Use of sodium humate in the preparation of a medicament for treating obesity

Abstract

The application discloses application of sodium humate in preparation of a medicine for treating obesity, and relates to the technical field of biological medicine. The application finds that the sodium humate has the effect of treating obesity, and further combined with near-infrared photothermal treatment can effectively improve the treatment effect on obesity. It is detected that the sodium humate has excellent photothermal conversion performance and good photothermal stability, and this characteristic lays a key foundation for the sodium humate in combination with the near-infrared photothermal treatment of obesity. Experiments prove that the sodium humate can realize efficient photothermal conversion in the fat area, and provides a precise action mode for local fat intervention. Animal experiments prove that the sodium humate and the sodium humate combined with photothermal treatment can effectively reduce the degree of obesity. Meanwhile, the combined treatment can improve the distribution of adipose tissue, realize remodeling of the adipose phenotype, and especially can promote the browning of white adipose tissue.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of sodium humate in the preparation of a drug for treating obesity. BACKGROUND

[0002] Obesity is a complex metabolic disease characterized by excessive energy deposition in the form of triglycerides in white adipose tissue. In recent years, its prevalence has continued to rise. Obesity not only affects an individual's physical health, but also often leads to insulin resistance, cardiovascular disease and other systemic metabolic disorders, further increasing the risk of diseases such as diabetes, hypertension and non-alcoholic fatty liver disease.

[0003] Although the medical community has conducted extensive research on the pathological mechanisms of obesity, there are still significant limitations in treatment methods. Currently, the most commonly used intervention method is still life intervention, such as diet control and exercise, but this method highly depends on the patient's self-control ability, and the actual effect is often difficult to maintain. For severe obese patients, invasive methods such as gastric resection, gastric bypass surgery and fat tissue resection can bring obvious weight loss effect, but they are accompanied by infection risk, postoperative complications and nutritional absorption disorders, which limit their widespread application. In terms of drug treatment, existing anti-obesity drugs mainly include pancreatic lipase inhibitors (such as orlistat), GLP-1 receptor agonists (such as liraglutide, benaglutiide, semeglutide) and GIP / GLP-1 receptor dual agonists (such as tirzepatide). These drugs can achieve short-term weight loss to some extent, but still have many shortcomings, such as common gastrointestinal adverse reactions (such as nausea, diarrhea), potential risk of pancreatitis, and weight rebound after drug discontinuation, which affect patient compliance and long-term efficacy.

[0004] Therefore, there is an urgent need in the field of obesity treatment to develop new treatment strategies that are safer, more effective and sustainable, in order to effectively control and manage obesity and its related metabolic disorders, and to provide better clinical solutions for patients. SUMMARY

[0005] The purpose of the present application is to provide the application of sodium humate in the preparation of a drug for treating obesity, in order to solve the problems existing in the prior art. The present application researches and finds that sodium humate (HA) has the effect of treating obesity, and further combined with near-infrared (NIR) photothermal therapy can effectively improve the treatment effect on obesity.

[0006] To achieve the above purpose, the present application provides the following scheme:

[0007] The present application provides the application of sodium humate in the preparation of a drug for treating obesity.

[0008] Further, the drug is a photothermal conversion drug based on photothermal therapy.

[0009] Further, the drug treats obesity by reducing body weight, lowering body fat rate, improving fat distribution and / or browning white fat.

[0010] The application also provides use of sodium humate in preparation of a drug for reducing liver injury caused by a high-fat diet.

[0011] Further, the drug is a photothermal conversion drug based on photothermal therapy.

[0012] The application discloses the following technical effects:

[0013] The application researches and finds that sodium humate has an effect of treating obesity, and further combined with near-infrared photothermal therapy can effectively improve the treatment effect on obesity. It is detected that HA has excellent photothermal conversion performance and good photothermal stability, and this characteristic lays a key foundation for the combination of HA and near-infrared photothermal therapy for obesity. Experiments prove that HA can efficiently realize photothermal conversion in the fat area, and provide a precise action mode for local fat intervention. Animal experiments prove that HA and HA combined with photothermal therapy can effectively reduce the degree of obesity. Meanwhile, the combined therapy can improve fat tissue distribution, realize fat phenotype remodeling, and especially promote browning of white fat. In the cell experiment, HA treatment makes the size of lipid droplets in adipocytes significantly reduced, and presents a morphology similar to that of brown adipocytes. HA and NIR combined treatment can increase the expression levels of heat production related proteins UCP1 and PGC-1a in mature white adipocytes, and verify the effect of promoting browning of white fat and improving heat production function from the cell level.

[0014] In addition, the application finds that HA has excellent safety while playing a therapeutic role. HA and HA combined with photothermal therapy can reduce the level of liver injury caused by a high-fat diet, and has no obvious damage to main organs and no significant toxicity to adipose-derived stem cells (ADSCs), which reflects good biocompatibility and safety.

