Application of RLN-3 in preparation of medicine for preventing and treating diabetic adipose tissue inflammation

Abstract

The invention relates to application of RLN-3 in preparation of a medicine for preventing and treating diabetic adipose tissue inflammation, and belongs to the technical field of medicines. In order to solve the problem that an existing medicine is poor in treatment effect on diabetic adipose tissue inflammation, the invention provides application of RLN-3 in preparation of a medicine for preventing and treating diabetic adipose tissue inflammation, and the medicine for preventing and treating diabetic adipose tissue inflammation takes RLN-3 as a unique active component or one of the active components. Cell and animal experiments prove that RLN-3 can promote the transformation of macrophage phenotype from M1 type to M2 type; expression of anti-inflammatory factors in adipose tissue and serum is up-regulated, and diabetic adipose tissue inflammatory response and adipose tissue fibrosis are inhibited. The new clinical application of RLN-3 is developed based on the effect of RLN-3 in inhibiting adipose tissue inflammatory response, and RLN-3 is safe and non-toxic, so that RLN-3 is expected to be developed into a new drug for safely and effectively improving insulin resistance and reducing blood sugar level.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to the application of RLN-3 in preparing a drug for preventing and treating diabetic adipose tissue inflammation. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by high blood sugar levels, often accompanied by insulin resistance and pancreatic beta cell dysfunction. Insulin resistance means that the biological effect of insulin in lowering blood sugar cannot be fully exerted in the body, that is, the body's sensitivity to insulin decreases, which will lead to increased blood sugar and further aggravate the condition of diabetes.

[0003] In the pathogenesis of diabetes, adipose tissue inflammation plays a key role in diabetic insulin resistance. Under inflammatory conditions, adipocytes produce a large number of inflammatory factors, such as TNF-α, IL-10, etc. TNF-α is the first proinflammatory cytokine found to be associated with insulin resistance. It interferes with the phosphorylation of insulin receptor substrates, damages the insulin signaling pathway, and leads to decreased insulin sensitivity. Inflammatory factors such as IL-10 also participate in the occurrence of insulin resistance through similar mechanisms. Adipose tissue is rich in macrophages, which will change from the anti-inflammatory M2 type to the pro-inflammatory M1 type under inflammatory conditions. M1 macrophages secrete a large number of inflammatory factors, further exacerbating the inflammatory response and insulin resistance.

[0004] Diabetic adipose tissue inflammation is significantly different from other types of inflammation. It is mainly manifested as chronic low-grade inflammation under the environment of hyperglycemia and lipotoxicity. In addition, diabetic patients are usually accompanied by a variety of metabolic abnormalities, and simple anti-inflammatory treatment is difficult to comprehensively improve the condition. Although existing technologies and drugs have made certain progress in the prevention and treatment of diabetes and its related adipose tissue inflammation, there are still certain defects. For example, the adverse reactions of insulin sensitizers. Although this type of drug can increase insulin sensitivity, adverse reactions such as weight gain and heart failure may occur during its use, thus limiting its clinical application. Summary of the invention

[0005] In order to solve the problem that existing drugs have poor therapeutic effects on diabetic adipose tissue inflammation, the present invention provides the use of RLN-3 in preparing drugs for preventing and treating diabetic adipose tissue inflammation.

[0006] The technical solution of the present invention:

[0007] Application of relaxin RLN-3 in the preparation of drugs for preventing and treating diabetic adipose tissue inflammation.

[0008] Furthermore, the drug for preventing and treating diabetic adipose tissue inflammation contains RLN-3 as the only active ingredient or one of the active ingredients.

[0009] Furthermore, the content of RLN-3 in the drug for preventing and treating diabetic adipose tissue inflammation is 0.1wt% to 99wt%.

[0010] Furthermore, the drug for preventing and treating diabetic adipose tissue inflammation also includes pharmaceutically acceptable excipients.

[0011] Furthermore, the drug for preventing and treating diabetic adipose tissue inflammation is a parenteral dosage form.

[0012] Furthermore, the drug for preventing and treating diabetic adipose tissue inflammation is in the form of an injectable dosage form.

[0013] Furthermore, the drug for preventing and treating diabetic adipose tissue inflammation inhibits the inflammatory response of diabetic adipose tissue by promoting the transformation of macrophage phenotype from M1 to M2.

[0014] Furthermore, the drug for preventing and treating diabetic adipose tissue inflammation inhibits the inflammatory response of diabetic adipose tissue by upregulating the expression of anti-inflammatory factors.

[0015] Furthermore, the drug for preventing and treating diabetic adipose tissue inflammation inhibits diabetic adipose tissue fibrosis by inhibiting the inflammatory response of diabetic adipose tissue.

