CN106924736B

CN106924736B - Application of gp130 inhibitor in preparation of products for intervening and treating blood sugar metabolism diseases caused by obesity

Info

Publication number: CN106924736B
Application number: CN201710320342.5A
Authority: CN
Inventors: 张俊杰; 孙书宏; 宋建稳
Original assignee: Beijing Normal University
Current assignee: Beijing Normal University
Priority date: 2017-05-09
Filing date: 2017-05-09
Publication date: 2020-01-03
Anticipated expiration: 2037-05-09
Also published as: CN106924736A

Abstract

The invention discloses an application of a gp130 inhibitor in preparing products for intervening and treating blood sugar metabolism diseases caused by obesity, and provides an application of a substance inhibiting gp130 expression and/or activity in at least one of the following 1) -4): 1) preparing products for intervening and/or treating blood sugar metabolism diseases caused by animal obesity; 2) preparing products for inhibiting the expression of ATX in adipose tissues or adipocytes; 3) preparing a product for reducing plasma LPA and/or ATX levels; 4) the preparation of products for inhibiting STAT3 activation in adipocytes. The invention discovers that the expression of ATX in fat cells can be reduced by the treatment of gp130 inhibitor, and the abnormal blood sugar metabolism related to obesity can be obviously improved. Based on the research results, a new strategy for intervention and treatment of obesity-induced type II diabetes by taking gp130 as a target is provided.

Description

Application of gp130 inhibitor in preparation of products for intervening and treating blood sugar metabolism diseases caused by obesity

Technical Field

The invention relates to the technical field of biology, in particular to application of a gp130 inhibitor in preparing products for intervening and treating blood sugar metabolism diseases caused by obesity.

Background

With the improvement of the quality of life of people, obesity has gradually become one of the most serious chronic diseases in the 21 st century. Research shows that obesity is directly related to diabetes mellitus, cardiovascular diseases, cancer and other diseases. With the progress of science, particularly in the last two decades of research, it has been found that adipose tissue is not only an energy storage tissue but also a multifunctional active secretory organ. It has been found that adipocytes can secrete various cytokines including interleukins (interleukins, IL), interferons (interferon, IFN), Tumor Necrosis Factors (TNF), and participate in various physiological and pathological processes such as chronic inflammatory response and tumorigenesis.

Autotaxin (ATX) is a member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, NPP2, a secreted glycoprotein. It has lysoPLD activity and its main function is to catalyze the hydrolysis of Lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). ATX is a key enzyme catalyzing the production of LPA, a bioactive phospholipid molecule that can exert biological functions via LPA receptors on cell surfaces. ATX is a key enzyme for generating LPA, is highly expressed in adipocytes, conditionally knocks down ATX gene in adipocytes, reduces ATX content in mouse plasma by 50%, and reduces LPA concentration in plasma by 38%, which indicates that adipose tissue is the main source of ATX in vivo. When High-fat Diet (HFD) is fed to cause obesity, the expression of ATX in mouse fat cells is remarkably increased, the content of LPA in blood plasma is remarkably increased, and insulin resistance symptoms are shown. Adipocyte ATX-specific knockout (FATX-KO) mice fed HFD had significantly reduced symptoms of insulin resistance compared to wild-type mice. The expression of ATX is also obviously increased in fat cells of db/db diabetes model mice with hereditary obesity, and is closely related to the degree of insulin resistance. Clinical studies found that the expression of ATX is significantly increased in visceral fat in obese patients compared to normal; the expression level of ATX in adipose tissues is higher in obese people with diabetes or exhibiting symptoms of impaired glucose tolerance than in obese patients with normal glucose metabolism. All these studies indicate that obesity can lead to abnormal increase of ATX expression in adipocytes, and thus the up-regulation of ATX/LPA levels is closely related to glucose metabolism disorder in obese individuals.

