Ultrasound-assisted preparation of sweet corn cob polysaccharide selenium nanoparticles alleviates symptoms of chronic fatigue syndrome

Abstract

Chronic fatigue syndrome (CFS) is a long-term chronic condition that predisposes individuals to oxidative stress and disruption of the gut microbiota. In this study, sweet corn cob polysaccharide selenium nanoparticles (U-SCPSeNPs) with relatively small particle sizes were prepared using an ultrasound-assisted method. Transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffractometry (XRD), Fourier transform infrared spectroscopy (FTIR), and ultraviolet-visible spectroscopy (UV-Vis) were used to characterize the U-SCPSeNPs and determine the monosaccharide composition of the U-SCPSeNPs. The U-SCPSeNPs were used to improve the CFS of the mice. The results showed that the ultrasound-assisted method reduced the particle size of the SeNPs, and U-SCPSeNPs with a particle size of 76.74 nm and a selenium content of 186.83 ± 7.80 mg g⁻¹ were obtained at an ultrasonication time of 40 min. Sweet corn cob (SCP) bound to the SeNPs through hydrogen bonding. In terms of energy production, the production capacity of Na⁺–K⁺-ATP, Mg²⁺-ATP, and Ca²⁺-ATP was enhanced by U-SCPSeSCP in CFS mice; In terms of oxidative stress, the levels of SOD and MDA were decreased and CAT and GSH-Px were increased by SCPSeSCP. U-SCPSeSCP improved the diversity and abundance of the gut microbiota in CFS mice, and decreasing the relative abundance of Firmicutes increased the relative abundance of Bacteroidota at the phylum level. This study provides a reference for synthesizing polysaccharide SeNPs and assessing the ability of U-SCPSeNPs to alleviate CFS.

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1. Introduction

Chronic fatigue syndrome (CFS) is a complex disorder that may cause immune, inflammatory, neurological, and mitochondrial dysfunction and disturbances in the gut microbiota.^1–3 People with CFS suffer from cognitive impairment and memory loss, and CFS can seriously affect the quality of life of patients; for example, it can cause difficulty in concentrating, sore throat, muscle aches, and headaches.⁴ The direct and indirect healthcare costs incurred by the United States because of CFS are 17–24 billion dollars each year.⁵ These factors make a comprehensive assessment of CFS even more urgent.

Patients with CFS often suffer from oxidative stress, systemic inflammation, impaired adenosine triphosphate (ATP) production, and disturbances in the gut microbiota.^6,7 Therefore, treating these conditions can considerably improve the condition of patients. Selenium (Se) is an essential trace element with excellent antioxidant and anti-inflammatory properties;^8,9 therefore, Se may improve CFS. As Se is toxic, selecting an appropriate form of Se for medical administration is necessary. The main forms of Se are inorganic Se, organic Se, and Se nanoparticles.¹⁰ Compared with inorganic and organic Se, nano-Se has lower toxicity and greater bioavailability; however, owing to the instability of nano-Se, it is prone to aggregation and deposition, which affects the activity of Se nanoparticles.¹¹ The complex branching structure of polysaccharides can effectively disperse sE nanoparticles, and polysaccharides contain many –OH and C–O–H groups, which can bind to SeNPs.¹² The combination of polysaccharide and Se nanoparticles not only further decreases the toxicity of Se but also considerably increases the functional activity of the polysaccharide and Se; thus, polysaccharide and Se nanoparticles constitute a potent combination.¹³ As polysaccharides modulate gut microbes and increase the production of short-chain fatty acids (SCFAs), they may ameliorate CFS-induced changes in the gut microbiota.^14,15 Sweet corn cobs are rich in carbohydrates, fibers, and some minerals, making them ideal for polysaccharide extraction. Sweet corn cob polysaccharide (SCP) is a non-starch polysaccharide with a molecular weight of 175.453 kDa and a complex three-dimensional structure. As SCP has good antioxidant capacity, it is a highly suitable candidate for integration with nano-Se and for application in the treatment of CFS.^16,17

Ultrasonic waves are sound waves with a frequency of more than 20 kHz. Under the action of ultrasonic waves, tiny bubbles inside the liquid are generated, grow and collapse, resulting in a “cavitation phenomenon”. Ultrasound can effectively prevent the aggregation of particles and promote their uniform dispersion, thus reducing the particle size. Ultrasonic treatment is widely used in the preparation of nanoparticles because of its simplicity, efficiency and environmental friendliness.¹⁸

In this study, sweet corn cob polysaccharide Se nanoparticles were prepared using a redox method, and the particle size of the SeNPs was further reduced by ultrasound treatment to obtain U-SCPSeNPs. Characterization was conducted via TES, EDX, ICP-MS, XRD, FTIR, and UV-VIS, as well as determination of the monosaccharide composition of the U-SCPSeNPs. A mouse model of CFS was constructed to study the therapeutic effect of U-SCPSeNPs on CFS, and finally, the regulatory effect of U-SCPSeNPs on the gut microbiota of CFS mice was investigated.