[0015] In summary, HA combined with photothermal therapy realizes multiple effects such as weight loss, body fat rate reduction, fat distribution improvement and white fat browning by targeting the photothermal effect of the fat area, and guarantees the safety of main organs of the body, thereby providing a new efficient and safe way for obesity treatment. BRIEF DESCRIPTION OF DRAWINGS

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 Images show the speciation of sodium humate; where A: HA powder image; B: HA solutions dissolved in different media; C: HA solutions in different media after 60 days.

[0018] Figure 2 Particle size analysis diagram of HA solution;

[0019] Figure 3 The zeta potential diagram for HA;

[0020] Figure 4 The Fourier transform infrared (FLIR) spectrum of HA;

[0021] Figure 5 Vis-NIR absorption spectra of HA solutions at different concentrations;

[0022] Figure 6 To investigate the near-infrared (808 nm, 0.5 W / cm) spectra of HA solutions of different concentrations. 2 The results of the photothermal conversion performance test under irradiation are shown in the figure.

[0023] Figure 7 The heating / cooling curves of a 0.5 mg / mL HA solution after three repeated NIR irradiations are shown.

[0024] Figure 8 Figure 1 shows the experimental results of using sodium humate combined with photothermal therapy to treat obesity induced by a high-fat diet in mice. A: Flowchart of the animal experiment; B and C: Near-infrared (808 nm, 0.5 W / cm²) thermal imaging of obese mice after injection of PBS buffer or HA solution into the subcutaneous white adipose tissue in the groin area. 2 D: Thermal imaging of local temperature in mice irradiated for 180 s (B) and line graph of temperature change (∆T) (C); E: Comparison of body size of mice in each group after treatment; F: Change in body weight of mice during treatment; G: Body fat percentage of mice in each group after treatment; H: Comparison of size (G) and weight (H) of inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), and brown adipose tissue (BAT) among mice in each group after treatment; n = 3, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001;

[0025] Figure 9 Figure 6 is a HE staining diagram of adipose tissue of mice for sodium humate combined with photothermal for treating high-fat diet-induced mouse obesity experiments;

[0026] Figure 10 Figure 6 is a HE staining diagram of adipose tissue of mice for sodium humate combined with photothermal for treating high-fat diet-induced mouse obesity experiments;

[0027] Figure 11 Figure 6 is a HE staining diagram of adipose tissue of mice for sodium humate combined with photothermal for treating high-fat diet-induced mouse obesity experiments;

[0028] Figure 12 Figure 6 is a HE staining diagram of adipose tissue of mice for sodium humate combined with photothermal for treating high-fat diet-induced mouse obesity experiments; DETAILED DESCRIPTION

[0029] The detailed description set forth below is intended as a description of various example embodiments of the application and is not intended to represent the only embodiments in which the application can be practiced. The term "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0030] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where a range of values is provided, it is understood that each intervening value, to the upper and lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also encompassed by the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials useful in connection to the documents. In the event of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification controls.

[0032] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application. The specification and examples are illustrative only.

[0033] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended and do not limit the object to which they are applied to the items recited following the terms.

[0034] Example 1

[0035] I. Characterization and photothermal properties of sodium humate (HA)

[0036] 1. Preparation of HA solution

[0037] HA powder (0.1 g) was weighed with a precision electronic analytical balance, dissolved with 1x PBS buffer, and a 5 mg / mL HA solution was prepared. Centrifugation at 1000 rpm for 3 min at room temperature to remove large insoluble particles. After sterilization in an autoclave, it was stored at 4°C for later use. Figure 1

[0038] 2. Stability analysis of HA solution

[0039] Sodium humate powder was dissolved in different media (pure water, PBS, DMEM-F12 culture medium), and the state just after dissolution and the state after 60 days of placement were recorded. Sodium humate can be dissolved in various aqueous media and has good stability (Figs. B and C). Figure 1

[0040] ​​3. Nanoparticle tracking analysis (NTA) analysis of HA particle size and quantity

[0041] (1) Take 30 μL of HA solution (10 mg / mL) and dilute 500 times with 15 mL of pure water.

[0042] (2) Put the sample on the machine, inject it into the nanoparticle tracking analyzer, and measure the average speed of the particles by the Stokes-Einstein equation to estimate the particle size and quantity. The nanoparticle size is mostly distributed between 100-150 nm ( Figure 2 ).

[0043] 4. Zeta potential

[0044] (1) Take 200 μL of HA solution (10 mg / mL) and dilute 6 times with 1 mL of pure water.

[0045] (2) Put the sample on the machine to detect the Zeta potential. The HA solution particles are negatively charged, with a potential of about -45 mV ( Figure 3 ).