The IL-6 family of cytokines is a generic term for a number of cytokines, including IL-6, LIF, IL-11, CT-1, CNTF, OSM, etc., which activate downstream signaling pathways to perform biological functions by binding to specific receptors on the cell surface. gp130 is a subunit common to IL-6 family cytokine receptors, and when stimulated by an external IL-6 family cytokine, gp130 forms a dimer by itself or forms a dimer with another cytokine-specific transmembrane Receptor, and then binds to an outer membrane specific Receptor (such as IL6 Receptor-alpha) to form a Receptor complex, thereby mediating and activating downstream signaling pathways such as JAK-STAT, PI3K-Akt and MAPK. The IL-6 family of factors all require the gp130 receptor to be involved in mediating their biological functions, and therefore the IL-6 family of cytokines is also known as gp130 cytokine. Adipocytes can express and secrete multiple IL-6 family cytokines such as IL-6, IL-11, LIF, CNTF and CT-1. Fat is the major tissue for IL-6 production, and 1/3 IL-6 is produced by adipose tissue in healthy humans. Adipose tissues of obese individuals are in a chronic inflammatory state, and are greatly infiltrated by immune cells, so that the expression level of proinflammatory cytokines such as IL-6, TNF-alpha and the like in the adipose tissues is remarkably increased, and the method is one of important factors causing the dysfunction of the obese adipose tissues and causing the occurrence of obesity-related diseases.

Disclosure of Invention

It is an object of the present invention to provide the use of a substance that inhibits gp130 expression and/or activity or a substance that blocks gp 130-mediated JAK-STAT3 signaling pathway.

The substance inhibiting gp130 expression and/or activity or the substance blocking gp 130-mediated JAK-STAT3 signal pathway provided by the invention is applied to at least one of the following 1) -4):

1) preparing products for intervening and/or treating blood sugar metabolism diseases caused by obesity; suitable for human and any non-human animal

2) Preparing a product for inhibiting the expression of ATX in adipose tissue or adipocytes;

3) preparing a product for reducing plasma LPA and/or ATX levels;

4) the preparation of products for inhibiting STAT3 activation in adipocytes.

In the above application, the disease of blood glucose metabolism is type II diabetes.

In the application, the inhibition of the expression of ATX in the adipose tissues or the adipocytes is realized by inhibiting gp 130-mediated activation of STAT3 in a JAK-STAT3 signal pathway, namely, reducing the phosphorylation level of STAT3 (P-STAT3 level).

In the above applications, the substance inhibiting gp130 expression and/or activity or blocking gp 130-mediated JAK-STAT3 signaling pathway may be any substance capable of inhibiting gp130 known to those skilled in the art without any creative effort in combination with the prior art, such as small molecule compounds, interfering RNA of suppressors, and antibodies.

The substance for inhibiting gp130 expression and/or activity or the substance for blocking gp 130-mediated JAK-STAT3 signal channel is a gp130 inhibitor or siRNA for interfering gp130 expression.

In the above application, the gp130 inhibitor is SC 144;

or the nucleotide sequence of the siRNA interfering gp130 expression is sequence 1.

The application of gp130 or its ligand in preparing the product for increasing the expression of ATX in fat cells is also the protection scope of the present invention.

In the above application, the gp130 ligand is an IL-6 family cytokine;

or, the IL6 family cytokine is IL-6, LIF, CT-1 or CNT.

In the application, the IL-6 family cytokine can improve the expression of ATX in the fat cell by activating a gp130-STAT3 signal channel in the fat cell.

In the above application, the product is a medicament.

The application of gp130 as a target point in designing or preparing products for intervening and treating obesity-induced type II diabetes is also the protection scope of the invention;

or inhibiting the expression of STAT3 in the preparation of products inhibiting the expression of ATX.

Research work finds that gp 130-mediated JAKs-STAT3 signal pathway plays an important role in the expression of ATX in adipocytes. IL-6 family cytokines (e.g., IL-6, LIF, CT-1, or CNTF) can significantly up-regulate the expression of ATX in adipocytes through the gp130 signaling pathway; the gp130 signal channel is inhibited by using a gp130 specific inhibitor Sc144, so that the expression of ATX in adipocytes can be obviously reduced; knock-down of gp130 in adipocytes using gp 130-specific sirnas can also significantly down-regulate ATX expression in adipocytes.

Animal experiments show that compared with Normal Diet (ND) mice, the fat tissue of fat mice fed with high fat food (HFD) has obviously increased expression level of ATX, the plasma content of ATX and LPA of the fat mice is also obviously increased, and the type II diabetes characteristics such as insulin antagonism and hyperglycemia tolerance are generated. The gp130 specific inhibitor SC144 is used for treating high fat food feeding (HFD) to obtain an obese mouse, which can obviously reduce the expression of ATX in adipose tissues, reduce the content of ATX and LPA in blood plasma, and obviously improve type II diabetes symptoms such as insulin antagonism and impaired glucose tolerance caused by obesity.

