Medicine for treating obesity based on lycium barbarum polysaccharide LBP supplement

Abstract

The invention belongs to the technical field of medicines for treating obesity, and discloses application of a lycium barbarum polysaccharide LBP supplement in preparation of medicines for treating obesity. Lycium barbarum polysaccharide (LBP) supplements function by reducing accumulation of AT and LDs in adipocytes. The present invention provides powerful evidence that LBP supplements are beneficial to reducing accumulation of AT and LDs in adipose cells, based on concentration, duration, and the manner in which LBP is administered. In addition, SIRT1 is a key factor identified as modulating the LBP stimulated fat reduction process. According to the present invention, the ATF6 is adopted as the transcription inhibition factor so as to reduce the SIRT1 activity; the invention provides a sex LBP which not only targets SIRT1, but also targets ATF6 to show the effect, which can further improve UPR and ER stress, and is a treatment strategy for obesity.

Description

Technical Field

[0001] The present invention belongs to the technical field of drugs for treating obesity, and in particular relates to an application of a Lycium barbarum polysaccharide (LBP) supplement in the preparation of drugs for treating obesity. Background Art

[0002] Currently, obesity, a serious global epidemic that is linked to high morbidity and mortality, imposes a huge burden on individuals and public health. The most recent WHO global report on obesity shows that overweight (BMI ≥ 25 kg / m2) is present in 39% of males and 39% of females aged over 2016. In 2016, obesity (BMI ≥ 30 kg / m2) was present in 11% of males and 15% of females. That is to say, there are almost one billion overweight adults worldwide, of whom more than 500 million are obese (World Obesity Atlas). Given the overall inefficiency and potential adverse effects of anti-obesity drugs, there has been a growing interest in discovering alternative fat-reducing nutritional strategies, especially through the use of natural dietary products. Currently, most of these natural products are considered as alternatives to anti-obesity drugs. These natural products include Lycium barbarum polysaccharides (LBP), which is a purine-based medical plant with a long history of purine-based background in traditional Chinese medicine, also called "Goji Berry".

[0003] Studies on humans and rodents have reported that LBP exerts numerous beneficial effects, including cardiovascular preservation, anticancer effects, and antioxidant effects. LBP has also been reported to improve glycemic control in animals and prevent obesity. LBP induces a reduction in body fat by inhibiting the fat accumulation process. However, the mechanisms involved remain poorly characterized. Lipid droplets (LD) are dynamic subcellular organs of purine-based species whose development is strongly associated with obesity. LDs are neutral lipid cores widely distributed in different tissues, such as adipose and liver, composed of cholesterol esters (CE) and triglycerides (TG). LDs are believed to originate from the endoplasmic reticulum (ER) where TG is synthesized.

[0004] As a member of the CIDE protein family, adipocyte-specific protein 27 (Fsp27) is essential for monosomal LD ​​generation in adipocytes by regulating lipid transfer via atypical LD ​​fusions through purine-directed neutral LDLD contact sites (LDCs). Fsp27 shows upregulated expression in adipose tissue (AT) and its absence causes drastically reduced lipid storage. On the contrary, ectopic expression of Fsp27 leads to increased LDs size. However, little is known about the sufficient role played by Fsp27 in the amelioration of LDs accumulation in AT by LBP treatment. It is well known that excessive lipid intake leads to ER stress and the unfolded protein response (UPR).

[0005] Previous studies have shown that LBP protects against obesity via ERS pathway signaling. It activates the transcript Fakor-6 (ATF6), one of the three ER stress sensors and a basic region leucine zipper (bZip) transcription factor, which has a role in lipid accumulation. Under normal conditions, ATF6 remains inactive.

[0006] Under ER stress, the UPR cascade that stimulates ATF6 is activated. From these studies, ATF6 is involved in the accumulation of LDs.

[0007] Through the above analysis, the problems and defects of the existing technology are as follows: the existing technology has not yet studied the application of Lycium barbarum polysaccharide LBP in the treatment of obesity. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides an application of a Lycium barbarum polysaccharide LBP supplement in the preparation of a drug for treating obesity.

[0009] The present invention is achieved by preparing a drug for treating obesity, which contains an effective dose of Lycium barbarum polysaccharide LBP supplement as an active ingredient and can achieve the effect of treating obesity by regulating the accumulation of AT (fat cells) and LDs (fat droplets) in adipose tissue.

[0010] The present invention also provides a health food for preventing or improving obesity, comprising an appropriate amount of Lycium barbarum polysaccharide LBP supplement as a key ingredient. The health food helps prevent or improve obesity and improves physical health by regulating the expression of ATF6 and SIRT1 in fat cells.

[0011] The present invention also provides a dietary supplement for improving metabolic syndrome, which contains a certain proportion of Lycium barbarum polysaccharide LBP supplement. The dietary supplement can effectively reduce the accumulation of AT and LDs in adipose tissue, thereby alleviating metabolic abnormalities caused by obesity and promoting physical health.

[0012] The present invention also provides a pharmaceutical formula for regulating blood lipids, comprising a certain amount of Lycium barbarum polysaccharide LBP supplement. The pharmaceutical formula regulates the expression levels of ATF6 and SIRT1, regulates lipid metabolism, reduces lipid deposition in adipocytes, and lowers blood lipid levels, thereby achieving the effect of preventing or treating obesity-related diseases.

[0013] The present invention also provides an application for preparing a drug for treating obesity, wherein the drug is administered orally to a patient at 100 mg / kg body weight of Lycium barbarum polysaccharide LBP twice daily for 6 months to treat obesity. The Lycium barbarum polysaccharide LBP improves endoplasmic reticulum stress in cells by targeting ATF6, and reduces Fsp27 accumulation in adipocytes through a TF6 / SIRT1-dependent mechanism, thereby reducing fat storage. The efficacy is evaluated by measuring the patient's weight, BMI, waist circumference, and TG levels in the blood before and after treatment.

