Carbon dots from purple sweet potato as a promising anti-inflammatory biomaterial for alleviating the LPS-induced inflammation in macrophages

Abstract

This study synthesizes carbon dots derived from crude extracts of purple sweet potato (CPP-CDs) and evaluates its anti-inflammatory effects in a lipopolysaccharide (LPS) -induced acute inflammation model. Characterization revealed that CPP-CDs possess a uniform spherical structure and excellent photoluminescent properties. In vitro, CPP-CDs significantly inhibited the expression of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), reduced the accumulation of reactive oxygen species (ROS), suppressed pyroptosis, and facilitated the polarization of macrophages from the M1 phenotype to the M2 phenotype. In vivo, CPP-CDs significantly improved the survival rates of LPS-treated mice, mitigated tissue damage, and suppressed the levels of pro-inflammatory cytokines. Mechanistic studies indicated that CPP-CDs exert anti-inflammatory effects through the inhibition of the TLR4/NF-κB signaling pathway and the modulation of the NLRP3 inflammasome. Additionally, CPP-CDs exhibited excellent biocompatibility, with no significant toxicity observed in mice. This study provides strong evidence supporting the application of CPP-CDs as a novel anti-inflammatory material, highlighting their potential for acute inflammation treatment and expanding the possibilities for the development of carbon-dot-based anti-inflammatory therapies.

Keywords: Anti-inflammatory material; Carbon dots; NLRP3 inflammasome; Purple sweet potato; Reactive oxygen species; TLR4/NF-κB signaling pathway.

Scheme 1

Synthesis of CPP-CDs and their application in acute inflammation treatment. a. Purple sweet potato tubers were utilized to synthesize CPP-CDs via a one-step hydrothermal method. b. Mouse bone marrow-derived macrophages were isolated and cultured to establish an LPS-induced inflammation model; CPP-CDs effectively ameliorated the inflammatory response through multiple anti-inflammatory pathways. c. Following intraperitoneal administration, CPP-CDs effectively alleviated systemic inflammation, reduced tissue damage, and improved survival in mice

Fig. 1

Effects of three carbon dots on inhibiting LPS-induced inflammation. a-c. Left: Images of the raw material extracts for the three types of carbon dots. Right: Images of the three types of carbon dots under UV light. CDs: CPP-CDs, CDs1:CPSPSL-CDs, CDs2: SPR-CDs. d-f. Relative gene expression levels of IL-1β, IL-6, and TNF-α measured by RT-qPCR. BMDMs were treated with LPS (1 µg/mL, 4 h), low-concentration CPP-CDs (L, 0.5 mg/mL, 8 h), and high-concentration CPP-CDs (H, 1 mg/mL, 8 h) (n = 3 biological replicates). g. Relative gene expression levels of IL-1β, IL-6, and TNF-α measured by RT-qPCR. BMDMs were treated with LPS (1 µg/mL, 4 h), PSPCE and CPP-CDs (both 1 mg/mL, 8 h) (n = 3 biological replicates). d-g. Data were analyzed by One-way ANOVA for multiple group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

Synthesis and characterization of CPP-CDs. a. TEM images of CPP-CDs showing an overall size of 50 nm and individual particles measuring 5 nm. b. Size distribution analysis of CPP-CDs. c. Fluorescence excitation and emission spectra of CPP-CDs. d. FTIR spectra of CPP-CDs. e. XRD patterns of CPP-CDs. f. Full survey XPS spectra of CPP-CDs. g. High-resolution XPS spectrum of C 1s for CPP-CDs. h. High-resolution XPS spectrum of O 1s for CPP-CDs

Fig. 3

Effects of CPP-CDs on LPS-induced inflammation. a. ELISA measurements of IL-1β, IL-6, and TNF-α protein levels in BMDMs culture supernatants. Experimental groups were treated with LPS (1 µg/mL, 4 h), Nigericin (Nig, 5µM, 45 min), and various concentrations of CPP-CDs (n = 4 biological replicates). b-c. ATP levels were measured using an ATP assay kit to assess intracellular and extracellular ATP levels in BMDMs (n = 4 biological replicates). d. ROS levels in BMDMs were measured using a ROS detection probe (n = 3 biological replicates). e. Morphological changes in BMDMs were observed under an optical microscope for each treatment group. Red arrows indicate pyroptotic cells (n = 3 biological replicates). Scale bar: 20 μm. f. LDH release levels in BMDMs for each treatment group, represented as the percentage of LDH activity in the cell lysate (n = 4 biological replicates). g. The relative expression of M2 macrophage markers ARG1 and CD206 was measured by RT-qPCR in BMDMs treated with various concentrations of CPP-CDs (n = 3 biological replicates). a-g. Data are expressed as mean ± standard error of the mean (SEM). One-way or Two-way ANOVA was used for multiple group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

In vivo anti-inflammatory effects of CPP-CDs in an LPS-induced mouse model. a. Mouse model construction process: Mice were divided into groups and given intraperitoneal injections of low (L: 15 mg/kg) or high (H: 30 mg/kg) concentrations of CPP-CDs, followed by LPS injection (5 mg/kg) 2 h later. b. Survival rate of mice monitored for 72 h after LPS and CPP-CDs treatment (n = 6, two independent experiments). c. Colon and liver tissues were collected 24 h after LPS and CPP-CDs treatment and stained with H&E to observe tissue damage (magnification: 15×). d. Pathological scoring of colonic lesions and liver inflammation. Colon score was based on weight loss, rectal bleeding, and stool consistency; liver score was based on the number and area of inflammatory foci around the central vein (n = 6, two independent experiments). e. ELISA measurements of IL-1β, IL-6, and TNF-α levels in mouse serum (n = 4 biological replicates). b, d-e. All data are expressed as mean ± standard error of the mean (SEM). One-way or Two-way ANOVA was used for multiple group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