[0016] Beneficial effects of the present invention:

[0017] The present invention provides an application of relaxin RLN-3 (Relaxin-3) in drugs for preventing and treating inflammation in diabetic adipose tissue. The present invention confirms through animal experiments that RLN-3 can inhibit the M1 phenotype transformation of macrophages, promote the increase of macrophage lactate levels, and stimulate the transformation of macrophage phenotype from M1 to M2; it can reduce the expression of pro-inflammatory factors in adipose tissue and serum and upregulate the expression of anti-inflammatory factors, thereby inhibiting the inflammatory response of diabetic adipose tissue and adipose tissue fibrosis. The present invention develops new clinical uses of RLN-3 based on its efficacy in inhibiting the inflammatory response of adipose tissue. Since RLN-3 is safe and non-toxic, it is expected to be developed into a new generation of safe and effective new drugs for improving insulin resistance and lowering blood sugar levels. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a Western blot of macrophage phenotype markers in each group of Example 1;

[0019] Figure 2 This is a comparison chart of the protein expression of macrophage phenotype markers in each group of Example 1, A is iNOS, B is CD86, C is Arg1, and D is CD206;

[0020] Figure 3 This is a comparison of intracellular and extracellular lactic acid concentrations of macrophages in each group of Example 1, A is the intracellular lactic acid concentration, and B is the extracellular lactic acid concentration;

[0021] Figure 4 The comparison diagram of lactic acidification degree of macrophages in each group of Example 1, A is a comparison diagram of lactic acid level, and B is a comparison diagram of H3K18la expression;

[0022] Figure 5 Western blot images of proteins in the upstream mechanism of lactate production in macrophages of each group in Example 1;

[0023] Figure 6 This is a comparison chart of protein expression of proteins of the upstream mechanism of macrophage lactate production in each group of Example 1, A is LDHA, B is PKM1, and C is PKM2;

[0024] Figure 7 It is a comparison chart of inflammatory factors in the serum and adipose tissue of each group of rats in Example 2, A is TNF-α in rat adipose tissue, B is IL-1β in rat adipose tissue, C is IL-10 in rat adipose tissue, D is TNF-α in rat serum, E is IL-1β in rat serum, and F is IL-10 in rat serum;

[0025] Figure 8 HE staining and MASSON staining of rats in each group in Example 2, A is HE staining, B is MASSON staining;

[0026] Fig. 9 These are immunofluorescence comparison photos of M1 phenotype markers of adipose tissue macrophages in each group of rats in Example 2;

[0027] Fig.10 These are immunofluorescence comparison photos of M2 phenotype markers of adipose tissue macrophages in each group of rats in Example 2. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0029] 1. Experimental animals used in the animal experiments of the present invention:

[0030] Thirty healthy male SD rats, 6 weeks old, SPF microbial grade, were purchased from the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University. The rats were kept under standard conditions, with the indoor temperature controlled at about 22±2°C, the humidity controlled at about 60±10%, 12 hours of light per day, 12 hours of dark cycle control (lighting time was 06:00-18:00), free drinking water, and regular ultraviolet light disinfection.

[0031] 2. The macrophage cell line used in the cell experiment of the present invention: mouse mononuclear macrophage leukemia cells (RAW264.7).

[0032] 3. The relaxin RLN-3 used in the animal test and cell test of the present invention was purchased from Phoenix Pharmaceuticals Inc. The RLN-3 used in the example includes an A chain and a B chain, and the A chain and the B chain are cross-supported by three disulfide bonds;

[0033] The amino acid sequence of the A chain is DVLAGLSSSCCKWGCSKSEISSLC;

[0034] The amino acid sequence of the B chain is RAAPYGVRLCGREFIRAVIFTCR;

[0035] The three disulfide bonds are A chain Cys10-A chain Cys15; A chain Cys11-B chain Cys10 and A chain Cys24-B chain Cys22.

[0036] 4. Method for preparing the main reagents in the animal test and cell test of the present invention:

[0037] (1) TBST buffer

[0038] 100 ml of TBS 10× buffer and 200.5 ml of 20% Tween were added to deionized water to make up to 1000 ml, and the mixture was shaken and stirred to mix well. The mixture was mixed well before each use and stored at room temperature.

[0039] (2) Electrophoresis buffer

[0040] 3.03 g of Tris-Base (PH = 8.3), 14.4 g of glycine, 1.0 g of SDS, add deionized water to make up to 1000 ml, stir evenly with a magnetic stirrer, store at room temperature, and can be reused 2-3 times.

[0041] (3) Electrotransfer buffer

[0042] 3.03 g of Tris-base (PH=8.8), 14.4 g of glycine, 200 ml of anhydrous methanol, add deionized water to make up to 1000 ml, stir evenly with a magnetic stirrer, and store at 4°C.

[0043] (4) Blocking solution

[0044] Add 5g of skimmed milk powder for sealing into 100ml of prepared TBST solution, stir with a magnetic stirrer until dissolved evenly, and store at -20℃. It can be reused 3-4 times.

[0045] (5) Horseradish enzyme labeled goat anti-rabbit, anti-mouse IgG antibody dilution

[0046] Horseradish enzyme-labeled goat anti-rabbit and anti-mouse IgG antibodies were prepared in a 1:5000 ratio with TBST buffer.