[0014] The present invention also provides an application for preparing a health food for preventing obesity, characterized in that the health food contains 50 mg of wolfberry polysaccharide LBP per serving, and it is recommended to consume two servings per day, which is suitable for adults with a family history of obesity or initial signs of obesity; the wolfberry polysaccharide LBP inhibits ATF6 expression, reduces ATF6-mediated SIRT1 mRNA transcription inhibition, alleviates endoplasmic reticulum stress in cells, and helps control body weight and fat accumulation.

[0015] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0016] First, the present invention provides strong evidence that LBP supplementation is beneficial in reducing the accumulation of LDs in AT and adipocytes, based on the concentration, duration, and mode of taking LBP. In addition, SIRT1 is identified as a key factor that regulates the LBP-stimulated fat-lowering process. This work of the present invention provides ATF6 as a transcriptional repressor to alleviate SIRT1 activity. The present invention proposes a sexual LBP that shows this effect not only by targeting SIRT1, but also by targeting ATF6, which will further improve UPR and ER stress and is a therapeutic strategy for obesity.

[0017] The present invention proves that LBP can significantly reduce the TG level of HFD-fed mice. The present invention proves for the first time that the BW and TG level of mice are positively correlated with the LBP dosage.

[0018] Third, the application of the Lycium barbarum polysaccharide LBP supplement provided by the present invention in the treatment and prevention of obesity and its specific mechanism of action.

[0019] Obesity is becoming more and more serious worldwide. Traditional drug treatments or dietary control methods often have side effects or poor results. Therefore, finding a safe and effective natural product as a treatment or prevention of obesity has become an important technical issue.

[0020] Technological advancements:

[0021] 1. Discovery of new application areas:

[0022] Lycium barbarum polysaccharide LBP supplements can be used to treat and prevent obesity, which opens up a new direction for its application in the field of health food or medicine.

[0023] 2. In-depth exploration of the mechanism of action:

[0024] The specific mechanism of action of Lycium barbarum polysaccharide LBP supplementation in the treatment of obesity includes reducing the accumulation of adipocytes and fat droplets, inhibiting SIRT1 expression through ATF6, improving endoplasmic reticulum stress response (UPR and ER stress), regulating the accumulation of Fsp27, and alleviating ATF6-mediated SIRT1 transcriptional inhibition, etc. In-depth exploration of these mechanisms provides theoretical support for the application of Lycium barbarum polysaccharide LBP supplementation in the treatment of obesity.

[0025] 3. Quantification of treatment effects:

[0026] It is mentioned that Lycium barbarum polysaccharide LBP supplementation works by affecting TG (triglyceride) levels, which provides a specific biomarker for evaluating its therapeutic effect.

[0027] 4. Determination of effective dose:

[0028] The effective dose of Lycium barbarum polysaccharide LBP supplementation was determined to be 100 mg / kg / d, which provides clear guidance for practical application.

[0029] The present invention has achieved significant technological progress by discovering a new application of Lycium barbarum polysaccharide LBP supplement in the treatment and prevention of obesity, and by deeply exploring its mechanism of action and determining its effective dose, thus providing a new and natural solution to the problem of obesity. This not only helps obese patients find safer and more effective treatment methods, but also provides new ideas and directions for the research and development of related health foods or drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of 4-6 week old male C57BL / 6 mice provided in an example of the present invention randomly divided into 4 groups (n=5 each), and fed with chow diet, high fat diet (HFD), HFD+50 mg / kg / d LBP and HFD+100 mg / kg / d LBP;

[0031] Figure 2 is a schematic diagram of mouse body weight (BW) provided in an embodiment of the present invention;

[0032] Figure 3 It is a schematic diagram of detecting plasma triglyceride (TG) using a TG content detection kit provided in an embodiment of the present invention;

[0033] Figure 4 Schematic diagram of H&E (hematoxylin and eosin) stained mouse adipose tissue specimen provided by an embodiment of the present invention;

[0034] Figure 5 It is a schematic diagram of measuring the size of fat cells as the cross-sectional area on randomly selected H&E stained sections using Image J software provided in an embodiment of the present invention;

[0035] Figure 6 are different group MRI images provided by embodiments of the present invention;

[0036] Figure 7 4-6 weeks old male C57BL / 6 mice (n=5) provided in the embodiment of the present invention were fed CHOW or HFD for 2 weeks; 30 days thereafter, the mice fed with HFD were intravenously injected with pAAV-ZsGreen-shRNA-mSIRT1 plasmid once every five days with or without LBP (400 mg / kg / day) and with or without nail gavage;

[0037] Figure 8 is a schematic diagram of mouse weight provided by an embodiment of the present invention;

[0038] Fig. 9 It is a schematic diagram of the detection level of plasma triglyceride (TG) using a TG content detection kit provided in an embodiment of the present invention;

[0039] Fig.10 Schematic diagram of H&E (hematoxylin and eosin) staining of mouse adipose tissue specimens provided in an embodiment of the present invention;

[0040] Fig.11 is a schematic diagram of measuring the cross-sectional area of ​​a randomly selected H&E stained slice of a fat cell provided in an embodiment of the present invention using Image J software;

[0041] Fig.12 are different groups of MRI images provided by embodiments of the present invention;

[0042] Fig.13 The present invention provides an embodiment of the present invention, which shows that at a concentration greater than 50 μM, LBP treatment significantly inhibits the cell viability of PA after 24 hours of contact;

[0043] Fig.14This is a graph showing that exposure to LBP attenuates the viability of adipocytes in a dose-dependent manner on days 1 to 2 of differentiation provided by an embodiment of the present invention;

[0044] Fig.15 This is a graph showing that exposure to LBP attenuates the viability of adipocytes in a dose-dependent manner at 3-4 days of differentiation provided by an embodiment of the present invention;

[0045] Fig.16 This is a graph showing that exposure to LBP attenuates the viability of adipocytes in a dose-dependent manner at 5-6 days of differentiation, as provided in an embodiment of the present invention;

[0046] Fig.17 It is provided in the embodiment of the present invention that if LBP treatment is not performed, the cell viability decreases on 6 days of differentiation;

[0047] Fig.18 is a cell viability diagram of differentiated adipocytes provided by an embodiment of the present invention;

