indicate PLA MPs phagocytosed by macrophages; red arrows indicate undigested PLA MPs), HE staining (black arrows indicate inflammatory infiltration in the kidneys), and IL-1β expression in mouse kidneys. n = 5 mice per group. *P < 0.05. PLA MPs, polylactic acid microplastics; HE, hematoxylin-eosin; IL-1β, interleukin 1β

MSR1 and CD36 are key membrane receptors potentially involved in macrophage phagocytosis of PLA MPs

We next investigated the molecular mechanisms underlying macrophage phagocytosis of PLA MPs using an in vitro RAW264.7 macrophage model. Consistent with the in vivo findings, the number of phagocytosed PLA oligomer MPs was significantly higher than that of polymer MPs (Fig. 3A and B). To investigate the underlying mechanisms, we performed transcriptome analysis on macrophages exposed to PLA oligomer or polymer MPs. Principal component analysis (PCA) showed distinct transcriptional separation of both oligomer and polymer exposure groups from the control group (Fig. 3C). K-means clustering of gene expression profiles identified six distinct clusters with divergent expression patterns across the experimental groups (Fig. 3D). Notably, cluster 6 exhibited dose-dependent upregulation in the oligomer MP groups and showed overall higher expression levels than the polymer MP groups (Fig. 3D). To prioritize transcriptional changes most relevant to the stronger biological effects of oligomer MPs, we focused on cluster 6 because it was the only cluster showing both oligomer-specific and dose-dependent upregulation (Fig. 3D). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that genes in this cluster were mainly involved in the phagosome formation pathway, suggesting a pivotal role in activating macrophage phagocytic and immune responses after PLA oligomer MPs exposure (Fig. 3E). We therefore further examined differentially expressed phagosome-related genes within this cluster and identified six significantly upregulated candidate genes (Fig. 3F). To validate the transcriptomic findings, we performed qPCR analysis of the six candidate genes. Among them, only two macrophage scavenger receptors, Msr1 and Cd36, exhibited dose-dependent increases that were markedly higher than those in the polymer group (Fig. 3G-L). Further, western blot analysis confirmed that the protein levels of MSR1 and CD36 followed the same trend (Fig. 3M-O). Collectively, these results identified MSR1 and CD36 as key molecules potentially involved in macrophage phagocytosis of PLA MPs.

Fig. 3.

Transcriptomic identification and validation of key molecules involved in macrophage phagocytosis of PLA MPs. A Representative images of RAW264.7 macrophages phagocytizing PLA oligomer and polymer MPs; B Quantification of phagocytosed PLA MPs; C PCA of transcriptomic profiles (n = 5); D K-means clustering of the transcriptome profiles (n = 5); E KEGG enrichment analysis of Cluster 6; (F) Volcano plot of differentially expressed genes related to the phagosome formation pathway (n = 5); G–L Validation of mRNA relative expression through qPCR for Msr1, Cd36, Atp6v0d2, Rab7b, Tlr2 and Itga5; M–O Representative western blot bands and relative protein levels of MSR1 and CD36. n = 3 per group unless otherwise indicated. *P < 0.05. PLA, polylactic acid microplastics; qPCR, quantitative polymerase chain reaction; RNA-seq, RNA sequencing; PCA, principal component analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes

Membrane receptors MSR1 and CD36 mediate macrophage differential phagocytic capacity for PLA MPs

To validate the functional role of MSR1 and CD36, RAW264.7 macrophages were transfected with siRNA targeting Msr1 or Cd36, followed by exposure to either PLA oligomer or polymer MPs. Two independent siRNAs were used for each gene, both effectively suppressing their respective mRNA expression (Figure S4A and S4B). The corresponding siRNAs significantly attenuated the PLA oligomer or polymer MP-induced upregulation of Msr1 and Cd36 expression in RAW264.7 macrophages (Fig. 4A and D). Microscopic observation revealed that silencing either receptor substantially reduced macrophage phagocytosis of both PLA oligomer and polymer MPs (Fig. 4B-C and E-F). Notably, knockdown of Msr1 or Cd36 abolished the differential phagocytic capacity observed between oligomer and polymer MPs (Fig. 4B-C and E-F). These results demonstrated that the macrophage scavenger receptors MSR1 and CD36 involved in the phagocytosis of both PLA oligomer and polymer MPs, and that the enhanced phagocytosis of oligomer MPs arises from their greater ability to upregulate the expression of these membrane receptors.