[0047] (6) 10% FBS high glucose DMEM medium

[0048] 50ml fetal bovine serum, 445ml high-glucose DMEM medium, 1ml penicillin-streptomycin solution (100×), take the above reagents out of the 4℃ refrigerator, rewarm them, and prepare them in a clean bench after irradiation with ultraviolet light for 30 minutes. Store at 4℃ after preparation and use within a short period of time.

[0049] (7) 10% FBS low-glucose DMEM medium

[0050] 50ml fetal bovine serum, 445ml low-glucose DMEM medium, 1ml penicillin-streptomycin solution (100×), take the above reagents out of the 4℃ refrigerator, rewarm them, and prepare them in a clean bench after irradiation with ultraviolet light for 30 minutes. Store at 4℃ after preparation and use within a short period of time.

[0051] (8) 33.3mmol / L high glucose DMEM medium

[0052] Weigh 0.25 g of D-glucose powder and add it to 50 ml of the prepared 10% FBS low-glucose DMEM medium. Mix well, filter and use, and store at 4°C.

[0053] (9) Preparation of palmitic acid solution

[0054] 1) Prepare 40% d-BSA solution: Weigh 1.2 g d-BSA and place it in 3 ml PBS buffer preheated to 55°C. Centrifuge at 8000 rpm for 20 minutes to obtain 3 ml of a completely dissolved 40% d-BSA solution, which is a brown-yellow clear liquid.

[0055] 2) Prepare 40mM PA solution: Dissolve 0.04g NaOH in 10ml deionized water to prepare 10ml 0.1mol / L NaOH solution. Add 0.0307g PA to the above 3ml NaOH solution and saponify in a 75℃ water bath for about 30 minutes to obtain a colorless, clear and transparent liquid, which is 40mM palmitic acid saponified liquid.

[0056] 3) Rapidly add the prepared PA to the PBS solution to obtain 6 ml of 2 mM PA + 20% PBS solution, shake well, and place in a water bath below 55°C for 30 minutes to dissolve to obtain a brown-yellow clear solution.

[0057] 4) After filtering with a bacterial filter in a clean bench, store in a 4°C refrigerator. The working concentration can be diluted 40× with 10% FBS and 33.3mmol / L high-glucose DMEM medium to obtain 500μmol / LPA+0.1%BSA+

[0058] High glucose DMEM medium with 10% FBS.

[0059] V. Statistical Analysis of Animal and Cell Experiments of the Present Invention

[0060] The experimental measurement data were expressed as mean ± standard deviation and analyzed using SPSS26.0 statistical software. One-way ANOVA was used for variance analysis of multi-component data, and Tukey's multiple comparisons were performed with P < 0.05 as the difference being statistically significant (*, P < 0.05; **, P < 0.01).

[0061] Example 1

[0062] This example demonstrates that RLN-3 can regulate the transformation of macrophage phenotypes through RAW264.7 cell experiments.

[0063] 1. Cultivation of RAW264.7 cells

[0064] 1. Cell recovery: Irradiate the cell recovery tool with UV light in the clean bench for 30 minutes, and place the cell culture medium stored at 4°C at room temperature. Heat the water bath to 37°C, place the RAW264.7 cells stored in liquid nitrogen in a 37°C water bath, and gently shake the cryotube until it dissolves.

[0065] Place the dissolved cells in a clean bench, use a pipette irradiated with ultraviolet light to suck the cell freezing solution into a 15ml centrifuge tube, then slowly add 2ml of low-glucose medium, and mix the cells and low-glucose medium by blowing. Place in a high-speed centrifuge at 1000 rpm for 5 minutes. Discard the supernatant after centrifugation, retain the cell pellet, add 4ml of low-glucose medium, gently blow the medium and cell pellet to mix, use a pipette to transfer the medium mixed with cells to a 25cm cell culture bottle, shake it in the "8" method, and put it in a cell culture incubator. After 24 hours, observe the cell morphology and density. If the cell morphology is good and the density exceeds 90%, proceed to the next step.

[0066] 2. Cell passaging: Rewarm the low-glucose culture medium and PBS buffer at room temperature, discard the original culture medium, add 3ml PBS, gently shake the culture bottle, clean the residual culture medium on the cell surface, and repeat the operation twice. Continue to add 3ml of fresh low-glucose culture medium to the culture bottle, blow the cell surface with the culture medium to make the adherent cells fall off, blow 30-50 times, observe under a microscope that most of the cells have fallen off, transfer the liquid in the culture bottle to a 15ml centrifuge tube, balance centrifuge, high speed 1000 rpm, centrifuge for 5 minutes. Discard the supernatant after centrifugation and add 3ml of fresh low-glucose culture medium, gently blow the culture medium and cell pellet and mix them, and pass them at a ratio of 1:3. Place in a cell culture incubator and continue to culture.

