Ulcerative colitis is a chronic non-specific inflammatory disease of the colon and rectum and is a type of inflammatory bowel disease (IBD). The aim of this paper was to investigate the effect of Haematococcus carbon dots (HP-CDs) on dextran sulfate sodium (DSS)-induced ulcerative colitis in mice. HP-CDs were prepared from Haematococcus (HP) powder, firstly by hydrothermal synthesis of Rhodococcus amphitrite. The effect of HP-CDs on the mouse model of DSSinduced ulcerative colitis was then investigated in terms of histology and pathology. Effects. The results showed that HP-CDs significantly attenuated colitis and reduced body weight loss, Disease Activity Index score and colonic atrophy. In addition, HP-CDs reduced MPO activity and the expression of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, in colonic tissues.

• Carbon dots

• Haematococcus pluvialis

• Ulcerative colitis

• Anti-inflammation

Liu Y,Wang M, Ge G,Wang Y, Wang Z (2024) Preventive Effect of Hemocyanobacterial Carbon Points on Acute Ulcerative Colitis Induced by Dextran Sodium Sulfate. JSM Gastroenterol Hepatol 11(1): 1129.

Inflammatory bowel disease (IBD) is a chronic relaping disease that includes ulcerative colitis (UC) and Crohn’s disease (CD). UC is an immune disorder that tends to recur and persist, and the main symptoms typically present as abdominal pain, diarrhea, bloody stools, weight loss, and even systemic complications [1-3]. Dextrose Sodium Sulfate (DSS) is an irritant agent that tends to damage physical barrier of intestines and irritate mucous membranes, which can lead to overexpression of immune cells, damage to the intestines, and ultimately cause inflammation [4,5]. Also, other prescribed drugs have been given to alleviate the symptoms, for example, aminosalicylic acid, glucocorticoids, and biologics. However, these drugs hardly cure UC completely and carry variable degrees of side effects [6,7]. Therefore, the search for and development of effective functional ingredients or therapeutic drugs for colitis with low toxicity and side effects not only avoids the above situation, but also facilitates the development of new drugs.

Carbon dots (CDs) are a class of carbon-based nanomaterials with diameters of below 10 nm [8,9]. It has various bioactivities including  anti-inflammatory,  anticancer,  antimicrobial and antioxidant properties [10-13]. CDs, as a subclass of nanoparticles, exhibit the advantages of small size and high permeability, which can enhance permeability in the intestinal tract [14]. With the rise of green chemistry, microalgae have been used as precursors for the preparation of CDs [9,15-18]. Haematococcus pluvialis (HP) has been recognized as a new food ingredient in China. It is a source of many anti-inflammatory components, such as astaxanthin. Many vivo studies have shown that astaxanthin relieves inflammation in tissues and organs and plays a significant role in the intervention of DSS-induced colitis and acetic acid-induced gastric ulcers [19-21]. Interestingly, there is no report about HP as a precursor to synthesize carbon dots for the prevention of ulcerative colitis.

Here, we first prepared carbon dots by hydrothermal method using Haematococcus spp. as the precursor, then characterized the physicochemical properties of the carbon dots, and then verified the physicochemical properties and preventive effects of HP-CDs by a DSS-induced ulcerative colitis model in mice.

Chmicals and reagents

Haematococcus pluvialis (Yunnan Alphy Biotech Corporation); Dextran sulfate sodium (4000 Da), 5-aminosalicylic acid (5-ASA) (Shanghai Yuanye Science and Technology Co., Ltd); Myeloperoxidase assay kit and inflammatory cytokine kits of IL- 6, IL-1β, and TNF-α ( Nanjing Jiancheng Bioengineering Institute).

Synthesis of HP-CDs

The synthesis of carbon dots was carried out as previously published with some modifications [22]. 1 g of Haematococcus pluvialis powder was mixed with 50 mL of deionized water, and then the mixture was dispersed in a polytetrafluoroethylene- sealed autoclave heated at 180°C for 6.5 h. Finally, particulate impurities were removed from the reaction product using a 0.22 μm microporous filter membrane, and the filtrate was lyophilized to obtain Haematococcus pluvialis carbon dots (HP-CDs), which were stored at 4 °C for further experiments.

Characterize of HP-CDs

Appropriate amount of HP-CDs powder was taken and diluted with deionized water and the aqueous solution of HP- CDs was scanned at full wavelength (200 nm-700 nm) by UV- Vis spectrophotometer. The functional group information of the samples could be obtained by analyzing an appropriate amount of lyophilized carbon dots powder samples on an infrared spectrometer with a wave number range of 4000-400 cm-1 and an instrumental resolution of 4 cm-1. The prepared solution of HP-CDs was taken and the particle size, polydispersity index (PDI), and zeta potential of HP-CDs were determined at room temperature using Malvern ZEN 3600 dynamic light scattering instrument. The fluorescence spectra of the HP-CDs samples were determined using a fluorescence spectrophotometer by diluting the HP-CDs to a suitable concentration at room temperature.