[0046] 5. Fourier transform infrared spectroscopy

[0047] (1) In a dry room at room temperature, add dry HA powder and appropriate amount of potassium bromide powder to a mortar, grind thoroughly several times, then put it into a tablet press to press it into a transparent sheet, and finally put it on the machine for detection.

[0048] (2) Before testing, collect the background first, remove the background noise, then collect the infrared spectrum of the sample, resolution: 4 cm -1 , scanning times 32 times, test wave band 400-4000 cm -1 . The chemical structure of HA contains active functional groups such as carboxyl, quinone and phenolic hydroxyl groups ( Figure 4 ).

[0049] 6. Visible-near infrared absorption spectrum

[0050] (1) Dissolve HA powder with pure water to prepare HA solutions of different concentrations (0, 0.05, 0.1, 0.25, 0.5, 1, 2 mg / mL), respectively, and take 200 μL into a 96-well plate.

[0051] (2) Use SpectraMax iD3 multifunctional microplate reader to detect the absorbance at 600 nm-900 nm. Compared with pure water, HA shows a wide spectrum of absorption characteristics in the near-infrared region ( Figure 5 ).

[0052] 7. In vitro photothermal properties and stability of HA solution

[0053] (1) Prepare 1 mL of 0.05, 0.1, 0.25, 0.5, 1, 2 mg / mL HA solution in 1.5 mL EP tube.

[0054] (2) Fix the collimator (10 mm in diameter) of the fiber-coupled laser (808 nm) on the height of the stand, so that the distance between the collimator and the liquid water surface is 10 cm. Set the current to make the output power 0.5 W / cm 2 , irradiate the liquid, and detect the temperature change every 30 s within 3 min using the FLIR infrared thermal imager. Compared with pure water, the temperature change of the HA solution after NIR irradiation is higher, and shows concentration and time-dependent characteristics (Fig. 2B), which proves that HA has excellent photothermal conversion performance. Figure 6

[0055] (3) Take 0.05 mg / mL as the determination concentration. First, turn on the infrared laser to determine the temperature rise of the liquid within three minutes, and record the temperature every 30 s. After three minutes, remove the infrared light, and record the temperature change after removal, record the temperature every 30 s within the first 10 minutes, and then record it once every minute until the temperature drops to 24℃. Subsequently, repeat the above operation twice, record the temperature change, and make a cold-heat cycle curve. After three repeated heating / cooling cycles, the photothermal conversion performance of the HA solution is not reduced (Fig. 2C), which proves that HA has good photothermal stability. Figure 7

[0056] Two, in situ injection of HA in mouse inguinal white adipose tissue

[0057] (1) Take a sterile insulin syringe, and suck 100 μL PBS or HA solution into the syringe, and inject it into the mouse's inguinal area parallel to the abdominal surface.

[0058] (2) After checking that there is no problem, put the mouse back in the cage.

[0059] (3) After 24 h, fix the fiber collimator of the infrared laser at a height of 10 cm from the mouse's inguinal area, and give NIR (808 nm, 0.5 W / cm 2 ) irradiation, and use the infrared thermal imager to record the temperature change every 30 s. The results are shown in Figs. 3B and 3C. The results show that the subcutaneous white adipose tissue area of the mouse injected with HA has a significant temperature rise after NIR irradiation. Figure 8

[0060] Three, the effect and molecular mechanism of HA combined with photothermal therapy on high-fat diet-induced mouse obesity

[0061] 1. Construction and treatment of mouse model

[0062] ​​​Thirty male SPF-grade C57BL / 6J mice, aged 6-8 weeks, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After one week of acclimatization in the animal facility, they were divided into five groups (NCD, HFD, HFD+HA, HFD+NIR, and HFD+HA+NIR), with six mice in each group. The NCD group was fed a 10 kcal% fat-based diet (XTCON50J from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.), while the HFD, HFD+HA, HFD+NIR, and HFD+HA+NIR groups were fed a 60 kcal% fat-based diet (XTHF60 from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.). Body weight was recorded weekly. After 10 weeks of feeding, treatment began, with the treatment methods for each group as follows: Figure 8 As shown in A and Table 1.

[0063] Table 1 Experimental Groups

[0064]

[0065] The body weight changes of each treatment group before and after each cycle were recorded, and the treatment lasted for seven cycles. After seven cycles of treatment, the body weight of mice in the HFD+HA+NIR group showed a significant decreasing trend. Figure 8 (E).

[0066] 2. Measurement of mouse body fat using the EchoMRI-100H animal body composition analyzer

[0067] Body fat percentage in mice of each treatment group was measured using an EchoMRI-100H animal body composition analyzer. Results showed that the body fat percentage in the HA combined with photothermal therapy group was significantly reduced, approaching that of the NCD (normal control) group. Figure 8 (Middle F).