2. Materials and methods

2.1. Materials

Sweet corn cobs (Zea mays L. saccharata Sturt) were obtained from Hao Wei Agriculture Co., Ltd (Harbin, Heilongjiang, China). Na₂SeO₃ and Vc were obtained from Shanghai Yuan Ye. (Shanghai, China). Salidroside was obtained from Evergreen Biologicals Ltd (Shanxi, Xi'an, China). Standard monosaccharides were obtained from Macklin Biochemical Technology Co., Ltd (Shanghai, China). Kits for determining liver glycogen, blood urea nitrogen (BUN), lactic acid (LAC), ATP, superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and glutathione peroxidase (GSH-Px) were obtained from Nanjing Jiancheng Bioengineering Institute, China.

2.2. Extraction of the SCP

SCPs were extracted following a previously described method with slight modifications.¹⁶ Briefly, sweet corn kernels were defatted and deproteinized via the Sevage method and ethanol precipitation to obtain crude SCPs, which were then purified using DEAE-52 cellulose and Sephadex G-200 columns and freeze-dried to obtain SCPs.

2.3. Ultrasound-assisted preparation of sweet corn cob polysaccharide Se nanoparticles (U-SCPSeNPs)

First, 40 mg of SCP was dissolved in 40 mL of ultrapure water. Then, 4 mL of Na₂SeO₃ (2 mg mL⁻¹) and 4 mL of vitamin C (Vc) solution (9 mg mL⁻¹) were added, and the reaction was conducted for 4 h at 45 °C. When the reaction stopped, the mixture was dialyzed for 48 h in a dialysis bag (molecular weight = 3 kDa). The solutions were subjected to ultrasonic treatment (KQ-250DE, Shumei, China) at 250 W for 0 min, 20 min, 40 min, or 60 min, respectively. During ultrasonication, the temperature was controlled below 40 °C using an ice bath, and the samples were then dialyzed in a dialysis bag (molecular weight = 3 kDa) for 48 h. Water changes were performed every 8–12 h. Vc was added to the replaced ultrapure water to verify the presence of the Na₂SeO₃ residue, and dialysis was completed if the ultrapure water did not turn red. Finally, the samples were freeze-dried to obtain U-SCPSeNPs 0, U-SCPSeNPs 20, U-SCPSeNPs 40, and U-SCPSeNPs 60, respectively.

2.4. Characterization of the U-SCPSeNPs

2.4.1. Particle size. First, 10 mg of U-SCPSeNPs were weighed, added to 10 mL of ultrapure water, and dissolved. The mixture was subsequently filtered through an aqueous membrane. The particle size was determined via dynamic light scattering (Malvern Zetasizer Nano ZS 90, Malvern Instruments Ltd, UK) with the following parameters: particle refractive index = 1.590, particle absorption coefficient = 0.01, water refractive index = 1.33, and temperature = 25 °C.¹⁹

2.4.2. TEM and EDX. The solution containing U-SCPSeNPs was placed on a copper mesh for drying and then observed using a transmission electron microscope (HT7700, HITACHI, Japan) with an accelerating voltage of 20 V.²⁰ The elemental composition of the U-SCPSeNPs was analyzed via EDX.²¹

2.4.3. ICP-MS. The Se content of the U-SCPSeNPs was determined via inductively coupled plasma-mass spectrometry (Thermo, USA).²²

2.4.4. XRD. X-ray diffraction (PW3040/60, PANalytical B.V. Netherlands) was used to determine the crystal types of the U-SCPSeNPs at a scanning speed of 2° min⁻¹ and scanning angles (2θ) of 5–90°.²³

2.4.5. FTIR. The U-SCPSeNPs were mixed and milled with KBr and analyzed using a Fourier transform infrared spectrometer (PerkinElmer, USA); a KBr flake was used as the background.²⁴

2.4.6. UV-Vis. The U-SCPSeNPs were dissolved in ultrapure water, scanned using a UV-visible spectrometer (PerkinElmer, USA) at 200–800 nm, and zeroed with ultrapure water.²⁵

2.4.7. Monosaccharide composition. High-performance liquid chromatography (Agilent 1100, USA) was performed to determine L-glucuronic acid (L-GA), D-mannuronic acid (D-ManA), D-mannose (D-Man), D-glucosamine (D-GlcN), D-ribose (D-Rib), L-rhamnose (L-Rham), D-glucuronic acid (D-GlcUA), D-galacturonic acid (D-GalUA), D-galactosamine (D-GalN), D-glucose (D-Glc), D-galactose (D-Gal), D-xylose (D-Xyl), L-arabinose (L-Ara), and L-fructose (L-Fuc) for the 14 markers and, later, to assess the monosaccharide composition of U-SCPSeNPs.²⁶ The detection conditions were as follows: C18 column 250 mm × 4.6 mm × 5 μm, mobile phase A was phosphate buffer, mobile phase B was acetonitrile, the column temperature was 30 °C, the flow rate was 1 mL min⁻¹, and the injection volume was 5 μL.