[0067] 2. RAW264.7 Cell Grouping

[0068] To study the regulation of RLN-3 (Relaxin-3) on the phenotypic transformation of macrophages induced by high glucose and palmitic acid, RAW264.7 cells were divided into four groups:

[0069] 1) CON group: cultured in low-glucose medium for 12 hours;

[0070] 2) HG+PA group (high glucose HG: 33.3mmol / L + palmitic acid PA: 500umol / L): cultured in high glucose combined with palmitic acid medium for 12 hours;

[0071] 3) HG+PA+Relaxin-3 group (Relaxin-3: 100 ng / ml, high glucose HG: 33.3 mmol / L + palmitic acid PA: 500 umol / L): After pretreatment with Relaxin-3 for 30 minutes, the cells were cultured in high glucose combined with palmitic acid medium for 12 hours;

[0072] 4) Relaxin-3 group: After pretreatment with Relaxin-3 for 30 minutes, the cells were cultured in low-glucose medium for 12 hours.

[0073] 3. Western Blot Detection of Related Protein Expression

[0074] 1. Protein Extraction from Cell Samples

[0075] 1) Observe the morphology and density of cells under a microscope, calculate the cell lysis buffer required for the cells, and prepare the cell lysis buffer in advance. The ratio of the lysis buffer to the protease inhibitor is 1000:10.

[0076] 2) Place the cells from which protein extraction is required on ice, discard the cell culture medium, wash the residual culture medium on the cell surface with 4°C PBS, and add an appropriate amount of cell lysis solution (100-200μl). Gently scrape the cells with a disposable cell scraper, collect them in a 1.5ml EP tube, lyse and shake on ice for half an hour.

[0077] 3) Place in a high-speed centrifuge at 13500 rpm / min, 4°C, for 20 minutes.

[0078] 4) Use a micropipette with an appropriate range to absorb the supernatant after centrifugation, add it to an EP tube, and mark it. The protein concentration to be measured.

[0079] 2. Preparation of protein standard curve and determination of protein concentration (BCA method)

[0080] 1) Prepare BCA working solution: solution A: solution B = 50:1;

[0081] 2) Add deionized water to the standard wells of the 96-well plate, and add 19 μl of deionized water to the 20 μl, 19 μl, 18 μl, 16 μl, 12 μl, 8 μl, 4 μl, and 0 μl sample wells;

[0082] 3) Dilute the protein standard. In a 96-well plate, add 0μl, 1μl, 2μl, 4μl, 8μl, 12μl, 16μl, and 20μl of the diluted standard to the standard wells, and add 1μl of cell protein to the sample wells.

[0083] 4) Add 200 μl of BCA working solution to each well and incubate at 37°C for 30 minutes in the dark.

[0084] 5) Measure the absorbance at A540nm using an enzyme reader, record the reading, and calculate the protein concentration based on the standard curve.

[0085] 6) Protein denaturation: 5× Loading Buffer and the obtained cell protein were added to the supernatant at a ratio of 1:4, and placed at 100° C. for 10 minutes to denature the cell protein, thereby obtaining a protein sample.

[0086] 3. Protein index detection

[0087] 1) After cleaning the glass plate with deionized water, fix it on the gel making machine, and test the water between the flat glass plate and the concave glass plate with deionized water for 10 minutes to observe whether the seal between the glass plates is good. If the glass plates are well sealed, prepare the separation gel.

[0088] 2) Rewarm the lower layer glue, lower layer glue buffer and coagulant at room temperature. The ratio of lower layer glue to lower layer glue buffer is 1:1. Take the appropriate amount of the above liquid and add it to the beaker to mix well. Add the liquid to the gap of the glass plate and shake it slightly to avoid bubbles.

[0089] 3) Use a pipette to quickly add anhydrous ethanol to the gap of the glass plate to seal the glue. Observe that there are no bubbles between the anhydrous ethanol and the lower layer of glue. Solidify at room temperature for 15-20 minutes.

[0090] 4) After 15-20 minutes, the lower layer of gel solidifies and the upper layer of anhydrous ethanol is discarded.

[0091] 5) Prepare the upper layer of glue according to the required amount, mix the upper layer of glue and add it to the solidified lower layer of glue, quickly insert the comb and observe whether there are any bubbles, and solidify it at room temperature for 15-20 minutes;

[0092] 6) After the upper layer of gel solidifies, remove the glass plate from the gel dispenser, place it in the electrophoresis tank, add the prepared electrophoresis solution, and slowly pull out the comb;

[0093] 7) Protein loading: Take out the extracted protein sample from -20℃ and let it warm up at room temperature. Add 3μl of marker to the lanes on both sides as a mark, and then load the sample in sequence according to the experimental grouping, adding the calculated protein loading amount.

[0094] 8) Protein electrophoresis: After loading, add sufficient electrophoresis solution to the electrophoresis tank, connect the electrophoresis instrument, set the voltage to 80V, and adjust the electrophoresis time according to the molecular weight of the protein.

