Application of lactobacillus paracasei JLPF-131 preparation in preparation of medicine for improving obesity induced by optical rhythm disorder

Abstract

The invention relates to the technical field of probiotics, in particular to application of a lactobacillus paracasei JLPF-131 preparation in preparation of a medicine for improving obesity induced by optical rhythm disorder, and the lactobacillus paracasei JLPF-131 preparation comprises inactivated thalli of lactobacillus paracasei JLPF-131; the lactobacillus paracasei JLPF-131 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 8, 2019, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO.18098. The lactobacillus paracasei JLPF-131 has the advantages that the lactobacillus paracasei JLPF-131 can be used for preparing the lactobacillus paracasei JLPF-131; the lactobacillus paracasei JLPF-131 preparation provided by the invention can prevent obesity caused by prolonged light exposure time.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and in particular to application of a Lactobacillus paracasei JLPF-131 preparation in the preparation of a medicine for improving obesity induced by photorhythmia. Background Art

[0002] In modern society, shift work and a vibrant nightlife increase the duration and intensity of people's nighttime light exposure. Studies have shown that nighttime light exposure is a direct factor influencing the disruption of endogenous circadian clocks and circadian rhythm disorders. Disrupting physiological processes controlled by endogenous circadian clocks can lead to a variety of adverse diseases, including obesity. Photorhythm disorder-induced obesity is a special type of obesity caused by abnormal light cycles that interfere with the body's circadian rhythm system. The human circadian clock receives environmental light signals through the retina to regulate metabolic rhythms. When exposed to unnatural light conditions such as nighttime light and shift work for a long time, the synergistic effect of the circadian clock and metabolic system is disrupted, leading to abnormal adipose tissue metabolism, increased insulin resistance, and imbalanced appetite regulation, which in turn causes obesity. Unlike ordinary obesity, which is mainly caused by excessive energy intake, insufficient exercise, or genetic factors, the core pathological mechanism of photorhythm disorder-induced obesity lies in the interference of environmental light with the circadian rhythm system. Even if these patients maintain normal eating and exercise habits, they may still experience metabolic disorders and weight gain.

[0003] At present, the prevention and treatment of obesity induced by photorhythm disorders mainly include adjusting the lighting environment, improving lifestyle and using drug intervention. For example, wearing blue light blocking glasses to reduce light stimulation at night, or using melatonin supplements to assist in regulating the biological clock. However, existing prevention and treatment methods have significant limitations: adjusting the lighting environment and improving lifestyle require patients to maintain a high degree of self-discipline for a long time, with poor compliance, and it is difficult to completely reverse the disordered biological rhythm; although drug intervention can regulate metabolism to a certain extent, it may cause adverse reactions such as drowsiness and headaches, and long-term use also has potential drug resistance and safety risks, which cannot meet the urgent clinical demand for safe and effective intervention methods. Summary of the Invention

[0004] In response to the technical problems that existing prevention and treatment methods for obesity induced by photorhythm disorders have poor compliance and difficulty in reversing disordered biological rhythms, as well as adverse reactions, potential drug resistance and safety risks caused by drug intervention, the present invention provides a use of a Lactobacillus paracasei JLPF-131 preparation in the preparation of a medicine for improving obesity induced by photorhythm disorders.

[0005] The technical solutions of the present invention are as follows: A Lactobacillus paracasei JLPF-131 preparation is used in the preparation of a medicine for improving obesity induced by light rhythm disorder, wherein the Lactobacillus paracasei JLPF-131 preparation comprises inactivated cells of Lactobacillus paracasei JLPF-131; Lactobacillus paracasei ( Lactobacillus paracasei ) JLPF-131 was deposited in the General Microbiology Center of China Culture Collection Administration on July 8, 2019. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCCNO.18098.

[0006] Furthermore, the activated Lactobacillus paracasei JLPF-131 was inoculated into MRS liquid culture medium and cultured at 37° C. for 24-48 hours to obtain a bacterial solution; the bacterial solution was heat-inactivated and then centrifuged to collect the bacteria; the bacteria were resuspended in physiological saline and freeze-dried to obtain a Lactobacillus paracasei JLPF-131 preparation.

[0007] Furthermore, the number of bacteria in the Lactobacillus paracasei JLPF-131 preparation was 1.0×10 8 cfu / g.

