Application of lactobacillus rhamnosus SDSP202418 in preparation of medicine or food for improving exercise-induced fatigue

Abstract

The invention provides application of lactobacillus rhamnosus SDSP202418 in preparation of a medicine or food for improving exercise-induced fatigue, and relates to the technical field of microorganisms. The lactobacillus rhamnosus SDSP202418 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number of the lactobacillus rhamnosus SDSP202418 is CGMCC No.29613. The research finds that the lactobacillus rhamnosus SDSP202418 can effectively improve the exercise-induced fatigue, and has a wide application prospect in the field of medicines or foods for improving the exercise-induced fatigue.

Description

Technical Field

[0001] The invention provides application of Lactobacillus rhamnosus SDSP202418 in preparing a medicine or food for improving sports fatigue, belonging to the technical field of microorganisms. Background Art

[0002] The most direct and objective manifestation of body fatigue is the decline in exercise endurance, and the time to exercise exhaustion is the most commonly used indicator of body endurance.

[0003] Biochemical indicators that indicate the occurrence of sports fatigue include urea and urea nitrogen. Serum urea (UA) is a metabolite of protein and amino acid decomposition in the body and is a common indicator for evaluating the degree of fatigue after exercise. Intense exercise and heavy physical labor will accelerate nucleotide metabolism. Urea is produced after the decomposition and conversion of nucleotides and nucleosides. At this time, the blood urea content increases. At present, animal experiments have confirmed that supplementing with specific probiotics can alleviate sports fatigue to a certain extent. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an application of Lactobacillus rhamnosus SDSP202418 in the preparation of a medicine or food for improving sports fatigue. Previous studies have confirmed that Lactobacillus rhamnosus has a significant antioxidant effect. However, few studies have investigated the effect of Lactobacillus rhamnosus on improving sports fatigue. The present invention is to isolate a new lactic acid bacterium Lactobacillus rhamnosus SDSP202418 from traditional shrimp paste, and explore the mechanism of Lactobacillus rhamnosus SDSP202418 in improving running fatigue. This study provides a theoretical basis for the development and preparation of medicines or foods for improving sports fatigue.

[0005] The Lactobacillus rhamnosus SDSP202418 strain provided by the invention is separated from Shandong shrimp paste, and is stored in the China General Microbiological Culture Collection Center (CGMCC, Beijing, China), with a number of CGMCC No.29613.

[0006] In order to explore the effect of Lactobacillus rhamnosus SDSP202418 on improving exercise-induced fatigue in mice, 24 mice were randomly divided into 4 groups: normal group (Nor), exercise model control group (Con), positive control group (vitamin C, Vc, 200 mg / kg), SDSP18 group (Lactobacillus rhamnosus SDSP202418, 1×10 9The mouse exercise model was established by 3 weeks of running training, during which Vc and Lactobacillus rhamnosus SDSP202418 were supplemented daily. The body weight and major organ weights, running exhaustion time, serum biochemical indexes (blood glucose, LA, BUN, UA, LDH, AST and ALT), gene expression of antioxidant enzymes (Cu / Zn SOD, Mn SOD and CAT) in liver and skeletal muscle, and gene expression of MyHC I, MyHC IIa, MyHC IIb, MyHC IIx, PGC-1α and Sirt1 in skeletal muscle were measured.

[0007] Compared with the control group, supplementation with Lactobacillus rhamnosus SDSP202418 can effectively improve sports-induced fatigue and has broad application prospects in the field of drugs or foods for improving sports-induced fatigue. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Effects of Lactobacillus rhamnosus SDSP202418 on body weight (A) and blood glucose (B) of mice;

[0009] Figure 2 The effect of Lactobacillus rhamnosus SDSP202418 on the exhaustion time (A) and organ index (B) of mice;

[0010] Figure 3 Effects of Lactobacillus rhamnosus SDSP202418 on the antioxidant capacity of serum (A), liver (B) and skeletal muscle (CD) in mice;