[0048] Fig.19 This is a diagram showing the effect of LBP on adipocyte differentiation for 1 to 2 days provided in an embodiment of the present invention;

[0049] Fig. 20 This is a diagram showing the effect of LBP on adipocyte differentiation for 3 to 4 days provided in an embodiment of the present invention;

[0050] Fig.21 This is a diagram showing the effect of LBP on adipocyte differentiation for 5 to 6 days provided in an embodiment of the present invention;

[0051] Fig. 22 is a graph of non-LBP treated cell viability provided by an embodiment of the present invention;

[0052] Fig.23 The immunofluorescence experiment provided in the embodiment of the present invention confirmed that the accumulation of LDs in the differentiation group was higher than that in the control group;

[0053] Fig.24 Is the flesh staining provided by the embodiment of the present invention to determine the LDs accumulation less cells in LBP intervention to differentiate moderate treatment cell map;

[0054] Fig.25 is a graph showing the reduction of SIRT1 at mRNA and protein levels by HFD feeding provided in an embodiment of the present invention;

[0055] Fig.26 It is a graph of relative mRNA levels of SIRT1, ATF6, Fsp27 and SIRT1KD samples under different conditions (CHOW, HFD, WT, HFD+LBP) provided by an embodiment of the present invention;

[0056] Fig. 27This is a diagram showing that the LDs-related genes ATF6 and Fsp27 in the AT of HFD-fed mice are significantly increased at the mRNA and protein levels provided by an embodiment of the present invention;

[0057] Fig.28 is a gene expression diagram of LDs related to the inhibition of SIRT1 provided in an embodiment of the present invention;

[0058] Fig.29 is the ratio of proteins (SIRT1, ATF6, Fsp27) to actin (ACTIN) provided in the embodiments of the present invention, which may be used to evaluate the expression level of these proteins during cell differentiation;

[0059] Fig.30 is a diagram showing different concentrations of "LBP" and "ROSI" and their effects on cell differentiation provided by an embodiment of the present invention;

[0060] Fig.31 is a graph showing the effects of different concentrations of "ROSI" and "LBP" on cell differentiation provided by an embodiment of the present invention;

[0061] Fig.32 is a graph showing the relative mRNA levels of "SIRT1" and "p3xFLAGATF6" under different treatment conditions provided by an embodiment of the present invention;

[0062] Fig.33 is an expression graph showing "p3xFLAGATF6" at different "ROSI" and "LBP" concentrations provided by an embodiment of the present invention;

[0063] Fig.34 is a graph showing a comparison of the mRNA level of the Fsp27 gene with a control group (which may be undifferentiated or normal state) provided in an embodiment of the present invention;

[0064] Fig.35 The expression of three proteins, SIRT1, ATF6 and Fsp27, provided in the embodiment of the present invention in the differentiated state, with actin (ACTIN) as a control graph;

[0065] Fig.36 is the ratio of SIRT1 protein to actin (ACTIN) provided in the embodiment of the present invention, which may be used to evaluate the relative expression level of SIRT1 in cells;

[0066] Fig.37 is a ratio diagram of Fsp27 protein to actin (ACTIN) provided in an embodiment of the present invention;

[0067] Fig.38is a comparison chart of the mRNA level of the SIRT1 gene provided in the embodiment of the present invention and the control group;

[0068] Fig.39 This is a schematic diagram of transfection of ATF6 or its corresponding control siRNA by measuring the relative mRNA levels of the genes shown by qRTPCR after the siRNA provided in the examples of the present invention. DETAILED DESCRIPTION

[0069] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0070] The following are two specific examples showing how to use Lycium barbarum polysaccharide LBP supplements in the treatment and prevention of obesity:

[0071] Example 1: Application of drugs for treating obesity

[0072] In this example, the research team used a drug formulation with Lycium barbarum polysaccharide LBP supplement as the main ingredient to treat patients who have been diagnosed with obesity. The treatment plan is as follows:

[0073] Patient screening: 30 obese patients with a BMI greater than 30 were selected.

[0074] Dosage and Administration: Patients were given 100 mg / kg body weight of Lycium barbarum polysaccharide LBP orally twice daily for 6 months.

[0075] Mechanism of action: Lycium barbarum polysaccharide LBP improves intracellular endoplasmic reticulum stress by targeting ATF6 and reduces Fsp27 accumulation in adipocytes through an ATF6 / SIRT1-dependent mechanism, thereby reducing fat storage.

[0076] Effect evaluation: The efficacy of Lycium barbarum polysaccharide LBP was evaluated by measuring the patients' weight, BMI, waist circumference and blood TG levels before and after treatment.

[0077] Example 2: Application of health food for preventing obesity

[0078] In this example, the R&D team developed a health food containing Lycium barbarum polysaccharide LBP, aiming to help people at higher risk prevent obesity. The product development and application plan is as follows:

[0079] Product development: Develop a health food containing 50 mg of Lycium barbarum polysaccharide LBP per serving, with a recommended daily intake of two servings.

[0080] Target population: Adults with a family history of obesity or showing signs of obesity.

[0081] Mechanism of action: Lycium barbarum polysaccharide LBP inhibits ATF6 expression, thereby reducing ATF6-mediated SIRT1 mRNA transcriptional inhibition, alleviating endoplasmic reticulum stress in cells, and helping to control body weight and fat accumulation.

[0082] Consumer guidance: It is recommended that the target population take one serving of health food each morning and evening, and use it continuously in combination with proper diet and exercise to achieve the best preventive effect.

[0083] These two examples demonstrate the diversity and potential of the application of Lycium barbarum polysaccharide LBP in the clinical and health food fields, and provide innovative directions for the treatment and prevention of obesity. The Lycium barbarum polysaccharide LBP supplement provided in the embodiment of the present invention reduces the accumulation of LDs in AT and adipocytes, inhibits the expression of SIRT1 through ATF6, improves UPR and ER stress by targeting SIRT1 and ATF6, and regulates the accumulation of Fsp27 by using the ATF6 / SIRT1-dependent mechanism.