[0008] Furthermore, the Lactobacillus paracasei JLPF-131 preparation also includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients are glucose, erythritol or maltodextrin.

[0009] Furthermore, improving obesity induced by light rhythm disturbance includes preventing obesity caused by prolonged light exposure.

[0010] Furthermore, preventing obesity caused by prolonged light exposure includes reducing body weight; or / and, increasing satiety; or / and, reducing blood sugar levels; or / and, reducing body fat; or / and, reducing insulin resistance index.

[0011] Furthermore, prevention of obesity caused by prolonged light exposure includes increasing the proportion of CD68+ macrophages, increasing the expression level of NOD2, and decreasing the expression levels of TNF-α, IL-6 and / or IL-1β.

[0012] Furthermore, preventing obesity caused by prolonged light exposure includes regulating and lowering AST, ALT, TC and TG levels, increasing FGF21 hormone levels, or / and increasing melatonin levels.

[0013] Furthermore, the prevention of obesity caused by prolonged light exposure included increasing the phosphorylation level of AMPKα1, decreasing the protein expression of ACC, and increasing the protein expression of CPT1α.

[0014] Furthermore, preventing obesity caused by prolonged light exposure includes increasing GLP-1 and PYY levels and regulating the expression of POMC and NPY genes.

[0015] The beneficial effects of the present invention are: The present invention provides a use of a Lactobacillus paracasei JLPF-131 preparation in the preparation of a drug for ameliorating photorhythmia-induced obesity. The Lactobacillus paracasei JLPF-131 preparation exhibits significant efficacy in ameliorating photorhythmia-induced obesity. Experimental data indicate that the Lactobacillus paracasei JLPF-131 preparation can be used to prepare a drug for preventing obesity caused by prolonged light exposure. Specifically, at the metabolic regulation level, the preparation effectively regulates the body's energy metabolism balance by reducing body weight and body fat, lowering blood sugar levels, reducing the insulin resistance index, and improving satiety. The preparation also regulates and lowers AST, ALT, TC, and TG levels, while increasing FGF21 hormone levels and melatonin levels, further improving lipid metabolism and liver function. The preparation also increases the phosphorylation level of AMPKα1, reduces ACC protein expression, and increases CPT1α protein expression, promoting fat oxidation and decomposition and inhibiting fat synthesis, thereby effectively preventing obesity. In terms of inflammation regulation and immune improvement, this preparation can increase the proportion of CD68+ macrophages, upregulate NOD2 expression, and reduce the expression levels of TNF-α, IL-6, and / or IL-1β, helping to regulate inflammatory responses and alleviate the inflammatory state caused by photorhythm disorders, thereby preventing obesity. In terms of hormone and gene regulation, it can increase GLP-1 and PYY levels, regulate the expression of POMC and NPY genes, and further regulate appetite and metabolism by regulating the expression of intestinal hormones and related genes, synergistically playing a role in preventing obesity. DETAILED DESCRIPTION

[0016] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work should fall within the scope of protection of the present invention.

[0017] The Lactobacillus paracasei JLPF-131 used in the present invention has been disclosed in patent application CN112501089A (a strain of Lactobacillus paracasei JLPF-131 for improving hypertension and its use in a preparation).

[0018] Example 1 Screening of strains capable of removing active oxygen (1) Preparation of inactivated bacterial solution Lactobacillus paracasei JLPF-131 and 21 strains isolated and purified at the same time (for the isolation and purification methods, see CN112501089A) were activated and inoculated into MRS liquid culture medium, cultured at 37°C for 24 hours, and heat-inactivated (105°C, 30 minutes) to obtain inactivated bacterial liquid.

[0019] Wherein, the preparation method of MRS liquid culture medium is as follows: Take 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of potassium hydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, and 0.2 g of magnesium sulfate heptahydrate, dissolve them in distilled water, make up to 1 L, adjust the pH to 6.8, sterilize at 121°C and 0.1 MPa for 20 min, let cool, and then divide into aseptic packages for later use.

[0020] (2) Strain screening Place 400 μL of inactivated bacterial solution in a culture tube, add 1 mL of 0.2 mM DPPH-ethanol solution, incubate in the dark at room temperature for 60 minutes, and then measure the absorbance (A) at 517 nm using a spectrophotometer. Place 400 μL of distilled water in a culture tube, add 1 mL of 0.2 mM DPPH-ethanol solution, incubate in the dark at room temperature for 60 minutes, and then measure the absorbance (A0) at 517 nm using a spectrophotometer. Calculate the ROS scavenging rate based on the test results using the following formula:

[0021] The clearance rate of the inactivated bacterial liquid of 22 strains of bacteria was tested respectively. The clearance rate of the inactivated bacterial liquid of Lactobacillus paracasei JLPF-131 was the best, which was 72%.