[0095] 9) Protein transfer: Pour the electrotransfer buffer into the tray, put the "sandwich" clip into the tray with the white side facing down, and put the fiber pad, 3M filter paper, methanol-activated PVDF membrane, lower layer of gel containing the target protein band, 3M filter paper, fiber pad in sequence, clamp the clip tightly to ensure that there are no bubbles between each layer. Place the clip in the electrotransfer tank in the correct direction and add the electrotransfer solution. Place the electrotransfer tank in ice, connect the electrotransfer instrument, and set the electrotransfer conditions: 200V. The electrotransfer time is adjusted according to the molecular weight of the protein.

[0096] 10) Blocking: After the transfer is completed, place the membrane in a special skim milk blocking solution, shake it slowly on a shaker, and incubate it at room temperature for two hours.

[0097] 11) After the blocking time is over, transfer the membrane to TBST buffer and wash it quickly for 10 minutes to clean the remaining blocking solution on the PVDF membrane.

[0098] 12) Incubate with primary antibody: dilute the primary antibody with TBST buffer according to the ratio, place the membrane in the diluted primary antibody, and shake at 4°C overnight;

[0099] 13) Washing the primary antibody: Recover the primary antibody and wash the membrane quickly on a shaker in TBST buffer for 10 minutes x 3 times;

[0100] 14) Incubate with secondary antibody: Place the membrane in the prepared secondary antibody, place on a shaker, and incubate at room temperature for 1 hour;

[0101] 15) Washing the secondary antibody: wash the membrane three times with TBST buffer, 10 minutes each time;

[0102] 16) Prepare the luminescent solution: add developer A and developer B in a volume ratio of 1:1 and place in a dark place;

[0103] 17) Development: Immerse the mixed solution on the membrane to ensure that the membrane is evenly covered, react at room temperature for 1 minute, and place it in the BIO-RAD gel imager for development.

[0104] 4. Lactic acid content detection

[0105] 1) Prepare the required reagents in advance according to the instructions: enzyme working solution, color developing agent;

[0106] 2) Use a potent cell lysis buffer to lyse macrophages for 30 minutes, extract 20 μl of cell fluid, and add it to a 5 ml EP tube; add 20 μl of the supernatant of macrophages to each 5 ml EP tube; add 1 ml of enzyme working solution and 200 μl of color developer to the above cell fluid and supernatant, mix well, and react in a 37°C water bath for 10 minutes.

[0107] 3) Add 2 ml of stop solution to each tube and mix thoroughly;

[0108] 4) Take 250 μl of the reaction solution from each tube and place it in a 96-well plate, read the results with an ELISA reader at a wavelength of 530 nm.

[0109] Figure 1 This is a Western blot of macrophage phenotype markers in each group of Example 1;

[0110] Figure 2 This is a comparison chart of the protein expression of macrophage phenotype markers in each group of Example 1, A is iNOS, B is CD86, C is Arg1, and D is CD206;

[0111] like Figure 1 and Figure 2As shown in the figure, compared with the control group, the expression of iNOS and CD86, the phenotypic markers of M1 macrophages in the HG+PA group, was significantly increased. After the addition of RLN-3 (Relaxin-3), the upregulation of the above indicators was reversed. Compared with the control group, the expression of Arg1 and CD206, the phenotypic markers of M2 macrophages, was upregulated under the induction of high glucose and palmitic acid, and the expression was further increased after exogenous administration of RLN-3 (Relaxin-3). These results suggest that RLN-3 (Relaxin-3) can inhibit the M1 phenotypic transformation of macrophages and promote the M2 phenotypic transformation.

[0112] Figure 3 This is a comparison of intracellular and extracellular lactic acid concentrations of macrophages in each group of Example 1, A is the intracellular lactic acid concentration, and B is the extracellular lactic acid concentration; Figure 4 1 is a comparison chart of the lactic acidification degree of macrophages in each group of Example 1, A is a comparison chart of lactic acid levels, and B is a comparison chart of H3K18la expression.

[0113] like Figure 3 and Figure 4 As shown in the figure, compared with the control group, the levels of lactic acid inside and outside the cells increased under the induction of high sugar combined with palmitic acid, and the exogenous administration of RLN-3 (Relaxin-3) promoted the further increase of lactic acid levels. Subsequently, the expression of pan-lactylation increased, further promoting the increase of H3K18la expression, stimulating the transformation of macrophages from M1 to M2, and increasing the expression of Arg1, thereby exerting its anti-inflammatory effect.

[0114] Figure 5 Western blot images of proteins in the upstream mechanism of lactate production in macrophages of each group in Example 1; Figure 6 This is a comparison chart of the protein expression of the upstream mechanism proteins of macrophage lactate production in each group of Example 1, A is LDHA, B is PKM1, and C is PKM2.