Experiments of the invention disclose a molecular mechanism of high expression of ATX in adipocytes, namely that the expression of ATX in adipocytes is activated by IL-6 family cytokines through gp 130-mediated signal pathway self-feedback, and the gp130 inhibitor treatment is found to be capable of down-regulating the expression of ATX in adipocytes, thereby remarkably improving obesity-related blood glucose metabolism disorder. Based on the research results, a new strategy for intervention and treatment of obesity-induced type II diabetes by taking gp130 as a target is provided.

Drawings

FIG. 1 shows that the expression of ATX in adipocytes depends on the gp130-JAK-STAT3 signaling pathway.

FIG. 2 is a graph showing that IL-6 family cytokines up-regulate the expression of ATX in adipocytes via gp130 signaling pathway.

Figure 3 is a graph showing that oral administration of gp130 inhibitor SC144 down-regulates the expression level of ATX in adipose tissue and the level of ATX in plasma of HFD obese mice.

Figure 4 is a graph showing that oral administration of gp130 inhibitor SC144 down-regulates LPA levels in plasma of HFD obese mice.

Fig. 5 shows that oral administration of gp130 inhibitor SC144 significantly improves insulin antagonism and impaired glucose tolerance in HFD obese mice.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Part of the materials are as follows:

table 1 shows the reagents and antibodies

The methods employed in the following examples are partly as follows:

first, mRNA level detection

1. RNA extraction

1) Extracting cell RNA:

a) cell samples were collected, washed 2 times with PBS solution, and centrifuged to remove supernatant.

b) 1ml of Trizol solution was added to the precipitate, and the mixture was uniformly blown and allowed to stand at room temperature for 5 minutes.

c) Chloroform (0.2ml/1ml Trizol) was added, the tubes were capped and shaken vigorously by hand for 15 seconds.

d) The mixture was allowed to stand at room temperature for 5 minutes, 12000g and centrifuged at 4 ℃ for 15 minutes.

e) The upper aqueous phase was transferred to a new centrifuge tube, 0.5ml isopropanol was added, mixed well and left for 10 minutes.

f)12000g, 4 ℃ centrifugal 10 minutes, abandon the supernatant.

g) Adding 1ml of 75% ethanol, washing once, 7500g, centrifuging at 4 ℃ for 5 minutes, removing supernatant, and drying precipitate for 10 minutes.

h) Adding 20 μ l DEPC water to dissolve the precipitate, and placing in a 60 deg.C water bath to dissolve for 10 min.

i) The RNA concentration was measured by a spectrophotometer and the prepared samples were frozen in a freezer at-80 ℃ for storage.

2) Tissue RNA extraction:

a) shearing tissue with sterilized small scissors, adding 1ml Trizol, and homogenizing with homogenizer

b) Chloroform (0.4ml/1ml Trizol) was added, the tubes were capped and shaken vigorously by hand for 15 seconds.

c) The mixture was allowed to stand at room temperature for 5 minutes, 12000g and centrifuged at 4 ℃ for 15 minutes.

d) The upper aqueous phase was transferred to a new centrifuge tube, 0.5ml isopropanol was added, mixed well and left for 10 minutes.

e)12000g, 4 ℃ centrifugal 10 minutes, abandon the supernatant.

f) Adding 1ml of 75% ethanol, washing once, 7500g, centrifuging at 4 ℃ for 5 minutes, removing supernatant, and drying precipitate for 10 minutes.

g) Adding 40 μ l DEPC water to dissolve the precipitate, and placing in a 60 deg.C water bath to dissolve for 10 min.

h) The RNA concentration was measured by a spectrophotometer and the prepared samples were frozen in a freezer at-80 ℃ for storage.

2、RT-qPCR

Mu.g of RNA was taken, 2. mu.l of the primer was added thereto, treated at 70 ℃ for 10 minutes, and immediately cooled on ice.