[0022] Lactobacillus paracasei JLPF-131 was sent for identification by Sangon Biotech (Shanghai) Co., Ltd. The primers used in the identification process are as follows: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (as shown in SEQ ID NO: 1); 1492R:5'-GGTTACCTTGTTACGACTT-3' (as shown in sequence 2).

[0023] The Lactobacillus paracasei ( Lactobacillus paracasei

[0024] Further research will be conducted on Lactobacillus paracasei JLPF-131.

[0025] Example 2 Preparation of Lactobacillus paracasei JLPF-131 Preparation Frozen Lactobacillus paracasei JLPF-131 was activated on a 0.5% CaCO3 MRS plate. The activated Lactobacillus paracasei JLPF-131 was inoculated into MRS liquid medium at a 1% (w / w) inoculum and cultured at 37°C for 24 hours to obtain a bacterial suspension. The bacterial suspension was heat-inactivated (105°C, 30 minutes) and centrifuged. The cells were collected, washed with sterile saline, and resuspended in 15% (w / w) saline to obtain a suspension. The concentration of the suspension was adjusted to 1.0-2.0 × 10 10 cfu / mL, a bacterial suspension was obtained, and the bacterial suspension was freeze-dried to obtain the Lactobacillus paracasei JLPF-131 preparation. The number of bacteria in the Lactobacillus paracasei JLPF-131 preparation was 1.0×10 8 cfu / g.

[0026] The preparation method of MRS plate medium containing 0.5% CaCO3 is as follows: Take 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of potassium hydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, 0.2 g of magnesium sulfate heptahydrate, 5 g of CaCO3, and 15 g of agar, dissolve them in distilled water, make up to 1 L, adjust the pH to 6.8, and sterilize at 121°C and 0.1 MPa for 20 min. Then, under sterile conditions, pour the sterilized culture medium into a sterilized plate and let it cool for use.

[0027] The preparation method of MRS liquid culture medium is as follows: Take 10 g of peptone, 5 g of beef powder, 5 g of sodium acetate trihydrate, 2 g of potassium hydrogen phosphate heptahydrate, 1 mL of Tween-80, 0.05 g of manganese sulfate tetrahydrate, 2 g of triammonium citrate, 20 g of glucose, and 0.2 g of magnesium sulfate heptahydrate, dissolve them in distilled water, make up to 1 L, adjust the pH to 6.8, sterilize at 121°C and 0.1 MPa for 20 min, let cool, and then divide into aseptic packages for later use.

[0028] Example 3 Preparation of Lactobacillus paracasei JLPF-131 Preparation Frozen Lactobacillus paracasei JLPF-131 was activated on a 0.5% CaCO3 MRS plate. The activated Lactobacillus paracasei JLPF-131 was inoculated into MRS liquid medium at a 1% (w / w) inoculum and cultured at 37°C for 24 hours to obtain a bacterial suspension. The bacterial suspension was heat-inactivated (105°C, 30 minutes) and centrifuged. The cells were collected, washed with sterile saline, and resuspended in 15% (w / w) saline to obtain a suspension. The concentration of the suspension was adjusted to 1.0-2.0 × 10 10 cfu / mL, a bacterial suspension was obtained, which was freeze-dried and mixed with maltodextrin (purchased from Baolingbao Biotechnology Co., Ltd.) to prepare a Lactobacillus paracasei JLPF-131 preparation. The number of cells in the Lactobacillus paracasei JLPF-131 preparation was 1.0×10 8 cfu / g.

[0029] Experimental Example 1 Effects of Lactobacillus paracasei JLPF-131 Preparation on Body Weight, Blood Glucose, Food Intake and Other Indicators in Mice 1. Preparation Prepare bacterial coating solution: add 0.1 g gelatin to 1000 mL of purified water, heat in a 60°C water bath, and store at room temperature after the gelatin is completely dissolved.