2.5. Animal experiments

Male five-week-old ICR mice (n = 64) weighing 20 ± 2 g were purchased from Yisi Laboratory Animal Centre (Changchun, Jilin, China), License No. SCXK [ji]-2020-0002, Quality Certificate No. 202000034164. The mice were first acclimatized by feeding for seven days under the following conditions: 12 h/12 h day/night cycle, 50 ± 10% humidity, 24 ± 2 °C room temperature, and adequate ventilation. All the mice were provided free access to food and water during acclimation. All experimental animals were treated humanely, and all experimental manipulations were approved by the Ethics Committee of Harbin Business University (Approval No. HSDU2020-065, Harbin, China).

2.5.1. Design of animal experiments. After acclimation, the mice were subjected to CFS modeling, and after 28 days of modeling, they were randomly divided into eight groups (n = 8 mice per group) as follows: normal control (NC), model control (MC), salidroside positive control (PC), SCP, U-SCPSeNP 0, U-SCPSeNP 20, U-SCPSeNP 40, and U-SCPSeNP 60, respectively. The administered dose was 200 mg kg⁻¹ in all cases, and equal quantities of saline were given to the NC and MC groups.

Except for the mice in the NC group, the mice in all the other groups were subjected to the following methods for modeling CFS.²⁷ The mice were kept in specific rat cages (20 × 9 × 8 cm) to simulate a crowded environment (Fig. 1). The mice were stimulated to run on a treadmill at a speed of 10 m min⁻¹ for 1 h. Then, they were allowed to rest for 1 h and immobilized in 50 mL centrifuge tubes (11.6 × 2.7 cm) for 6 h. Next, they were exposed to noise disturbance for 4 h after the immobilization period ended. The first behavioral test was performed on the mice after day 28. First, a tail suspension experiment, in which the mice were suspended for 6 min, was performed, during which the struggle time, swaying time, and immobility time were recorded. After the mice were given sufficient rest, an open field experiment was performed, in which the mice were placed in the open field, and the total distance traveled, the trajectory of the mice, the average speed, and the remaining time were recorded. After the first behavioral test, the mice were treated with the administered drug for 28 days, and the modeling experiments for CFS were continued. The mice underwent a second behavioral test on day 28 after drug administration, and the equipment for the behavioral test was purchased from SANS Biotechnology Co., Ltd (Nanjing, Jiangsu, China). When the experiment ended, the mice were sacrificed, blood was collected from the heart, and the plasma was centrifuged at 3000 rpm for 10 min to obtain the serum. The liver was stored at −80 °C, and feces were obtained from the colon and stored at −80 °C.

Fig. 1 Animal experimental design.

2.5.2 Measurement of CFS-related indicators. Liver glycogen, BUN, LAC, ATP, SOD, MDA, CAT, and GSH-Px levels were determined following the instructions provided with the kit.

2.5.3. Analysis of the gut microbiota. DNA was extracted from the fecal samples using a DNA extraction kit, and the purity and concentration of the DNA were tested using Nanodrop One (Thermo Fisher Scientific, USA). When the purity and concentration of the DNA reached standard values, PCR amplification of the primers was conducted using a PCR instrument (Bio-Rad S1000, Bio-Rad Laboratory, CA), after which the PCR products were detected via electrophoresis. After passing the test, the PCR products were further purified. Finally, the database was established according to the ALFA-SEQDNALibrary PrepKit, and 16S rDNA sequencing was performed. This part was tested by Guangdong Megger Gene Technology Co. (Shenzhen, Guangdong, China).

2.6. Statistical analysis

The figures in this article were made using Origin 2021 (Origin Lab, Northampton, USA). The data were analyzed via IBM SPSS version 26.0 (IBM Corp., Armonk, NY, USA). The results are expressed as the means ± standard deviations (SDs), and group differences were assessed via one-way ANOVA. All differences were considered statistically significant at P < 0.05.

3. Results

3.1. TEM, EDS, and Se content

The TEM results are shown in Fig. 2A, it was demonstrated that all SeNPs presented spherical shape. After sonication for 20 min and 40 min, the SeNPs decreased in size. After sonication for 60 min, the SeNPs became significantly larger. The particle size distribution of the U-SCPSeNPs is shown in Fig. 2B. The particle size distributions of U-SCPSeNPs 0, 20, 40, and 60 were 129.19, 107.89, 76.74, and 153.23 nm, respectively. Therefore, U-SCPSeNP 40 had the smallest particle size.

Fig. 2 TEM images (A), particle size distributions (B) and elemental compositions (C–E) of U-SPCSeNPs.