Mixing was carried out according to the following system: 5. mu.l of M-MLV 5 Xbuffer, 1. mu.l of dNTPs (10mM), 0.5. mu.l of RNAse inhibitor, 1. mu.l of M-MLV reverse transcriptase, and DEPC water were added to a final volume of 25. mu.l, and the mixed solution was reacted in a water bath at 42 ℃ for 1 hour, inactivated at 72 ℃ for 10 minutes, and frozen at-20 ℃ for storage in a refrigerator. According to an operation manual of IQSYBR Green Supermix (Bio-Rad), an iCycleiQreal-time PCR instrument of Bio-Rad is used for detecting the target gene, and the result is detected by the analysis software of Bio-RadiQ 5.

3. The primer sequences are shown in Table 2.

TABLE 2

Secondly, detecting the expression level of the protein

1. Protein extraction

1) Extracting cell protein:

a) collecting cells, centrifuging at 1500rpm for 5 minutes, removing supernatant, and washing cell precipitate twice with precooled PBS;

b) sucking the residual PBS, adding a proper amount of cell lysate RIPA, uniformly blowing and stirring, and performing ice bath lysis for 30 minutes;

c) centrifuging at 12000rpm at 4 deg.C for 20 min, and transferring the supernatant to a new centrifuge tube;

2) tissue protein extraction:

a) the tissue was collected, washed clean with PBS, minced with sterile small scissors, and broken with a homogenizer after addition of an appropriate amount of RIPA (containing protease inhibitors).

b) Cracking in ice bath for 30 min;

c) the mixture was centrifuged at 12000rpm at 4 ℃ for 20 minutes, and the supernatant was transferred to a new centrifuge tube.

2. BCA protein concentration quantification kit description

1) Mixing the solution A and the solution B according to a volume ratio of 50: 1, preparing a BCA working solution, and fully and uniformly mixing;

2) protein standard is diluted to different concentration gradients in equal proportion

3) Adding 20 mul of measurement sample into 200 mul of BCA working solution, fully and uniformly mixing, and reacting for 30 minutes at 37 ℃;

4) and (5) measuring by using a spectrophotometer in a BCA measuring mode, and drawing a standard curve.

3. Immunoblot hybridization (western blot)

1) Electrophoresis

Preparing SDS-PAGE albumin glue, adding 40 mu g of sample protein into a loading buffer solution, carrying out 5-minute boiling water bath, carrying out centrifugation at 12000rpm for 2 minutes, cooling, and carrying out centrifugation to obtain a supernatant and loading. The electrophoresis is carried out by using 80V constant voltage electrophoresis, and the electrophoresis is carried out by changing the voltage to 120V after the bromophenol blue enters the separation gel.

2) Rotary film

Activating the PVDF membrane by methanol for 15 seconds, then soaking the PVDF membrane in distilled water for 5 minutes, and then soaking the PVDF membrane in a membrane conversion buffer solution for 15 minutes. The membrane transfer was performed according to the Bio-Rad Wet-Transferometer method at 120V and 300mA for 60 minutes.

3) Blocking and antibody incubation

(1) Taking out the PVDF membrane, washing the PVDF membrane once by TTBS (transthyretin-sodium bromide), and putting the PVDF membrane into blocking solution (5% BSA) to incubate for 1 hour at room temperature;

(2) washing with TTBS for 5 min 3 times, and incubating at 4 deg.C overnight (about 8 hr) in primary antibody dilution;

(3) washing with TTBS for 5 min for 3 times, and incubating in secondary antibody diluent at room temperature for 1 hr;

4) the membrane was removed, washed 3 times with TTBS and developed by ECL luminescence.

III, siRNA transfection

1. Transfection

1) The synthesized siRNA was dissolved in 150. mu.l RNase-free water to a concentration (20. mu.M) according to the Gilmax's instructions, and was stored in aliquots frozen at-80 ℃.

2) siRNA transfection was performed according to Lipofectamine2000 instructions, using the following specific procedure (35 mm dish as an example):

(1) replacing fresh culture medium before cell transfection;

(2) the siRNA was diluted with 250. mu.l of Opti-MEM medium to a final concentration of 25nM or 50 nM; at the same time, 5. mu.l Lipofectamine2000 was diluted with 250. mu.l Opti-MEM medium, gently mixed, and left at room temperature for 5 minutes;

(3) the siRNA and Lipofectamine2000 were gently mixed, left at room temperature for 20 minutes, and then added to the cell culture medium and mixed well.