[0030] 2. Grouping and Dosing 48 6-8 week old C57BL / 6J male mice were randomly selected and fed adaptively for one week. They were then randomly divided into four groups: blank control group, model group, low treatment group, and high treatment group. The light control requirements for each group of mice are as follows: Daytime (7:00-19:00): All four groups were exposed to 200 lux white light; At night (19:00-7:00): the model group, low treatment group and high treatment group were exposed to 5lux 480nm blue light, and the blank control group was placed in a dark environment (0lux).

[0031] During the light control period, the drug administration procedures for each group were as follows: Blank control group: 100 μL bacterial coating solution was gavaged daily; Model group: 100µL bacterial coating solution was gavaged daily; Low treatment group: The Lactobacillus paracasei JLPF-131 preparation prepared in Example 2 was dissolved in the bacterial coating solution to prepare a low concentration of inactivated Lactobacillus paracasei JLPF-131 coating solution (1×10 4 CFU / mL), 100 μL was administered orally daily; High treatment group: The Lactobacillus paracasei JLPF-131 preparation prepared in Example 2 was dissolved in the bacterial coating solution to prepare a high concentration of inactivated Lactobacillus paracasei JLPF-131 coating solution (1×10 9 CFU / mL), 100 µL was administered orally every day.

[0032] During the light control period, the hourly temperature of each group was 22±1℃, the humidity was 50%-60%, and the same feed and drinking water conditions were given.

[0033] 3. Phenotypic indicator detection (1) Body weight test: Measure the body weight of each group of mice before the start of the experiment and measure the body weight of the mice again after 8 weeks.

[0034] (2) Food intake detection: The weekly food intake of each group of mice was recorded, and the weekly food intake of each mouse was counted to detect the effect of drug administration on the relief of polyphagia symptoms.

[0035] (3) Blood glucose test: After 8 weeks of fasting overnight, blood was collected from the tail vein to measure the blood glucose of mice.

[0036] (4) Oral glucose tolerance test (OGTT, 2 g / kg glucose).

[0037] (5) Dual-energy X-ray absorptiometry (DEXA) was used to measure body fat percentage.

[0038] 4. Test results (1) Weight test results As shown in Table 1, before treatment (week 0), there was no significant difference in the body weight of the four groups of mice (P ≥ 0.05). After treatment (after 8 weeks), the body weight of the model group mice was significantly higher than that of the blank group (P < 0.05), indicating that the light rhythm disorder-induced obesity model was successfully established. The body weight of mice in the low- and high-treatment groups decreased significantly compared with the model group (P < 0.05), indicating that the Lactobacillus paracasei JLPF-131 preparation has the effect of treating rhythm disorder-induced obesity. The body weight of mice in the high-treatment group did not differ significantly from that of the blank group (P ≥ 0.05), indicating that the high-treatment group had a better therapeutic effect than the low-treatment group.

[0039] Table 1 Body weight test results of each group

[0040] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05), n = 12.

[0041] (2) Food intake detection Table 2 Food intake test results of each group

[0042] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05), n = 12.

[0043] As shown in Table 2, before treatment (week 0), there was no significant difference in the food intake of the four groups of mice (P≥0.05). After treatment (after 8 weeks), the daily food intake of the mice in the low-treatment group and the high-treatment group was significantly reduced compared with the model group (P<0.05). Therefore, oral administration of the Lactobacillus paracasei JLPF-131 preparation increased the satiety of the mice and reduced their daily food intake.

[0044] (3) Blood glucose test results Table 3 Blood glucose test results of each group

[0045] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05), n = 12.

[0046] As shown in Table 3, before treatment (week 0), there was no significant difference in the blood glucose levels of the four groups of mice (P ≥ 0.05). After treatment (after 8 weeks), the blood glucose levels of the mice in the low-treatment and high-treatment groups were significantly lower than those in the model group. Therefore, oral administration of the Lactobacillus paracasei JLPF-131 preparation would reduce the blood glucose level in the mice. There was no significant difference in the blood glucose levels of the mice in the high-treatment group compared with the blank group (P ≥ 0.05), indicating that the treatment effect of the high-treatment group was better than that of the low-treatment group.

[0047] (4) Body fat percentage and sugar resistance test results Table 4 Body fat percentage and anti-glycemic ability test results of each group

[0048] Note: Different lowercase letters in the same column indicate significant differences ( P <0.05), n=12.