Fluorescence Quantum Yield of HP-CD

The QY of the BS-CDs was measured according to a previously reported method [14]. Rhodamine B (RhB) was chosen as a reference and its QY at 340 nm was 0.31.The QY of BS-CD was calculated according to the following equation:

where sm represents the sample, st represents the standard (or reference), n represents the refractive index of the solvent, PLarea represents the integral area of the fluorescence intensity, and OD represents thecorresponding absorption value.

Antioxidant of HP-CDs

The scavenging ability of HP-CDs on DPPH radicals was tested with reference to the previously reported method [23].

Two ml of HP-CDs solution with different concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg/mL) were taken separately, and 2 ml of DPPH (0.1 m mol/L) solution was added and stirred well, and the reaction was carried out for 30 min at room temperature with protection from light, and absorbance values were measured at 517 nm, with Vc as a positive control. The effect of the HP-CDs solution on the DPPH radicals the scavenging rate was calculated as follows:

Where Ac is the absorbance of the DPPH solution without sample,Ai is the absorbance of the sample in the presence of DPPH solution. Aj is the absorbance of the sample without DPPH solution.

Stability

In order to verify the particle size and fluorescence stability of HP-CDs aqueous solution. Take appropriate amount of diluted HP-CDs solution and irradiate it under 365 nm UV lamp for 6 h. Incubate it in water bath at different temperatures (20, 30, 40, 50, 60, 70?) for 2 h to store the particle size and potential for one month; and measure its fluorescence intensity at 340 nm.

Phytotoxicity of HP-CDs

The HP-CDs powder obtained by lyophilization was prepared into solutions with different concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg/ml) for cultivation of mung beans, and the germination of mung beans was observed at room temperature after 4 days to analyze the biotoxicity of HP-CDs.

Animal experiments

All Eight-week-old ICR male mice were housed in an SPF- grade environment with temperature control at 25 ± 2°C, humidity control at 55%, and a dark/light cycle for 12 h. All mice studies were approved by the Animal Research and Ethics Committee of Shanghai Ocean University (approval number: SHOU-DW-2023-074) and complied with international guidelines. The mice were randomized into Cont, DSS, 5-ASA, HP (Haematococcus pluvialis), LCD (low dose of HP-CDs), and HCD (high dose of HP-CDs) groups (n = 6). The method of Li and Tao was adopted with appropriate modifications [24,25]. Except for the Cont group, mice in the other groups drank 3% w/v DSS solution freely to induce acute ulcerative colitis. During the molding, mice of the Cont group were given distilled water (10 mL/kg) once a day. The other groups were given with 3% DSS, 5-ASA (50 mg/mL), HP (100 mg/mL), LCD (100 mg/mL),and HCD (200 mg/mL) once a day at the same dose. Mice were monitored for symptoms such as body weight changes, mental status, and fecal occult blood during the induction of colitis. After 7 d of DSS induction, all mice were executed and the colon was removed for further study.

HE staining

The colon tissue was washed with saline and fixed in 10% neutral formalin solution. The colon tissues were dehydrated with different gradients of ethanol and xylene, then embedded in paraffin wax according to the conventional method, and finally sectioned with a pathology slicer. After staining, the pathological morphology of the sections was observed under light microscope.

Inflammatory cytokine analysis of HP-CDs

The levels of IL-1β, IL-6, and TNF-α were detected following the directions of the Elisa kits.

Statistical analysis

All data were presented as the mean ± standard deviation. Histograms and curves were created using GraphPad Prism 8.0 and Origin 2023. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test has been used (*p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001 vs. Cont group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. DSS group).

Characterization of HP-CDs

As shown in Figure 1(a),

Figure 1: Characterization of HP-CDs. (a) Ultraviolet spectroscopy; (b) infrared spectroscopy; (c-d) fluorescence spectroscopy.

