Therapeutic potential of naturally derived carbon dots in sepsis-associated acute kidney injury

Abstract

Background: Sepsis is a life-threatening infectious disease characterized by an uncontrolled inflammatory response and consequent multi-organ dysfunction. The kidneys, as primary excretory organs with high blood flow, are particularly susceptible to damage during sepsis. Nonetheless, the existing treatment options for sepsis-associated acute kidney injury (SA-AKI) are still restricted. Nanomedicine, especially carbon dots (CDs), has attracted considerable interest lately for outstanding biomedical characteristics.

Methods: To avoid the generation of toxic effects, the natural CDs derived from Ziziphi Spinosae Semen (Z-CDs) were synthesized employing a hydrothermal method. The free radical scavenging capabilities of Z-CDs were evaluated by utilizing ABTS assay, NBT method, and Fenton reaction. A lipopolysaccharide (LPS)-stimulated RAW 264.7 cell model was used to explore the therapeutic potential of Z-CDs on cellular oxidative stress and inflammation. The CuSO₄-induced zebrafish inflammation model and LPS-exposed SA-AKI mouse model were employed to assess the therapeutic efficacy of Z-CDs in vivo.

Results: The synthesized Z-CDs exhibited distinctive unsaturated surface functional groups, which confer exceptional biocompatibility and the ability to scavenge free radicals. Moreover, Z-CDs were particularly effective in eliminating excess reactive oxygen species (ROS) in cells, thus protecting mitochondrial function from oxidative damage. Notably, Z-CDs have demonstrated significant therapeutic benefits in protecting kidney tissue in SA-AKI mouse model with minimizing side effects. In mechanism, Z-CDs effectively reduced ROS production, thereby alleviating inflammatory responses in macrophages through the suppression of the NF-κB pathway.

Conclusions: This study developed a multifunctional nanomedicine derived from traditional medicinal herb, providing a promising pathway for the advancement of innovative drug therapies to treat SA-AKI.

Keywords: Acute kidney injury; Antioxidant; Carbon dots; NF-κB pathway; Phytochemicals; Sepsis.

Scheme 1

Schematic illustration demonstrates the function of Z-CDs for high ROS scavenging activity in the treatment of SA-AKI

Fig. 1

Characterization of Z-CDs. A TEM image of Z-CDs. Scale bar: 50 nm. B Size distribution of Z-CDs. C The high-resolution TEM image of Z-CDs. D AFM image of Z-CDs. Scale bar: 600 nm. E XRD pattern of Z-CDs. F Ultraviolet absorption spectra (black line), fluorescence excitation (red line), and emission (blue line) spectra of Z-CDs. Inset: Images of Z-CDs taken under daylight (left) and 365 nm UV lamp (right). G FTIR spectra of Z-CDs. H Full scan XPS spectrum of Z-CDs. I–K High-resolution C 1 s, O 1 s, and N 1 s XPS spectra of Z-CDs

Fig. 2

Z-CDs exert excellent ROS scavenging activities of in vitro. A The total antioxidant capacity of Z-CDs was measured by using the ABTS method. B The UV–vis absorption spectra of ABTS^•+ solutions after mixing with Z-CDs at different concentrations. C The elimination O2^•− capacity of Z-CDs of different concentrations. D The •OH scavenging capacity of Z-CDs was detected based on the Fenton reaction. E UV–vis absorption spectra of TMB solutions after mixing with Z-CDs. F Schematic diagram of the free radicals scavenging activity of the Z-CDs. Data are expressed as mean ± SD, n = 3

Fig. 3

Z-CDs alleviate cellular oxidative damage by eliminating mitochondrial ROS. A, B Detection of cellular ROS in RAW 264.7 cells with or without Z-CDs for 24 h using DCFH-DA probe. Scale bar: 20 μm. C, D The mitochondrial superoxide in RAW 264.7 cells were measured by using MitoSOX red probe. Scale bar: 20 μm. E, F Assessment of cellular MDA and SOD levels. Data are expressed as mean ± SD, n = 3. ^###P < 0.001, vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001, vs LPS group

Fig. 4

Z-CDs exert anti-inflammatory effects by inhibiting the NF-κB pathway. A Quantification of the NO production in RAW 264.7 cells after being administered with Z-CDs. B Representative images of COX- 2, iNOS, and NLRP3 protein levels in RAW 264.7 cells. C–E Quantitative analysis of the indicated protein levels in (B). F–H The expression of IL- 6, IL- 1β, and TNF-α were measured by qRT-PCR. I–K Representative profiles and quantitative analysis of phosphorylated IκBα (Ser32) and phosphorylated P65 (Ser536) with or without Z-CDs treatment. Data are expressed as mean ± SD, n = 3. ^###P < 0.001, vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001, vs. LPS group

Fig. 5

Z-CDs exert as a therapeutic agent against SA-AKI in vivo by eliminating ROS and improving inflammatory response. A Phenotypes and quantitative analysis of neutrophil distribution and tail neutrophil spread to lateral line in zebrafish. The yellow box indicates the neutrophil spreading area (neutrophils of 72 hpf transgenic zebrafish exhibiting red fluorescence). B Quantification of the number of neutrophils from (A). Each dot represents one zebrafish, n = 5. C Schematic diagram of the animal experimental program. D–E Quantitative determination of serum levels of CREA and BUN, indicators of kidney impairment in mice. Each dot represents one mouse, n = 5. F–G Expression of mRNA levels of IL- 6 and IL- 1β, inflammation-related indicators, in mouse kidney tissues by qPCR. n = 5. H Representative images of the histologic morphology of kidney tissue assessed by H&E staining. n = 5. Scale bar: 100 μm. Data are expressed as mean ± SD. ^###P < 0.001, vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001, vs. LPS group

Fig. 6

Biosafety valuation of Z-CDs. A–C CCK- 8 analysis was performed with various concentrations of Z-CDs in RAW264.7, HUVEC, and HK2 cells. n = 3. D Quantitative analysis of body weight. n = 6. E–H Analysis of peripheral blood in indicated mice. n = 6. I–L Serum levels of ALT, AST, CERA, BUN in indicated mice. n = 5. M Representative images of the histologic morphology of different tissues (heart, liver, spleen, lungs, and kidneys) were evaluated by H&E staining. n = 5. Scale bar: 100 μm. Data are expressed as mean ± SD