The EDX results are shown in Fig. 2C and D. C, O, and Se are the main components of U-SCPSeNPs, and the detection of Se in U-SCPSeNPs indicates the success of the preparation of SeNPs. The elemental contents in Fig. 2E are reflected in Table 1, with SCP, U-SCPSeNPs 0, U-SCPSeNPs 20, U-SCPSeNPs 40, and U-SCPSeNPs 60 exhibiting elemental C contents of 58.5%, 46.4%, 49.7%, 46.5%, and 55.4%, respectively; O contents of 37.7%, 40.0%, 33.5%, 34.6% and 32.7%, respectively; and Se contents of 3.8%, 13.6%, 16.8%, 18.9%, and 11.9%, respectively. After ultrasound treatment, the content of O was decreased, and the maximum content of Se was recorded after sonication for 40 min, whereas the content of Se decreased and that of C increased when the sonication time reached 60 min.

Table 1 Proportion of C, O, and Se in the EDX analysis

Element	SCP weight (%)	SCPSeNPs weight (%)	U-SCPSeNPs 20 weight (%)	U-SCPSeNPs 40 weight (%)	U-SCPSeNPs 60 weight (%)
C	58.5	46.4	49.7	46.5	55.4
O	37.7	40.0	33.5	34.6	32.7
Se	3.8	13.6	16.8	18.9	11.9

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The Se contents of U-SCPSeNPs 0, U-SCPSeNPs 20, U-SCPSeNPs 40, and U-SCPSeNPs 60 detected by ICP-MS were 134.56 ± 8.88 mg g⁻¹, 170.90 ± 8.91 mg g⁻¹, 186.83 ± 7.80 mg g⁻¹, and 116.10 ± 6.41 mg g⁻¹, respectively, which matched the EDS results.

3.2. Structural characterization of U-SCPSeNPs

The results of the XRD assay are shown in Fig. 3A. The SCP and U-SCPSeNPs showed broadened peaks, indicating that the SeNPs were present in an amorphous state, which suggested that the SeNPs existed at the nanoscale.²⁸ The FTIR results are shown in Fig. 3B, where SCP showed characteristic absorption peaks at 3305, 2929, 1640, 1151, and 1016 cm⁻¹, which corresponded to O–H, C–H, C=O, and C–O–C, respectively. After ultrasonication, the U-SCPSeNPs also presented the above characteristic absorption peaks, but the difference was that the O–H characteristic absorption peaks of the U-SCPSeNPs redshifted, which matched most of the results of the oxidative synthesis of SeNPs²⁹ and revealed that ultrasonication did not affect the binding of the SCPs to the SeNPs. Additionally, SCP binds to the SeNPs through O–H hydrogen bonding. The UV-Vis results are shown in Fig. 3C. No absorption peak for SCP was detected at 200–800 nm, which agreed with the basic characteristics of polysaccharides, whereas U-SCPSeNPs presented an absorption peak at 250–300 nm, which also indicated the formation of SeSCP.³⁰ The height of the absorption peak was related to the Se content, which matched the EDS results that the U-SCPSeNPs had the highest Se content.

Fig. 3 XRD pattern (A), FTIR spectra (B), UV-Vis absorption spectra of U-SCPSeNPs (C) and monosaccharide composition (D) of U-SPCSeNPs. A diagram illustrating the mechanism of synthesis of U-SPCSeNPs (E and F). Note: Peaks 1–14 in D represent the following compounds: 1: L-GA, 2: D-ManA, 3: D-Man, 4: D-GlcN, 5: D-Rib, 6: L-Rham, 7: D-GlcUA, 8: D-GalUA, 9: GalN, 10: D-Glc, 11: D-Gal, 12: D-Xyl, 13: L-Ara, and 14: L-Fuc.

The monosaccharide composition of the U-SCPSeNPs is shown in Fig. 3D. By controlling the retention time of the 14 monosaccharides, we found that the SCP and U-SCPSeNPs consisted of Glc only and that ultrasonication and selenation did not change the monosaccharide composition of the SCP. According to our structural analysis of SCP conducted in another study, as shown in Fig. S1–3,† the SCP main chain consists of α-D-Glc-(1 →,→ 4), and the branched chain consists of α-D-Glc-(1 →,→ 6).¹ Combining the results of FTIR, XRD, and monosaccharide composition, we deduced that SCP binds to SeNPs in the form shown in Fig. 3E and F. A large quantity of –OH present in Glc in SCP decreases Na₂SeO₃ to SeNPs via ascorbic acid, and SeNPs form hydrogen bonds with –OH, thus preventing the aggregation of SeNPs.