Fig. 4.

Phagocytic capacity of macrophages toward PLA oligomer and polymer MPs following knockdown of Msr1 or Cd36. A Relative mRNA expression levels of Msr1; B Quantitative results and C representative images of macrophage phagocytosis of PLA oligomer and polymer MPs after Msr1 knockdown; D Relative mRNA expression levels of Cd36; E Quantitative results and F representative images of macrophage phagocytosis of PLA oligomer and polymer MPs after Cd36 knockdown. n = 3 per group. *P < 0.05. PLA MPs, polylactic acid microplastics

MSR1 mediates stronger macrophage recruitment and inflammation induced by PLA oligomer than polymer MPs

We next sought to elucidate the mechanism underlying the macrophage accumulation observed in vivo following exposure to PLA oligomer and polymer MPs. Although both forms of PLA MPs modulated the mRNA expression of multiple chemokines, chemokine ligand 2 (Ccl2) and chemokine ligand 5 (Ccl5) were more strongly upregulated by oligomer MPs than by polymer MPs (Fig. 5A), mirroring the macrophage enrichment phenotype observed in vivo. In addition, the extracellular protein levels of CCL2 also showed a similar pattern (Fig. 5B), while CCL5 did not exhibit this trend (Figure S5A). To determine whether CCL2 is responsible for PLA oligomer MP–induced macrophage recruitment, RAW264.7 macrophages were co-treated with a chemokine receptor 2 (CCR2) inhibitor during PLA oligomer MPs exposure. Increasing concentrations of the inhibitor caused a dose-dependent reduction in macrophage migration toward PLA oligomer MPs (Fig. 5C and D), confirming that CCL2 mediates oligomer-induced macrophage recruitment.

Fig. 5.

Effects of PLA oligomers and polymers MP exposure on macrophage recruitment and inflammation activation in RAW264.7 macrophages. A Relative mRNA expression of canonical chemokines; B Relative CCL2 levels in the culture medium; C Representative images and D quantification of RAW264.7 macrophage migration in a transwell assay after 24-hour co-treatment with PLA oligomer MPs and the CCR2 inhibitor INCB3284 (abbreviated as IN); E CCL2 concentration in the culture medium after Msr1 and Cd36 knockdown followed by 24-hour exposure to PLA oligomer MPs; F, G Relative mRNA expression of Il-1β and Tnf-α in RAW264.7 macrophages after 24-hour exposure to PLA oligomer MPs; H, I Relative mRNA expression of Il-1β and Tnf-α in RAW264.7 macrophages after Msr1 knockdown followed by 24-hour exposure to PLA oligomer MPs; J, K Relative mRNA expression of Il-1β and Tnf-α in RAW264.7 macrophages after Cd36 knockdown followed by 24-hour exposure to PLA oligomer MPs. n = 3 per group. *P < 0.05. CCL2, chemokine ligand 2; CCR2, chemokine receptor 2; IL-1β, interleukin 1β; PLA MPs, polylactic acid microplastics