[0049] As shown in Table 4, the body fat rate and insulin resistance index of mice in the low-treatment group and the high-treatment group were significantly lower than those in the model group (P < 0.05), and the insulin resistance index of mice in the high-treatment group was not significantly different from that in the blank group (P ≥ 0.05), indicating that the anti-glycemic ability of mice was significantly enhanced and the body fat rate was significantly decreased after treatment with the Lactobacillus paracasei JLPF-131 preparation, and the effect was better when administered by oral gavage at a high concentration.

[0050] Experimental Example 2 Effects of Lactobacillus paracasei JLPF-131 Preparation on CD68+ Macrophage Levels, NOD2 Receptor and Inflammatory Factor Levels 1. Detection of CD68+ macrophage levels Tissue isolation: At the end of the eighth week, mice were fasted overnight and sacrificed by cervical dislocation in Experimental Example 1. Liver tissue was obtained (this was performed simultaneously with blood sampling in Experimental Example 3). Liver tissue was mechanically minced and digested with collagenase IV (1 mg / mL) at 37°C for 30 minutes. Single-cell suspensions were obtained by filtration through a 70 μm filter.

[0051] Erythrocyte lysis: Treat the single-cell suspension with ACK lysis buffer (150 mmol / L NH4Cl, 10 mmol / L KHCO3, 0.1 mmol / L EDTA) for 5 minutes, centrifuge and remove the supernatant to remove interference from erythrocytes in the liver.

[0052] Cell labeling: Single cells after erythrocyte lysis were stained with anti-CD45-FITC (pan-leukocyte marker) and anti-F4 / 80-APC (macrophage marker) antibodies at 4°C. After incubation in the dark for 20 minutes, the cells were washed three times with PBS to remove unbound antibodies and reduce background interference.

[0053] Gating strategy: Exclude debris: Circle the live cell population in the FSC / SSC scatter plot.

[0054] Macrophage sorting: F4 / 80+ population (total macrophages) was screened from CD45+ cells.

[0055] CD68 subset analysis: Anti-CD68-PE antibody was used to further stain the cells to distinguish between CD68+ (mature macrophages) and CD68- (immature or inflammatory type) subsets.

[0056] Data acquisition: At least 10,000 events were acquired using a flow cytometer (BD FACSCanto II), and the proportion of CD68+ cells was analyzed using FlowJo software.

[0057] 2. Detection of NOD2 receptor and inflammatory factor levels (qPCR method) RNA extraction: Mouse liver tissue was lysed using TRIzol reagent, and the upper aqueous phase was collected by centrifugation after chloroform layer separation. RNA was precipitated with isopropanol, washed with 75% ethanol, and then dissolved in RNase-free water.

[0058] Reverse transcription: 1 μg of RNA was reverse transcribed into cDNA using HiScript RT SuperMix (reaction conditions: 25°C, 5 min, 50°C, 15 min, 85°C, 5 s).

[0059] Primer design: TNF-α: F: 5'-CAGGCGGTGCCTATGTCTC-3' (as shown in sequence 4); R: 5'-CGATCACCCCGAAGTTCAGT-3' (as shown in Sequence 5).

[0060] IL-6: F: 5'-TAGTCCTTCCTACCCCAATTTCC-3' (as shown in SEQ ID NO: 6); R: 5'-TTGGTCCTTAGCCACTCCTTC-3' (as shown in SEQ ID NO: 7).

[0061] IL-1β: F: 5'-GCAACTGTTCCTGAACTCAACT-3' (as shown in SEQ ID NO: 8); R: 5'-ATCTTTTGGGGTCCGTCAACT-3' (as shown in Sequence 9).

[0062] Internal reference GAPDH: F: 5'-AGGTCGGTGTGAACGGATTTG-3' (as shown in SEQ ID NO: 10); R: 5'-TGTAGACCATGTAGTTGAGGTCA-3' (as shown in SEQ ID NO: 11).

[0063] qPCR amplification: Reaction system: AceQ SYBR Green Master Mix 10 μL, cDNA 1 μL, primers 0.5 μM each, ddH2O to 20 μL.

[0064] Program: 95°C, 5 min → 40 cycles (95°C, 10 sec, 60°C, 30 sec) → melting curve analysis.

[0065] Data Analysis: The relative mRNA expression was calculated using the 2-ΔΔCt method, with the blank control group as the benchmark (expression = 1).

[0066] 3. Test results Table 5 Inflammation level results of each group

[0067] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05), n = 12.

