4.2. Method

The extraction of LBP was based on the previous method [54], and some modifications were made. Lycium barbarum was cleaned, dried, and crushed according to 1:20 (g/mL), and then, 90% ethanol was added at 70 °C for 2 h, followed by degreasing of the pigment. The residue was made in a ratio of 1:25 (g/mL) by adding distilled water at 90 °C for 4 h; filter supernatant was made in a ratio of 1:4 (V:V) by adding anhydrous ethanol and precipitating overnight. The precipitate was dissolved in a small amount of distilled water, and a sevage reagent (chloroform: n-butanol = 4:1) was added according to a ratio of 1:5 (V:V). The protein was separated by a liquid funnel and repeated three times. The residue was concentrated by rotary evaporation to allow viscosity and freeze-dried at −80 °C and 2.5 Pa for 1 d.

The polysaccharide content was determined by a phenol–sulfuric acid method [55]. Standard curve preparation: 100 mg/mL glucose reference solution; 0.1, 0.2, 0.4, 0.8, and 1.0 mL were placed in test tube, supplemented with distilled water to 2.0 mL. After shaking with 1.0 mL 6% phenol, 5.0 mL concentrated sulfuric acid was rapidly dropped and immediately shaken. After being placed for 5 min, the mixture was heated in a boiling water bath for 15 min and cooled to room temperature (25 °C). The absorbance (A) was measured at 490 nm, and the standard curve was obtained with the mass concentration of glucose as the abscissa and the absorbance as the ordinate. The regression equation is Y = 6.3804X − 0.0279, R² = 0.9992.

Determination of polysaccharide content: LBP was configured to 1 mg/mL aqueous solution, polysaccharide solution 2.0 mL, and determination of polysaccharide content. Each sample was subjected to three parallel experiments. The determination method is the same as above, using the standard curve to calculate the polysaccharide content.

The yield of polysaccharide is calculated as follows:

M_t is the quality of the total polysaccharide, M_s is the quality of the sample, and C_p is the content of polysaccharide.

The final yield of LBP was 4.73%.

LBP was prepared into an aqueous solution with a mass concentration of 20 mg/mL, and a 30 mL crude polysaccharide solution was taken. The supernatant was separated by DEAE-52 cellulose column chromatography and eluted with distilled water. The elution flow rate was maintained at 1 mL/min. One tube was collected every 6 min, and the change in sugar content was detected by the phenol–sulfuric acid method [56]. The eluent was collected, and the polysaccharide content was detected by the phenol–sulfuric acid method after freeze-drying. The final purified polysaccharide content was 97.22%.

The chemical structure of LBP was analyzed by Fourier transform infrared spectroscopy [57]. Precisely weighed LBP (2 mg) and potassium bromide (200 mg) were pressed into tablets, and a blank control using potassium bromide powder was pressed into tablets; these tablets were separately placed in Fourier transform infrared spectrometer FT-IR650 (Tianjin Port East Technology Development Co., Ltd., Tianjin, China) scanning records.

The monosaccharide composition of LBP was determined by ion chromatography [58]. Then, 5 mg of LBP was precision weighed in ampoan ule bottle, 2 mL 3 mol/L trifluoroacetic acid as added, and the mixture was hydrolyzed at 120 °C for 3 h. The acid hydrolysis solution was transferred to the tube and dried by nitrogen blowing. Next, 5 mL of deionized water was added, dissolved and mixed. Then, 50 μL was added to 950 μL deionized water and centrifuged at 12,000 rpm for 5 min. The supernatant was analyzed by ion chromatography (Thermo Fisher, Shanghai, China). Chromatographic conditions: chromatographic column: Dionex Carbopac^TM PA20 (3 × 150 mm); mobile phase: A: H₂O; B: 15 mM NaOH:100 mM NaOAC (1:1); flow rate: 0.3 mL/min; sample size: 5 μL; column temperature: 30 °C; detector: Electrochemical detector.

The results were compared with 16 monosaccharide standards (fucose (Fuc), aminogalactose hydrochloride (GalN), rhamnose (Rha), arabinose (Ara), glucosamine hydrochloride (GlcN), galactose (Gal), glucose (Glu), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galactose aldehyde acid (GalA), glucuronic acid (GlcA), N-acetyl-D-glucosamine (GlcNAc), guluronic acid (GulA), and mannose aldehyde acid (ManA)) to determine the monosaccharide species and molar ratio.

LBPT were composed of 20% LBP and 80% excipients (35% lactose, 3% sucralose, 2% ethanol, 20% citric acid and 20% sodium bicarbonate). After the powder raw materials were mixed evenly, wolfberry polysaccharide effervescent tablets were pressed in a single press.