3.3. Behavioral tests in CFS mice

The results of the behavioral tests in the CFS mice are shown in Tables 2 and 3 and Fig. 4. After 28 days of forced exercise, restraint stress, and noise, the mice developed fatigue.²⁷ The first tail suspension experiment revealed that the struggling duration of the model mice was significantly shorter than that of the mice in the NC group, whereas the duration of shaking and immobility was significantly greater than that of the mice in the NC group (P < 0.05). The results of the first open field experiment revealed that the total distance traveled and average speed of movement of the mice were significantly lower than those of the mice in the NC group, and the resting time was significantly greater than that of the mice in the NC group (P < 0.05). Additionally, the distance traveled by the model mice was significantly shorter, as shown in the trajectory graph in Fig. 4A, indicating successful modeling.³¹

Fig. 4 Trajectory diagram of the first open field experiment (A). Trajectory diagram of the second open field experiment (B). The data are expressed as the mean ± SD (n = 8). Significant differences (P < 0.05) are indicated with different letters and the letter order indicates a decreasing average.

Table 2 Results of the tail suspension experiment

Group (n = 8)	The first tail suspension experiment				The second tail suspension experiment
	Time (s)	Struggling time (s)	Shaking time (s)	Immobility time (s)	Time (s)	Struggling time (s)	Shaking time (s)	Immobility time (s)
Significant differences (P < 0.05) are indicated with different letters and the letter order indicates a decreasing average.
NC	360	336.69 ± 2.88^a	10.90 ± 1.33^a	12.40 ± 1.60^b	360	332.15 ± 1.55^a	15.89 ± 1.28^e	11.97 ± 0.63^g
MC	360	286.45 ± 4.84^b	27.02 ± 2.74^b	46.53 ± 2.11^a	360	269.80 ± 2.57^f	37.54 ± 1.91^a	52.66 ± 2.09^a
PC	360	288.02 ± 2.38^b	27.68 ± 1.33^b	44.29 ± 1.84^a	360	318.86 ± 0.96^b	22.34 ± 0.59^d	18.81 ± 0.99^f
SCP	360	288.71 ± 3.62^b	26.36 ± 1.28^b	44.93 ± 2.43^a	360	291.60 ± 2.32^e	28.68 ± 1.39^b	39.72 ± 0.95^b
U-SCPSeNPs 0	360	287.96 ± 2.05^b	25.55 ± 0.76^b	46.49 ± 1.30^a	360	303.98 ± 1.47^c	28.80 ± 0.37^b	27.22 ± 1.12^d
U-SCPSeNPs 20	360	286.85 ± 2.38^b	27.26 ± 0.82^b	45.89 ± 2.24^a	360	308.56 ± 0.56^c	26.34 ± 0.60^bc	25.11 ± 1.13^de
U-SCPSeNPs 40	360	286.00 ± 2.11^b	27.25 ± 1.68^b	46.75 ± 0.43^a	360	314.73 ± 1.77^b	23.00 ± 1.50^cd	22.27 ± 1.17^e
U-SCPSeNPs 60	360	286.88 ± 2.23^b	27.48 ± 1.46^b	45.64 ± 0.80^a	360	297.89 ± 2.04^d	28.16 ± 1.67^b	33.95 ± 1.02^c

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Table 3 Results of the open field experiment

Group (n = 8)	The first open field experiment				The second open field experiment
	Time (s)	Total distance traveled (mm)	Average velocity (mm s^−1)	Rest time (s)	Time (s)	Total distance traveled (mm)	Average velocity (mm s^−1)	Rest time (s)
Significant differences (P < 0.05) are indicated with different letters and the letter order indicates a decreasing average.
NC	300	16 625 ± 212.22^a	55.42 ± 0.71^a	7.77 ± 0.42^b	300	17 021 ± 289.92^a	56.74 ± 0.97^a	7.62 ± 1.05^d
MC	300	10 417 ± 330.09^b	34.72 ± 1.10^b	23.63 ± 1.35^a	300	9096 ± 295.35^h	30.32 ± 0.98^h	32.49 ± 1.60^a
PC	300	10 477 ± 211.03^b	34.92 ± 0.70^b	23.66 ± 0.91^a	300	15 517 ± 198.66^b	51.72 ± 0.66^b	12.65 ± 0.83^c
SCP	300	10 374 ± 203.71^b	34.58 ± 0.68^b	23.26 ± 1.22^a	300	11 634 ± 158.01^g	38.78 ± 0.53^g	19.21 ± 0.52^b
U-SCPSeNPs 0	300	10 441 ± 331.74^b	34.75 ± 1.04^b	23.44 ± 0.97^a	300	13 023 ± 174.57^e	43.41 ± 0.58^e	16.98 ± 0.72^b
U-SCPSeNPs 20	300	10 448 ± 352.87^b	34.82 ± 1.16^b	23.72 ± 1.60^a	300	13 984 ± 278.79^d	46.61 ± 0.93^d	14.28 ± 0.32^c
U-SCPSeNPs 40	300	10 522 ± 252.34^b	35.07 ± 0.84^b	23.44 ± 0.61^a	300	14 743 ± 119.00^c	49.14 ± 0.40^c	13.19 ± 0.49^c
U-SCPSeNPs 60	300	10 629 ± 313.93^b	35.43 ± 1.05^b	23.82 ± 1.25^a	300	12 303 ± 248.69^f	41.01 ± 0.83^f	18.45 ± 0.95^b

