CN108079304B - Application of Nemo-like protein kinase in preparation of medicine for treating type II diabetes - Google Patents

Abstract

The invention discloses application of Nemo-like protein kinase in preparation of a medicament for treating type II diabetes, belonging to new application of nemo.A wild type C57 mouse is taken as a research object, expression of N L K protein is found to be enhanced under high glucose stimulation, and the N L K protein is shown to be glucose response protein.A liver specificity knockout mouse (N L K-HKO) of an N L K gene and a littermate negative control mouse are taken as experimental objects, and regulation and control effects of the N L K gene on blood glucose are researched by detecting abdominal glucose tolerance, liver glycogen storage and insulin sensitivity.

Description

Application of Nemo-like protein kinase in preparation of medicine for treating type II diabetes

Technical Field

The invention belongs to the field of gene function and application, and relates to application of Nemo-like protein kinase (N L K) serving as a drug target in screening drugs for treating type II diabetes and application of an N L K inhibitor in preparing drugs for treating type II diabetes.

Background

Diabetes mellitus is a lifelong metabolic disease characterized by chronic hyperglycemia and hyperinsulinemia, which are caused by multiple causes. Diabetes can cause various concurrent diseases, and seriously threatens the life health of patients. At present, the death rate of diabetes is second to cardiovascular and cerebrovascular diseases and cancers, and according to the statistics of the world health organization, the complications of the diabetes are more than 100, including cardiovascular diseases, stroke, nephropathy and the like. Patients who have had amputations due to diabetes are 10-20 times of non-diabetic patients. Clinical data show that about 10 years after the diabetes mellitus happens, 30-40% of patients can have at least one complication, and later-stage drug treatment is difficult to reverse, so that the prevention and early treatment of the diabetes mellitus are the best method for avoiding the diabetes mellitus complications.

The liver is the center for regulating the metabolism of sugar, fat and protein, and the three energy metabolites need to be processed in the liver for the organism to use. Three major metabolic disorders, especially disorders of carbohydrate and fat metabolism, directly affect the development and development of type II diabetes. The liver is an important place for regulating the steady balance of blood sugar, and when the concentration of blood sugar is increased, the liver takes in glucose in blood to promote glycogen synthesis and fat conversion so as to reduce the blood sugar; in contrast, when the blood glucose concentration decreases, glycogenolysis and glyconeogenesis are promoted, and the produced glucose is transported into the blood, thereby maintaining blood glucose homeostasis.

The international diabetes union (IDF) proposes five main points of modern diabetes treatment, namely diet control, exercise therapy, blood sugar monitoring, drug therapy and diabetes education, oral hypoglycemic drugs occupy a large proportion in the drug therapy market of the type II diabetes, and according to different drug targeting, clinically used antidiabetic drugs are mainly divided into two types, namely drugs targeting peripheral organs (pancreas, muscles, small intestine and the like) and drugs targeting liver tissues, commonly used drugs comprise insulin, metformin, thiazolidinedione drugs which are recently marketed, α -glucosidase inhibitors and the like.

Nemo-like protein kinase (N L K) is an evolutionarily well-conserved serine/threonine protein kinase [1] N L K protein in mammals can phosphorylate a plurality of transcription factors (TCF/TEF, Foxo, Notch1, etc.) and regulate early embryonic development processes of organisms [2-4] recently, studies have shown that N L K is also closely related to various life processes such as tumor development and development [5], adipogenesis [6] and hematopoietic function [7 ].

Reference documents:

[1]T.Ishitani,S.Ishitani,Nemo-like kinase,a multifaceted cellsignaling regulator,Cell Signal,2013,25:190-197.

[2]S.Ota,S.Ishitani,N.Shimizu,K.Matsumoto,M.Itoh,T.Ishitani,NLKpositively regulates Wnt/beta-catenin signalling by phosphorylating LEF1 inneural progenitor cells,EMBO J,2012,31:1904-1915.