[0084] The effective dose of the Lycium barbarum polysaccharide LBP supplement provided by the embodiment of the present invention is 100 mg / kg / d.

[0085] The present invention hypothesizes that LBP alleviates LDs accumulation in adipose tissue (AT) by inhibiting Fsp27 via an ATF6 / SIRT1-dependent mechanism. LD accumulation in mouse AT was induced using a high-fat diet (HFD). 3T3-L1 preadipocytes (PA) were differentiated, and the effects of LBP and its mechanisms were studied in vivo and in vitro. In addition, LBP was used to identify LDs-related genes (ATF6 and Fsp27). HFD differentiation resulted in a significant increase in the accumulation of LDs in adipose tissue and adipocytes, respectively, which were unusually suppressed by LBP supplementation. LBP statistically activated SIRT1 and reduced the expression of ATF6 and Fsp27.

[0086] Interestingly, upon overexpression or repression of ATF6, the inhibitory effect of LBP was either abolished or exacerbated.

[0087] Furthermore, SIRT1 levels are transcriptionally regulated by LBP through an opposing action mediated by ATF6. Thus, the improvement of obesity by LBP supplementation is mediated by improving LD accumulation, in part, through an ATF6 / SIRT1-dependent mechanism.

[0088] The present invention found that another natural dietary product, Equol, can alleviate atherosclerosis by inhibiting ER stress in vascular endothelial cells, which is partly mediated by downregulation of ATF6 expression. But the effect of LBP on ATF6 expression in AT was evaluated. In addition, LBP is known to engage in the occupational purine-based small molecule siRNA 1 (SIRT1) activator, which requires nicotinamide adenine dinucleotide (NAD+) for its deacetylation activity. Sufficient NAD+-dependent SIRT1 vigorously cooperates with its actions to use cellular energy.

[0089] SIRT1, induced by calorie restriction and exercise, is crucial for regulating lipid and glucose homeostasis. Specific sufficient ends connect SIRT1 to cellular energetics and energy homeostasis, and SIRT1 has become a molecular target of interest for metabolic disorders such as obesity. SIRT1 is a known negative regulator of the ER stress response. SIRT1 has been shown to deacetylate X-box binding protein 1 (XBP1) and inhibit its transcriptional activity, thereby enhancing ER stress-induced apoptosis; while in mammals, SIRT1 also attenuates PERK-eIF2α-dependent translational repression. However, the underlying mechanisms of sufficient

[0090] The involvement of ATF6 and SIRT1 in LBP-induced inhibition of LD accumulation has never been explored. We found that LBP can improve LD accumulation in mouse AT and adipocytes in vitro through an ATF6 / SIRT1-dependent mechanism.

[0091] 1. Data and Methods

[0092] 1.1 Reagents

[0093] Sufficiently pure LBP (SP9311, Sorabio Science & Technology, Beijing, China) was determined to be >90% by high performance liquid chromatography (HPLC). Cell culture materials (e.g., fetal bovine serum [FBS], culture medium, and Dulbecco's Modified Eagle's Medium [DMEM]) were provided by Hyclone (Carlsbad, CA, USA).

[0094] Isobutylformylxanthine (IBMX), dexamethasone (DEX), and insulin were ordered from Sigma-Aldrich (St. Louis, MO, USA). Antibodies (Abs) against SIRT1 (2028, Cell Signaling Technology, Inc., Danvers, MA, USA); antibodies (Abs) against ATF6 and Fsp27 (PA5-20215, PA1-46128, ThermoFisher, Rockford, IL, USA); ACTINAb (TA09, Zhongshan Golden Bridge Biotechnology, Beijing, China); Triazole reagent (Hangzhou Biotechnology); construction of ATF6 siRNA and its control siRNA (GeneCopoeia, Guangzhou, China); synthesis of pAAV-ZsGreen-shRNAmSIRT1 (shSIRT1) plasmid and its control vector (Life Technologies, Shanghai, China); p3×FLAGATF6 plasmid and corresponding control vector.

[0095] 1.2 Animal experiments

[0096] Male C57BL / 6 mice, 8 weeks old (n=20) and 4-6 weeks old (n=25), were provided by the Medical Laboratory Animal Center of the Third Military Medical University.

[0097] The use and care of these animals followed the institutional guidelines for the use of laboratory animals in this study, and the experiments were performed with the approval of the SHSMU Laboratory Animal Care Ethics Committee (no. 2021-0063).

[0098] All mice were housed in ventilated cages in a specific pathogen-free (SPF) animal facility with a controlled environmental setting (21-22 degrees Celsius, 30%-60% humidity, 12:12 h light-dark cycle, and food and water AD Libitum). Change indium mouse activity.

[0099] Monitor body condition and body weight (BW).

[0100] Protocol 1: BW-matched C57BL / 6 mice were randomly divided into 4 groups and fed with Chinese canine diet (CHOW), high fat diet (HFD), HFD with 50 Mg / kg / d of LBP and HFD with 100 Mg / kg / d of LBP, respectively. Adequate Chinese canine diet and HFD diet were obtained from Medical Co., Ltd. (Jiangsu, China), respectively. LBP was diluted and supplemented with distilled water and was given once every 24 hours at 8:00 a.m. for 12 weeks, during which adequate Chinese canine and HFD groups were given adequate distilled water and adequate LBP volume. Adequate BW was checked every week.

[0101] Protocol 2: Initially, C57BL / 6 mice were fed with CHOW or HFD (containing 60% fat) for two weeks. Thereafter, for 30 days, HFD-fed mice were treated with or without LBP (400 mg / kg / day), and with Oregon-purine-free kinase intravenous injection of pAAV-ZsGreen-shRNA mSIRT1 plasmid once every five days. shSIRT1 (12.5 μg) + non-liposomal nanoparticle transfection reagent (25 μl; Entranster-In Vivo; Englin Biosystems, Beijing, China) were mixed and with 10% glucose solution was used in each injection according to the supplier's protocol. BW was checked every two days.

[0102] Mice were starved overnight and then sacrificed. Serum obtained by fixation and centrifugation (3000×g, 10 min, 4°C) was refrigerated at -80°C until biochemical parameter testing.

