Recombinant CLCF1 proteins for treatment of metabolic diseases

Abstract

The invention discloses application of recombinant CLCF1 protein in treatment of metabolic diseases, and belongs to the field of biological medicine. According to the invention, obese mice accompanied with abnormal glycolipid metabolism are taken as research objects, and the function of the CLCF1 recombinant protein in mice with metabolic syndromes is researched by a method of intraperitoneal injection of the CLCF1 recombinant protein. The result shows that compared with a control mouse, the 1 mg / kg CLCF1 recombinant protein has the advantage that the weight, hyperlipemia, hyperglycemia and liver fatty degeneration of the obese mouse are obviously reduced when being used for treating the obese mouse. Aiming at the functions of the CLCF1, the CLCF1 can be used for preparing medicines for treating obesity-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a method for treating metabolism-related diseases by using a recombinant protein of cardiotrophin-like cytokine 1 (recombinant CLCF1, rCLCF1). Background Art

[0002] "Metabolic disease" is a general term for type 2 diabetes mellitus (T2DM), hyperlipidemia, obesity, non-alcoholic fatty liver disease (NAFLD) and hypertension, also known as metabolic syndrome (MS). Multiple diseases may occur simultaneously, they have common risk factors, and are associated with an increased risk of cardiovascular disease, cancer and premature death. Over the past two decades, the prevalence of metabolic diseases has continued to rise, and due to their fatal complications, they have imposed a heavy medical and social burden on the world. However, there is still a lack of effective prevention and treatment strategies.

[0003] Obesity is a global epidemic and a major risk factor for metabolic diseases including diabetes, hyperlipidemia, non-alcoholic fatty liver disease, stroke, and myocardial infarction, and is associated with a shortened life expectancy. Obesity affects 200 million people worldwide, placing a great pressure on world public health. Obesity is caused by an imbalance between energy intake and energy expenditure. Although lifestyle interventions (diet and exercise) have a significant impact on weight management, achieving long-term weight loss success remains extremely challenging. Various approaches, including bariatric surgery and medications, have been used for obesity treatment in clinical settings. However, these conventional options are still suboptimal and carry the risk of adverse effects. Therefore, treatments with higher efficacy and low side effects are urgently needed.

[0004] Cardiotrophin-like cytokine factor 1 (CLCF1) is a secreted protein, a member of the interleukin 6 cytokine family, and the second ligand of the ciliary neurotrophic factor receptor (CNTFR) trimer complex. Studies have shown that CNTFR shares several structural, anatomical, and signaling properties with the leptin receptor. Both have overlapping distributions in the hypothalamus, and both can lead to phosphorylation and activation of STAT3 and mTOR after ligand activation, suggesting that CLCF1 may have the ability to affect metabolic balance similar to leptin. This study found that recombinant CLCF1 protein can significantly improve the symptoms associated with metabolic syndrome without obvious adverse reactions. Based on previous reports and our research results, we believe that recombinant CLCF1 protein may become a promising new drug for the treatment of metabolic syndrome in the future. Summary of the invention

[0005] The present invention relates to an application of a recombinant CLCF1 protein in the preparation of a drug for treating metabolism-related diseases, which is mainly reflected in that the recombinant CLCF1 protein can reduce obesity, lower hypertriglyceridemia, hypercholesterolemia and hyperglycemia, and alleviate liver fatty degeneration.

[0006] The present invention uses a metabolic syndrome mouse model caused by high-fat diet feeding, and treats it by intraperitoneal administration of 1 mg / kg recombinant CLCF1 protein (once a day for one week), and maintains high-fat diet feeding during the treatment period. It was found that the recombinant CLCF1 protein treatment reduced the body weight, white adipose tissue (WAT) weight and liver fatty degeneration degree of obese mice, and reduced blood triglycerides, blood cholesterol and random blood sugar. Therefore, the recombinant CLCF1 protein has the effect of alleviating metabolic syndrome-related diseases, and it can provide a novel method for treating metabolic-related diseases.

[0007] The invention relates to application of recombinant CLCF1 protein in preparing medicine for treating obesity.

[0008] The present invention relates to application of a recombinant CLCF1 protein in preparing a drug for reducing hyperglycemia.

[0009] The present invention relates to application of a recombinant CLCF1 protein in preparing a drug for reducing hyperlipidemia.

[0010] The present invention relates to application of a recombinant CLCF1 protein in preparing a medicine for alleviating fatty degeneration of the liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Recombinant CLCF1 protein reduces the body weight, blood lipid and blood glucose levels of obese mice. A is the body weight statistics of mice in the PBS and 1mg / kg CLCF1 groups before and after treatment; B is the weight statistics of white adipose tissue in the PBS and 1mg / kg CLCF1 groups; C is the serum triglyceride and cholesterol levels of mice in the PBS and 1mg / kg CLCF1 groups. D is the blood glucose level of mice in the PBS and 1mg / kg CLCF1 groups before and after treatment. All data are expressed as Mean±SEM. *P<0.05, **P<0.01.