the solution exhibits blue fluorescence under the excitation of 365 nm UV light, and there is an obvious absorption peak at 274 nm. According to the results of previous studies, the 250-300 nm absorption band represents the typical absorption of the aromatic series, which belongs to the π-π* conjugate leap of C=C, indicating the formation of carbon structure in the graphite core [26,27]. The absorption peak at 3201 cm-1 belongs to the O-H stretching vibration , and the sharp absorption peak at 2925 cm-1 may indicate the C-H stretching vibration [28]. Generally, the broader O-H vibrational bands indicate the presence of multiple hydroxyl structures in the samples, the absorption peak at 1379 cm-1 is attributed to the C-N bending vibration [2,29], and the absorption peak at 1031 cm-1 may indicate the C-N bending vibration [17], and the sharp absorption peak at 2925 cm-1 may indicate the C-H stretching vibration [28]. As shown in Figure 1(c), HP- CDs exhibited excitation-dependent fluorescence when the excitation wavelength was between 300-430 nm, showing strong fluorescence emission at 410 nm. In summary, an electron can reach the ground state through multiple photon emission pathways leading to excitation-related emission, a property determined by the size effect of the nanoparticles and their apparent functional groups [30,31]. The fluorescence excitation spectra of HP-CDs were scanned and shown in Figure 1(d), which yielded the optimal excitation wavelength of HP-CDs to be 340 nm. The emission spectra of HP-CDs showed strong fluorescence signals with a symmetric wavelength range and a large width of excitation and emission peaks, which indicated that the HP-CDs possessed excellent fluorescence properties. The fluorescence quantum yield reflects the ability of the substance to emit fluorescence, and the fluorescence quantum yield of HP-CDs was measured to be 24.39% according to the method of Zhang et al [32].

Antioxidant of HP-CDs

Based on the data in Figures 2-6,

Figure 2: Antioxidant capacity of HP-CDs. (a) DPPH; (b) ABTS

Figure 3: Stability of HP-CDs.(a) Light stability; (b-c) heat stability;(d-f) storage stability

Figure 4: Growth of mung bean sprouts.(a) Sunlight; (b) ultraviolet light.

Figure 5: Basic characterization of animals. (a) Schematic diagram of animal experiment grouping; (b) occult blood in the stool; (c) Weight change; (d) Change in DAI

Figure 6: Histological evaluation. (a) HE staining; (b-d) Inflammatory factor levels.

it can be seen that the scavenging effect of HP-CDs solution on DPPH radicals was concentration-dependent in the concentration range of 0.2-1.2 mg/mL. The linear equation obtained by linear fitting of the experimental data in Figure 2.The calculated IC50 was 0.3189 mg/mL, which indicated that HP-CDs had a good scavenging effect on DPPH radicals. The scavenging effect of HP-CDs solution on ABTS free radicals was also concentration-dependent in the range of 0.2-1.2 mg/mL concentration, and the higher the concentration of HP-CDs solution, the better its scavenging effect on ABTS free radicals. The scavenging rate of HP-CDs was 85.33% at a concentration of 1.2 mg/mL, and the IC50 of HP-CDs was 0.49 mg/mL, which indicated that HP-CDs had a better scavenging effect on ABTS radicals. From the antioxidant experiments we can easily see that HP-CDs have good antioxidant properties, and it is hypothesized that this carbon point can be developed as a new drug.

Light stability, heat stability, storage stability

The prepared HP-CDs solution was placed in a 365 nm UV dark box for 6h irradiation, and the results are shown in Figure 3(a). The fluorescence intensity of HP-CDs solution was more stable during UV irradiation up to 6h, which indicated that UV lamp irradiation could not affect the fluorescence stability of HP- CDs solution. In order to investigate the effect of temperature on the stability of HP-CDs, we determined the change of fluorescence intensity of HP-CDs after heating for 2h at 20-70? under variable temperature and 70? under constant temperature. As can be seen from Figure 3(b), the fluorescence intensity of HP-CDs solution was slightly decreased after heating for 2h at a constant temperature of 70 ?. When HP-CDs was heated at variable temperature, the fluorescence intensity basically remained stable. Neither constant temperature nor variable temperature can affect the fluorescence intensity of HP-CDs solution, and these data indicate that HP-CDs has good thermal stability. Figure 3(e) shows the changes of particle size and PDI of HP-CDs during 30 days of storage, and it can be found that the particle size and PDI values of HP-CDs were relatively stable during the rest of the storage time, except for the larger values of HP-CDs at the 18th day. Although the PDI value at the 18th day appeared to be larger, its value was 0.308, and it is generally considered that a PDI value of less than 0.5 can be considered as a good dispersion of nanoparticles in suspension. As shown in the figure, the emission peaks of the HP-CDs solution remained almost unchanged with a slight decrease in fluorescence intensity when the solution was stored at 4 °C for one month, which indicates that the fluorescence properties of the carbon dots are relatively stable and can be stored for a long time.

Phytotoxicity of HP-CDs

From Figures 4, it was found that mung beans could grow and germinate in different concentrations of HP-CDs solutions with good growth conditions. Even in 1mg/ml concentration of HP- CDs solution, mung bean could still survive. Under the irradiation of ultraviolet lamp, blue fluorescence could be clearly produced inside the mung bean sprouts, indicating that the HP-CDs solution could be absorbed by mung beans and was not toxic.