[0103] Afterwards enough liver tissue was carefully removed from the abdomen for weighing, slicing and subsequent homogenization using RIPA lysis buffer (20 mg liver tissue / 300 μl RIPA) for western blot analysis. As for other tissues, they were immediately stored in liquid nitrogen at -80 °C for other assays like plasma TG determination and real-time RT-PCR. Plasma LBP concentrations were determined by HPLC as described above.

[0104] 1.3 Histological analysis

[0105] The harvested tissues were fixed with 4% paraformaldehyde and embedded in paraffin. 5-micron sections were then stained with hematoxylin and eosin (H&E). All sections were analyzed in minute detail. The cross-sectional area of ​​a randomly selected H&E-stained section was measured using ImageJ software for a fat cell of sufficient size.

[0106] 1.4 Magnetic resonance imaging of mice

[0107] A radiologist blinded to group assignment performed MRI measurements of mice using a 7T MRI scanner (70 / 16 PharmaScan, Bruker). Anesthesia for surgery was performed with 3% isoflurane.

[0108] During MRI scanning, mice were placed in a plastic cradle with teeth on the jaw and plastic screws indium their ear canals. At that time came continuous physiological monitoring achieved using an MRI-compatible system (SA Instruments).

[0109] The experimental parameters were as follows: Spin echo sequence: TR / TE = 150 / 5.8 ms, Field of view (FOV) = 31.50 x 20.25 mm2, Matrix size = 210 x 135. The entire abdominal AT of each mouse was covered with axial slices of 1 mm thickness but without spacing.

[0110] 1.5 Cell cultivation and intervention

[0111] 3T3-Ll preadipocytes (PA) ordered from American Type Culture Collection were cultured at 37°C in DMEM containing 25 mM glucose (DMEM-H), penicillin (100 U / ml), sodium pyruvate (1 mM), streptomycin (100 μg / ml) and 10% FBS in a humidified atmosphere of 5% CO. To induce differentiation, cells were stimulated for differentiation two days after confluence using DMEM supplemented with 10% FBS, insulin (167 nM), DEX (1 M) and IBMX (0.5 mM) for 48 hours.

[0112] They were then bathed in 10% FBS / DMEM + insulin (167nM) for another 48 hours and then in 10% FBS / DMEM for another 96 hours, when 90% of the cells would have matured adipocytes and accumulated LDs. All stimulations were guided by the 3T3-L1 adipocytes cultured in DMEM alone for a period of time during which no additions were described.

[0113] Sufficient 3T3-LlPA, differentiating adipocytes and mature adipocytes were treated with a series of concentrations (0.1, 1, 10, 25, 50, 100 μM) of LBP for 24 hours for further experiments. Rosiglitazone (ROSI) was used as a positive control, which increased lipid accumulation and promoted sufficient differentiation of adipocytes.

[0114] 1.6 Cell viability measurement

[0115] The viability of 3T3-LlPA was measured using the Cell Counting Kit-8. Cells were seeded in wells (5 × 10 3 Cells / well in 96-well microplates (Corning Life Sciences, Acton, MA; 3650) were given 24 Planck constants and were then specific for the treatments related to the As mentioned above. Cells were cultured at 37°C for 1 hour after which sufficient cells were counted in 10ul / well of Kit8 solution, after which viability was determined by absorbance measurement (450 nm) using a monochrome microplate reader (SafireII; Tecan Group GmbH, Mannedorf, Switzerland). A sufficient percentage of viable cells to go to the adequate control group (set As 100%) was sufficient visually measured by the density at 450nm.

[0116] 1.7 RNA interference and lentiviral transfection

[0117] p3×FLAG-ATF6 plasmid and its corresponding control pCMV-3FLAG-7.1 vector, provided by Adegen (#11975), were transfected into 3T3-Ll adipocytes as described with Lipofectamine 2000 (Invitrogen, 11668-027). For siRNA analysis, 3T3-Ll adipocytes were treated with corresponding siRNA transfection using Lipofectamine 2000 24 hours after cell seeding. Afterwards, different concentrations of LBP were used for 24 hours of cell exposure.

[0118] 1.7 Western Movies

[0119] Mature adipocytes (1×10 6 ) were cultured in 100 mm size culture dishes 2 was treated with arsenic indicated. Whole cell lysate products, prepared and together with ice-cold RIPA buffer, were treated with sodium dodecyl sulfate polyacrylamide gel electrophoresis as described previously 28 and transferred to polyvinyl fluoride (PVDF) membranes and incubated at 4°C, overnight with gentle shaking using appropriate primary Abs. This was followed by membrane culture and together with purine scapegoat anti-mouse / rabbit IgG horseradish peroxidase conjugated Ab (Bio-Rad). Fusion FX5 spectrometer manufactured by VilberLourmat Inc., France was responsible for the detection of conjugated proteins.

[0120] 1.8 RNA isolation and qRT-PCR experiments

[0121] After isolating total RNA from mature adipocytes using Trizol reagent, 1 μg aliquot from each sample was reverse transcribed. See Table 1 for primer sequences used for amplification.

[0122] Using an iQ5 machine (American Bio-Rad), 25 μl of the reaction mixture was added and the target gene was amplified as follows: denaturation (95 °C, 30 sec); 40 cycles of 5 sec at 95 °C, 34 sec at 60 °C; extension at 95 °C for 15 sec and at 30 °C for 6 sec. Amplification reactions, by adequate relative threshold cycle (Ct) formula, were normalized against GAPDH mRNA, a control.

[0123] Table 1. Primers for Real-time RT-PCR

[0124]

[0125] The sufficient luciferase activity size was carried out outside 48 Planck constants after LBP treatment with arsenic according to the teaching of this sufficient dual luciferase reporter assay system (Promega, Madison, WI, USA). The potential of ATF6 modulation region in human SIRT1 promoter was predicted using three online algorithms Genomatix, UCSC and JASPAR for LBP treatment.