[0068] 3. Collection of mouse serum, adipose tissue, and major organs

[0069] (1) First, anesthetize the mice, weigh the mice, and calculate the dosage of anesthetic. The dosage of 1% sodium pentobarbital is 8 μL / g.

[0070] (2) Using the same steel ruler as a reference, place the mouse on a piece of white paper and take a rough picture. Figure 8 (D). Mice in the HFD group were more rounded and shorter, while mice treated with HA combined with photothermal therapy were more slender.

[0071] (3) After the mice were dissected, the peritoneal cavity was exposed, the pleura was cut open, and the heart blood was sucked up with a clean 1 mL syringe. After the needle was removed, it was punched into a 1.5 mL EP tube. After standing at room temperature for 1 h, centrifugation (4°C, 3000 rpm, 10 min) was performed, and the separated serum was temporarily stored at -80°C. The mouse inguinal white adipose tissue (iWAT), epididymal white adipose tissue (eWAT), brown adipose tissue (BAT), and the mouse major organs (heart, liver, lung, spleen, kidney) were taken out with scissors and forceps and placed in PBS buffer on ice. According to different experimental requirements, the organs were fixed or stored at -80°C for subsequent use.

[0072] (4) The separated iWAT, eWAT and BAT were placed on a film and photographed (Fig. 5B). Figure 8 The weights of the three kinds of fat of each group of mice were recorded, and the ratio of the weight to the body weight was calculated (Fig. 5C). Figure 8 The results showed that the volume and weight of white adipose tissue in the HFD group increased, but recovered after HA combined with photothermal therapy, close to that of normal mice; the volume and weight of brown adipose tissue in the HFD group decreased, but increased after HA combined with photothermal therapy, proving that HA combined with photothermal therapy can improve the distribution of adipose tissue.

[0073] 4. Hematoxylin-eosin (HE) staining of adipose tissue

[0074] The preparation of tissue sections and HE staining were entrusted to Hunan Aifang Biotechnology Co., Ltd., and the specific steps were as follows:

[0075] (1) Sampling and fixation: the fresh tissues of mice were rinsed with pre-cooled PBS buffer to remove blood or impurities. A small piece was immediately put into 4% paraformaldehyde fixing solution for room temperature fixation for more than 24 h.

[0076] (2) The tissue embedding was performed according to the steps shown in Table 2:

[0077] Table 2 Specific operation of tissue embedding

[0078]

[0079] (3) Paraffin section: the wax block was placed on a paraffin section machine for sectioning, with a thickness of 4 μm. The section was floated on a 40°C water surface of a section spreading machine to flatten the tissue, and the glass slide was used to take out the tissue. The section was baked in a 65°C oven. After water baking and wax baking, the section was taken out and stored at room temperature for standby.

[0080] (4) De-waxing and hydration: xylene, 2 times, 10 min each time; anhydrous ethanol→95% ethanol→85% ethanol→75% ethanol→distilled water, 5 min for each grade.

[0081] (4) HE staining: drop hematoxylin staining solution to cover the tissue, stain for 5 min → rinse with running water to remove the floating color → 1% hydrochloric acid alcohol differentiation for a few seconds, rinse with running water → 1% ammonia water solution for 1 min, rinse with running water → eosin staining solution, 2 min, and then wash off the floating color with distilled water.

[0082] (5) Dehydration and mounting: 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol for 1 min each → xylene, 1 min → xylene, 1 min → neutral gum mounting.

[0083] (6) Microscopic observation, image acquisition and analysis. The results showed that the white adipose tissue and brown adipocytes of the HFD group were increased, while the adipocytes were reduced and restored to normal morphology after HA combined with photothermal therapy, among which the iWAT appeared multi-chamber adipocytes, and the "whitening of brown adipose tissue" morphological change, proving that HA combined with photothermal therapy can achieve adipose phenotype remodeling of diet-induced obese mice. Figure 9 ).

[0084] 5. Total protein extraction of tissue

[0085] (1) Prepare cell tissue quick lysis solution (RIPA) and protease inhibitor benzylsulfonyl fluoride (PMSF), and vortex mix them in a volume-mass ratio of 100:1, and place on ice.

[0086] (2) Take the tissue sample from the-80℃ refrigerator and place it in a 1.5 mL EP tube, and add the corresponding lysis solution in a ratio of tissue weight:lysis solution = 1:9 (g:mL).

[0087] (3) Grind the tissue with a grinder and place it on ice for lysis for 30 min.

[0088] (4) Pre-cool the centrifuge at 4℃, 14000 rpm, and centrifuge for 30 min; collect the supernatant after centrifugation; fat tissue needs to be centrifuged twice to remove the upper fat.

[0089] (5) Perform BCA protein quantitative determination on the supernatant and perform different treatments according to the experimental purpose.