3) Cells were harvested 48 hours after transfection, and RNA or total protein of the cells was extracted and subjected to the experiment.

2. siRNA sequences

siRNAs were synthesized from Gimerax Shanghai according to the sequences in Table 3.

Table 3 shows siRNA sequences

gp130siRNA	5'-ACGACUUAGGACUAUCUUAAA-3' (sequence 1)
		STAT1siRNA	5′-GCCAAGGAGGAGAUCUCCGAAGAUA-3′
STAT3siRNA
		5′-CUGGAUAACUUCAUUAGCA-3′
NC siRNA			5’-UUCUCCGAACGUGUCACGU-3’

Fourthly, extraction of lipid and mass spectrum detection

Lipid extraction: 0.5ml PBS was added to the glass centrifuge tube, 100. mu.l 14:0LPA (5. mu.M) was added as an internal standard, followed by 3ml methanol/chloroform (2: 1). To the above solution were added 50. mu.l of plasma and 15. mu.l of 6N HCl, vortexed, and then allowed to stand on ice for 10 minutes. Adding 1ml chloroform and 1.3ml distilled water, mixing, centrifuging at 1750g room temperature for 10 minutes, taking the lower organic phase, drying by nitrogen, dissolving with 150 μ l methanol/water (9: 1), sampling, and detecting on a machine.

Separation and detection of lipids: lipid molecules were separated by liquid chromatography using a liquid chromatography-mass spectrometry (LC-MS) method, and quantitative detection was performed by mass spectrometry. The specific method comprises the following steps: lipid separation was performed using a C18HPLC column (5 μm, 2.1mm ID x 20mm, TR-0121-C185) containing two flow phases, flow phase A: isopropanol/acetonitrile/formic acid containing 10mM ammonium formate (90:10:0.1, v/v/v); and (3) flow phase B: methanol/H₂O/NH₄OH (90:10:0.1, v/v/v). The process comprises the following steps: (1) perfusing with 100 μ L/min-100% of flow phase B for 3 min; (2) increasing the flow rate from 100 mu L/min to 300 mu L/min within 1min, and simultaneously changing the flow phase from 100% flow phase B to 50% flow phase B; (3) perfusing with 300 μ L/min-50% of the flow phase B for 6 min; (4) reducing the flow rate from 300 mu L/min to 100 mu L/min within 1min, and simultaneously changing the flow phase from 50% flow phase B to 100% flow phase B; (5) perfusing with 100 μ L/min-100% of flow phase B for 1 min. Quantitative detection of LPA was performed using a QTRap 4500 mass spectrometer (API4500QTRap mass spectrometer).

Fifth, the blood sugar of the experimental animal is measured

Starving the mice overnight with water continuously after grain breaking, and then performing an insulin tolerance test and a glucose tolerance test, wherein the specific flow comprises the following steps: 1) insulin tolerance test: clipping a mouse tail to take blood, detecting and recording the blood sugar level at the time point of 0h, injecting insulin (0.75U/kg) into the abdominal cavity, detecting the blood sugar content of the mouse every 15 minutes, and recording the blood sugar change within 120 minutes; 2) glucose tolerance test: blood glucose levels were recorded at the time of 0h by clipping the mouse tail and blood sampling, glucose (1g/kg) was injected intraperitoneally, and then the blood glucose content of the mice was measured every 15 minutes, and the change in blood glucose was recorded within 120 minutes. A Roche (Roche) blood glucose meter and blood glucose test strips were purchased for blood glucose testing.

Example 1 obtaining and detecting adipocytes

First, obtaining adipocytes

3T3-L1 murine preadipocytes were purchased from cell banks of the university of cooperative medicine.