This study used 4-week-old male SD rats. Animals were maintained in a 12/12 h light/dark cycle, turned on at 7:00 a.m. and off at 7:00 p.m., with free access to normal food and water. After one week of adaptive culture, the rats were randomly divided into 8 groups (Figure 3A):

After 28 consecutive days of daily drug intervention, groups (SC) to (HET) were assigned for forced swimming test, and rats in the group (SC) were used as non-swimming control. After the swimming experiment, rats in all groups were sacrificed, and blood, skeletal muscle, liver, heart and kidney were collected. Blood samples were centrifuged at 3500 rpm for 10 min at 4 °C to separate serum. All samples are stored at −80 °C for subsequent detection.

Forced swimming tests were performed 1 h after the last drug intervention, as described earlier, with minor modifications [59]. Rats were placed separately in a swimming pool (150 × 280 × 280 cm) filled with water (25 ± 2 °C), and lead wires (5% of body weight) were connected to the tail roots of each rat. When uncoordinated movement was observed in each rat, and the rats failed to return to the water within 5 s, the exhaustive swimming time was recorded.

The activity of CK, AST, AST and AST, as well as the content of BUN, BLA and Glu in serum, were measured and calculated using certain kits. Liver tissue was homogenized with 0.9% saline, and LG content was determined and calculated using a kit. The muscle was homogenized with 0.9% saline, and the activities of CAT, SOD and PK, as well as the contents of MDA, GSH, ATP and MG, were measured and calculated with the kits.

A small part of the liver tissue was fixed with 10% neutral buffer formalin solution for 24 h, and then dehydrated by different grades of ethanol (80%, 95% and 100%), transparent in xylene and covered with paraffin. A 0.2 μm slice was then cut from paraffin-embedded tissue using a slicer (Leica Instruments GmbH, Wetzlar, Germany). The slides were prepared by dewaxing, stained with H&E, and sealed with neutral gum. Microphotographs were obtained using a Nikon DS-Ri2 microscope (Nikon Corporation, Tokyo, Japan).

Another part of the liver tissue was fixed with 2.5% glutaraldehyde and 4% paraformaldehyde at 4 °C for 2 h, rinsed with 0.1 M phosphate buffer, and then fixed with 1% osmic acid at 4 °C for 2 h. After rinsing with 0.1 M phosphate buffer, different grades of acetone (30%, 50%, 70%, 90% and 100%) were dehydrated. After embedding, sections were stained with uranium acetate and lead citrate, and finally observed using a JEM-1400 PLUS transmission electron microscope (Japan Electronics Corporation, Tokyo, Japan).

Total RNA was extracted from rat muscle tissue by using TRIzol and reverse transcribed. A Roche LightCycler 96 real-time fluorescence polymerase chain reaction system (Roche, Shanghai, China) was used for a quantitative real-time polymerase chain reaction, and amplification was carried out in 20 μL reaction volumes containing 1×SYBR Green. The relative expression of each gene was calculated compared with β-actin. The primers used for RT-PCR are shown in Table 1.

RT-PCR primer sequences.

Extract muscle protein using RIPA lysate. Protein concentration was determined by the BCA method. β-actin, Nrf2, KEAP1, NQO1, HO-1, AMPKα1, pAMPKα1, PGC1-1α, Nrf1 and TFAM proteins were isolated by SDS-PAGE gel electrophoresis system (WIX Technology Beijing Co., Ltd., Beijing, China). The protein was transferred to 0.45 μm NC membrane by an electro-transfer membrane system (Shanghai Peiqing Technology Co., Ltd., Shanghai, China) and blocked in 5% BSA for 1 h. The NC membrane was incubated with anti-β-actin (1:1000), Nrf2 (1:1000), Keap1 (1:1000), NQO1 (1:1000), HO-1 (1:1000). AMPKα1 (1:1000), p-AMPKα1 (1:1000), PGC-1α (1:1000), NRF1 (1:1000), TFAM (1:1000) were incubated overnight at 4 °C. After washing with 1×TBST, HRP Goat Anti-Rabbit IgG (1:4000) was incubated for 1 h at 25 °C. After washing with 1×TBST, ECL chemiluminescence solution was used for detection in an ODYSSEY Fc dual-color infrared laser imaging system (LI-COR, Lincoln, NE, USA).

The SPSS 17 software (IBM SPSS Modeler, Chicago, IL, USA) was used for statistical analysis of the results. Data were analyzed by one-way ANOVA to evaluate the difference between the two groups. A person correlation analysis was used to evaluate the correlation between different parameters. The heatmap and PCA graph was drawn by Omicshare (GeneDenovo Biotechnology Co Ltd., Guangzhou, China). The data are expressed as the mean ± standard deviation (mean ± SD). Results with * p < 0.05, ** p < 0.01, and *** p < 0.001 were considered significant.