[0090] 6. Proteomic analysis of mouse iWAT

[0091] (1) Sample preparation and standardization: take one sample from each group of the above extracted inguinal white adipose tissue; after quantification by BCA method, uniformly adjust the concentration to 1 μg / μL with lysis solution, and detect 100 μg / sample (send sample on ice).

[0092] (2) Mass spectrometry detection: The sample was sent to the Wenzhou Medical University Large Instrument Platform, and after enzymatic hydrolysis and liquid chromatography separation, it was detected by machine. A quadrupole-electric field orbitrap-linear ion trap three-in-one (Orbitrap Fusion Lumos) ultra-high resolution mass spectrometer was used for detection to obtain the original protein spectrum identification data.

[0093] (3) Data quality control and filtering: The corresponding protein sequence number was searched in the UniProt database. According to the strict quality control standard shown in Table 3:

[0094] Table 3 Data quality control

[0095]

[0096] Finally, 2808 reliable proteins were obtained for subsequent analysis.

[0097] (4) Hierarchical clustering of basic protein expression: The heat map constructed by the hierarchical clustering algorithm generated by the BioLadder platform of the online bioanalysis platform. The protein expression patterns of each group of samples showed significant grouping characteristics (Fig. 2 Figure 10 A). Among them, the overall proteomic characteristics of the HFD+HA group and the HFD+HA+NIR group showed a high degree of correlation, while the proteomic characteristics of the HFD group and the HFD+NIR group were highly similar, suggesting that single NIR intervention may be difficult to reverse the abnormal protein expression induced by high-fat diet.

[0098] (5) Bioinformatics analysis: 1403 differentially expressed proteins with significant differences (p < 0.05, fold change > 2) were selected. GO analysis was performed on them using the DAVID database, which was mainly enriched in lipid metabolism-related biological behaviors (Biological Process), and the expression of specific brown adipocyte differentiation (such as UCP1) changed (Fig. 3 Figure 10 B).

[0099] 7. Western Blotting

[0100] (1) Protein sample preparation: After BCA quantification, the tissue sample was added to 5x loading buffer and lysis buffer to adjust to the same concentration, denatured at 100°C in a metal bath for 5 min, and instantaneously separated for use. It can be stored at -30°C;

[0101] (2) Gel preparation: Prepare 10% SDS-PAGE gel;

[0102] (3) Sample loading: Fix the two SDS-PAGE gel plates with a clamp from front to back, and place them in the electrophoresis tank according to the positive and negative poles. Add the newly prepared 1x SDS electrophoresis buffer diluted with ddH2O into the inner tank, and add the recovered electrophoresis buffer into the outer tank. Carefully remove the comb, and carefully add the denatured protein sample into the sample well, and mark (Marker) on both sides;

[0103] (4) Electrophoresis: Set the conditions as follows: concentrated gel 70 V, 40 min; separation gel 110 V, 60-70 min. Start electrophoresis after setting;

[0104] (5) Membrane transfer: Take out the pre-cooled 1x membrane transfer solution, pour it into a tray. Rinse the SDS of the gel plate with running water and immerse it in the membrane transfer solution. Prepare the membrane transfer clamp with the black side facing down, and assemble it in a sandwich structure "sponge-filter paper-gel-PVDF membrane-filter paper-sponge". Note that the PVDF membrane needs to be activated with methanol; there should be no air bubbles between the layers. Place the membrane transfer clamp in the electrophoresis tank after clamping, pay attention to the correspondence of the positive and negative poles, add the membrane transfer solution, and add ice bricks to keep the membrane transfer solution at low temperature. Set 250 mA for 100 min for membrane transfer after ensuring that everything is correct;

[0105] (6) Blocking: Prepare 5% skimmed milk, and place the polyvinylidene fluoride (PVDF) membrane after membrane transfer into the milk, and block it in a shaking incubator at room temperature for 2 hours;

[0106] (7) Incubation of the first antibody: After blocking, wash the strip in 1x TBST in a shaking incubator for 3 times, 5 min each time. According to the size of the desired protein and the position of the marker, cut the strip and place it in the corresponding first antibody tube, and incubate it in a shaking incubator at 4°C overnight;

[0107] (8) Incubation of the second antibody: Take out the strip from the first antibody, wash it in 1x TBST in a shaking incubator for 3 times, 5 min each time, then place it in the corresponding second antibody tube, and incubate it in a shaking incubator at room temperature for 70 min;

[0108] (9) Exposure: Take out the strip from the second antibody, wash it in 1x TBST in a shaking incubator for 3 times, 15 min each time. Prepare ECL developing solution (A liquid:B liquid = 1:1), and after washing the strip, gently absorb the water with filter paper, transfer it to the exposure instrument, drop the developing solution on the strip with a sample gun, close the door, and click to obtain the image.

