Carbon dots induce pathological damage to the intestine via causing intestinal flora dysbiosis and intestinal inflammation

Abstract

Background: Carbon dots (CDs), as excellent antibacterial nanomaterials, have gained great attention in treating infection-induced diseases such as periodontitis and stomatitis. Given the eventual exposure of CDs to the intestine, elucidating the effect of CDs on intestinal health is required for the safety evaluation of CDs.

Results: Herein, CDs extracted from ε-poly-L-lysine (PL) were chosen to explore the modulation effect of CDs on probiotic behavior in vitro and intestinal remodeling in vivo. Results verify that PL-CDs negatively regulate Lactobacillus rhamnosus (L. rhamnosus) growth via increasing reactive oxygen species (ROS) production and reducing the antioxidant activity, which subsequently destroys membrane permeability and integrity. PL-CDs are also inclined to inhibit cell viability and accelerate cell apoptosis. In vivo, the gavage of PL-CDs is verified to induce inflammatory infiltration and barrier damage in mice. Moreover, PL-CDs are found to increase the Firmicutes to Bacteroidota (F/B) ratio and the relative abundance of Lachnospiraceae while decreasing that of Muribaculaceae.

Conclusion: Overall, these evidences indicate that PL-CDs may inevitably result in intestinal flora dysbiosis via inhibiting probiotic growth and simultaneously activating intestinal inflammation, thus causing pathological damage to the intestine, which provides an effective and insightful reference for the potential risk of CDs from the perspective of intestinal remodeling.

Keywords: Carbon dots; Inflammation; Intestinal flora; Probiotics; Reactive oxygen species.

Fig. 1

Successful preparation and characterization of PL-CDs. a TEM image of PL-CDs. Insets are the particle size distribution and high-resolution TEM (HRTEM) image of PL-CDs. b Zeta potential of PL and PL-CDs. c FT-IR spectra of PL and PL-CDs. d UV–vis spectra of PL-CDs

Fig. 2

The antibacterial effect of PL-CDs incubated with L. rhamnosus for 24 and 48 h. a, b Photographs of L. rhamnosus incubated with PL-CDs at different concentrations and the quantified bacterial inhibition rate. c, d Growth curves of L. rhamnosus and pH changes of solution induced by PL-CDs at different concentrations during 24 h. ^ap < 0.05 (400 μg/mL vs. Control); ^bp < 0.05 (200 μg/mL vs. Control); ^cp < 0.05 (100 μg/mL vs. Control); ^dp < 0.05 (50 μg/mL vs. Control)

Fig. 3

The antibacterial effect of PL-CDs incubated with L. rhamnosus for 24 h in the presence or absence of light. a Photographs of L. rhamnosus incubated PL-CDs at different concentrations for 24 h under dark or light conditions. b The quantified bacterial count from Image J

Fig. 4

Effects of PL-CDs on morphology and internal structure of L. rhamnosus. a, b SEM images of L. rhamnosus incubated with PL-CDs at different concentrations for 24 h, and the quantified probiotic elongation (n = 50). c TEM images of L. rhamnosus incubated with PL-CDs (400 μg/mL) for 24 h. Blue and purple arrows represent the cell wall and cell membrane of L. rhamnosus, respectively; Green wireframe represents the cytoplasmic distribution

Fig. 5

The antibacterial mechanism of PL-CDs against L. rhamnosus. a, b ROS generation induced by PL-CDs under light or dark conditions. c The absorbance of DPBF at 410 nm. d, e Intracellular ROS production of L. rhamnosus induced by PL-CDs under light or dark conditions. f–h The enzyme activities of CAT, GSH-PX, and MDA affected by PL-CDs. i Detection of membrane permeability of L. rhamnosus. j, k DNA content detected by NanoDrop and electrophoresis

Fig. 6

Toxic effects of PL-CDs on cells. a, b Live-dead staining of Caco-2 and L929 incubated with PL-CDs for 24 h. c, d The quantified percentage of living cells from image J. e–h Effect of PL-CDs on cell apoptosis of Caco-2 and L929 for 24 h, and the quantified Annexin-FITC/PI percentage

Fig. 7

Intestinal pathological damage and inflammation caused by PL-CDs. a Monitoring of mouse weight affected by PL-CDs. b, c Images of the colon and the quantified colon length. d–f Images of H&E, AB-PAS, and PAS staining of colon tissue. g–i mRNA and protein expressions of tight junction proteins (ZO-1 and occludin) by qRT-PCR and Western blot. j–q The mRNA and protein expressions of pro-inflammatory (IL-1β and TNF-α) and anti-inflammatory factors (IL-10 and IFN-γ) by qRT-PCR and ELISA

Fig. 8

Intestinal flora imbalance induced by PL-CDs. a, b Multi samples rarefaction curves and Shannon curves. c, d Relative abundance of intestinal flora at the phylum level and at the family level, n = 4

Fig. 9

Model of PL-CDs induced intestinal toxicity