Gastroprotective effects of Nelumbinis Rhizomatis Nodus carbonisata-derived carbon dots on ethanol-induced gastric ulcers in rats

Gastric ulcers is a common gastrointestinal digestive system disease. Considering the frequency of human gastric ulcers, the side effects and cost of some existing synthetic drugs, the use of natural products is an important choice for many people. The aim of present study was to explore gastroprotective effects of nelumbinis rhizomatis nodus carbonisata carbon dots (NRNC-CDs) on ethanol-induced gastric ulcers in rats. The NRNC-CDs were synthesized via high temperature calcinations treatment at 350 ℃ for 1 h were characterized by various spectroscopic and electron microscopy techniques for their structural, morphological, and optical properties. In vitro cytotoxicity of CDs for the human gastric epithelial cells line (GES-1 cells) was assessed by the CCK-8 assay. Furthermore, the study evaluated gastroprotective effects of NRNC-CDs on ethanol-induced gastric ulcers in rats, followed by a preliminary study on the possible mechanisms of gastroprotection.


Background
Gastric ulcers is a common gastrointestinal digestive system disease, which occurs owing to an imbalance between the defensive function and aggressive factors of the mucosal barrier [1]. These aggressive factors mainly include physical stress, heavy tobacco inhalation, excessive alcohol or caffeine, non-steroidal anti-in ammatory drugs, and microorganism infection [2]. Especially ethanol, the common factor existed in our daily life, plays a vital roles in the development of gastric cell necrosis and vascular injury, which was further induce gastric damage and disease [3]. At present, clinical strategies for management of treating gastric ulcer is to use proton pump inhibitors and H 2 receptor antagonists, which achieved an effective ulcer healing process by inhibiting gastric acid secretion, neutralize gastric acid and inhibit cell apoptosis through cytoprotection [4,5]. However, the use of these drugs are cause some side effects, such as arrhythmias, nausea, constipation and hypergastrinemia, etc [5,6]. Accordingly, it is necessary to develop novel anti-gastric ulcer products, as a promising therapeutic resource, which may emerge fewer side effects.
Carbon dots (CDs) are a new class of nanoscale materials with abundant surface functional groups (amino, carbonyl, carboxyl, or hydroxyl), and have attracted tremendous attention because of their possessed multiple considerable bene ts such as photochemical stability, ease of functionalization, high biocompatibility, hypotoxicity and hydrophilicity, etc. [7,8]. Moreover, it have been reported that administration of CDs have no signi cant effect on blood biochemistry and hematology analysis in vivo, and caused no histopathological abnormalities on major organs [9]. Although the drug is administered in different ways, the CDs could be excreted from the body e ciently and rapidly [10].Thus, these unique properties make CDs a promising candidate in the clinical applications [11][12][13].
Nelumbinis Rhizomatis Nodus (NRN), known as "Oujie" in Chinese, is originated from the dried rhizome node part of Nelumbo nucifera Gaertn. NRN is often made into a charcoal processed product for use, as name Nelumbinis Rhizomatis Nodus carbonisata (NRNC). Herein, in the study, Nelumbinis Rhizomatis Nodus carbonisata as carbon source were treated to prepare the CDs by a simple, eco-friendly method, which was named the Nelumbinis Rhizomatis Nodus carbonisata carbon dots(NRNC-CDs). Furthermore, the study assessed the protection effects of NRNC-CDs on ethanol-induced gastric ulcer in rat.
Accordingly, Some histopathological damage and biochemical indexes were evaluated.

Characterization of the NRNC-CDs
The TEM image (Fig. 1A) clearly indicates that the formed NRNC-CDs were spherical morphology with homogeneous dispersion. The particle size distribution calculated from hundred particles of CDs reveal that narrowly distributed CDs have an average diameter of 2.89±0.82nm. The HRTEM image ( Fig.1B and   1C) were shows that NRNC-CDs revealed the lattice spacing of 0.29 nm. The XRD patterns of NRNC-CDs presented the broad diffraction peak at 22.0 • associated with the formation of amorphous graphitic carbon in the Fig.1D, which was consistent with the TEM results.
The spectral properties of NRNC-CDs were shown in Fig.2A. The UV-Vis absorption band was observed at 270 nm, which was contributed to the π−π * transition of C=C on the surface of CDs [14]. The excitation and emission uorescence spectrum of NRNC-CDs indicated that the maximum excitation exhibited at 354nm and the maximum emission at 456 nm. The quantum yield (QY) of NRNC-CDs was calculated to be 1.28%. The surface functional groups of NRNC-CDs were analyzed by the FTIR spectrum, as shown in Fig.2B, the peak was corresponded at 3441 cm −1 , which resulted from the stretching vibration of O-H or N-H. The strong characteristic absorption peaks at 2932 cm -1 corresponds to the existence of the-CH 2stretch. The absorption peaks at 1629 cm -1 and 1400 cm -1 were assigned to C=O and C-N, respectively.
The peaks at 1047 cm -1 corresponded to the symmetric stretching vibrations of C-O-C [15,16].
The XPS was employed to evaluate the surface composition of NRNC-CDs. The XPS full scan spectrum of NRNC-CDs ( Fig.2C) displayed that the NRNC-CDs were mainly composed by C, O and N elements.