[0012] Figure 2 Recombinant CLCF1 protein improves liver steatosis in obese mice. A is a representative picture of H&E staining and Oil Red O staining of the liver of mice in the PBS and 1 mg / kg CLCF1 groups; B is the levels of serum liver function indicators alanine aminotransferase and aspartate aminotransferase in mice in the PBS and 1 mg / kg CLCF1 groups. All data are expressed as Mean ± SEM. *P < 0.05, **P < 0.01. Specific embodiments

[0013] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0014] Example 1: Expression and purification of recombinant CLCF1 protein

[0015] The human CLCF1 coding sequence was cloned by PCR and constructed into the plasmid pcDNA3.1. The recombinant plasmid was transformed into XL1-blue competent cells, and a single colony was picked and inoculated into 5 ml of 2YT medium containing ampicillin. It was cultured overnight at 37°C and 250 rpm, and then a large amount of plasmid was extracted. The expression plasmid was transfected into the prepared 293T cells using the transfection reagent PEI. After transfection, the new culture medium was replaced 6-8 hours after transfection. The supernatant was collected once 48 hours after transfection and once at 72 hours after transfection, and the cell precipitate was removed by centrifugation. The above liquid was concentrated to a volume of about 20 ml using an ultrafiltration tube, and then the protein was dialyzed in a dialysis bag at 4°C with a magnetic stirrer for 24 hours. During the continuous dialysis process, the dialysate was replaced at least 3 times, with an interval of 8-12 hours each time. The dialyzed protein supernatant was incubated with nickel chelate (Ni-NTA) for 1 hour, and the bound protein was eluted with 20, 50, 250 and 500 mM imidazole, respectively. The eluted fractions were collected and analyzed by 10% (vol / vol) SDS-PAGE.

[0016] Full-length sequence of recombinant CLCF1 protein:

[0017]

[0018] Example 2: Experimental animals and feeding

[0019] 8-week-old, wild-type, male C57BL / 6J mice, weighing 22±2g, were purchased from Beijing Sibeifu Biotechnology Co., Ltd. All experimental animals were housed in the Experimental Animal Center of Tianjin Medical University in an SPF-grade environment with a humidity of 40-60%, a room temperature of 24±2℃, a light intensity of 12h / d, and free access to water and food.

[0020] Example 3: Obesity mouse model

[0021] Wild-type, male C57BL / 6J mice were fed a high-fat diet (HFD) containing 60% fat for 8 weeks to induce an obesity model. Experimental animal grouping: After 8 weeks of high-fat diet feeding, the mice were randomly divided into 2 groups: PBS treatment group (PBS): an equal amount of PBS (300 μl) was intraperitoneally injected once a day for one week, and HFD feeding continued during this period; 1 mg / kg CLCF1 protein treatment group (1 mg / kg CLCF1): each mouse was intraperitoneally injected with recombinant CLCF1 protein at a dose of 1 mg / kg according to body weight, once a day for one week, and HFD feeding continued during this period. Body weight was weighed and blood glucose levels in the diet were tested before and after the treatment. After the treatment of the above groups, the animals were anesthetized, the eyeballs were removed and blood was collected, and then left to stand for 2-4 hours. After centrifugation at 3000 rpm for 20 minutes, the upper serum was collected. Serum alanine aminotransferase and aspartate aminotransferase levels were detected using a blood biochemical analyzer. Blood cholesterol and triglyceride levels were detected using commercial kits. H&E staining and oil red staining were used to observe liver fatty degeneration.

[0022] Example 4: Detection of fatty degeneration and lipid droplets in the liver of obese mice

[0023] 1. Liver pathology testing

[0024] 1) Prepare 4% paraformaldehyde in advance, pour 10 ml of 4% paraformaldehyde into a 15 ml centrifuge tube and mark it (mouse group, number, tissue type, date of sampling).

[0025] 2) Sampling: After the mice were euthanized, fresh livers were obtained. A piece of liver lobe was cut and fixed in pre-prepared 4% paraformaldehyde, and then paraffin-embedded and sectioned after fixation for 48 hours.

[0026] 3) Place the slices in a fume hood and dewax them twice with xylene, 10 min each time. Rehydrate with gradient ethanol (100%, 95%, 85%, 75%), 5 min each time. Soak in distilled water for 2 min.