[3]H.Kojima,T.Sasaki,T.Ishitani,S.Iemura,H.Zhao,S.Kaneko,H.Kunimoto,T.Natsume,K.Matsumoto,K.Nakajima,STAT3 regulates Nemo-like kinase bymediating its interaction with IL-6-stimulated TGFbeta-activated kinase 1 forSTAT3Ser-727 phosphorylation,Proc Natl Acad Sci U S A,2005,102:4524-4529.

[4]T.Ishitani,T.Hirao,M.Suzuki,M.Isoda,S.Ishitani,K.Harigaya,M.Kitagawa,K.Matsumoto,M.Itoh,Nemo-like kinase suppresses Notch signalling byinterfering with formation of the Notch active transcriptional complex,NatCell Biol,2010,12:278-285.

[5]Y.Huang,Y.Yang,Y.He,J.Li,The emerging role of Nemo-like kinase(NLK)in the regulation of cancers,Tumour Biol,2015,36:9147-9152.

[6]P.Supakankul,S.Mekchay,Association of NLK polymorphisms withintramuscular fat content and fatty acid composition traits in pigs,Meat Sci,2016,118:61-65.

[7]M.Kortenjann,M.Nehls,A.J.Smith,R.Carsetti,J.Schuler,G.Kohler,T.Boehm,Abnormal bone marrow stroma in mice deficient for nemo-like kinase,Nlk,Eur J Immunol,2001,31:3580-3587.

[8]S.Z.Li,H.H.Zhang,J.B.Liang,Y.Song,B.X.Jin,N.N.Xing,G.C.Fan,R.L.Du,X.D.Zhang,Nemo-like kinase(NLK)negatively regulates NF-kappa B activitythrough disrupting the interaction of TAK1 with IKKbeta,Biochim Biophys Acta,2014,1843:1365-1372.

disclosure of Invention

In order to solve the defects and shortcomings of the prior art, the invention aims to research the correlation between the expression of the N L K gene and type II diabetes, provide a new strategy for regulating and controlling blood sugar by targeting the N L K gene, and further apply the N L K gene to the treatment of type II diabetes.

The purpose of the invention is realized by the following technical scheme:

according to the invention, firstly, a wild C57 mouse is taken as a research object, and the expression of the N L K protein is enhanced under high glucose stimulation, which indicates that the N L K protein is a glucose response protein.

Meanwhile, the N L K gene liver specificity knockout mouse (N L K-HKO) and the littermate negative control mouse are taken as experimental objects, and the regulation and control effect of the N L K gene on blood glucose is researched by detecting abdominal glucose tolerance, liver glycogen storage and insulin sensitivity.

On the basis, the invention provides the application of the Nemo-like protein kinase as a drug target in screening drugs for preventing, relieving and/or treating type II diabetes.

In a second aspect of the invention, there is provided the use of an inhibitor of Nemo-like protein kinase in the manufacture of a medicament for the prevention, alleviation and/or treatment of type ii diabetes.

Preferably, the inhibitor of Nemo-like protein kinase is an inhibitor that inhibits the activity or protein level of a Nemo-like protein kinase protein, or an inhibitor that inhibits the mRNA level of a Nemo-like protein kinase, the inhibitory activity of which is reversible or irreversible.

Preferably, the inhibitor for inhibiting the protein activity or protein level of Nemo-like protein kinase comprises an antibody of Nemo-like protein kinase, a protein, polypeptide, enzyme, natural compound, synthetic compound, organic matter, inorganic matter for inhibiting the protein activity or protein level of Nemo-like protein kinase; the inhibitor for inhibiting the protein activity or protein level of Nemo-like protein kinase refers to a substance that can bind to Nemo-like protein kinase but does not produce a biological response upon binding, or the inhibitor can block, inhibit or attenuate a response mediated by an agonist and can compete with the agonist for binding to Nemo-like protein kinase.