[0126] Based on the predicted overlap, a 1.2 kb human SIRT1 promoter sequence (-2000 to +100 bp) was synthesized for cloning into the XhoI and HindIII sites of the pGL3-basic vector

[0127] The plasmid, called pAAV-ZsGreen-shRNAmSIRT1 (shSIRT1), was used from Life Technologies (Shanghai, China). The obtained construct was further verified by DNA sequencing.

[0128] By referring to the suggestion of lipofectamine2000, 3T3-L1 adipocytes were transfected with sufficient shSIRT1 Oregon pGL3-basic SIRT1 plasmid forward and together with purine base pRL-TK plasmid (Promega, USA), followed by transfection of sufficient ATF6 and together with a siRNA Oregon p3×FLAG-ATF6 plasmid respectively. Luciferase activity was measured and the ratio of an activity of firefly luciferase activity to that of Renilla luciferase activity was calculated. For each measurement, arsenic wells arsenic each assay that was performed on adipocytes were determined in triplicate.

[0129] 1.9 Chromatin immunoprecipitation (ChIP)

[0130] The present invention used ChIP kit (Millipore) to perform ChIP assays, referring to previous studies. Briefly, following PA (0.2mM) Oregon vehicle control (0.1% ethanol) treatment of 24 h 3T3-L1 adipocytes was fixed with 1% formaldehyde for 10 min at 37°C to crosslink nuclear proteins to DNA. Cells were then aggregated to lyse the cells and a SDS cell lysis buffer was added.

[0131] DNA was cut on chromatin at lengths of 200-1000 bp and then immunoprecipitated with 2 μg of Ab against ATF6 (negative control: IgG). After DNA precipitation, they were processed for PCR Oregon qPCR amplification using primers for paired overlapping sufficient SIRT1 promoter region of ATF6 (~1340 to ~1333), and together with total DNA extracts (input) Arsenic positive PCR control.

[0132] 2.0 Statistics and Analysis

[0133] Unless otherwise stated, data from different groups are presented as ± standard error. Multiple groups (≥3) and comparisons between groups were made by one-way ANOVA and two-tailed unpaired t test. Significance of in vivo findings was determined by repeated measures ANOVA. P < 0.05 indicated statistical significance.

[0134] 3. Results

[0135] LBP protects against lipid accumulation in a purine-based HFD-fed mouse model. Animals were randomized into 4 groups and fed a purine-based HFD in the presence or absence of LBP for 12 weeks. Figure 1 The HFD-fed mice gained significantly more weight than the chow group starting at week 8 and maintained the highest weight of all groups throughout the experiment ( Figure 2 At week 12 of mortality, BW of mice fed HFD and with 50 and 100 Mg / kg / d of LBP were (30.3 ± 1.64) g and (27.58 ± 1.57) g, respectively, compared with (31.4 ± 2.4) g of HFD-fed mice (P < 0.05).

[0136] There was negligible difference in daily caloric intake between the HFD-fed groups with or without LBP supplementation. And LBP at 100 mg / kg / day tissue LED depurinylation resulted in more clear reductions in plasma TG ( Figure 3 As expected, the size of adipocytes in visceral adipose tissue (VAT) of HFD-fed mice was much larger than that of Chinese dogs ( Figure 4-Figure 5 ). However, sufficient adipocytes were obtained from mice treated with LBP, which appeared smaller in multi-ocular LDs compared with the chow group, and the choow group showed larger, monocular LDs.

[0137] Interestingly, administration of LBP also resulted in crown-like structures (CLS), a characteristic of macrophage infiltration surrounded by dead adipocytes in the AT of obese animals and humans 30 . Here, CLS was particularly observed in the AT of mice fed FDR and 100 mg / kg / day LBP, compared with mice that were fed a purine-based HFD, whereas mice nude to 50 mg / kg / day LBP showed no such differences ( Figure 4 ).

[0138] Abdominal MRI of live mice at week 11 further confirmed these findings ( Figure 6In conclusion, LBP administration in HFD-fed mice resulted in improved lipid accumulation in AT, smaller LD size, and smaller number and mass of LDs in AT.

[0139] H&E stained sections were measured using ImageJ software. Quantitative data obtained after three repeated measurements are expressed as mean ± SD; statistical significance between the two labeled treatment groups is indicated as *P < 0.05, P < 0.01, and *P < 0.001, and no significance is indicated by NS.

[0140] LBP inhibits lipid accumulation in AT via SIRT1 in mice fed a short-term HFD. In the first two weeks, mice were fed either normal mouse chow or a HFD diet. Then, HFD-fed animals were gavaged for 30 days, and together with Oregon in the absence of 400mg / kg / dLBP.

[0141] At the same time, shSIRT1 plasmid was either injected into the nail vein every five days or not ( Figure 7 ). The BW of mice was measured every other day.

[0142] Compared with control mice, the biomass of the HFD-fed group increased, which was significantly inhibited by LBP intervention ( Figure 8 ). To clarify the role of SIRT1 in regulating adipose lipid storage, SIRT1 shRNA (shSIRT1) was applied to knockdown (KD) SIRT1 expression in vivo due to LBP administration. As shown in Figure 2B, adequate HFD-fed WT mice that were administered LBP had lower BW than adequate related groups without LBP treatment. At the same time, SIRT1 KD mice on a purine-based HFD gained more BW than HFD-fed WT ones, while LBP in the former induced a slight, but not significant, decrease in body weight compared with the latter ( Figure 8 ).

[0143] HFD feeding resulted in a significant increase in plasma TG levels compared with chow mice ( Fig. 9 ). And the LBP-induced reduction in TG levels in HFD-fed WT mice was particularly abolished in HFD-fed SIRT1 KD animals, implying that SIRT1 has a great influence on the LBP-induced improvement in lipid metabolism ( Fig. 9 ).

[0144] Histological analysis showed that LBP supplementation with 400mg / kg / d of LFD-fed mice resulted in fewer and smaller adipocytes, as well as the occurrence of CLS, indicating a significant inhibitory effect of LBP on HFD-induced adipocyte accumulation ( Fig.10).

[0145] Larger and increased numbers of adipocytes were observed in the AT of HFD-fed SIRT1 KD mice, whereas LBP-induced improvement in LD accumulation in AT in HFD-fed mice was particularly abolished in SIRT1 KD mice ( Figure 11-Figure 12 ).