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After 28 days of treatment, the suspension experiment and open field experiments were performed again. In the tail suspension experiment, the groups showed different degrees of improvement in struggle time, but the degree of improvement was significantly lower than that of the NC group (P < 0.05); moreover, the U-SPCSeNP 40 group had no significant difference in struggle time from the PC group (P > 0.05) and exhibited shaking time and immobility time similar to those of the PC group. In the open field experiments, the total distance traveled and mean speed also increased in all the groups, and the remaining time was also significantly lower than that in the MC group (P < 0.05). As shown in the trajectory diagram in Fig. 4B, the distance traveled by each group of mice also increased significantly, and the U-SPCSeNPs 40 group was the closest to the PC group among the administered groups in terms of the total distance traveled, the mean speed, and the remaining time. SCP and U-SCPSeNPs improved the CFS in mice, and U-SPCSeNP 40 had a more prominent effect.

3.4. Effects of U-SCPSeNPs on basal biochemical indices in CFS mice

The changes in the weights of the mice are shown in Fig. 5A. After modeling, the weights of the mice in each group decreased and were significantly lower than those of the mice in the NC group (P < 0.05). After treatment, the weight of the mice increased, and the weight of the mice in each group was significantly greater than that of the mice in the MC group at the 28^th day (P < 0.05), which illustrated the alleviation of CFS in the mice after treatment. The liver glycogen content of the mice was significantly lower in the MC group than in the other groups (P < 0.05) (Fig. 5B), which indicated that the hepatic glycogen production and storage capacity of the CFS mice were affected and that the hepatic glycogen content of the other groups was significantly greater but still lower than that of the NC group after treatment (P < 0.05). The levels of BUN and LAC in the blood of the mice are shown in Fig. 5C and D. The BUN and LAC levels of the mice in the MC group were significantly greater than those in the other groups (P < 0.05), which indicated that CFS might lead to renal function impairment and the inability to excrete BUN in time.³² The reduction in LAC levels after treatment indicated remission of CFS in the mice. The ability of the MC group to produce Na⁺–K⁺-ATP, Mg²⁺-ATP, and Ca²⁺-ATP in the blood is shown in Fig. 5E–G. The ability of the MC group to produce ATP was significantly lower than that of the other groups (P < 0.05), which indicated that CFS can affect the production of ATP, whereas the ability to produce ATP was restored after the drug was administered.³³ The oxidative stress-related indices are shown in Fig. 5H–K. CFS significantly increased the content of SOD and MDA, while significantly decreased the content of CAT and GSH-Px (P < 0.05), which indicated that the mice with CFS were in a state of oxidative stress in vivo.³⁴ These results also revealed that salidroside improved the condition of CFS mice, while the U-SCPSeNP 40 group was closest to the PC group.

Fig. 5 Effects of U-SCPSeNPs on body weight (A), live glycogen (B), BUN (C), LAC (D), Na⁺–K⁺-ATP (E), Mg²⁺-ATP (F), Ca²⁺-ATP (G), SOD (H), MDA (I), CAT (J), and GSH-Px (K) in mice. The data are expressed as the mean ± SD (n = 8). Significant differences (P < 0.05) are indicated with different letters and the letter order indicates a decreasing average.

3.5. Effect of U-SCPSeNPs on gut microbiota diversity in CFS mice

The α diversity of the gut microbiota is shown in Fig. 6A and B. The Chao1 index explains the abundance of the gut flora, whereas the Shannon index explains the diversity of the gut microbiota. The Chao1 and Shannon indices of the gut microbiota were significantly lower in the MC group compared to other groups (P < 0.05), indicating that CFS affects both diversity and abundance of gut microbiota in mice. In contrast, there was no significant difference between the Chao1 and Shannon indices of U-SCPSeNP group and NC group (P > 0.05), suggesting that U-SCPSeNPs can help alleviate disruptions caused by CFS on gut microbiota.

Fig. 6 Chao1 (A) and Shannon (B) indices of alpha diversity in the gut microbiota; NMDS (C) and PCoA (D) analyses of β diversity in the gut microbiota. The data are expressed as the mean ± SD (n = 6). Significant differences (P < 0.05) are indicated with different letters, and the letter order indicates a decreasing average.

The β diversity of the gut microbiota is shown in Fig. 6C and D. NMDS and PCoA can reveal species diversity among samples, but their calculation methods are different, with NMDS using the Bray–Curtis algorithm and PCoA using the Euclidean algorithm. The stress value of 0.1236 obtained from the NMDS analysis indicates that the result is a satisfactory fitting model, and the NC and U-SCPSeNP groups are significantly distant from the PC group in the figure; similar results were observed through PCoA. These results suggest the possibility of a large change in the gut microbiota of the PC group of mice due to the combined effect of CFS and salidroside. Additionally, the closer proximity of the NC and U-SCPSeNP groups in NMDS and PCoA suggested that the composition of the gut microbiota in the two groups was more similar, indicating that the U-SCPSeNPs alleviated CFS-induced disorders of the gut microbiota.