To further explore whether the membrane receptors MSR1 and CD36 contribute to this process, we silenced Msr1 or Cd36 using siRNA. Knockdown of Msr1 markedly reduced the PLA oligomer-induced extracellular release of CCL2, whereas Cd36 silencing produced no comparable effects (Fig. 5E). These results demonstrate that MSR1, but not CD36, indeed contributes to the CCL2 production and secretion stimulated by PLA oligomer MPs. Alongside their stronger chemotactic activity, PLA oligomer MPs elicited a more robust inflammatory response than polymer MPs. This was evidenced by significantly higher mRNA levels of Il-1β and Tnf-α in RAW264.7 macrophages exposed to oligomer MPs than to polymer MPs (Fig. 5F and G). Notably, Msr1, but not Cd36, knockdown significantly blunted this cytokine induction (Fig. 5H-K). Together, these results demonstrated that MSR1 serves as a pivotal upstream receptor linking PLA oligomer recognition to CCL2–dependent macrophage recruitment and cytokine activation, forming the molecular basis of the heightened inflammatory potential of oligomeric MPs.

PI3K/AKT signaling mediates MSR1-dependent inflammatory responses to PLA oligomer MPs

We next sought to elucidate how MSR1 transduces PLA oligomer recognition into inflammatory activation. Transcriptomic enrichment analysis of RAW264.7 macrophages revealed that the PI3K/AKT signaling pathway was markedly activated by PLA oligomer MPs (Fig. 3E). Nearly all genes associated with this pathway exhibited a dose-dependent upregulation following oligomer exposure (Fig. 6A), indicating broad activation of PI3K/AKT signaling. Western blot analysis further confirmed a dose-dependent increase in AKT phosphorylation in response to PLA oligomer MPs, whereas polymer MPs elicited only minimal activation (Fig. 6B). To verify the functional relevance of this pathway, we next applied the PI3K/AKT inhibitor LY294002 to RAW264.7 macrophages during PLA oligomer MP exposure. LY294002 effectively suppressed AKT phosphorylation induced by PLA oligomer MPs (Fig. 6C). Notably, blockade of PI3K/AKT signaling significantly reversed the oligomer-induced upregulation of Il-1β and Tnf-α mRNA expression (Fig. 6D and E), as well as the extracellular secretion of CCL2 (Fig. 6F), indicating that PI3K/AKT activation is required for the full induction of inflammatory cytokines and chemokines in response to PLA oligomer MPs. Furthermore, Msr1 knockdown, but not Cd36 knockdown, attenuated AKT phosphorylation in macrophages exposed to PLA oligomer MPs (Fig. 6G and H). Together, these results demonstrated that MSR1 activation triggers PI3K/AKT signaling, thereby amplifying inflammatory cytokine production and chemokine release in response to PLA oligomer MPs.

Fig. 6.

Effects of PI3K/AKT inhibition on PLA oligomer MP–induced inflammatory activation in RAW264.7 macrophages. A Heatmap showing the expression of all PI3K/AKT pathway–related genes in RAW264.7 macrophages after 24-hour exposure to PLA oligomer or polymer MPs; B Representative western blot bands and quantification of phosphorylated AKT in RAW264.7 macrophages; C Representative western blot bands and relative protein levels of phosphorylated AKT; D, E Relative mRNA expression of Il-1β and Tnf-α in RAW264.7 macrophages; F CCL2 concentration in the culture medium; G, H Representative western blot bands and quantification of phosphorylated AKT. n = 3 per group. *P < 0.05. CCL2, chemokine ligand 2; PLA MPs, polylactic acid microplastics; IL-1β, interleukin 1β

Inhibiting the MSR1-PI3K/AKT pathway effectively alleviates PLA oligomer-induced kidney injury