[0011] Figure 4 Effects of Lactobacillus rhamnosus SDSP202418 on the mRNA expression levels of MyHC I (A), MyHC IIa (B), and MyHC IIx (C) in muscle;

[0012] Figure 5 Effects of Lactobacillus rhamnosus SDSP202418 on the mRNA expression levels of PGC-1α (A) and Sirt1 (B) in muscle;

[0013] Figure 6 Effects of Lactobacillus rhamnosus SDSP202418 on histopathology in (A) liver and (B) skeletal muscle. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical scheme and advantages of the present invention clearer, the preferred embodiments of the present invention are further described in detail in conjunction with the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0015] 1 Materials and reagents

[0016] Experimental strain: Lactobacillus rhamnosus SDSP202418 was isolated from Shandong shrimp paste. It was deposited in the China General Microbiological Culture Collection Center (CGMCC, Beijing, China) with the number CGMCC No.29613 and the preservation date of January 12, 2024. Its Latin name is Lacticaseibacillus rhamnosus.

[0017] Table 1 Experimental materials and reagents

[0018]

[0019]

[0020] 2. Instruments and Equipment

[0021] Table 2 Instruments and equipment

[0022]

[0023] 3 Experimental methods

[0024] 3.1 Animal Model

[0025] The experiment used 6-week-old Kunming mice weighing between 20-22g, which were purchased from the Experimental Animal Center of Chongqing Medical University. The mice were placed in a warm room at 20-24°C, 50% relative humidity, and a 12h / 12h light-dark cycle. All mice were provided with a standard diet, food, and water. The mice were randomly divided into four groups, with 6 mice in each group. The normal group and the control group mice were gavaged with 200μL of 0.9% saline. The vitamin C (Vc) group and the SDSP18 group were gavaged with 0.1mL / 10g (mouse body weight) of 200mg / kg vitamin C and rhamnosus lactobacillus SDSP202418 (1.0×10 9 CFU / mL).

[0026] The mice in the exercise control group and the exercise drug group underwent a one-day adaptive treadmill training (slope of 0°) at a speed of 10m / min, and exercised for 10min every day. After one week of adaptive training, the intensive exercise training model was to set the speed of the mouse exercise treadmill to 10m / min, with a treadmill slope of 5°, 10 minutes a day, and 6 consecutive days a week. After 2 weeks of continuous training, the mice were subjected to fatigue testing on the second day after the high-intensity training. The test slope was 15° and the running speed was 20m / min. Under electrical stimulation, the exhaustion judgment standard was that the mouse prostrated on all fours, could not continue to exercise under acoustic and optical stimulation, and could not complete the righting reflex. Finally, blood was collected from the mouse eyeballs, and the liver, kidney, heart, spleen, lung and skeletal muscle were dissected for subsequent analysis.

[0027] 3.2 Determination of serum cytokine and antioxidant levels in mice

[0028] Strictly follow the method in the kit instructions to determine the levels of glucose, BUN and UA in mouse serum and the activities of LDH, ALT, AST and CAT.

[0029] 3.3 H&E staining of mouse liver and skeletal muscle tissue sections

[0030] Mouse liver and skeletal muscle tissues were obtained and fixed with 4% paraformaldehyde. Skeletal muscle tissue blocks with a thickness of about 2 μm were cut, dehydrated, transparent, waxed and embedded. After embedding, conventional sections with a thickness of 4 μm were continuously cut on a slicer and fixed on a slide. After dewaxing by immersion in alcohol and xylene, they were immersed in hematoxylin staining solution, quickly bleached with distilled water, stained with eosin staining solution, dehydrated, transparently mounted, and the morphological changes of skeletal muscle tissue were observed under an optical microscope.

[0031] 3.4 Determination of mRNA in mouse liver and skeletal muscle tissue

[0032] RNA was extracted using Trizol reagent.

[0033] (1) First-strand cDNA synthesis (RNA reverse transcription) was performed strictly according to the instructions of the kit (HiScript SuperMix).