The method for inducing the 3T3-L1 preadipocytes to differentiate to generate the adipocytes is disclosed in the cocktail method provided by the reference document, and the specific process is as follows:

3T3-L1 preadipocytes were cultured using DMEN (Macgene) medium containing 10% New bovine serum (Hyclone) to which was added 2mM L-glutamine (Life Technologies), 100. mu.g/ml streptomycin (Life Technologies) and 100U/ml penicillin (Life Technologies). The differentiation process was initiated when 3T3-L1 preadipocytes grew mitotically to 0 days exposure to the inhibitor. Mature adipocytes were obtained after inducing differentiation for 8 days by culturing in 10% fetal bovine serum (Hyclone) DMEM + insulin (insulin, 1. mu.g/ml) + Desmin (Dex, 0.25. mu.M) + 3-isobutyl-1-methylxanthine (IBMX,0.5mM) in complex medium for 48 hours, and then in 10% fetal bovine serum (Hyclone) DMEM + insulin (insulin, 1. mu.g/ml) in complex medium, and changing the medium fresh every 48 hours until triglyceride lipid droplets were produced in 3T3-L1 cells.

Detection of adipocytes

1. Lipid droplet accumulation assay

The differentiation and maturation of preadipocytes 3T3-L1 are induced in vitro, and after 8 days of induction, obvious lipid drop accumulation in the cells is observed by oil red staining (figure 1A), which proves that mature adipocytes are obtained by in vitro induction.

2. PPAR-gamma expression assay

The protein of the mature adipocytes was extracted, immunoblot hybridization was performed, and the primary antibody was an antibody against PPAR- γ (a marker gene for adipocyte cell differentiation), and the results are shown in the right panel of fig. 1B, where it can be seen that the adipocytes express PPAR- γ, demonstrating that mature adipocytes were obtained.

3. Detection of mRNA and protein expression levels of ATX and P-STAT3 and STAT3 levels in adipocytes and preadipocytes.

Total RNAs of the mature adipocytes and preadipocytes were extracted as templates, respectively, and RT-PCR was performed using ATX primers (Table 2) (the method was as described above) to detect the expression level of ATX mRNA in the cells.

Detecting the level of secreted ATX protein in the culture medium of mature adipocytes and preadipocytes by immunoblotting; lysates of mature adipocytes and preadipocytes were prepared separately, and levels of P-STAT3 and STAT3 in the cells were detected by immunoblot hybridization. The primary antibody for immunoblot detection is an anti-ATX, P-STAT3, or STAT3 antibody.

The results are shown in fig. 1B, where P-STAT3 levels were elevated in mature adipocytes compared to preadipocytes, and both mRNA and protein expression levels of ATX were significantly increased.

Example 2 application of blocking gp130-STAT3 signal pathway in inhibition of adipocyte ATX expression

First, gp130 inhibitor treatment and gp130 knockdown using specific siRNA can reduce ATX expression in adipocytes

1. gp130 inhibitor treatment

gp130 inhibitor SC144 treatment group: SC144 dissolved in DMSO was added to the cell culture medium and mature adipocytes were treated with SC144 at a final concentration of 10 μ M for 24 hours;

DMSO control group: mature adipocytes were treated with the same volume of DMSO as the experimental group by adding to the cell culture medium for 24 hours.

The RNA of the gp130 inhibitor SC 144-treated cells and DMSO-control cells were extracted as templates, and RT-PCR (method described above) was performed using ATX primers (Table 2) to detect the expression level of ATX mRNA in the cells.

Detecting the level of ATX protein secreted in the culture medium of cells of the gp130 inhibitor SC 144-treated group and the DMSO control group by immunoblotting; lysates from two sets of cells were prepared separately and the levels of P-STAT3 and STAT3 in the cells were detected by immunoblot hybridization. The primary antibody for immunoblot detection is an anti-ATX, P-STAT3, or STAT3 antibody.

As a result, as shown in fig. 1C, the extent of activation of gp130 downstream STAT3 (i.e., the level of P-STAT 3) was significantly reduced in cells of the gp130 inhibitor SC 144-treated group, and the mRNA and protein expression levels of ATX were significantly suppressed, compared to the DMSO control group.

2. gp130siRNA treatment

NC siRNA control group: NC siRNA was transfected into mature adipocytes using Lipofectamine2000 as before, with siRNA transfection concentration of 100nM, and siRNA treatment of mature adipocytes for 48 hours.

gp130siRNA treatment group gp130siRNA was transfected into mature adipocytes using Lipofectamine2000 at an siRNA transfection concentration of 100nM, and mature adipocytes were treated with siRNA for 48 hours.

The RNA of the cells of the gp130 siRNA-treated group and NC siRNA control group were extracted as templates, and RT-PCR was performed using ATX primers (Table 2) (the method was as described above) to detect the expression level of ATX mRNA in the cells.