[0146] MRI scans also consistently showed that HFD-fed SIRT1KD mice had much higher fat mass than WT mice, and that the LBP-induced fat mass benefit was abolished by SIRT1KD in vivo ( Fig.13 ). Overall, LBP administration prevented HFD-induced AT lipid accumulation, which was significantly abolished by SIRT1 knockdown in vivo, implicating the involvement of SIRT1 in LBP-induced amelioration of LD accumulation in AT.

[0147] LBP suppressed 3T3-L1 adipocyte differentiation and reduced LD storage, as described previously, in an adipocyte cell model. At concentrations greater than 50 μM, LBP treatment significantly inhibited cell viability after 24 h of PA exposure ( Fig.13 ).

[0148] During differentiation, cell viability was determined by adding additional differentiation medium each time. These results indicate that exposure to LBP attenuates adipocyte viability in a dose-dependent manner ( Figure 14-16 ).

[0149] Without LBP treatment, cell viability decreased on the 6th differentiation date ( Fig.17 ), is due to the terminal increase of differentiation and rounding of sufficient adipocytes which leads to their floating in the medium. Cell viability of differentiated adipocytes ( Fig.18 ) and mature adipocytes ( Fig.19 ) remained unchanged after 25 μM LBP treatment, indicating that LBP exerts a stronger effect at the early stage of adipocyte differentiation.

[0150] As for TG content, no significant changes were detected in PA after exposure to LBP24 for a long time ( Fig.18 In addition, increased lipid accumulation was observed during the difference ( Figure 18-Figure 21 ).

[0151] Meanwhile, significantly lower TG contents of at least 25% were detected in differentiated and mature cells treated with LBP when compared with non-LBP-treated cells ( Figure 18-Figure 22 ). Indium is added, arsenic is Fig.23As shown, an immunofluorescence experiment confirmed that the accumulation of LDs in the differentiation group was higher than that in the control group.

[0152] Therefore, considering all experiments completed, the present invention demonstrates that 25 μM of LBP is suitable for use in the follow-up experiments to achieve the maximum biological effect.

[0153] In addition, in vivo staining identified cells with less LD accumulation in LBP-treated cells compared with differentiated moderately treated cells ( Fig.24 ). In conclusion, LBP intervention can delay differentiation and reduce LDs accumulation in adipocytes.

[0154] Figure 3LBP suppressed 3T3-L1 adipocyte differentiation and reduced LD storage. Statistical significance of the data (mean ± SD) between the two marker treatment groups is indicated by *P < 0.05, P < 0.01, and **P < 0.001.

[0155] LBP inhibitors LDs-associated gene expression in mouse adipocytes and adipocytes

[0156] LBP administration improved LDs accumulation in mice fed with HFD and reduced LDs lipid accumulation in adipocytes. Next, we explored the sufficient effects of LBP on LDs-related genes in mouse AT and adipocytes. We found that HFD feeding reduced SIRT1 mRNA and protein levels in AT, which were significantly inhibited by LBP administration ( Fig.25 and Fig.26 ).

[0157] The LDs-related genes ATF6 and Fsp27 were significantly increased at both mRNA and protein levels in the AT of HFD-fed mice, which was significantly inhibited by LBP administration. However, the sufficient LBP-induced reduction of ATF6 and Fsp27 was abolished by SIRT1 knockdown in mice, implying an important and significant effect of SIRT1 on the repression of LDs-related gene expression followed by LBP administration ( Fig. 27 and Fig.28 ). In addition, the present invention investigated the effect of LBP on LDs-related genes in 3T3-L1 adipocytes. At the mRNA and protein levels, a significant increase in SIRT1 was observed in 3T3-L1 adipocytes treated with LBP (Figure Figure 29-Figure 39 ), accompanied by enhanced SIRT1 activity.

[0158] The present invention explored SIRT1 and ATF6 in adipocytes, followed by LBP intervention. To identify the ability of ATF6 to regulate SIRT1 transcription, adipocytes were transfected with pGL3-Basic-SIRT1 plasmid with firefly luciferase activity, and then co-transfected with siRNA against ATF6 or p3×FLAG-ATF6 plasmid and pRLTK plasmid, respectively. According to the dual luciferase assay, silencing ATF6 significantly enhanced LBP-induced SIRT1 transcriptional activity, while overexpression of ATF6 aggravated the inhibition of SIRT1 transcriptional activity. These results indicate that the regulation of SIRT1 mRNA transcription by LBP treatment is mediated by ATF6. To further determine whether ATF6 inhibits SIRT1 mRNA expression by directly binding to its promoter, ChIP assay was performed in adipocytes transfected with p3×FLAG-ATF6 plasmid. The results showed that ATF6 Ab precipitated with the promoter DNA of SIRT1, indicating that endogenous ATF6 and SIRT1 can be co-localized in the proximal region of the SIRT1 promoter. Thus, LBP binds to the SIRT1 promoter in adipocytes by directly binding to ATF6.

[0159] The present study of the present invention is the first to demonstrate that BW and TG levels in mice are positively correlated with the dose of LBP administered. The beneficial effect was more significant in mice induced by 100 mg / kg / dLBP compared to 50 mg / kg / d. A. Previous studies have found that BW2 is reduced after 5 weeks of receiving a supplemented diet (HFD or not supplemented with 20 or 40 mg LBP). Consistent with these results, the present invention provides SIRT1KD mice fed HFD with LBP (400 mg / kg body weight / day) for 30 days, with a slight decrease in BW and TG content. However, the higher dose of LBP did not cause a significant reduction in the WT group as expected, indicating that duration affects the effect of LBP. In the first experiment, the present invention treated mice with both HFD diet and LBP, while the present invention provided LBP after 2 weeks of HFD in the second experiment, indicating that LBP intake needs to be as early as possible. The most important finding of this animal model is the confirmation of the key role of SIRT1 in the body fat-lowering effect induced by LBP. As shown for 30 days of work, the benefits of LBP are almost abolished without SIRT1. In conclusion, the differences found in the present invention can be attributed to differences in various aspects of the experimental design, namely, the length of the experimental period, LBP dose, LBP administration method, animal strain and most importantly, SIRT1.