Preferably, the antibodies to the Nemo-like protein kinase include, but are not limited to, monoclonal antibodies, synthetic antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain fv (scFv), including bispecific scFv, single chain antibodies, Fab fragments, F (ab') fragments, disulfide linked fv (sdfv), and epitope binding fragments of any of the above. In particular, antibodies for use in the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. The immunoglobulin molecules used in the present invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Preferably, the antibody is a human or humanized monoclonal antibody.

As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or from a mouse or other animal in which antibodies are expressed from human genes.

Preferably, the inhibitor for inhibiting the mRNA level of Nemo-like protein kinase may be an antisense nucleic acid sequence thereof, siRNA, miRNA, shRNA, dsRNA, or other protein, polypeptide, enzyme, compound capable of inhibiting the mRNA level of Nemo-like protein kinase.

The dosage form of the medicament of the invention can be in the form of oral preparations, such as tablets, capsules, pills, powders, granules, suspensions, syrups and the like; it can also be administered by injection, such as injection solution, powder for injection, etc., by intravenous, intraperitoneal, subcutaneous or intramuscular route. All dosage forms used are well known to those of ordinary skill in the pharmaceutical arts.

The medicament of the present invention can be administered to any animal which may develop or has developed fatty liver and related diseases. These animals include both human and non-human animals, such as pets or livestock, and the like.

The agents of the invention may be administered to a subject by routes known in the art, including, but not limited to, oral, parenteral, subcutaneous, intramuscular, intravenous, intraperitoneal, intrahepatic, intramyocardial, intrarenal, vaginal, rectal, buccal, sublingual, intranasal, transdermal and the like.

The dosage administered will depend on the age, health and weight of the recipient, the type of drug combination, the frequency of treatment, the route of administration, etc. The drug may be administered in a single daily dose, or the total daily dose may be administered in divided doses of two, three or four times daily. The dose may be administered one or more times, and the administration time may range from a single day to several months or longer.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention discovers a new function of N L K, namely N L K has the function of reducing blood sugar.

(2) Based on the function of N L K in the blood sugar reducing process, the N L K protein provides a target for developing medicines for preventing, relieving and/or treating type II diabetes.

(3) The N L K inhibitor can be used for preparing medicines for preventing, relieving and/or treating type II diabetes.

Drawings

FIG. 1 is a graph showing the results of N L K protein expression after glucose induction.

FIG. 2 shows the IPGTT assay results for WT and N L K-HKO mice (0.01. ltoreq. p < 0.05, p < 0.01).

FIG. 3 shows the statistics of the AUC of IPGTT in WT and N L K-HKO mice (. times.p < 0.01).

FIG. 4 shows PAS staining results of liver tissues of WT and N L K-HKO mice.

FIG. 5 shows the results of insulin signaling pathway activation assays for WT and N L K-HKO liver tissues stimulated by insulin.

Detailed Description

The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.

Reagents related to the test are purchased in markets at home and abroad or are prepared according to a formula in a specification; the experimental methods not specifically described are all the conventional ones known in the art.

Experimental animals and breeding:

the experimental animal species, sex, week age and source are C57B L/6 (WT) mice and hepatocyte-specific N L K knockout mice (N L K-HKO), male, 8 week old C57B L/6 mice purchased from Beijing Huafukang Biotech, Inc., and N L K-HKO mice were bred with N L K-flox/flox (presented by the Wanlin Argian laboratory of the institute of Bass medicine, Chinese academy of medicine) and Alb-Cre instrumental mice.

The experimental animal feed formula comprises: high-sugar feed (purchased from southbound telofil feed technologies ltd): percentage of heat: protein (20%); carbohydrate (70%); fat (10%), total caloric mass ratio: 3.85 kcal/g.

Animal feeding and environmental conditions: all experimental mice were housed in the SPF-rated animal house (license number: SYXK (Ew): 2009-0053) of the cardiovascular disease institute of Wuhan university. The mouse is illuminated alternately every 12 hours at the temperature of 24 +/-2 ℃ and the humidity of 40-70 percent, and the mouse can eat freely by drinking water.