[0068] As shown in Table 5, compared with the model group, the proportion of CD68+ macrophages in the obese mice in the low-treatment group and the high-treatment group was significantly increased after treatment (8 weeks later) (P < 0.05), indicating that treatment with the Lactobacillus paracasei JLPF-131 preparation can promote macrophage maturation and inhibit inflammatory responses. In addition, after treatment, TNF-α mRNA, IL-6 mRNA, and IL-1β mRNA in the obese mice in the low-treatment group and the high-treatment group were significantly decreased (P < 0.05), and NOD2 mRNA was significantly increased (P < 0.05), indicating that the Lactobacillus paracasei JLPF-131 preparation activates the NOD2 receptor, regulates the negative feedback mechanism of the NF-κB pathway (such as inhibiting its activity), thereby reducing the expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β, and alleviating the inflammatory response in obese mice.

[0069] Experimental Example 3 Effects of Lactobacillus paracasei JLPF-131 Preparation on Hormone Levels in Mice At the end of the eighth week, after an overnight fast, approximately 1 mL of blood was collected from the orbital cavity the next day and centrifuged at 3500 rpm for 15 minutes. The upper serum sample was frozen, and 500 μL of blood was collected from the heart and placed in an anticoagulant tube containing sodium heparin for routine blood tests and biochemical markers. Some of these blood samples were assayed for the following parameters using kits from Nanjing Jiancheng Bioengineering Co., Ltd., strictly following the manufacturer's instructions: aspartate aminotransferase (AST), alanine aminotransferase activity (ALT), cholesterol (TC), triglycerides (TG), fibroblast growth factor 21 (FGF21), and melatonin (MT). The results are shown in Table 6.

[0070] Table 6 Test results of blood samples taken from the hearts of mice in each group

[0071] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05), n = 12.

[0072] As shown in Table 6, the AST, ALT, TC, and TG levels in the low- and high-treatment groups were significantly lower than those in the model group, indicating that the Lactobacillus paracasei JLPF-131 preparation can effectively alleviate liver damage caused by obesity in mice and reduce blood lipid levels. The FGF21 and MT levels in the low- and high-treatment groups were significantly higher than those in the model group, indicating that the Lactobacillus paracasei JLPF-131 preparation can increase FGF21 hormone levels in mice, inhibit lipogenesis, and increase thermogenesis. The Lactobacillus paracasei JLPF-131 preparation can also increase melatonin levels in mice. In particular, the melatonin level in the high-treatment group was close to that of the blank group, indicating a significant therapeutic effect.

[0073] Experimental Example 4 Western blot detection of AMPK phosphorylation and downstream ACC and CPT1α expression in mouse liver Liver tissue (obtained from tissue isolation in Experimental Example 2) was lysed, and protein was extracted as a protein sample. The protein sample was mixed with 4× SDS buffer at a ratio of 3:1 and denatured in a boiling water bath for 10 minutes. The denatured protein was loaded onto a 10% SDS-PAGE electrophoresis. The separated proteins were transferred to a PVDF membrane, blocked with 5% skim milk powder for 2 hours at room temperature, and incubated with the primary antibody at 4°C overnight. The PVDF membrane was washed with PBST and incubated with a secondary antibody (HRP-conjugated rabbit secondary antibody) for 1 hour at room temperature. The membrane was then washed with PBST and visualized with ECL chemiluminescence. The grayscale value of each band was determined using ImageJ software. The results are shown in Table 7.

[0074] Table 7 AMPK phosphorylation and downstream ACC and CPT1α expression results of each group

[0075] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05), n = 12.

[0076] As shown in Table 7, the Lactobacillus paracasei JLPF-131 preparation can increase the phosphorylation level of AMPKα1 and regulate the downstream metabolic pathway. Specifically, the increase in p-AMPKα1 (phosphorylated AMP-activated protein kinase α1) reduced the protein expression of ACC (acetyl-CoA carboxylase), inhibited the activity of ACC, and increased the protein expression of CPT1α (carnitine palmitoyltransferase 1α), thereby promoting fatty acid β-oxidation and regulating fat metabolism. In particular, the Lactobacillus paracasei JLPF-131 preparation in the high treatment group increased the phosphorylation level of AMPKα1 and regulated the downstream metabolic pathway. The ACC / CPT1α pathway efficiently promoted fatty acid β-oxidation, promoted lipolysis, and restored fat metabolism to normal physiological levels (equivalent to the blank group, P≥0.05).