3.6. Effects of U-SCPSeNPs on the composition of the gut microbiota in CFS mice

The species composition at the phylum level of the gut microbiota is shown in Fig. 7A, with the phylum level in mice consisting mainly of Firmicutes and Bacteroidota. The relative abundances of the species Firmicutes and Bacteroidota are shown in Fig. 7B and C. The relative abundance of Firmicutes was significantly lower (P < 0.05) than that of the MC, PC, and SCP groups in the U-SCPSeNP group but did not differ significantly from that of the NC group, whereas the relative abundance of Bacteroidota was significantly greater than that of the MC, PC, and SCP groups but not significantly different from that of the NC group (P > 0.05). These findings suggest that U-SCPSeNPs decreased the relative abundance of Firmicutes and increased the relative abundance of Bacteroidota.

Fig. 7 Phylum-level species composition (A). The relative abundances of Firmicutes (B) and Bacteroidota (C). Genus-level species composition (D). The relative abundances of Lactobacillus (E), Staphylococcus (F), Jeotgalicoccus (G), and Bacteroides (H); LDA value histogram (I) and species evolutionary branching diagram (J) of LEfSE analysis.

The species composition at the genus level of the gut microbiota is shown in Fig. 7D, where the gut microbiota at the genus level of the mice consists mainly of Lactobacillus, Staphylococcus, Jeotgalicoccus, and Bacteroides. The relative abundances of Lactobacillus, Staphylococcus, Jeotgalicoccus, and Bacteroides are shown in Fig. 7E–H. Among the Lactobacillus groups, no significant difference was recorded (P > 0.05). Staphylococcus and Jeotgalicoccus were significantly more abundant in the MC, PC, and SCP groups than in those of the NC and U-SCPSeNP groups (P < 0.05), whereas no significant difference was detected between the NC and U-SCPSeNP groups (P > 0.05). The relative abundance of Bacteroides in the NC group was significantly greater than that of the MC, PC, and SCP groups (P < 0.05). More unassigned bacteria were present in the U-SCPSeNP group.

A histogram of the distribution of LDA values of the gut microbiota and a branching diagram of species evolution are shown in Fig. 7I and J. U-SCPSeNPs significantly increased Deferribacteres at the class level; Bacteroidales and Deferribacterales at the order level; Muribaculaceae, Tannerellaceae and Deferribacteraceae at the family level; Parabacteroides, Mucispirillum and Paraprevotella at the genus level; and Tidjanibacter massiliensis and Alistipes sp. cv. 1 at the species level.

4. Discussion

The biological functions and stability of SeNPs are related to their particle size, and ultrasound treatment can help obtain SeNPs with smaller particle sizes, which contributes to the study of Se nanoparticles.³⁵ The mechanism underlying the reduction in the size of SeNPs by sound may involve the dispersal of SeNPs more thoroughly by ultrasound, and the dispersed SeNPs may be attracted by the formation of hydrogen bonds with the –OH groups of SCP, preventing them from aggregating. Second, SCPs have a complex secondary structure. In the absence of sonication, SCP may be entangled in the secondary structure, and after sonication, the secondary structure may be unfolded to attract more SeNPs. The U-SCPSeNPs had the smallest particle size when the ultrasound treatment lasted 40 min, whereas the particle size of the U-SCPSeNPs increased when the ultrasound treatment lasted for 60 min, probably because the treatment broke the hydrogen bonds formed between the SCP and the SeNPs, causing the SeNPs that were originally attracted by the hydrogen bonds to be released and aggregate with other SeNPs. We also found that U-SCPSeNPs with smaller particle sizes also have higher Se contents, probably because SeNPs with smaller particle sizes can make better use of the –OH of SCP and form hydrogen bonds with more –OH, leading to an increase in the Se content.