[0115] Glycolysis is the key process of lactate production. The three key enzymes in this process are hexokinase, phosphofructokinase-1 and pyruvate kinase (PK). Pyruvate kinase is the key enzyme that regulates the last step of glycolysis, including muscle-type pyruvate kinase (PKM), which is divided into PKM1 and PKM2. The main function of lactate dehydrogenase (LDH) is to catalyze the oxidation of lactate to pyruvate, while lactate dehydrogenase A (LDHA) has a higher affinity for pyruvate.

[0116] like Figure 5 and Figure 6As shown in the figure, under the induction of high sugar combined with palmitic acid, LDHA expression was upregulated after administration of exogenous Relaxin-3. Compared with the control group, PKM2 expression increased under the induction of high sugar combined with palmitic acid, and downregulated after administration of exogenous RLN-3 (Relaxin-3); PKM1 expression was downregulated under the induction of high sugar combined with palmitic acid, and upregulated after administration of exogenous RLN-3 (Relaxin-3).

[0117] Example 2

[0118] The inflammatory response of adipose tissue is an important part of the mechanism of type 2 diabetes and insulin resistance, and secretes a variety of pro-inflammatory and anti-inflammatory factors. In order to further verify the role of H3 relaxin, we conducted in vivo experiments. This example confirmed that RLN-3 can inhibit the inflammatory response and adipose tissue fibrosis in diabetic adipose tissue through type 2 diabetes rat model experiments.

[0119] 1. Construction of type 2 diabetes rat model

[0120] Adult male SD rats (180-220 g) aged 8 weeks were purchased and randomly divided into a control group (CON group), a type 2 diabetic rat group (DM group), and a type 2 diabetes + Relaxin-3 group (DM + R3 group).

[0121] The CON group was fed with a standard normal diet, and the DM group and the DM+R3 group were fed with a high-fat diet (D12492, fat energy ratio = 60% kcal, protein energy ratio = 20% kcal, carbohydrate ratio = 20% kcal).

[0122] Four weeks later, the rats in the DM and DM+R3 groups were injected with a single intraperitoneal vein injection of STZ (35 mg / kg, Sigma-Aldrich, St. Louis, MO, USA, dissolved in 0.1 mol / L citric acid-sodium citrate solution, pH 4.4), and the rats in the control group were injected with an equal dose of citric acid-sodium citrate solution.

[0123] One week after STZ injection, the three groups of SD rats were tested for tail vein blood glucose with a blood glucose meter. Two consecutive blood glucose levels ≥16.7mmol / L indicated successful modeling and the data were recorded. After STZ injection, high-fat diet was continued for 8 weeks, for a total of 12 weeks, and then the rats were collected.

[0124] 2. Intraperitoneal administration of rats in the type 2 diabetes + Relaxin-3 group

[0125] Six weeks after STZ injection, the DM+R3 group was given an intraperitoneal injection of Relaxin-3 (2 μg / kg / d), and the CON and DM groups were given an intraperitoneal injection of an equal volume of saline for 2 consecutive weeks.

[0126] 3. Collection of Rat Adipose Tissue

[0127] 1) Anesthesia: Rats were anesthetized by intraperitoneal injection of 0.01 ml / g 2.5% aflototin solution;

[0128] 2) Fixation: After the anesthetized rats were unresponsive, they were fixed on a fixation table in the supine position;

[0129] 3) Blood sampling from the abdominal aorta: Use sterilized surgical scissors to cut the abdominal cavity along the midline of the abdomen to fully expose the abdominal aorta, puncture the abdominal aorta at the centripetal end of the bifurcation, with the needle tip facing downward, the needle insertion angle is about 25°-30°, the depth is preferably 5mm, and blood is slowly drawn;

[0130] 4) Separate the fat from the rat epididymis, place in PBS buffer at 4°C, remove the residual blood around it, wipe dry with filter paper, and package;

[0131] 5) Part of the obtained adipose tissue is subjected to pathological testing, and part is subjected to ELISA testing.

[0132] 4. Paraffin Embedding and Sectioning

[0133] 1) Fresh adipose tissue was placed in 4% paraformaldehyde at room temperature overnight.

[0134] 2) Dehydrate the adipose tissue specimens in a gradient of alcohols: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, alcohol benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, wax I for 1 hour, wax II for 1 hour, and wax III for 1 hour.

[0135] 3) Embed the wax-soaked fat tissue in an embedding machine. Place the melted wax in the embedding frame, and take the tissue out of the dehydration box and put it into the embedding frame before the wax solidifies. Cool it at -20℃, take it out after the wax solidifies, and trim the wax block.

[0136] 4) Place the trimmed wax block on a paraffin slicer and slice it to a thickness of about 4 μm. Float the slices on 40°C warm water on the slicer to spread the tissue, then pick them up with a glass slide and place them in a 60°C oven overnight.