[0160] The present invention demonstrates that LBP can significantly reduce TG levels in HFD-fed mice. It is known that TG is synthesized in the ER and fills the core of growing LDs39. LDs are lipid storage depots, and their dynamic changes such as biogenesis and growth are crucial for regulating lipid homeostasis. Here, the present invention found that Fsp27, a fat-specific protein 27 located at the LD-LD contact site, is essential for controlling LDs growth and fusion, leading to LDs accumulation and obesity. Similarly, evidence suggests that AT is a major target mediating the metabolic effects of the presence of Fsp27. The present invention observed that LBP reduced Fsp27 in AT of HFD-fed mice. The ER regulates a variety of cellular processes involved in inflammation, insulin signaling, and nutrient metabolism through the UPR. Sustained activation of the UPR has been associated with a variety of metabolic disorders. Chronic UPR activation has been observed in liver and / or AT in dietary and genetic obese mouse models as well as obese mouse models and non-alcoholic fatty liver disease populations. ATF6 has been indicated to be upregulated in the presence of HFD, indicating that HFD leads to activation of the UPR. LBP significantly downregulated the activity of ATF6 in HFD mice. In contrast, in SIRT1 KD mice, the LDs accumulation effect of Fsp27 was enhanced and the lipid-lowering effect of LBP was abolished. The present invention hypothesizes that SIRT1 acts as a key regulator of LBP-promoted LDs reduction, which further improves UPR and ER stress.

[0161] In accordance with previous observations, adipocytes incubated with LBP at 400 μM. In 3T3-L1 mature and mature adipocytes, LBP significantly reduced cell viability and dose-dependently decreased lipid accumulation and intracellular TG levels. Regarding 3T3-L1 differentiated PA, exposure to 0.1 and 10 μM LBP did not alter TG content. In contrast, LBP at a concentration of 25 μM reduced TG. At the same time, early exposure of 3T3-L1 adipocytes triggered a significant decrease in intracellular TG content. This result is consistent with what was achieved in vivo.

[0162] The expression of genes such as SIRT1, ATF6 and Fsp27 and LDs were analyzed in mature adipocytes treated with 25 μM LBP. It significantly reduced ATF6 and Fsp27 at the mRNA and protein levels in vivo and in vitro, but enhanced SIRT1 activity as expected. ATF6 was involved in the reduction of LBP-mediated LD accumulation. First, ATF6 is one of the UPR signaling factors that promotes the growth of LDs, because its depletion significantly reduced the accumulation of LDs while its ectopic expression significantly promoted the accumulation of LDs.

[0163] LBP is proposed to drive its beneficial effects by targeting and activating SIRT1, demonstrating that ER stress-activated transcription factor ATF6 appears dispensable for SIRT1. Here, the present invention presents a different result, namely that ATF6 plays a role in suppressing SIRT1 expression. Silencing ATF6 promotes upregulation of SIRT1, whereas overexpression of ATF6 leads to SIRT1 deficiency. Based on the above evidence, it is hypothesized that ATF6 has a transcriptional effect on SIRT1. ATF6, belonging to the ATF subfamily 46 of bZIP transcription factors, is considered to be a transcriptional activator or repressor.

[0164] ATF6 is considered to be the multidrug resistance phenotype of gastric cancer cells by deactivating SIRT147. The present invention discovered a potential binding site promoter region of ATF6 in SIRT1, and directly inactivated the direct binding transcriptional activity of SIRT1 through ATF6.

[0165] In summary, the present invention provides strong evidence that LBP supplementation is beneficial in reducing the accumulation of LDs in AT and adipocytes based on the concentration, duration, and way of taking LBP. In addition, SIRT1 was identified as a key factor regulating the LBP-stimulated fat-lowering process. This work of the present invention provides ATF6 as a transcriptional repressor to alleviate SIRT1 activity. The present invention proposes a sexual LBP that shows this effect not only by targeting SIRT1, but also by targeting ATF6, which will further improve UPR and ER stress and is a therapeutic strategy for obesity.

[0166] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A drug for treating obesity, characterized in that: This medicine contains an effective dose of Lycium barbarum polysaccharides LBP supplement as an active ingredient.

2. A health food for preventing or improving obesity, characterized in that: Contains an appropriate amount of Lycium barbarum polysaccharides LBP supplement as the key ingredient.

3. A dietary supplement for improving metabolic syndrome, characterized in that: Contains a certain proportion of Lycium barbarum polysaccharide LBP supplement.

4. A pharmaceutical formulation for regulating blood lipids, characterized in that: The pharmaceutical formula comprises a certain amount of Lycium barbarum polysaccharide LBP supplement. The pharmaceutical formula regulates the expression levels of ATF6 and SIRT1, regulates lipid metabolism, reduces the deposition of lipids in fat cells, and lowers blood lipid levels, thereby achieving the effect of preventing or treating obesity-related diseases.

5. An application of preparing a drug for treating obesity, characterized in that: The drug treats obesity by giving patients 100 mg / kg body weight of Lycium barbarum polysaccharide LBP orally twice a day for 6 months. Lycium barbarum polysaccharide LBP improves endoplasmic reticulum stress in cells by targeting ATF6 and reduces Fsp27 accumulation in adipocytes through a TF6 / SIRT1-dependent mechanism, thereby reducing fat storage. The efficacy is evaluated by measuring the patient's weight, BMI, waist circumference and TG levels in the blood before and after treatment.

6. An application of preparing a health food for preventing obesity, characterized in that: The health food contains 50 mg of wolfberry polysaccharide LBP per serving, and it is recommended to consume two servings per day. It is suitable for adults with a family history of obesity or initial signs of obesity. The wolfberry polysaccharide LBP inhibits ATF6 expression, reduces ATF6-mediated SIRT1 mRNA transcription inhibition, alleviates endoplasmic reticulum stress in cells, and helps control body weight and fat accumulation.

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