To validate the functional role of the MSR1-PI3K/AKT signaling axis in vivo, we next examined whether pharmacological inhibition of this pathway could mitigate PLA oligomer–induced renal injury in mice. Animals were orally administered PLA oligomer MPs for 28 consecutive days and concomitantly treated with either the MSR1 inhibitor Fucoidan or the PI3K/AKT inhibitor LY294002 (Fig. 7A). Western blot analysis of kidney tissues revealed that Fucoidan, but not LY294002, significantly reduced MSR1 expression (Fig. 7B and C), and both inhibitors markedly suppressed AKT phosphorylation compared with the untreated PLA oligomer group (Fig. 7D and E). Consistently, inhibitor intervention markedly alleviated glomerular injury (Fig. 7F and L and S6 first column), reduced CD68⁺ macrophage infiltration (Fig. 7G and L and S6 second and third column), and diminished the accumulation of PLA oligomer MPs (Fig. 7H-I and L and S6 second and third columns). Inhibitor intervention also attenuated renal IL-1β and CCL2 expression (Fig. 7J-K and L and S6 fourth and fifth column), further confirming the suppression of inflammatory activation in vivo. Collectively, these results demonstrated that inhibition of the MSR1-PI3K/AKT signaling pathway effectively alleviates PLA oligomer–induced macrophage accumulation, inflammatory activation, and renal structural injury in vivo, highlighting this pathway as a potential therapeutic target for mitigating MP-associated kidney damage.

Fig. 7.

Intervention of the MSR1-PI3K/AKT pathway reduces PLA oligomer-induced renal injury. (A) Schematic diagram of Fucoidan and LY294002 intervention experiment design; B, C Representative western blot bands and relative protein levels of MSR1; (D, E) Representative western blot bands and relative protein levels of phosphorylated AKT; F Quantitative results of glomerular injury rates; G Relative quantitative results of CD68 positive cells; H Relative quantitative results of PLA counts; I Relative quantitative results of PLA counts in macrophage; J Relative quantitative results of fluorescence intensity of IL-1β in mouse kidneys; K Relative quantitative results of fluorescence intensity of CCL2 in mouse kidneys; L Representative images of HE staining (black arrows indicate inflammatory infiltration in the kidneys), CD68⁺ immunofluorescence, PLA autofluorescence (orange arrows indicate PLA MPs phagocytosed by macrophages; red arrows indicate undigested PLA MPs), IL-1β and CCL2 expression in mouse kidneys. n ≥ 5 mice per group. *P < 0.05. PLA MPs, polylactic acid microplastics; HE, hematoxylin-eosin; IL-1β, interleukin 1β; CCL2, chemokine ligand 2

Discussion

Biodegradable plastics are often presumed to be intrinsically safer than persistent polymers, yet an overlooked feature is that their chemical structure continues to degrade within biological environments, potentially generating transformation products with biological activities distinct from the parent material [9, 10, 23]. Here, we demonstrate that such in vivo degradation substantially reshapes the biological identity of PLA MPs, resulting in markedly enhanced renal accumulation and inflammation compared with their non-degraded forms. We show that PLA oligomer MPs are preferentially associated with MSR1-dependent macrophage responses and are more efficiently internalized by renal macrophages, triggering PI3K/AKT activation and a CCL2–dependent recruitment cascade that amplifies tissue inflammation (Fig. 8). These findings shift the toxicological evaluation of biodegradable MPs from the parent polymer to the evolving material states produced during degradation and highlight degradation-state profiling as a necessary dimension of biodegradable-material safety assessment.

A central mechanistic implication of our findings is that renal macrophages act not merely as passive phagocytes, but as selective interpreters of PLA degradation state. Renal macrophages serve as frontline sentinels for phagocytizing invading particles, and they can also capture particles that have excreted into urine [24, 25]. While this process is protective under physiological conditions, it can become detrimental under pathological conditions. The stimulation and activation of macrophages form a shared pathogenic pathway across diverse endogenous and exogenous particulates, orchestrating chemokine secretion and driving subsequent tubular injury and fibrosis [26]. In our study, renal macrophages internalized PLA oligomer MPs more efficiently than polymer MPs, leading to greater intracortical particle retention and providing a plausible mechanistic basis for the amplified renal injury of oligomers. This preferential phagocytosis may stem from intrinsic biophysical properties of oligomeric particles: their lower molecular weight increases the proportion of exposed chain ends and reactive moieties that facilitate receptor engagement, while their more flexible and loosely packed architecture may confer conformational adaptability that strengthens interactions with membrane receptors [27, 28]. This pattern parallels findings that oligomeric amyloid-β or α-synuclein more effectively disrupt membranes and activate microglia than fibrillar forms, and that low-molecular-weight hyaluronan fragments serve as danger signals, unlike their high-molecular-weight counterparts [29–31]. These observations support the notion that PLA oligomer MPs are perceived by renal macrophages as a more alarming danger signature than intact polymers, providing a mechanistic rationale for why biodegradation may paradoxically heighten, rather than diminish, the inflammatory and toxic potential of biodegradable MPs.