Colon and weight changes

To verify the function of HP and HP-CDs in DSS-induced UC, we established a typical DSS-induced UC model. As expected, mice in the DSS group appeared to persist weight loss after 3-4 d of liberally drinking 3% w/V DSS. This may be due to impaired digestion and absorption caused by intestinal injury and diarrhea, as well as reduced food intake in mice after drinking DSS. However, the intervention of HP (p < 0.01), LCD (p < 0.01) and HCD could significantly alleviate the rapid weight loss in mice. DAI score is the main parameter for evaluating the severity of UC [33]. DAI was assessed according to the scoring criteria established by previous authors with slight modifications [34]. In Figure 2b, after 4 d of modeling, all groups except the Cont group suffered from diarrhea and blood in the stool, which led to a great increase in the DAI score. Whereas, after the intervention of HP and HP-CDs, the DAI scores were remarkably lower than the DSS group.

Histological evaluation

The colonic tissues of mice in each group were prepared into pathological sections and then stained with H&E, and the results are shown in Figs. 4-8. Sections of the colon from control mice showed well-arranged crypts, intact epithelium without inflammatory cell infiltration, and no signs of congestion, edema, or ulceration in the submucosa [35]. However, in mice in the DSS group, we observed localized epithelial necrosis, disruption of the muscularis propria, detachment of colonocytes and cupping cells, altered or even disappeared crypt structure, and abundant submucosal edema with neutrophilic infiltration [36]. The colonic tissues of mice in the 5-ASA group were more structurally intact, and there was only a small amount of inflammatory cells abnormally infiltrated. Compared with the model group, mice in the HP group showed significant improvement in these injuries, especially in the extent of inflammatory cell infiltration. Notably, the administration of HP-CDs exhibited a significant protective effect against DSS-induced colonic injury, with microscopy showing epithelial cell production, restoration of colonic tissue integrity, increased presence of colonic cells and cupped cells, and decreased neutrophil infiltration [25]. The above results suggest that HP and HP-CDs are effective in protecting the structural integrity of the colon, thereby attenuating DSS induced colonic injury. Inflammatory mediators (cytokines and chemokines, etc.) have an important relationship with UC; excessive TNF-α disrupts the integrity of intestinal epithelial cells; IL-6 destroys the intestinal barrier by inhibiting T-cell apoptosis; and elevated levels of IL-1β are thought to be positively correlated with the degree of intestinal inflammation [5]. The results of inflammatory factor levels in colon tissues are shown in Fig. IL- 1β, IL-6 and TNF-α levels were significantly increased in the DSS group compared to the Cont group. The levels of inflammatory factors IL-1β, IL-6 and TNF-α were significantly decreased in the colonic tissues of mice in the HP group compared to the control group. LCD and HCD were able to down-regulate the expression of inflammatory factors such as TNF-α and IL-6 in the colonic tissues of mice (p<0.001). In addition, the level of inflammatory factor IL-6 was also significantly reduced in the colonic tissues of mice in the LCD (p<0.01) and HCD groups (p<0.001) compared to the DSS group. The above results suggest that HP and HP-CDs have a reparative effect on colonic epithelial barrier damage, which is consistent with the results of histopathologic sections. Notably, HCD showed stronger efficacy compared with the HP and LCD groups. In contrast, HP did not have a desirable effect on the downregulation of inflammatory factors in mouse colon tissues. We speculate that this was due to some adverse effects of the high dose of HP on the mice. Overall, the interventions of 5-ASA, HP, and different doses of HP-CDs showed different degrees of alleviation. These data suggest that HP-CDs play a preventive role against UC, which can be demonstrated by the reduction of inflammatory cell infiltration and cytokine modulation in DSS- induced mice.

In the present study, HP-CDs were prepared by hydrothermal synthesis using Rhodococcus rainieri as a carbon source.The basic characterization results showed the size distribution, motifs and optical properties. Plant experiments verified that HP-CDs inhibited plant growth at higher concentrations. In vivo experiments showed that HP-CDs have good anti-inflammatory activity. The results confirmed that HP-CDs could reduce the clinical symptoms and pathological damage of UC, and the mechanism might be related to the reduction of TNF-α, IL-1β, and IL-6 levels.

Author statement

All authors have agreed to the manuscript submission and confirm that the work has not been submitted simultaneously to another journal for consideration. In addition, all authors declare no conflicts of interest.

CRediT authorship contribution statement

Yuting Liu: Writing – original draft, Methodology, Investigation,Data curation. Wang: Investigation. Ge: Supervision. Wang: Supervision .Wang: Supervision. Ge: Supervision.. Zhang: Writing – review & editing,Resources, Project administration, Investigation, Conceptualization.

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