[0077] Experimental Example 5: Lactobacillus paracasei JLPF-131 preparation controls body weight in mice via the GLP-1 / PYY→NPY / POMC axis The ELISA kits of Shanghai ELISA Biological Co., Ltd. were used to detect GLP-1 (glucagon-like peptide-1) and PYY (peptide YY) in plasma (plasma prepared from the reserved blood sample in Experimental Example 3) in strict accordance with the instructions.

[0078] The expression of NPY and POMC genes in the mouse hypothalamus was detected using the same method as in Example 2.

[0079] Primer design: NPY: F: 5'-GCCAAATACTACACTGCCCTGAGAC-3' (as shown in SEQ ID NO: 12); R: 5'-TGTTATCGCCACCAAACAGCAG-3' (as shown in SEQ ID NO: 13).

[0080] POMC: F: 5'-TTCTCAGGCCAAGCGCTCCT-3' (as shown in SEQ ID NO: 14); R: 5'-TCGGCCTCCTCGACCATCTC-3' (as shown in SEQ ID NO: 15).

[0081] The results are shown in Table 8.

[0082] Table 8 Test results of each group

[0083] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05), n = 12.

[0084] As shown in Table 8, GLP-1 and PYY levels, as well as POMC gene expression, were significantly higher in the low- and high-treatment groups compared with the model group, with no significant difference between the high- and blank groups. NPY gene expression was significantly lower in the low- and high-treatment groups compared with the model group, with no significant difference between the high- and blank groups. This suggests that the Lactobacillus paracasei JLPF-131 preparation can activate intestinal L cells to secrete the satiety hormones GLP-1 and PYY. This signal is transmitted to the nucleus tractus solitarius (NTS) via vagal afferent fibers, thereby inhibiting hypothalamic NPY neurons and activating POMC neurons, thereby contributing to weight control.

[0085] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and substance of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be readily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. An application of a Lactobacillus paracasei JLPF-131 preparation in the preparation of a medicine for improving obesity induced by light rhythm disturbance, characterized in that: The Lactobacillus paracasei JLPF-131 preparation includes inactivated cells of Lactobacillus paracasei JLPF-131; Lactobacillus paracasei ) JLPF-131 was deposited in the General Microbiology Center of China Culture Collection Administration on July 8, 2019. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.18098.

2. The use according to claim 1, characterized in that The activated Lactobacillus paracasei JLPF-131 is inoculated into an MRS liquid culture medium and cultured at 37°C for 24-48 hours to obtain a bacterial solution; the bacterial solution is heat-inactivated and then centrifuged to collect the bacterial cells; the bacterial cells are resuspended in physiological saline and freeze-dried to obtain a Lactobacillus paracasei JLPF-131 preparation.

3. The use according to claim 1 or claim 2, characterized in that The number of Lactobacillus paracasei JLPF-131 cells was 1.0×10 8 cfu / g.

4. The use according to claim 1 or claim 2, characterized in that The Lactobacillus paracasei JLPF-131 preparation further comprises pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients are glucose, erythritol or maltodextrin.

5. The use according to claim 1, wherein Improvement of obesity induced by light rhythm disturbance includes prevention of obesity caused by prolonged light exposure.

6. The use according to claim 5, characterized in that Prevention of obesity caused by prolonged light exposure includes reducing body weight; or / and, increasing satiety; or / and, reducing blood sugar levels; or / and, reducing body fat; or / and, reducing insulin resistance index.

7. The use according to claim 5, characterized in that Prevention of obesity caused by prolonged light exposure includes increasing the proportion of CD68+ macrophages, increasing the expression level of NOD2, and reducing the expression levels of TNF-α, IL-6 and / or IL-1β.

8. The use according to claim 5, characterized in that Prevention of obesity caused by prolonged light exposure includes regulating and lowering AST, ALT, TC and TG levels, increasing FGF21 hormone levels, or / and increasing melatonin levels.

9. The use according to claim 5, characterized in that Prevention of obesity caused by prolonged light exposure includes increasing the phosphorylation level of AMPKα1, reducing the protein expression of ACC, and increasing the protein expression of CPT1α.

10. The use according to claim 5, characterized in that Preventing obesity caused by prolonged light exposure includes increasing GLP-1 and PYY levels and regulating the expression of POMC and NPY genes.

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