[0109] The results of the development show that the expression of UCP1 and PGC-1a in the inguinal white adipose tissue of the HFD+HA+NIR group is significantly higher than that of the HFD group, close to the level of the NCD group, proving that HA combined with local photothermal promotes the browning change of white adipose tissue in obese mice, and the expression of heat production related proteins in whole body fat (eWAT, BAT) also increases Figure 10 C).

[0110] 8. UCP1 immunohistochemistry

[0111] The tissue section preparation and UCP1 immunohistochemical staining were entrusted to Hunan Aifang Biotechnology Co., Ltd., and the specific steps were as follows:

[0112] Steps (1)-(4) were the same as "4. Hematoxylin-eosin (HE) staining of adipose tissue".

[0113] (5) Blocking endogenous peroxidase: soaking the tissue section in 3% hydrogen peroxide solution, incubating at room temperature for 15 min in the dark, and rinsing the slide twice with PBS buffer for 5 min each time;

[0114] (6) Antigen repair: placing the tissue section in a repair box filled with ethylenediaminetetraacetic acid (EDTA) antigen repair buffer (final concentration 0.01 mol / L), heating in a microwave oven, medium heat for 10 min, stop heating for 5 min, medium-low heat for 5 min, cooling at room temperature, and washing twice with PBS buffer on a shaker for 5 min each time;

[0115] (7) Blocking: adding 100 μL of non-immune goat serum to cover the tissue (draw a group of immunohistochemical circles on the edge), incubating at room temperature for 30 min, and then removing it;

[0116] (8) Primary antibody incubation: adding 1:400 diluted UCP1 primary antibody to cover the tissue, placing it in a wet box at 4°C overnight, and washing with PBST for 3 times, 5 min each time;

[0117] (9) Secondary antibody incubation: adding anti-rabbit antibody to cover the tissue, incubating at room temperature for 30 min, and washing with PBS for 3 times, 5 min each time;

[0118] (10) DAB reaction color development: adding DAB color developing solution (A liquid: B liquid = 50:1) into the immunohistochemical circle, observing the color of the section under the microscope, and immediately washing with tap water to stop the color development when it turns brown-yellow;

[0119] (11) Counterstaining: hematoxylin counterstaining for 1 min, and washing with running water for 2 min;

[0120] (12) Differentiation and return to blue: 1% hydrochloric acid alcohol differentiation for 2 s, and washing with running water for 15 min;

[0121] (13) Dehydration and transparency: 85%, 95%, and anhydrous ethanol, 5 min each time → xylene deparaffinization for 2 times, 5 min each time;

[0122] (14) Natural drying, neutral resin mounting, microscopic observation, and image collection.

[0123] The results showed that the density of UCP1 positive cells in the HFD group was reduced, but the expression of UCP1 was increased after HA combined with photothermal therapy (P < 0.05) Figure 10 Fig. 6C shows that the expression of UCP1 in the white adipose tissue of the HFD group was reduced, but the expression of UCP1 was increased after HA combined with photothermal therapy (P < 0.05), further proving that HA combined with photothermal therapy can promote the browning of white adipose tissue.

[0124] Four, liver injury and histocompatibility test of mice

[0125] 1. Serum alanine aminotransferase (ALT) detection

[0126] (1) Take a clean 96-well transparent plate, and add samples according to Table 4:

[0127] Table 4 ALT detection sample addition table

[0128]

[0129] (2) After incubating for 30 min in step 1 of (1), prepare a standard curve at the same time, and add samples according to Table 5:

[0130] Table 5 Preparation of ALT standard curve sample addition table

[0131]

[0132] Take the absolute OD value (OD value of each well minus the OD value of the zero well) as the horizontal axis and the enzyme activity as the vertical axis to draw a fitting curve. The absolute OD value of the above sample is substituted into the standard curve to obtain the ALT enzyme activity.

[0133] 2. Serum aspartate aminotransferase (AST) detection

[0134] (1) Take a clean 96-well transparent plate, and add samples according to Table 6:

[0135] Table 6 AST detection sample addition

[0136]

[0137] Calculate the absolute OD value = OD value of the determination well - OD value of the control well.

[0138] (2) After incubating for 30 min in step 1 of (1), prepare a standard curve at the same time, and add samples according to Table 7:

[0139] Table 7 Preparation of AST standard curve sample addition table

[0140]

[0141] Take the absolute OD value (OD value of each well minus the OD value of the zero well) as the horizontal axis and the enzyme activity as the vertical axis to draw a fitting curve. The absolute OD value of the above sample is substituted into the standard curve to obtain the AST enzyme activity.

[0142] 3. HE staining of major organs

[0143] The steps are the same as "III. 4. Hematoxylin-eosin staining of adipose tissue". The liver HE results show that normal liver can see the orderly arrangement of hepatocytes, and the structure of liver lobule is clear; while the liver of HFD group and HFD+NIR group has a large number of fat vacuoles, and the structure of liver lobule is disordered; the pathological changes of the two groups after HA treatment are reduced, and the normal liver morphology is restored. No obvious lesions are found in the major organs, indicating that HA and / or NIR irradiation will not cause damage to the major organs of mice Figure 11 C).