CPP-CDs modulate inflammatory signaling pathways. a. Western blot analysis of TLR4, p-P65, and P65 protein expression levels. BMDMs were treated with LPS (1 µg/mL, 4 h), low-concentration CPP-CDs (L, 0.5 mg/mL, 8 h), and high-concentration CPP-CDs (H, 1 mg/mL, 8 h). b. Immunofluorescence staining showing nuclear translocation of p-P65 in BMDMs (green) and DAPI-stained nuclei (blue) (n = 3 biological replicates). c. Western blot analysis of NLRP3, Cleaved-GSDMD, Pro-caspase-1, Cleaved-caspase-1, Pro-IL-1β, and Cleaved-IL-1β protein expression levels in BMDMs was performed using the same treatment as described in Fig. 5a. d. Immunofluorescence staining showing ASC speck formation and DAPI-stained nuclei (blue). BMDMs were treated with LPS (1 µg/mL, 4 h), Nig (5 µM, 45 min), and various concentrations of CPP-CDs (n = 3 biological replicates). e. Calcium ion influx in BMDMs measured using fluo-4 after LPS treatment for 10 min, with or without CPP-CDs pre-treatment for 6 h (n = 3 biological replicates). f, g. f (Upper panel): Net calcium flux measurements for different treatment groups using non-invasive microtest technology (NMT) in BMDMs, with each group tested for 5 min. g (Lower panel): Data are displayed as violin plots (n = 6 biological replicates). h. Western blot analysis of NLRP3, Cleaved-GSDMD, Pro-caspase-1, Cleaved-caspase-1, Pro-IL-1β, and Cleaved-IL-1β protein expression in BMDMs treated with LPS (1 µg/mL, 4 h), CPP-CDs (1 mg/mL, 8 h), and Ionomycin (5 µM, 1 h). i. ELISA measurements of IL-1β levels in BMDMs culture supernatants (n = 4 biological replicates). g, h, i. Data are expressed as mean ± standard error of the mean (SEM). One-way ANOVA was used for multiple group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Regulation of P2X  7R/NLRP3 interaction by CPP-CDs. a. Western blot analysis of P2 X 7R protein expression in different treatment groups. BMDMs were treated with LPS (1 µg/mL, 4 h), ATP (5 mM, 30 min), low -concentration CPP-CDs (L: 0.5 mg/mL), and high-concentration CPP-CDs (H: 1 mg/mL) (n = 3 biological replicates). b. Left: YO-Pro-1 staining showing changes in BMDMs membrane permeability. Green represents YO-Pro-1 staining, and blue represents DAPI-stained nuclei. Scale bar: 50 μm. Right: Relative fluorescence intensity of YO-Pro-1 and DAPI (n = 3 biological replicates). c. Western blot analysis of NLRP3, Cleaved-GSDMD, Pro-caspase-1, Cleaved-caspase-1, Pro-IL-1β, and Cleaved-IL-1β protein expression levels in BMDMs treated as indicated in Fig. 6a (n = 3 biological replicates). d. Interaction network prediction of P2 X 7R and NLRP3 proteins using the STRING database, showing potential direct binding relationships. e. Immunoprecipitation assays detecting the interaction between P2 X 7R and NLRP3 in different treatment groups. BMDMs were treated with LPS (1 µg/mL, 6 h), ATP (5 mM, 30 min), and different concentrations of CPP-CDs (L and H) (n = 3 biological replicates). f. Western blot analysis of NLRP3 protein stability after treatment with Cycloheximide in BMDMs (Chx, 20 µg/mL, 8 h) (n = 3 biological replicates). g. Western blot analysis of NLRP3 ubiquitination levels after immunoprecipitation of NLRP3. Treatment conditions were LPS (1 µg/mL, 6 h), ATP (5 mM, 30 min), and different concentrations of CPP-CDs, with MG132 (10 µM, 6 h) used to block protein degradation pathways in BMDMs (n = 3 biological replicates)

Fig. 7

Safety Assessment of CPP-CDs. a. Assessment of the impact of CPP-CDs administration on mouse body weight throughout the treatment period (n = 5, two independent experiments). b. Cytotoxicity evaluation of CPP-CDs on BMDMs using the CCK-8 assay. Cell viability was quantified at 0, 24, 48, and 72 h under different concentrations of CPP-CDs (0, 0.1, 0.5, and 1.0 mg/mL) based on OD450 values (n = 3, biological replicates). c. Morphological examination of the colon, liver, spleen, lung, and kidney tissues of mice from each group through H&E staining (magnification: 15×). d-h. Measurement of serum biomarkers, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (UA), urea (UREA), and creatinine (CREA). This analysis assessed the effects of CPP-CDs on key physiological indicators (n = 4, biological replicates). a-b, d-h. Data are expressed as mean ± standard error of the mean (SEM). One-way or Two-way ANOVA was used for multiple group comparisons. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8

Schematic representation of the mechanism through which CPP-CDs regulate LPS-induced inflammatory responses in macrophages