Carbon Dots Extracted from the Plant Gardenia jasminoides Ameliorates Ischemia-Reperfusion Injury

Abstract

Background: Ischemic stroke (IS) is probably the most important acute serious illness, where interdisciplinary approach is essential to offer the best chance for survival and functional recovery of patients. Carbon dots (CDs) with multifaceted advantages have provided hope for development brand-new nanodrug for treating thorny diseases. Methods: This study developed a green and environmentally responsible calcination method to prepare novel Gardenia jasminoides Carbonisata (GJC-CDs) as promising drug for ischemic stroke treatment. Results: In this work, we isolated and characterized for the first time a novel carbon dots (GJC-CDs) from the natural plant G. jasminoides. Results displayed that green GJC-based CDs with tiny sizes and abundant functional groups exhibited solubility, which may be beneficial for its settled biological activity. The neuroprotective effect of carbon dots from G. jasminoides were evaluated using the classical middle cerebral artery occlusion (MCAO) model. Assessing the infarct volume content of the ischemic cerebral hemisphere and determining the serum tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), reduced glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) levels of the mice in each group, it was evident that pre-administration of the drug by GJC-CDs significantly reduced the infarct volume as well as attenuated inflammatory responses and excessive oxidative stress in MCAO mice. Furthermore, in vitro cellular experiments demonstrated that GJC-CDs have good biosafety and anti-inflammatory and antioxidant capacity. Conclusions: Overall, GJC-CDs performs neuroprotective effect on cerebral ischemia and reperfusion injury, which not only provides evidence for further broadening the biological application of acute ischemic stroke but also offers novel strategy for the application of nanomedicine to treat acute diseases.

Keywords: Gardenia jasminoides; carbon dots; inflammation; ischemic stroke; oxidative stress.

Figure 1

Morphological and optical characterizations of GJC-CDs. (A) Transmission electron microscopy (TEM) images (30 k×, 20nm, arrows represent GJC-CDs observed under electron microscopy). (B) High-resolution transmission electron microscopy (HRTEM) images (500 k×, 2nm, arrows represent GJC-CDs observed under electron microscopy). (C) Particle size distribution histogram. (D) X-ray diffraction pattern. (E) Ultraviolet–visible spectrum. (F) Fourier transform infrared spectrum. (G) Fluorescence spectra for excitation and emission. (H) Fluorescence spectra of GJC-CDs with different excitation wavelengths.

Figure 2

The surface composition and elemental analysis of the prepared GJC-CDs by XPS. (A) X-ray photoelectron spectroscopy survey of GJC-CDs. (B) C1s. (C) O1s. (D) N1s.

Figure 3

Effect of GJC-CDs on cerebral infarct volume and neurologic function in MCAO mice. (A) TTC staining. (B) Percentage of infarct volume (n = 6). (C) Zea-Longa scores (n = 6, each dot represents the neurological functioning score of an MCAO mouse). Data were represented as means ± SD. ^#### p < 0.001 compared with the Sham group, * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the Model group.

Figure 4

Effect of GJC-CDs on cerebral blood circulation in MCAO mice. (A) Sham group; (B) Model group; (C) GJ group; (D) Low-dose GJC-CDs group; (E) Medium-dose GJC-CDs group; (F) High-dose GJC-CDs group; (G) The ratio of CBF in each group of mice (n = 6). Data were represented as means ± SD. ^### p < 0.001 compared with the Sham group, ** p < 0.01 and *** p < 0.001 compared with the Model group.

Figure 5

Effect of GJC-CDs on intracerebral pathological damage in MCAO mice. (A) Hematoxylin-eosin staining (n = 4). (B) Nissl staining (n = 4).

Figure 6

Effect of GJC-CDs on inflammation levels in MCAO mice. (A) TNF-α levels in serum (n = 6). (B) IL-1β levels in serum (n = 6). (C) IL-6 levels in serum (n = 6). (D) IL-10 levels in serum (n = 6). Data were represented as means ± SD. ^#### p < 0.0001 compared with the Sham group, * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the Model group.

Figure 7

Effect of GJC-CDs on oxidative stress levels in MCAO mice. (A) GSH levels in serum (n = 6). (B) MDA levels in serum (n = 6). (C) SOD levels in serum (n = 6). Data were represented as means ± SD. ^### p < 0.001, ^#### p < 0.0001 compared with the Sham group, * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the Model group.

Figure 8

Effect of different concentrations of LPS in BV2 microglia cells. (A,B) CCK-8 (n = 6). (C) NO–cell lysis assay (n = 6). (D) NO–cell supernatant assay (n = 6). (E,F) Morphology of BV2 cells stimulated at different times at 2 μg/mL LPS (200×, arrows show morphological changes in BV2 microglia cells in response to LPS stimulation). Data were represented as means ± SD. ^#### p < 0.0001 compared with the Blank group, **** p < 0.0001 compared with the Blank group.

Figure 9

Effect of GJC-CDs on LPS-induced inflammatory mediators and oxidative stress in BV2 microglia cells. (A,B) NO levels in supernatant (n = 6). (C) TNF-α levels in supernatant (n = 6). (D) IL-1β levels in supernatant (n = 6). (E) ROS fluorescence intensity. (F) SOD levels in supernatant (n = 6). Data were represented as means ± SD. ^#### p < 0.0001 compared with the Blank group; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with the 2 μg/mL LPS group.