[0034] (2) PCR reaction system: Prepare PCR reaction solution according to the instructions of the kit (SYBR Green PCR Master Mix). The primer sequences used are shown in Table 3. The expression level of the target gene was analyzed using the formula 2-ΔΔCT. β-actin was selected as the internal reference.

[0035] Table 3 Primer sequences of each gene

[0036]

[0037]

[0038] 3.5 Data processing

[0039] SPSS 22.0 statistical software was used for data analysis. One-way analysis of variance (ANOVA) was used to analyze the differences among the groups. The data were expressed as mean ± standard deviation. P < 0.05 indicated a significant difference among the groups.

[0040] 4 Results and analysis

[0041] 4.1 Effects of Lactobacillus rhamnosus SDSP202418 on body weight and blood glucose in mice

[0042] Depend on Figure 1 A shows that there was no significant difference in the initial body weight of the four groups of mice. After four consecutive weeks of supplementation with Lactobacillus rhamnosus SDSP202418, the weight of the four groups of mice increased to varying degrees. The control group was 0.4%, 2.6% and 1.2% less than the normal group, vitamin C group and SDSP18 group, respectively, but there was no significant difference (P>0.05). Blood sugar is the direct energy source for the body during exercise. Figure 1 As shown in Figure B, compared with the normal group, the blood glucose levels of the exercise mice were significantly reduced, especially those supplemented with vitamin C and probiotics. This may be due to the higher glucose consumption in the vitamin C and SDSP18 groups, which means that the mice supplemented with Lactobacillus rhamnosus SDSP202418 will obtain more sufficient energy supply during exercise, thereby enhancing exercise capacity and prolonging the duration of exercise.

[0043] 4.2 Effects of Lactobacillus rhamnosus SDSP202418 on exercise exhaustion time and organ indexes in mice

[0044] Exhaustion time is the most intuitive data to describe sports endurance. The normal group did not undergo fatigue experiments. Figure 2 A shows that after 3 weeks of daily running training, the exhaustion time of mice supplemented with rhamnosus lactobacillus SDSP202418 and vitamin C was 3.24 times and 2.01 times that of the control group, respectively, with significant differences (P<0.05). At the same time, the exhaustion time of mice supplemented with rhamnosus lactobacillus SDSP202418 was 2219s longer than that of the vitamin C positive group, indicating that supplementing with rhamnosus lactobacillus SDSP202418 can significantly improve endurance and delay fatigue.

[0045] The ratio of the main organs to body weight of mice is as follows Figure 2 As shown in B, compared with the normal group, there was no significant difference in the organ indexes of the heart, spleen and testis of mice in the vitamin C and SDSP18 groups, indicating that the intake of rhamnosus lactobacillus SDSP202418 during 2 weeks of running exercise had no adverse effects on the body weight and organ weight of mice. The liver index of the exercise group was significantly higher than that of the normal group, indicating that exercise fatigue can cause liver damage. The liver index of the SDSP18 group decreased to the level of the normal group, indicating that rhamnosus lactobacillus SDSP202418 has a good effect on improving the liver damage of mice with exercise fatigue. Compared with the normal group, the muscle index of the other three groups increased, among which the SDSP18 group increased significantly, indicating that adaptive exercise can improve the muscle index of mice, and probiotic intervention promotes the increase of muscle mass.

[0046] 4.3 Effects of Lactobacillus rhamnosus SDSP202418 on serum biochemical parameters in mice

[0047] The biochemical indices of mice after exhaustive exercise are shown in Table 4. Blood urea nitrogen (BUN) is a product of aerobic protein metabolism. The worse the body's ability to adapt to exercise load, the more obvious the increase in serum urea nitrogen. The serum BUN content in the vitamin C group and the SDSP18 group was 32.24% and 41.49% lower than that in the control group, respectively. LDH mainly catalyzes the reversible reaction between pyruvate and lactate and is an important enzyme in the body's anaerobic sugar metabolism pathway. Compared with the normal group, the LDH in the control group was significantly increased. Compared with the control group, the serum LDH in the vitamin C group and the SDSP18 group was significantly reduced by 24.6% and 42.7%, respectively. The above indicators comprehensively reflect that Lactobacillus rhamnosus SDSP202418 can relieve exercise fatigue.