Compared with those of normal mice, the body weights of CFS mice were lower, indicating an imbalance in energy regulation in the CFS. The liver stores glucose in the form of glycogen, which undergoes gluconeogenesis to release glucose into the bloodstream to produce energy, thus preventing protein and fat breakdown in the case of energy shortage.³⁶ The urea cycle in the liver is the main metabolic pathway for converting nitrogen to urea, and an increase in blood urea nitrogen levels indicates abnormal liver function in CFS mice.^37,38 During aerobic respiration, the breakdown of glucose into carbon dioxide and water releases a large amount of energy, whereas during anaerobic respiration, glucose is broken down into lactic acid. However, blood lactate can be converted into hepatic glycogen via gluconeogenesis, and liver injury results in persistently high blood lactate levels in CFS patients.^39,40 The accumulation of lactic acid in large quantities results in muscle soreness, the most intuitive abnormality experienced by CFS patients. Following energy production, ATP is catalytically hydrolyzed by ATPase to ADP and phosphate ions, releasing a large amount of energy. A decrease in ATPase activity in patients with CFS decreases energy production. ATPase provides metabolic substances to cells and metabolizes waste or toxic substances. It also regulates the ionic gradient inside and outside the cell.⁴¹ Therefore, ATPase activity plays a very important role in organisms. Na⁺–K⁺-ATPase and Mg²⁺-ATPase have optimal pH values of 7.5 and 8.0, respectively,⁴² and the pH of the blood is generally neutral (about 7.4), whereas the increase in the blood lactate content in patients with CFS may decrease the pH of the blood to a weakly acidic level. This, in turn, may decrease the activity of Na⁺–K⁺-ATPase and Mg²⁺-ATPase. Although the cause of CFS is unclear, prolonged oxidative stress can cause severe damage in CFS patients. SOD and MDA levels in CFS patients are high, whereas CAT and GSH-Px levels are lower than normal, which indicates that CFS patients cannot adequately manage oxidative stress.⁴³ U-SCPSeNPs alleviate CFS in mice, likely by decreasing the levels of SOD and MDA and increasing the levels of CAT and GSH-Px, thus decreasing the state of oxidative stress in CFS mice. This leads to the recovery of liver function and gluconeogenesis, thus decreasing urea nitrogen and lactic acid in the blood. When the normal state of blood is restored, ATPase enzyme activity also increases. In the experiments, U-SCPSeNP 40, with a smaller particle size and higher Se content, had a better effect on the CFS.

As CFS is a long-term chronic disease, it affects the gut microbiota.⁴⁴ The richness and diversity of the gut microbiota changed in mice with CFS, but no significant difference was recorded from those of the NC group in terms of β-diversity, suggesting that CFS may have caused those less-represented strains to change. Additionally, the β diversity of the PC group was significantly different from that of the NC group, indicating that the gut microbiota of the mice treated with both salidroside and CFS was significantly distinct; the effect of salidroside on the gut microbiota was probably not positive. At the phylum level, the microbiota consisted mainly of Firmicutes and Bacteroidota. CFS increased the abundance of Firmicutes and decreased the abundance of Bacteroidota, leading to an imbalance in the Firmicutes to Bacteroidota ratio. Assessing the composition of the microbiota at the genus level revealed that Firmicutes is mainly composed of Lactobacillus and Staphylococcus. Lactobacillus benefits gut health by ameliorating inflammation, regulating the balance of the gut microbiota, and increasing butyric acid production.^29,45–48 Most Staphylococcus species are pathogenic bacteria that cause inflammation.⁴⁹ Thus, U-SCPSeNPs may have inhibited the growth of Staphylococcus, thereby stabilizing the ratio of Firmicutes to Bacteroidota. Bacteroidota can degrade polysaccharides and increase the production of SCFAs, which benefits the health of the gut microecology.⁵⁰ Although U-SCPSeNPs increase the content of Bacteroidota at the portal level, many of these organisms are unclassified at the genus level; thus, the beneficial effects of U-SCPSeNPs on the gut microbiota need to be investigated in greater depth. Some studies have shown that CFS is accompanied by irritable bowel syndrome (IBS), and an increase in the level of unclassified Bacteroidota is the main marker for the absence of IBS.⁵¹

5. Conclusion

To summarize, SeNPs obtained by redox reactions can be dispersed using the ultrasound-assisted method, and then, SeNPs and SCP with complex structures can be bonded via hydrogen bonding to form U-SCPSeNPs; however, when the ultrasound treatment time exceeds a certain threshold, hydrogen bonds break, and the size of SeNPs increases. U-SCPSeNP 40 had a better effect on improving the CFS in mice, which suggests that smaller SeNPs with a higher Se content can more effectively improve the CFS. However, a strong association exists between smaller SeNPs and higher Se contents. Thus, a novel method is needed to prepare SeNPs with smaller particle sizes, and ultrasound-assisted methods need to be investigated in greater depth. CFS is a long-term chronic disease, and U-SCPSeNPs have a greater advantage over salidroside in terms of gut microbiota improvement. This study provides new ideas for preparing polysaccharide nanosized Se. This study also provides data to support the use of U-SCPSeNPs as functional foods to ameliorate CFS- and CFS-induced disorders of the gut microbiota.

Ethics statement

All animal experimental procedures followed animal ethics guidelines and were approved by the Animal Ethics Committee of Harbin University of Commerce (Approval No. HSDU2020-065, Harbin, China).

Author contributions

Jingyang Wang: writing – original draft, methodology. Xin Wang: supervision, writing – review & editing, project administration. Weiye Xiu: conceptualization. Chenchen Li: formal analysis. Shiyou Yu: formal analysis. Haobin Zhu: data curation. Chenxi Yang: methodology. Kechi Zhou: data curation. Yongqiang Ma: conceptualization.

Data availability

Data will be made available on request.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was financially Supported By Program for Young Talents of Basic Research in Universities of Heilongjiang Province (YQJH2024097). The authors are grateful to supports.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4fo04195j

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