[0137] 5. HE staining of adipose tissue

[0138] 1) Place tissue sections in xylene I, II, and III in sequence for 5 minutes each time;

[0139] 2) Place tissue sections into the following different concentrations of alcohol for 3-5 minutes each time for hydration;

[0140] 3) Stain the sections with hematoxylin for 5-8 minutes and rinse with tap water for 1-2 minutes;

[0141] 4) Immerse in dilute hydrochloric acid ethanol solution for differentiation for 2-3 seconds.

[0142] 5) Debluing with 1% ammonia water for 10 seconds, then rinse with tap water for about 1-3 minutes;

[0143] 6) Immerse in 0.5% eosin solution for staining for about 2 minutes;

[0144] 7) Dehydrate the tissue sections in 70%, 80%, 90% ethanol once each, 95% ethanol twice, and 100% ethanol three times, for 1 minute each time.

[0145] 8) Put each group of tissue sections into xylene for three times, each time for 3 minutes;

[0146] 9) Add neutral gum to the slide, seal it with a cover slip, and dry it in an oven.

[0147] VI. Masson staining of adipose tissue

[0148] 1) Dewax the sections and rinse with distilled water;

[0149] 2) Stain with Weigert iron hematoxylin for 5-10 minutes, then rinse with running water for several minutes;

[0150] 3) 1% hydrochloric acid alcohol for 15 seconds, rinse with running water for several minutes;

[0151] 4) Masson bluing solution turns blue, wash with water, and wash with distilled water for 1 minute;

[0152] 5) Stain with Ponceau acid fuchsin solution for 5-10 minutes, and rinse with running water for several minutes;

[0153] 6) Differentiate with 1% phosphomolybdic acid solution for about 5 minutes and spin dry;

[0154] 7) Stain with aniline blue solution for 5 minutes;

[0155] 8) Rinse the sections with 1% glacial acetic acid for 1 minute;

[0156] 9) After rapid water washing, rapid dehydration was performed with 95% ethanol for 5 minutes each time, twice in total, and dehydration was performed with anhydrous ethanol for 5 minutes each time, twice in total;

[0157] 10) Xylene clearing 2 times, 5 minutes each time;

[0158] 11) After air drying, add neutral gum and seal the slide;

[0159] 12) Observe and take photos under a light microscope;

[0160] 7. Adipose tissue immunofluorescence probe detection

[0161] 1) Dewaxing and hydration: Put the sections into xylene I for 10 minutes, xylene II for 10 minutes, xylene III for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, and rinse with PBS for 5 minutes;

[0162] 2) Repair: Place the slides in a box filled with EDTA antigen repair buffer, place in a microwave oven, heat on high for 5 minutes, cool for 10 minutes, repeat 2-3 times, and after natural cooling, place the slides on a decolorizing shaker in PBS and wash 3 times, 5 minutes each time;

[0163] 3) BSA blocking: After the slices are dried, use a tissue pen to draw a circle around the tissue, add 3% BSA for blocking and incubate for 30 minutes;

[0164] 4) Incubation with primary antibody: Gently shake off the blocking solution, add two primary antibodies prepared in a certain ratio on the slices, and incubate the slices flat in a humidified box at 4°C overnight.

[0165] 5) Incubate with secondary antibody, wash the slides in PBS 3 times, 5 minutes each time. After drying the sections, add secondary antibody of the same species as the primary antibody in the circle, cover the tissue, and incubate at room temperature in the dark for 50 minutes.

[0166] 6) DAPI counterstaining of cell nuclei: Wash the slides in PBS for 3 times, 5 minutes each time. After the sections are dried, add DAPI staining solution in the circle and incubate at room temperature for 10 minutes away from light;

[0167] 7) Sealing: Wash the slides in PBS for 3 times, 5 minutes each time. After drying, seal the slides with fluorescence quenching sealing medium;

[0168] 8) Place the slices under a microscope to take photos and collect images.

[0169] 8. ELISA detection of inflammatory factors in adipose tissue and serum

[0170] 1) Preparation of sample tissue homogenate: Wash 0.2-1g of tissue with pre-cooled saline, wipe it with filter paper and weigh it, then use a measuring cylinder to take 9 times the weight of the tissue with pre-cooled saline. Cut the tissue block into pieces in the saline as quickly as possible. Grind the cut tissue block for 10 seconds / time, with an interval of 30 seconds, repeat 3-4 times to obtain 10% tissue homogenate. Centrifuge the tissue homogenate at 3000 rpm for 10-15 minutes, and the supernatant is the sample to be tested.

[0171] 2) ELISA method to detect adipose tissue and serum inflammatory factors

[0172] Prepare reagents according to the instructions of the kit and calculate the required amount of reagents; package the well plate one day in advance and incubate in a 4°C refrigerator for 16-18 hours; wash the well plate and repeat four times; add 200μl of blocking solution to each well; seal the plate and incubate on a shaker at room temperature for 1 hour; wash the well plate and repeat four times; add 100μl of standard or sample to each well; seal the plate and incubate on a shaker at room temperature for 2 hours; wash the well plate and repeat four times; add 100μl of Avidin-HRP and seal the plate and incubate on a shaker at room temperature for 0.5 hour; wash the well plate and repeat five times; finally, wash in sequence and soak the wells in buffer for 30 seconds to 1 minute; add 100μl of TMB colorimetric solution and incubate in the dark for 15 minutes; add 100μl of Stop solution; read the absorbance at 450nm within 15 minutes.