[0048] The liver index of the control group increased significantly ( Figure 2 A), indicating that the liver of mice was abnormal after exercise fatigue. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are important indicators commonly used to reflect liver function in clinical and experimental settings. High levels often indicate impaired liver function. Therefore, the experiment further detected the serum ALT and AST levels. The ALB and AST levels in the serum of mice in the control group were significantly higher than those in the normal group. Rhamnosus lactobacillus SDSP202418 and vitamin C can significantly reduce the ALT and AST levels in the serum of mice with exercise fatigue. This shows that Rhamnosus lactobacillus SDSP202418 can improve abnormal liver function in mice after exercise fatigue.

[0049] Table 4 Effects of Lactobacillus rhamnosus SDSP202418 on serum biochemical parameters of mice

[0050]

[0051] The values ​​are expressed as mean ± standard deviation. Lowercase letters indicate significant differences among the groups (P<0.05).

[0052] 4.4 Effect of Lactobacillus rhamnosus SDSP202418 on the antioxidant capacity of mice

[0053] Body exercise will produce a large number of free radicals. Moderate exercise will activate the antioxidant defense system and eliminate free radicals. However, excessive exercise will reduce the body's ability to eliminate free radicals, leading to cell damage. Therefore, antioxidant factors are urgently needed to eliminate them.

[0054] Effects of Lactobacillus rhamnosus SDSP202418 on antioxidant factors in mouse serum Figure 3A. CAT is the body's main antioxidant defense system, and its activity can directly reflect the strength of the antioxidant capacity. Compared with the control group, the CAT activity of the SDSP18 group and the positive control group increased by 81.5% and 93.5%, respectively. This shows that Lactobacillus rhamnosus SDSP202418 can effectively improve the total antioxidant capacity of mice with exercise fatigue, reduce the degree of cellular oxidative damage, and enhance the body's antioxidant system capacity. mRNA expression of liver oxidative stress-related indicators such as Figure 3 As shown in B, the Cu / Zn SOD expression level of mice in the SDSP18 group was 1.7 times that of mice in the control group, and recovered to the same level as the normal group. This indicates that Lactobacillus rhamnosus SDSP202418 can improve the body's antioxidant capacity by increasing the expression of antioxidant enzymes in the liver, thereby alleviating fatigue. The mRNA expression levels of Mn SOD and CAT in skeletal muscle are shown in Figure 3 As shown in C and D. Rhamnosus lactobacillus SDSP202418 significantly promoted the expression of antioxidant enzyme genes, resulting in an increase of 53.06% and 65.2% in the content of Mn SOD and CAT compared with the control group, respectively. This shows that Rhamnosus lactobacillus SDSP202418 can regulate the expression of oxidative stress-related genes in skeletal muscle and improve exercise-induced skeletal muscle oxidative damage. 4.5 Rhamnosus lactobacillus SDSP202418 regulates muscle fiber type transformation

[0055] The composition of different muscle fibers plays a vital role in regulating muscle function and exercise endurance. Figure 4 As shown, vitamin C and Lactobacillus rhamnosus SDSP202418 significantly increased the expression of slow muscle fibers (MyHC I), which was 6 times and 13 times that of the control group. At the same time, Lactobacillus rhamnosus SDSP202418 significantly downregulated the expression of two fast muscle fibers (MyHC IIa and IIx).