[0027] 4) Use hematoxylin and eosin staining kit for staining. Stain with hematoxylin staining solution for about 3 minutes, and rinse with running water for 5 minutes.

[0028] 5) Use differentiation solution to differentiate for 1 min, and rinse with tap water twice, 5 min each time.

[0029] 6) Use eosin staining solution to stain for about 1 minute, discard the excess staining solution and then quickly dehydrate (lift and put down 2-3 times), 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol (I). 100% ethanol (II) immersion for 1 minute, xylene (I) 1 minute, xylene (II) 1 minute.

[0030] 7) Apply an appropriate amount of neutral gum to seal the slide and observe under a microscope.

[0031] 2. Oil Red O Staining of Liver

[0032] 1) After the mice were euthanized, fresh livers were taken, embedded with OCT medium, and frozen in a -80°C refrigerator.

[0033] 2) Use a cryostat to slice the tissue (5 μm) and place the slices onto a glass slide.

[0034] 3) Fix tissue sections with 60% isopropanol.

[0035] 4) Prepare Oil Red O working solution, that is, mix 0.5% Oil Red O stock solution and distilled water in a ratio of 3:2, and add the Oil Red O working solution dropwise to the tissue to completely immerse the tissue; stain at room temperature for 15 minutes.

[0036] 5) Place in 60% isopropanol for differentiation for 1 min.

[0037] 6) Use tap water to rinse the slices. Be careful not to use too much water to avoid damaging the slices.

[0038] 7) Stain with hematoxylin solution for 3 minutes.

[0039] 8) Rinse the slices with tap water for 5 minutes.

[0040] 9) Apply an appropriate amount of neutral gum to seal the slide and observe under a microscope.

[0041] Example 5: Detection of serum liver function indexes and blood lipid levels in obese model mice

[0042] 1. Serum liver function index detection: Use a fully automatic animal biochemical analyzer to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum.

[0043] 2. Determination of blood triglyceride levels

[0044] 1) The triglyceride content in mouse serum samples was determined using a liquid sample triglyceride enzymatic assay kit.

[0045] 2) Prepare the reaction working solution according to the kit instructions: Mix R1 reagent and R2 reagent in a ratio of 4:1 (i.e., 4 volumes of R1 reagent and 1 volume of R2 reagent), and prepare it before use. Each sample requires 190ul of reaction working solution.

[0046] 3) The standard (4 mM) was diluted serially to 1000 μmol / L, 500 μmol / L, 250 μmol / L, 125 μmol / L, 62.5 μmol / L, and 31.25 μmol / L.

[0047] 4) Add 190ul of working solution and 10ul of sample (buffer blank control, standard and sample to be tested) to a 96-well plate.

[0048] 5) Incubate at 37°C for 30 minutes.

[0049] 6) Use an ELISA reader to measure the OD value of each well at a wavelength of 550 nm. Prepare a standard curve and calculate the triglyceride concentration of the sample to be tested.

[0050] 3. Determination of blood cholesterol level

[0051] 1) The cholesterol content in mouse serum samples was detected using a liquid sample cholesterol enzymatic assay kit.

[0052] 2) Prepare the reaction working solution according to the kit instructions: Mix R1 reagent and R2 reagent in a ratio of 4:1 (i.e., 4 volumes of R1 reagent and 1 volume of R2 reagent), and prepare it before use. Each sample requires 190ul of reaction working solution.

[0053] 3) The standard (4 mM) was diluted serially to 2500 μmol / L, 1250 μmol / L, 625 μmol / L, 312.5 μmol / L, 156 μmol / L, and 78 μmol / L.

[0054] 4) Add 190ul of working solution and 10ul of sample (buffer blank control, standard and sample to be tested) to a 96-well plate.

[0055] 5) Incubate at 37°C for 30 minutes.

[0056] 6) Use an ELISA reader to measure the OD value of each well at a wavelength of 550 nm. Calculate the cholesterol concentration of the sample to be tested based on the standard curve.

Claims

1. Use of a recombinant CLCF1 protein in the treatment of metabolic-related diseases, wherein the metabolic-related diseases include obesity, hypertriglyceridemia, hypercholesterolemia, hyperglycemia and hepatic fatty degeneration.

2. Use of the recombinant CLCF1 protein according to claim 1 in the preparation of a drug for treating type II diabetes.

3. Use of the recombinant CLCF1 protein according to claim 1 in the preparation of a medicament for treating metabolic syndrome, the use according to any one of claims 1-2, wherein the recombinant CLCF1 protein: a. The full-length amino acid sequence of the recombinant CLCF1 protein is: b. A polypeptide derived from a in which the amino acid sequence in a is substituted, deleted or added with one or more amino acids and the activity remains unchanged.

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