Western blot：

(1) Tissue protein extraction

① to pre-cooled EP tube in dry ice, 3-4 steel balls were placed and a tissue sample weighed and quantified was added.

② adding PMSF into the lysate, mixing well, adding into the sample, and shaking up quickly.

③ samples were ground in a-80 ℃ pre-cooled grinder adapter with the grinding parameters set at 30Hz/s for 90 s.

④ after grinding, the steel ball is taken out after being placed on ice for 10 min.

⑤ ultrasonic cracking instrument cracks sample (5 KHz/time, 1s each time, 1s interval, 10 times repetition), and the sample is placed on ice for 10min after ultrasonic processing.

⑥ samples were placed in a 4 ℃ pre-cooled centrifuge and centrifuged at 12000rpm for 30 min.

⑦ the supernatant was aspirated and transferred to a new EP tube and centrifuged at 14000rpm/min for 10min at 4 ℃.

⑧ the supernatant was aspirated and transferred to a new EP tube for further centrifugation at 14000rpm for 5min at 4 ℃.

⑨ supernatants were aspirated accurately and Protein quantification was performed using the BCA Protein Assay Kit (Pierec, 23225).

(2) Protein extraction from cells

Adding the cell into a lysis solution, centrifuging after the cell is lysed, taking a supernatant, and quantitatively collecting a Protein sample by using a BCA Protein Assay Kit.

(3) Sample loading and electrophoresis

The required separation gel concentration is selected according to the size of the target protein, and generally 8% -10% of the separation gel can meet most experimental requirements. Preparing electrophoresis gel, and adding electrophoresis liquid into an electrophoresis tank. And loading the protein sample into an SDS-PAGE gel loading hole, and starting electrophoresis after the sample application is finished.

(4) Rotary film

Preparing a film transfer liquid, and precooling in advance; soaking the PVDF membrane in methanol for 1-2min before use; and (4) rotating the membrane, wherein the glue is on the negative electrode side, the membrane is on the positive electrode side, and the sponge and the filter paper are soaked in advance. The transfer membrane voltage was set to 250V, the current was set to 0.2A, and the transfer was 1.5 h.

(5) Sealing of

The protein membrane was placed in a prepared TBST, and the membrane-transfer solution was washed off. The protein membrane was placed in 5% skimmed milk powder (in TBST), shaken slowly on a shaker and sealed at room temperature for 1-4 h.

(6) Primary antibody incubation

After blocking, the protein membrane was washed 3 times with TBST, 5min each time, and then primary antibody was added and incubated overnight at 4 ℃.

(7) Incubation with secondary antibody

After primary antibody incubation, the membrane is washed 3 times by TBST, 5min each time, and a certain proportion of secondary antibody (in TBST) is added for incubation for 1h at room temperature.

(8) Protein detection

After incubation, wash 3 times with TBST for 5min each. Bands of interest were detected using a Bio-Rad Chemi Doc XRS + gel imaging system.

Example 1 glucose activates N L K expression

Experimental animals were grouped, C57B L/6 mice were Fasted (without water deprivation) from 5:00 pm to 9:00 am the next day, i.e., the experimental procedures were started after fasting for 16h, and were divided into three groups by the procedures, namely, Fasted group (Fasted 16h), refreshed group (high-sugar feed was fed for 6h after fasting for 16h), and i.p.glucose group (Fasted, i.e., glucose was intraperitoneally injected in an amount of 2g/Kg for 15 min).

Taking liver tissues:

(1) mice were weighed and then sacrificed by removing their necks quickly. The mice were fixed supine and their chest and abdomen hair were moistened with distilled water.

(2) The middle skin of the abdomen of the mouse is clamped by forceps pincers, the skin is cut to the lower part of the xiphoid process along the middle of the abdomen, the skin is cut to the tail end, the subcutaneous fascia, the muscle and the like are exposed layer by layer, the abdominal cavity is opened, and all the visceral organs are fully exposed.