[0056] The mRNA expression levels of genes related to the AMPK / PGC-1α pathway were as follows Figure 5 Compared with the control group, the SDSP18 group significantly upregulated the expression of PGC-1α and Sirt1 genes, increasing by 1.6 and 20 times, respectively. 4.6 Effects of Lactobacillus rhamnosus SDSP202418 on the morphological structure of mouse liver and skeletal muscle tissue

[0057] Figure 6Results of the evaluation of potential pathological changes in different liver and muscle tissues after programmed exercise training and probiotic supplementation are shown. In the normal group, hepatocytes were arranged radially with the central vein as the center, and the hepatic sinusoids and cords were arranged in an orderly manner. However, during forced exercise in the control group, the hepatocytes were arranged in a disordered manner, the nuclei and cytoplasm were condensed, and the nuclear membrane and nucleolus were ruptured. There was no significant change in the arrangement of the hepatic sinusoids and cords between the groups after supplementation with vitamin C or Lactobacillus rhamnosus SDSP202418 ( Figure 6 A), similar to the normal group, indicating that Lactobacillus rhamnosus SDSP202418 treatment can improve oxidative damage in mouse liver tissue.

[0058] Excessive exercise can lead to muscle tissue damage, which is manifested as blurred muscle bundle boundaries, irregular muscle fiber arrangement, interlaminar diffusion, enlarged gaps, connective tissue hyperplasia, inflammatory cell infiltration, and cell apoptosis. No Zenker degeneration, hyperplasia, or inflammatory cell infiltration was observed in the muscle tissue of the normal group. It was observed that larger tissue gaps appeared in the control group, which may be caused by muscle strain. It is worth noting that tissue cracks and necrotic muscle tissue were significantly reduced in the Vc group and the SDSP18 group. Supplementing the diet with Lactobacillus rhamnosus SDSP202418 or VC can help improve this phenomenon, reduce inflammatory infiltration, reduce muscle fiber gaps, prevent exercise-induced muscle tissue damage, and help improve the exercise endurance of mice ( Figure 6 b).

[0059] 5 Conclusion

[0060] The research of the present invention shows that supplementation of Lactobacillus rhamnosus SDSP202418 can restore the UA content of mice that have been subjected to long-term exhaustive exercise to normal levels. This indicates that Lactobacillus rhamnosus SDSP202418 can improve the energy supply and utilization capacity of the metabolic system and improve the body's exercise level. Blood urea nitrogen is the main end product of human protein metabolism. When the body exercises for a long time and cannot obtain enough energy through the decomposition of sugar and fat, proteins and amino acids in skeletal muscle participate in catabolism to provide energy and produce urea nitrogen. The results of this experiment show that supplementation of Lactobacillus rhamnosus SDSP202418 during 3 weeks of running training can significantly reduce the level of urea nitrogen in the serum of exercise mice, indicating that supplementation of probiotics can enhance the exercise capacity of exercise mice and improve the symptoms of exercise fatigue.

[0061] According to the contractile ability and energy metabolism characteristics of skeletal muscle fibers, skeletal muscle fibers are composed of slow muscle fibers and fast muscle fibers. Slow muscle fibers contract slowly and have strong exercise tolerance, while fast muscle fibers contract quickly and are prone to fatigue. Lactobacillus rhamnosus SDSP202418 can promote the formation of MyHC I in slow muscle fibers and reduce the formation of fast muscle fibers (MyHCIIa and MyHC IIx). The AMPK / PGC-1α pathway is an important regulatory pathway for mitochondrial function and muscle fiber type. PGC-1α and sirtuin1 (Sirt1) are two important downstream factors of AMPK signaling. It has been reported that peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) can activate the expression of slow muscle fiber genes, and overexpression of the PGC-1α gene can increase MyHC I. PGC-1α, as a transcriptional coactivator, is closely related to the energy metabolism of cells. Lactobacillus rhamnosus SDSP202418 can alleviate the phenomenon of downregulation of PGC-1α and Sirt1 gene expression caused by exercise. The results showed that Lactobacillus rhamnosus SDSP202418 promoted the formation of slow muscle fibers and reduced the formation of fast muscle fibers. SDSP18 regulated the transformation of skeletal muscle types by affecting myotube differentiation through the AMPK / PGC-1α pathway, improved skeletal muscle energy metabolism, and alleviated fatigue progression.

[0062] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. Application of Lactobacillus rhamnosus SDSP202418 in the preparation of a drug or food for improving sports fatigue, characterized in that: The Lactobacillus rhamnosus SDSP202418 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.29613.

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