Cell viability assay
As shown in Fig.3, the CCK-8 study revealed that the average cell viability was greater than 95% at a concentration of NRNC-CDs up to 500 μg/mL.The cell viability was higher than 100% when NRNC-CDs concentration was below 500 μg/mL. Cell viability gradually decreased as the NRNC-CDs concentration increased from 500 to 1000 μg/mL, which exerted low toxicity to GES-1 cells at a relatively high concentration.

Macroscopic evaluation of gastric lesions
The gross appearance of the rat stomachs after treatment with ethanol to induce gastric mucosal damage (Fig.4A). There was no congestion, edema, erosion or ulceration in the control group,but the model group rats showed severe damage in the gastric mucosa as evidenced by ulcerated and hemorrhagic lesions. As shown in Fig Effect of NRNC-CDs on antioxidant and anti-in ammatory activity in the gastric tissue homogenate The Fig.5 showed that the effect of NRNC-CDs on antioxidant and anti-in ammatory activity in the gastric tissue homogenate.The SOD, CAT, GSH and GSH-Px activities in the gastric tissue homogenate of the model group were signi cantly lower than those in the control group (P <0.05). As shown in Fig.5A, administration of NRNC-CDs at the doses of high and medium, similar to ranitidine group, exhibited a sigini cant increase in SOD level in stomach tissue(P < 0.05). Administration of NRNC-CDs and ranitidine increased gastric CAT level, compared with the model group (Fig.5B). The activities of GSH-Px and GSH in the high-, medium-and low doses of NRNC-CDs (high and medium) treatment group were signi cantly higher than those in the model group (P < 0.05) in the Fig.5C and 5D. Although the low dose did not achieve statistical signi cance, increase in SOD, GSH-Px and GSH productions were also observed compared with the model group.
As shown in Fig.5E and 5F, there was a respectively high level of TNF-α and IL-6 in the model group as compared with the control group (P < 0.01). However, NRNC-CDs at high, medium and low dose signi cantly declined the levels of the pro-in ammatory cytokines including TNF-α and IL-6 (P < 0.01).

Histopathological examination
Histological analyses of the gastric mucosa were depicted in Fig. 6. The microscopic The microscopic study of the control group (Fig.6A,a) shows typical gastric histoarchitecture with intact epithelium and glands. Ethanol administration caused extensive damage of gastric mucosa characterized by hyperemia, severe desquamation and loss of surface epithelial (Fig.6B,b). Pre-treatment with ranitidine (Fig. 6C,c) and NRNC-CDs( Fig. 6D-F,d-f) for 7 days had comparatively better protection of the gastric mucosa as seen by reduction in ulcer area, reduced or absence of hemorrhage and desquamation.Interestingly, treatment with NRNC-CDs signi cantly reduced the degeneration and hemorrhage induced by ethanol, indicating that protective action that was evident with the high dose of 5 mg/kg.