[0173] Figure 7 : is a comparison chart of inflammatory factors in the serum and adipose tissue of each group of rats in Example 2, A is TNF-α in rat adipose tissue, B is IL-1β in rat adipose tissue, C is IL-10 in rat adipose tissue, D is TNF-α in rat serum, E is IL-1β in rat serum, and F is IL-10 in rat serum; Figure 7 As shown in the results, compared with the control group, the levels of pro-inflammatory factors IL-1β and TNF-α in the adipose tissue and serum of rats with type 2 diabetes were significantly increased, and their levels decreased after the administration of exogenous RLN-3 (Relaxin-3). In contrast, compared with the control group, the anti-inflammatory factor IL-10 secreted by the adipose tissue and serum of rats with type 2 diabetes was significantly decreased, and the expression of IL-10 was upregulated after the treatment of exogenous RLN-3 (Relaxin-3). The results show that RLN-3 (Relaxin-3) can improve the inflammatory response of adipose tissue in rats with type 2 diabetes.

[0174] Figure 8 The HE staining and MASSON staining images of the rats in each group in Example 2, A is HE staining, B is MASSON staining; Figure 8 As shown, HE results showed that a large number of inflammatory cells infiltrated the adipose tissue of the DM group, while the structure of the adipose tissue of the type 2 diabetic rats after administration of RLN-3 (Relaxin-3) tended to be normal. Masson Trichome staining showed that the adipose tissue of the DM group rats had more fibrosis, and the fibrosis of the adipose tissue of the rats was improved after RLN-3 (Relaxin-3) treatment.

[0175] Fig. 9 These are immunofluorescence comparison photos of M1 phenotype markers of adipose tissue macrophages of rats in each group in Example 2; Fig.102 are immunofluorescence comparison photos of M2 phenotype markers of adipose tissue macrophages of rats in each group in Example 2; Fig. 9 and Fig.10 As shown, immunofluorescence double staining showed that the expression of M1 macrophage phenotype marker CD86 in adipose tissue of DM group was significantly increased, and CD86 expression decreased after RLN-3 (Relaxin-3) treatment.

[0176] The expression of CD206, a phenotype marker of M2 macrophages, was low in the DM group, and increased after RLN-3 (Relaxin-3) treatment.

Claims

1. Application of RLN-3 in the preparation of drugs for preventing and treating diabetic adipose tissue inflammation.

2. The use of RLN-3 according to claim 1 for preparing a drug for preventing and treating diabetic adipose tissue inflammation, characterized in that: The drug for preventing and treating diabetic adipose tissue inflammation has RLN-3 as the only active ingredient or one of the active ingredients.

3. The use of RLN-3 in the preparation of a drug for preventing and treating diabetic adipose tissue inflammation according to claim 2, characterized in that: The content of RLN-3 in the drug for preventing and treating diabetic adipose tissue inflammation is 0.1wt% to 99wt%.

4. The use of RLN-3 in the preparation of a drug for preventing and treating diabetic adipose tissue inflammation according to claim 4, characterized in that: The drug for preventing and treating diabetic adipose tissue inflammation also includes pharmaceutically acceptable excipients.

5. The use of RLN-3 according to claim 4 for preparing a drug for preventing and treating diabetic adipose tissue inflammation, characterized in that: The drug for preventing and treating diabetic adipose tissue inflammation is a non-gastrointestinal administration dosage form.

6. The use of RLN-3 in the preparation of a drug for preventing and treating diabetic adipose tissue inflammation according to claim 5, characterized in that: The drug for preventing and treating diabetic adipose tissue inflammation is in the form of an injection.

7. The use of RLN-3 in preparing a drug for preventing and treating diabetic adipose tissue inflammation according to claim 6, characterized in that: The drug for preventing and treating diabetic adipose tissue inflammation inhibits diabetic adipose tissue inflammatory response by promoting the transformation of macrophage phenotype from M1 type to M2 type.

8. The use of RLN-3 in preparing a drug for preventing and treating diabetic adipose tissue inflammation according to claim 7, characterized in that: The drug for preventing and treating diabetic adipose tissue inflammation inhibits the inflammatory response of diabetic adipose tissue by upregulating the expression of anti-inflammatory factors.

9. The use of RLN-3 according to claim 8 for preparing a drug for preventing and treating diabetic adipose tissue inflammation, characterized in that: The drug for preventing and treating diabetic adipose tissue inflammation inhibits diabetic adipose tissue fibrosis by inhibiting the inflammatory response of diabetic adipose tissue.

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