Materials
Dialysis membranes (molecular weight cut off was 1000 Da) were purchased from Beijing Ruida Henghui Technology Development Co., Ltd (Beijing, China).The cell counting kit (CCK)-8 was brought from Dojindo Molecular Technologies, Inc.(Kumamoto,Japan).The Dulbecco’s Modified Eagle Medium (DMEM) and the fetal bovine serum (FBS) were purchased from mediatech,Inc. (Conrning, USA). Absolute ethanol (EtOH) solution was purchased from Sigma-Aldrich Co. (St. Louis, MO,USA). Superoxide dismutase(SOD), catalase(CAT), glutathione(GSH) and glutathione peroxidase(GSH-Px) kits were purchased from Jiancheng Bioengineering Institute (Nanjing, China). The ELISA kits to measure tumor necrosis factor-α(TNF-α) were purchased from Proteintech Group, Inc(USA), and the ELISA kits to measure to interleukin 6 (IL-6) were purchased from Abcam(Amyjet scientific inc, UK). Deionized water was used as a solvent for all experiments.
Animals
A total of 50 male Sprague-Dawley (SD) rats (180-200 g) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Animals were acquired from the Laboratory Animal Centre, Beijing University of Chinese Medicine, and were bred in an environmentally controlled breeding room (temperature: 20-25ºC,) for 1 week prior to the start of the experiments and provided with standard laboratory food and water. Experimental protocols used in the present study were approved by the Animal Experimental Ethics Committee of Beijing University of Chinese Medicine (Beijing, China). All efforts were made to minimize animal suffering and to reduce the number of animals used in the experiments.
Preparation of carbon dots
CDs were prepared from NRN by using the modified pyrolysis method[38]. First, the NRN (80.0g) was placed in crucible, which was transferred into a muffle furnace (TL0612, Beijing Zhong Ke Aobo Technology Co., Ltd, China) and carbonized at 350℃ for 1h,yielding NRNC. After allowing the temperature to drop naturally to 30℃, the NRNC was grinded to powder and boiled twice in distilled water at 100℃ for 1h each time. The solution was cooled to room temperature and filtered with a 0.22μm filter membrane to remove any large particles, and that solutions was purified with a 1000 Da dialysis bag in deionized water for 7 day to remove the small molecules. Finally, the solution inside the dialysis membrane was collected for further characterization and usage.
Characterization of NRNC-CDs
The surface morphology of NRNC-CDs were obtained by transmission electron microscopy (TEM, Tecnai G2 F20 electron microscope). Structural details were obtained using a JEM-1230 high-resolution transmission electron microscopy (HRTEM; Japan Electron Optics Laboratory, Tokyo, Japan). The UV–vis absorption was determined using were recorded by spectroscopy (CECIL, Cambridge, UK) in the absorption wavelength 200-600 nm at 25℃. The fluorescence spectra (F-4500; Tokyo, Japan) were monitored with a molecular fluorescence spectrometer in a standard quartz cuvette. The functional groups of NRNC-CDs were recorded by performing Fourier transform infrared spectroscopy (FTIR) spectroscopy (Thermo Fisher Scientific, CA, USA ). X-ray photoelectron spectroscopy (XPS) of the sample was recorded using an ESCALAB 250Xi XPS (Thermo Fisher Scientific) using a mono x-ray source Al Kalpha 150 W. The X-ray diffraction (XRD) patterns were characterized on an x-ray diffractometer (Bruker AXS, Karlsruhe, Germany) using Cu Kα radiation (λ=1.5418 Å).
Quantum yield measurement
Quantum yield (QY) was measured with quinine sulfate in 0.1 M H2SO4 (quantum yield 54%) as a standard sample. The QY of NRNC-CDs was estimated according to the follow equation:
Where the ‘Φ’ is the fluorescence quantum yield, ‘I’ represents the integrated fluorescent intensity and ‘A’ stands for the absorbance. The ‘η’ is referred as the refractive index of the solvent, and the subscript ‘r’ refer to quinine sulfate.
Cell viability assay
In vitro cytotoxicity of NRNC-CDs for the human gastric epithelial cells line (GES-1 cells) was assessed by the CCK-8 assay. The GES-1 cells cultured in supplemented with 15% FBS and 1% penicillin-streptomycin (Mediatech, Manassas, VA, US ) incubated at 37℃ under humidified 5% CO2. Then, the culture medium was discarded and the GES-1 cells were incubated in the fresh culture medium containing NRNC-CDs with different doses of the NRNC-CDs solutions (1000, 500, 250, 125, 62.5, 31.25μg/mL) for another 24 h, and DMEM was selected as the positive. Next, the cells attached to each plate were treated with 10μL CCK-8 solution for 4 h incubation. Finally, the microplate reader (Biotek, VT, USA) was use to read absorbance at 450 nm.The cell viability was calculated according to the following equation:
where ‘ACDs’ and ‘Acontrol ’ are the absorbance of cells incubated in the presence and absence of NRNC-CDs, respectively.
Induction of acute gastric lesion by ethanol
All animals were randomly divided into six groups of ten rats each for intragastric administration. The groups were divided into: control group, gastric ulcer groups (model group), ranitidine control(50.00mg/kg), and the NRNC-CDs treatment groups(5, 2.5 and 1.25 mg/kg=high-dose,medium-dose and low-dose, respectively). All groups received prophylactic administration for 7 consecutive days. The rats were fasted within the 24 h ahead of the last administration. 2h after the final administration, acute gastric ulcer model was made by absolute ethanol (5.00mL/kg)[39]. Another one hour after ethanol instillation, the animals were sacrificed under anesthesia and stomachs were taken for further analysis. The stomachs of the anesthetized rats in each group were opened along a greater curvature and rinsed with normal saline, and then subjected to a general examination to assess any abnormal lesions.The superficial ulcer areaswere measured by image J analyzer software. The gastric ulcer index and the rate of protection of ulceration were calculated the following formula:
Measurement of related biochemical indexes in gastric tissues
Tissue samples of the stomachs were weighed, minced, and homogenized with cold PBS buffer (pH=7.4, v/v=1:9), a portion of each stomach tissue (0.5g) was cut into small pieces and 4.5 mL of cold PBS were added. Additionally, The mixture was homogenized on ice with a handheld homogenizer (S10, SCIENTZ, Ningbo). Some tissue homogenates were centrifuged for 2500rpm at 4 °C for 10 min, and then the supernatants were used to determine the levels of SOD, CAT, GSH and GSH-Px, respectively. Moreover, the other portion was centrifuged for 10 min at 5000 rpm to determine the level of TNF-α and IL-6 were analyzed by enzyme immunoassays.
Gastric histopathological assessments
The ulcerated stomachs were washed twice in ice-cold saline, and approximated regions of individual stomach (between cardiac and pylorus, the fundus) were sampled and cross-trimmed based on the lumen. Furthermore, all trimmed fundi were fixed in 10% (V/V) neutral buffered formalin for 24h, and paraffin sections were then cut to a thickness of 5 μm and stained with hematoxylin and eosin (H&E) for histological evaluation according to standard procedures.
Statistical analysis
All data was analyzed by the statistical package of social sciences (SPSS, version 20.0). The data with normally distribution and homogeneous variances were expressed as the mean ± standard deviation (SD). Data were analyzed using one-way analysis of variance. Differences were considered significant at values of P < 0.05.
Ethical approval
Experimental protocols used in the present study were approved by the Animal Experimental Ethics Committee of Beijing University of Chinese Medicine (Beijing, China).