[0144] The results of ALT and AST detection of serum of mice in each treatment group are shown in Figure 11 A and B, combined with the results of liver HE staining Figure 11 C) can be seen that the serum ALT and AST of mice in HFD group are increased, which confirms that high-fat diet can cause liver damage, while HA and HA combined with photothermal can reduce the level and reduce liver damage.

[0145] V. Extraction and differentiation of mouse adipose-derived stem cells (ADSCs)

[0146] 1. Primary isolation and extraction of ADSCs

[0147] (1) Anesthetize the mice with 8 μL / g of anesthetic, and after they are completely unconscious, soak them in 75% ethanol for 15 min and put them in a clean bench.

[0148] (2) Avoid hair contamination of the mice, and cut off the bilateral inguinal fat in parallel.

[0149] (3) Wash the inguinal fat tissue repeatedly 3 times in PBS buffer containing 1% penicillin-streptomycin solution (100x), and then wash it 3 times with PBS buffer.

[0150] (4) Under a microscope, try to remove blood vessels, lymph nodes, connective tissue, etc. as much as possible, and cut them into small pieces of about 1 cm 3 .

[0151] (5) Transfer to 2 times the volume of 0.2% collagenase solution, and digest in a constant temperature shaking incubator at 37°C. After 30 min, centrifuge at room temperature at 1000 rpm for 5 min, collect the cell precipitate, and resuspend the precipitate with DMEM-F12 complete medium. Continue to digest the remaining supernatant for 15 min, and cycle 2 times until the tissue appears cloudy or milky.

[0152] (6) Add an equal amount of DMEM-F12 complete medium to terminate digestion, filter through a 70 μm cell filter, and remove the undigested tissue.​

[0153] (7) Room temperature, 1000 rpm, centrifugation for 5 min, discard the supernatant. The precipitate is rinsed with PBS buffer, 1000 rpm, centrifugation for 5 min, discard the supernatant, repeat 2 times;

[0154] (8) Add red blood cell lysate, 37℃ for 5 min, then 1000 rpm, centrifugation for 5 min, discard the supernatant.

[0155] (9) Gently blow the DMEM-F12 complete medium into a single cell suspension and inoculate in a culture dish. Place in the incubator (condition setting is 37℃, 5% CO2) for culture. Change the liquid every other day.

[0156] 2. ADSCs cell differentiation induction

[0157] As shown in Figure A, the following cell experiments were carried out: Figure 12

[0158] (1) Cell inoculation and contact inhibition: inoculate into the culture plate at a density of 5×10 3 cells / cm 2 , monitor the cell growth state every day, maintain the contact inhibition state for 24 h (recorded as day 0).

[0159] (2) Absorb the original culture medium and replace it with 37℃ preheated adipogenic induction medium, place in the incubator for three days, observe the cell morphological changes during the treatment period.

[0160] (3) On the 3rd day, absorb the adipogenic induction medium and replace it with an equal volume of adipogenic maintenance medium, and re-induce in the incubator for four days.

[0161] (4) On the 7th day, discard the adipogenic maintenance medium and replace it with ordinary DMEM-F12 complete medium, and observe the differentiation of adipocytes under an inverted microscope every day, change the liquid every two days until the differentiation rate reaches more than 80%. Photograph the cell morphology before and after induction (Figure B), ADSCs before induction showed a spindle or fibroblast-like morphology, and mature adipocytes after induction by "cocktail method" showed large and transparent lipid droplets. Figure 12

[0162] Six, cytotoxicity (MTT) test

[0163] Using the standard MTT method, ADSCs cells were used as a model to study the cytotoxicity of HA+NIR ("±" means combined or not combined with NIR, and not combined with NIR means natural light).

[0164] ​​(1) Cell plating, plating requirements: 96-well plate, 3000 cells per well, 60 wells, and 5 control wells without cells, each well system 100 μL.

[0165] (2) After 12 hours of cell adhesion, the 96-well plate was taken out, and the cell state in each well was observed under a microscope. Then the original culture medium in the experimental wells was discarded, and HA solution with a final concentration of 0, 25, 50, 75, and 100 μg / mL (diluted with DMEM-F12 complete culture medium) was added, respectively, and finally returned to the incubator for 24 hours of incubation.

[0166] (3) After 24 hours, the 96-well plate was taken out, and then treated with white light or NIR (808 nm, 0.5 W / cm 2 , 10 min), and then cultured in the incubator for 12 hours before the next step.

[0167] (4) The 96-well plate was taken out, and the original culture medium was replaced with DMEM-F12 blank culture medium containing 0.5 μM MTT, and then placed in the incubator for 4 hours of incubation.