Our data further indicate that the inflammatory disparity between oligomeric and polymeric PLA MPs is determined not simply by the extent of phagocytosis, but by the signaling logic embedded in particle–receptor interactions. Although both MSR1 and CD36 contributed to the basal uptake of PLA particles, only MSR1 couples PLA oligomer MP recognition to PI3K–AKT activation and downstream CCL2–driven macrophage recruitment, thereby transforming particle uptake into an amplifying inflammatory program. This distinction indicates that the inflammatory potential of biodegradable MPs is governed not simply by the extent of phagocytosis but by the signaling logic encoded in specific receptor–ligand interactions. Scavenger receptors such as MSR1 and CD36 are known to exhibit remarkable context dependence, shifting between silent debris clearance and robust inflammatory activation depending on ligand type, tissue microenvironment, and metabolic state [32–35]. Our findings extend this principle by showing that different polymerization states of the same material can fundamentally alter receptor output, with oligomeric PLA species activating MSR1 while polymeric forms remain largely inert. These insights reposition MSR1 as a central determinant that biases macrophages toward inflammatory interpretation of PLA oligomer MPs.

This work also points toward several directions where deeper exploration could substantially advance the field. First, although the 2.5 mg/kg/day dose was selected to broadly approximate reported estimates of total environmental MP exposure, PLA-specific human exposure remains poorly defined and is likely lower than that of conventional plastics such as polyethylene, polypropylene, and polystyrene [36, 37]. Therefore, direct extrapolation of the present findings to human risk assessment requires caution. Future studies employing chronic low-dose exposure paradigms through realistic intake routes, including respiratory and gastrointestinal exposure, will be important for improving the translational relevance of biodegradable MP toxicology. Additionally, the PLA MPs examined here were chemically uniform and freshly generated, whereas MPs in natural settings undergo aging, oxidation, hydrolysis, and interactions with co-contaminants that may reshape their surface chemistry and receptor engagement [38]. Second, several in vivo analyses, including renal accumulation and renal functional marker assessment, were performed with a limited sample size and should therefore be interpreted as exploratory. Although increased BUN levels and histopathological changes suggest renal alterations after PLA oligomer MP exposure, serum creatinine remained unchanged, indicating that the current evidence supports mild or early-stage renal injury rather than overt renal functional impairment. Future studies with larger animal cohorts and more sensitive renal functional assays are needed to further validate these findings. Third, although our data support MSR1 as a key receptor involved in the inflammatory decoding of oligomeric PLA particles, the present study does not directly demonstrate physical binding between PLA particles and MSR1 or fully establish receptor specificity at the molecular interaction level. The interfacial features that may contribute to this MSR1-associated response remain to be defined, and future receptor–particle interaction assays, receptor-binding analysis, and genetic models will be needed to further validate the specificity and molecular basis of MSR1-dependent recognition. In addition, although our physicochemical characterization helped exclude hydrodynamic size, dispersion stability, zeta potential, and protein corona as dominant explanatory factors, other unexamined features, including surface characteristics and particle structural properties, may still contribute to the differential macrophage responses.