(3) Finding and taking down the liver of mouse rapidly, placing the taken-down liver specimen on a sterilized gauze, wiping off residual blood on the surface of liver, placing the liver in a sterile culture dish, cutting into pieces, and quick freezing in liquid nitrogen.

N L K expression detection, namely taking 3 groups of mouse liver tissues, and detecting the content of N L K protein in the liver tissues by Western blot, wherein the used primary antibody information is (N L K (#94350, CST) and GAPDH (#2118, CST)), and the secondary antibody is PeroxideAffinipure goat anti-rabbit-IgG (H + L) (#111-

The detection results are shown in fig. 1, the expression amount of the N L K protein in the refer group and the i.p. glucose group is significantly increased compared with the expression amount in the fast group, which indicates that the N L K protein is enhanced under high glucose stimulation, and indicates that the N L K protein is a glucose response protein.

Example 2 inhibition of development of type II diabetes by N L K Gene knockout

The experimental method comprises the steps of selecting 8-week-old male, WT mice and N L K-HKO mice for experiment, dividing the experimental mice into 2 groups, feeding the mice with common diet, performing an intercarrieagle glucose tolerance test (IPGTT) experiment by injecting glucose with the weight of 1.0g/kg in the 9 th week of the experiment, evaluating the glucose tolerance of the mouse body, taking materials at the end of the 10 th week, taking the mouse liver, fixing one part of the mouse liver in formaldehyde solution for pathological analysis, and quickly freezing the other part of the mouse liver in liquid nitrogen for Western blot detection.

1. Glucose Tolerance Test (IPGTT)

All mice to be tested were fasted (without water deprivation) from 5:00 pm to 9:00 am the next day, i.e. the experimental procedure was started 16h after fasting.

(1) Before measuring blood glucose, fasting body weight of the mice was measured, and the injection volume of glucose was calculated from 10. mu. L/g.

(2) The method comprises the steps of firstly detecting the abdominal blood glucose in 0min before glucose injection, and quickly injecting a glucose solution into the abdominal cavity after detection.

(3) The operation method of the intraperitoneal injection comprises the following steps: a. fixing the mice; the mouse is grabbed, the tail of the mouse is grabbed by the little finger and the ring finger of the left hand, the neck of the mouse is grabbed by the other three fingers, the head of the mouse is downward, and the abdomen of the mouse is fully exposed. b. Needle insertion positioning and injection: the syringe is held by the right hand when the needle is inserted from one side of the abdomen, the tip and the abdomen of the mouse form an included angle of 45 degrees, the needle is inserted and withdrawn, the needle head passes a small distance under the abdomen skin during injection, the needle head passes through the abdominal midline and then enters the abdomen at the other side of the abdomen, after the medicine is injected, the needle head is slowly pulled out, and the needle head is slightly rotated to prevent liquid leakage.

(4) And respectively measuring the blood sugar value of the mouse by cutting the tail at the time points of 15min, 30min, 60min, 90min and 120min after the intraperitoneal injection, and recording the blood sugar value and the detection time.

2. Mouse blood glucose level determination

(1) Preparing a blood glucose meter: checking a battery of a glucometer (Onedouch, Jones, USA), pressing a right switch, correctly putting the test paper into a left slot, displaying a number of a corresponding code of the test paper strip on a screen, and then displaying a blood dripping pattern to prompt the glucometer to enter a state to be tested.

(2) Fixing the mice: grasping the rat tail with the right hand, holding a towel with the left hand, folding the towel in half, pinching the folded part of the towel with the thumb and the forefinger, wrapping the head and the body of the rat into the towel in the palm, and fixing the root of the rat tail with the thumb and the forefinger.

(3) Tail shearing: the ophthalmic scissors can quickly cut off the rat tail at a position 0.1-0.2cm away from the tail end of the rat tail until blood drops automatically flow out.

(4) And (3) blood sugar detection: the edge of the glucometer test paper is touched with a blood drop, the blood is immersed in the test paper, and the glucometer counts down for 5 seconds to display the reading.