Discussion
The CDs have many fascinating properties and excellent biocompatibility with size of less than 10 nm [18]. The development of a green, simple and eco-friendly method for the preparation of bio-sourced CDs has become the focus of research with the development and in-depth study of CDs. It has been reported that many natural plants as precursors were prepared the green CDs, such as pear juice [19] ,gynostemma [20],wheat bran [21] and citrus fruit peels [22], etc. In this paper, the novel NRNC-CDs were prepared from NRN by a simple method, and this methode no added any toxic reagents or chemicals. These CDs are required no further tedious modi cation and puri cation steps [23][24][25]. In recent years, CDs are widely used in biomedical sciences, and the most studied property of CDs are their photoluminescence owing to its wide application in bioimaging, optoelectronicsand spectroscopic methods [26]. With continuous in-depth research on carbon dots, it has been found that it has great potential in treating diseases [27]. It was found in previous studies that pollen typhae carbonisata-derived CDs proved to be an effective substance for hemostasis [14], mulberry silkworm cocoon-derived CDs possess antiin ammatory [30] and fructus crataegi-derived CDs reduce postprandial blood glucose levels [31]. These CDs derived from different precursors, and they have different biological activities.
Considering the frequency of human gastric ulcers, the side effects and cost of some existing synthetic drugs, the use of natural products is an important choice for many people [28]. In the study, we were rstly studied the gastroprotective effect of NRNC-CDs in an experimental gastric injury model induced by ethanol. CDs were prepared from a green carbon sources(NRN). The choice of precursor plays a key role in the physical and chemical properties of carbon dots. NRN as a natrual materials contain basic elements such as carbon, nitrogen, oxygen, sulfur and phosphorus, which provide unique surface functional groups and properties for CDs. Moreover, this plant is abundant resources and economic, and it can be widely used in clinical. Besides, the NRNC-CDs exerted low toxicity to GES-1 cells by CCK-8 test. Accordinglly, further comprehensive researches of CDs on gastric ulcer is signi cance meaningful.
Ethanol -induced gastric ulcer induced was a classic model ,which is often used to screen substances with potential effects on gastric ulcer. As we all know, ethanol is considered to be a cause of gastric injury duo to it changes protective factors, including reducing mucus production and blood circulation in the mucosa [29,30]. Ethanol consumption can produce oxidative stress and acute in ammatory reaction in animals [31]. SOD, CAT and GSH constitute an endogenous antioxidant system, which can be used to remove excess oxygen-derived free radicals and maintain them at physiological levels [32]. GSH-Px also plays an important role in protecting against oxidative gastric mucosal injury [33]. In this experiments, we rstly showed that ethanol administration clearly altered the gastric mucosa by macroscopic evaluation of gastric lesions, and the NRNC-CDs signi cantly reduced the areas of gastric ulcer formation and ulcer index, suggesting the ability of these CDs in protecting gastric ulcer. In additon, we also showed in the present study that ethanol intoxication induced the depletion antioxidant enzyme activities such as SOD, CAT, GSH and GSH-Px.The stomach tissue of the body is full of blood vessels and has an adequate blood supply. When the stomach tissue is stimulated by foreign substances or conditioned, it can produce a large amount of oxygen free radicals. These substances can induce cell apoptosis and aggravate gastric mucosal damage [34]. The study results showed that NRNC-CDs remarkably reduced gastric mucosal damage and signi cantly increased the levels of SOD, CAT, GSH and GSH-Px, and that were reveal that the gastric homogenate of the NRNC-CDs pretreatment group potent gastric protection by alleviating oxidative stress.
The in ammation is an inevitable mediator in ethanol-induced gastric ulcers , which have accompanied the increased expression of pro-in ammatory cytokines, such as TNF-α and IL-6 [35]. Moreover, TNF-α and IL-6 levels were associated with the severity of gastric mucosal in ammation. There increasing IL-6 and TNF-α levels in gastric tissue can be attributable to the necrotizing effects of ethanol in the model group. These results of our study showed that pretreatment and treatment with NRNC-CDs could remarkably alleviate in ammation symptoms by suppressing the production of TNF-α and IL-6, which was consistent with previous ndings [36,37].
In summary, this experiment demonstrated that the NRNC-CDs have important ulcer protection properties in a rat model of gastric ulcer induced by ethanol. In the study, it was proved that NRNC-CDs also has an gastricprotection effect, and that were promoted ulcer healing by reducing oxidative stress and reducing the in ammatory response . Thus, NRNC-CDs may be developed a potential drug for the treatment of gastric ulcer.

Conclusions
In the work, a novel eco-friendly NRNC-CDs were prepar by a simple method, and their were found to be nontoxic toward GES-1 cells. Interestly, The results indicated that the NRNC-CDs were rstly proved the gastroprotective effects on ethanol-induced gastric ulcers in rats. The NRNC-CDs effectively reduced the ulcer index of the gastric mucosa and increased the percentage of ulcer inhibition macroscopically.
Furthermor the levels of SOD, CAT, GSH and GSH-Px increased and the levels of TNF-α and IL-6 were signi cantly decreased, which can prove that this protection may be related to the antioxidant properties of NRNC-CDs by activating some enzymatic antioxidant mechanisms and attenuating the in ammatory response.

Preparation of carbon dots
CDs were prepared from NRN by using the modi ed pyrolysis method [38]. First, the NRN (80.0g) was placed in crucible, which was transferred into a mu e 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 ltered with a 0.22μm lter membrane to remove any large particles, and that solutions was puri ed 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 uorescence spectra (F-4500; Tokyo, Japan) were monitored with a molecular uorescence spectrometer in a standard quartz cuvette. The functional groups of NRNC-CDs were recorded by performing Fourier transform infrared spectroscopy (FTIR) spectroscopy (Thermo Fisher Scienti c, CA, USA ). X-ray photoelectron spectroscopy (XPS) of the sample was recorded using an ESCALAB 250Xi XPS (Thermo Fisher Scienti c) 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 H 2 SO 4 (quantum yield 54%) as a standard sample. The QY of NRNC-CDs was estimated according to the follow equation: Where the 'Φ' is the uorescence quantum yield, 'I' represents the integrated uorescent 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. where 'A CDs ' and 'A control ' 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,mediumdose 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 nal administration, acute gastric ulcer model was made by absolute ethanol (5.00mL/kg) [39]. Another one hour after ethanol instillation, the animals were sacri ced 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 super cial 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 xed in 10% (V/V) neutral buffered formalin for 24h, and para n 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 signi cant 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).