Together, these findings establish the degradation state of PLA MPs as a critical determinant of their renal pathogenicity and define a mechanistic link between biodegradable polymer transformation and immune-mediated kidney injury. Oligomeric PLA particles generated during polymer breakdown preferentially engage MSR1-associated PI3K/AKT inflammatory signaling in renal macrophages, thereby engaging a self-reinforcing inflammatory program that promotes tissue injury. More broadly, our results indicate that the hazard profile of biodegradable MPs cannot be inferred from the parent polymer alone, but must be considered across the evolving particulate states generated during degradation. These findings highlight degradation-state profiling as an important dimension of biodegradable-material safety evaluation and provide a framework for understanding how material transformation reshapes biointerfacial interactions and organ-specific toxicity.

Conclusions

This study shows that the degradation state of PLA MPs is an important determinant of their renal toxicity. Compared with polymeric particles, oligomeric PLA particles exhibited greater renal accumulation and induced stronger renal inflammatory responses and structural injury, suggesting that polymer degradation generates particulate forms with distinct biological and toxicological properties. We further identified renal macrophages as key mediators of this difference and found that oligomeric particles preferentially activated an MSR1-PI3K/AKT–CCL2 signaling axis, leading to a self-amplifying inflammatory response. Inhibition of MSR1 or PI3K/AKT reduced macrophage uptake and inflammatory activation, thereby attenuating the toxicological difference between oligomeric and polymeric PLA MPs. Overall, these findings suggest that the safety of biodegradable MPs cannot be assessed solely on the basis of the parent polymer, but should also take into account the degradation-derived particulate states formed during breakdown. This study provides mechanistic insight into how biodegradable polymer degradation influences organ-specific toxicity.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

BUN

Blood urea nitrogen

CCL2

Chemokine ligand 2

CCL5

Chemokine ligand 5

CCR2

Chemokine receptor 2

CRE

Creatinine

DLS

Dynamic light scattering

DMEM

Dulbecco’s modified Eagle’s medium

ELISA

Enzyme-linked immunosorbent assay

FBS

Fetal bovine serum

HE

Hematoxylin-eosin

KEGG

Kyoto Encyclopedia of Genes and Genomes

MPs

Microplastics

MSR1

Macrophage scavenger receptor 1

PCA

Principal component analysis

PBS

Phosphate-buffered saline

PI3K/AKT

Phosphoinositide 3-kinase/protein kinase B

PLA

Polylactic acid

PVDF

Polyvinylidene fluoride

qPCR

Quantitative polymerase chain reaction

RNA-seq

RNA sequencing

SD

Standard deviation

SDS-PAGE

SDS-polyacrylamide gel electrophoresis

SEM

Scanning electron microscope

TBST

Tris-buffered saline with 0.1% Tween-20

Author contributions

X.C. and Y.D. contributed equally to this work as co-first authors. X.C. and Y.D. conceived the study, designed the experiments, and performed the conceptualization, methodology, data curation, formal analysis, visualization, and original draft writing. X.H., X.Y., Z.L., Y.H. and Y.Z. carried out the in vivo experiments and data analysis. H.L., L.M., and S.T. and H.X. performed the characterization experiments and analyzed the data. B.L. administered the project, and wrote and revised the manuscript. Z.H. provided resources, secured funding, administered the project, and revised the manuscript. All authors read and approved the final version of the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (Grant No. 82473661, 82273656, 82304177, 82504441), the Guangdong Basic Applied Basic Research Foundation (Grant No. 2025A1515011141), the Guangzhou Key Research and Development Program (Grant No. 2025B03J0133), Postdoctoral Researcher Funding Program of China (Grant No. GZC20250510), the Guangdong Provincial Key Laboratory of Tropical Disease Research (Grant No. 2017B030314035), as well as the Shenzhen Medical Research Fund (Grant No. A2502040).

Data availability

The RNA-seq datasets generated during the current study have been deposited in the National Genomics Data Center, China National Center for Bioinformation, under accession number PRJCA064731. Other data supporting the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All animals were obtained from the Guangdong Medical Laboratory Animal Center and were approved by the Ethics Committee for the Care and Use of Laboratory Animals of Southern Medical University (Permit No. SMUL202308006). Experiments followed the guidelines of the Chinese National Institutes of Health for laboratory animal care and use.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.