[0168] (5) Next, 100 μL of Formazan dissolving solution was directly added to each well, and then the Formazan was dissolved in the 37°C incubator.

[0169] (6) After the Formazan was completely dissolved, the SpectraMax iD3 multifunctional enzyme marker was used to detect the absorbance (OD value, represented by A) at 570 nm of each well, and the cell survival rate was calculated, with the formula as follows:

[0170] Cell survival rate (%) = (A 样本孔 - A 对照孔 ) / (A 空白孔 - A 对照孔 ) x 100%.

[0171] The results showed that whether there was 808 nm laser irradiation or not, HA with a concentration of 100 μg / mL, 75 μg / mL, 50 μg / mL, 25 μg / mL, and 10 μg / mL had no obvious toxicity to ADSCs cells (P>0.05 in the middle C). Figure 12

[0172] Seven, Effect of HA combined with photothermal on metabolism and differentiation of mature white adipocytes

[0173] 1. Oil red O staining of adipocytes

[0174] ​(1) When the adipocyte differentiation rate is ≥ 80%, replace the conventional DMEM-F12 complete culture medium with a solution of HA with a final concentration of 0, 50, 100 μg / mL (DMEM-F12 complete culture medium is prepared), and incubate for 24 h Figure 12 Central A.

[0175] (2) Half of the white light treatment is applied, and the other half is subjected to NIR (808 nm, 0.5 W / cm 2 , 10 min), and after the treatment, it is placed in the incubator for continuous culture for 12 h.

[0176] (3) The cell plate is taken out after 12 h, the old culture medium is aspirated, the cells are washed with PBS buffer for 3 times, and the cells are gently shaken to prevent cell detachment, and the PBS is discarded.

[0177] (4) 4% paraformaldehyde is added to each well for room temperature fixation for 30 min.

[0178] (5) The fixing solution is aspirated, and the PBS buffer is washed for 3 times and aspirated.

[0179] (6) 60% isopropanol solution is added along the side wall, and the dyeing is performed for 3 min.

[0180] (7) The isopropanol is aspirated, and the prepared oil red O working solution is added along the side wall, and the staining is performed at room temperature for 30 min.

[0181] (8) The oil red O working solution is aspirated, and the residual dyeing solution is washed with PBS buffer until the upper solution is colorless.

[0182] (9) The deposition of lipid droplets in the cells is observed under an inverted optical microscope, and the photographs are recorded.

[0183] The mature adipocytes induced by the "cocktail method" present large and transparent lipid droplets, and after the HA treatment, the size of the lipid droplets in the adipocytes is significantly reduced, and the morphology of the brown-like adipocytes is more presented Figure 12 Central D.

[0184] 2. Mitochondrial deep red fluorescent probe (MitoTracker Deep Red) staining

[0185] (1) Prepare preheated serum-free DMEM-F12 medium, and dilute the probe at 1:1000.

[0186] (4) Remove the old culture medium, and gently wash the cells with PBS buffer to remove the residual serum-containing culture medium.

[0187] (5) Add 300 μL of serum-free medium containing the probe to each well, and incubate at 37°C for 30 min.

[0188] (6) Discard the staining solution, wash with PBS buffer for 3 times, and add 300 μL of serum-free medium.

[0189] (7) Observe under an inverted fluorescence microscope.

[0190] Note: Avoid light throughout the process to prevent fluorescence quenching and affect the staining effect.

[0191] Through fluorescence microscope observation, it can be found that the number of mitochondria in mature adipocytes treated with HA+NIR is significantly increased Figure 12 Medium E).

[0192] 3. Protein extraction and Western Blotting of mature adipocytes

[0193] (1) Discard the original culture medium and wash with PBS buffer for 3 times.

[0194] (2) Add 60 μL of lysis buffer to each well, scrape with a cell scraper, and collect into a 1.5 mL EP tube.

[0195] (3) After lysis on ice for 30 min, centrifuge at 4°C, 14000 rpm for 30 min, and collect the supernatant after centrifugation. Determine the protein concentration by BCA method, adjust to 3 mg / mL, and denature at 100°C for 5 min.

[0196] (4) The steps of Western Blotting are shown in the section "3.7. Western Blotting".

[0197] Figure 12 Medium F shows that the expression levels of heat production related proteins UCP1 and PGC-1a in mature white adipocytes are increased by HA and NIR combined treatment, which also verifies at the cellular level that HA combined with local photothermal can promote the browning of white adipose tissue.

[0198] The above-described embodiments are only preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. Application of sodium humate in the preparation of drugs for treating obesity.

2. The application according to claim 1, characterized in that, The drug is a photothermal conversion drug based on photothermal therapy.

3. The application according to claim 1, characterized in that, The drug treats obesity by reducing weight, lowering body fat percentage, improving fat distribution, and / or browning white fat.

Images