3. Glycogen content assay in liver (PAS staining)

1) Liver dehydration, transparency, and waxing

A part of liver lobe tissues fixed in 10% neutral formalin is cut out and placed in a marked embedding frame, and washed for more than 30 minutes in a small flow running water, ① is dehydrated according to the following procedures that 75% alcohol (45 minutes) → 85% alcohol (45 minutes) → 95% alcohol (45 minutes) → absolute alcohol (1 hour), ② transparency: xylene (1 hour) → xylene (1 hour), ③ dipping wax (65 ℃), paraffin (1 hour) → paraffin (1 hour), after the tissue is washed, the embedding frame containing the tissues is placed in a machine basket, the program is started, and after the program is finished, the embedding tissue is taken out and the machine is cleaned for standby.

2) Liver glycogen PAS staining

Slicing with a microtome (slice thickness 5 μm) → slice baking 60min → xylene dewaxing 8min × 3 → hydration 100%, 95%, 70% alcohol 5min each → ddH2O washing, adding 0.5% periodic acid solution 5-10 min → distilled water washing, 70% alcohol washing, adding schiff reagent 15-30 min → running water washing 10min → hematoxylin staining (light staining) 2-3 min → ammonia rewet 30 sec → tap water washing 1 min → 70% put with one → 95% put with one → 100% alcohol 30s × 3 → xylene transparency 2min × 3 → sealing → photographing.

4. Insulin signal transduction pathway activity assay

WT and N L K HKO mice are fed with 10W normal diet, 0.75U/kg insulin is injected into the abdominal cavity, liver tissues are taken after completion, and the content of insulin signal transduction pathway related protein in the liver tissues is detected by Western Blot^Ser473(#4060)，p-AKT^Thr308(#2965)，AKT(#4691)，p-GSK3β(#9322)，GSK3β(#9315)，p-mTOR^Ser2448(#2971), mTOR (#2983), β -actin (#4970), all were purchased from CST.

The secondary antibodies were Peroxidase affinity goat anti-rabbitt-IgG (H + L) (# 111-.

The test results of the glucose tolerance test are shown in fig. 2 and 3, after glucose injection, the blood sugar levels of the WT mice and the N L K-HKO mice sharply increase to peak after 15min, and slowly decline with the time going to 60min after injection, but are still higher than the fasting blood sugar level (blood sugar at 0 min), and are recovered to the fasting blood sugar level at 120min, and the blood sugar level of the WT mice is always higher than the blood sugar level of the N L K-HKO mice from 0min to 120min (fig. 2).

As shown in FIG. 4, the PAS staining result of the liver is that the PAS staining degree in the section of the N L K-HKO mouse is obviously stronger than that of the WT mouse, which shows that the glycogen accumulation in the liver tissue of the N L K knockout mouse is obviously increased, and the N L K gene has an inhibiting effect on glycogen storage of the liver tissue.

The results of the detection of the insulin signaling pathway activity are shown in fig. 5, compared with the WT group, the phosphorylation levels of AKT and GSK3 β in the N L K-HKO mice were significantly increased, that is, the activity of the insulin signaling pathway was enhanced after the N L K gene was knocked out, and the N L K gene had the function of inhibiting the insulin signaling pathway.

The results show that compared with WT mice, N L K-HKO mice have enhanced glucose tolerance, increased liver glycogen storage amount, enhanced insulin signal pathway activation and N L K gene knockout inhibition of the occurrence and development of type II diabetes under high-sugar diet, and the results of the invention show that the N L K gene has an important deterioration effect in type II diabetes, and the inhibition of the expression of the N L K gene and the reduction of the content of the N L K protein can inhibit the occurrence of type II diabetes.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (1)

1. The application of the Nemo-like protein kinase as a drug target in screening drugs for preventing and/or treating type II diabetes is characterized in that the drugs for preventing and/or treating type II diabetes are drugs for inhibiting the expression of the Nemo-like protein kinase.

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