Detecting the level of ATX protein secreted in the culture medium of the cells of the gp130siRNA treated group and the NC siRNA control group by immunoblotting; lysates from two sets of cells were prepared separately and the levels of gp-130, P-STAT3, and STAT3 in the cells were detected by immunoblot hybridization. The primary antibody of the immunoblot detection is an anti-ATX antibody, a gp130 antibody, a P-STAT3 antibody or a STAT3 antibody.

The results are shown in fig. 1D, and compared with the NC siRNA control group cells, the extent of activation of gp130 downstream STAT3 (i.e., the level of P-STAT 3) was significantly reduced in the gp130siRNA treated group cells, and both mRNA and protein expression levels of ATX were significantly inhibited.

II, STAT3 inhibitor treatment and STAT3 knockdown using specific siRNA can reduce the expression of ATX in adipocytes

JAKs-STAT3 is an important downstream signaling pathway activated by gp 130.

1. STAT3siRNA treatment

NC siRNA control group: NC siRNA was transfected into mature adipocytes using Lipofectamine2000 at siRNA transfection concentration of 100nM, and mature adipocytes were treated with siRNA for 48 hours.

STAT1siRNA treatment group STAT1siRNA was transfected into mature adipocytes using Lipofectamine2000 at a siRNA transfection concentration of 100nM, and mature adipocytes were treated with siRNA for 48 hours.

STAT3siRNA treatment group STAT3siRNA was transfected into mature adipocytes using Lipofectamine2000 at a siRNA transfection concentration of 100nM, and the mature adipocytes were treated with siRNA for 48 hours.

And (3) transfecting the fat cells with NC siRNA, STAT1siRNA and STAT3siRNA respectively according to the siRNA transfection method to obtain cells of an NC RNAi group, a STAT1siRNA group and a STAT3siRNA group.

Total RNAs of the cells of the NC RNAi group, STAT1siRNA group and STAT3siRNA group were extracted as templates, respectively, and RT-PCR (method described above) was performed using ATX primers (Table 2) to detect the expression level of ATX mRNA in the cells.

Respectively detecting the levels of ATX protein secreted in the culture media of the cells of the NC RNAi group, the STAT1siRNA group and the STAT3siRNA group by immunoblotting; lysates of the cells of the NC RNAi group, STAT1siRNA group and STAT3siRNA group were prepared, and levels of STAT1 and STAT3 in the cells were detected by immunoblot hybridization. Primary antibodies were anti-ATX, STAT1, and STAT3 antibodies, respectively.

As a result, as shown in fig. 1E, knocking down STAT3 could down-regulate the mRNA and protein expression levels of ATX in adipocytes, whereas knocking down STAT1 could not.

2. STAT3 inhibitor S3I-201 treatment

STAT3 inhibitor treatment group, 20 μ M S3I-201, dissolved in DMSO, treated mature adipocytes for 24 hours);

DMSO control group: (mature adipocytes were treated with the same volume of DMSO as in the experimental group for 24 hours)

Total RNAs of the cells of the STAT3 inhibitor-treated group and the DMSO-control group were extracted as templates, respectively, and RT-PCR (method described above) was performed using ATX primers (Table 2) to detect the expression level of ATX mRNA in the cells. The levels of ATX protein secreted in the culture medium of cells of STAT3 inhibitor treated group and DMSO control group were detected by immunoblotting. Primary antibodies were anti-ATX antibodies, respectively.

Results as shown in figure 1F, the levels of mRNA and protein expression of ATX were significantly reduced in cells of the STAT3 inhibitor S3I-201 treated group compared to the DMSO control group.

Example 3 application of IL-6 family cytokines to upregulate adipocyte ATX expression

The IL-6 family cytokines include IL-6, LIF, CT-1, CNTF and the like. The IL-6 family cell factor can activate JAK-STAT, PI3K-Akt, MAPK and other downstream signal channels through gp130 receptors to regulate and control the expression of target genes.

The adipocytes obtained by in vitro induction in example 1 were treated as follows:

IL-6 group cells: the adipocytes were starved with serum for 8 hours, and the recombinant murine IL-6 was added to the cell culture medium of the adipocytes to a final concentration of 10ng/ml, and treated for 8 hours.

LIF group cells: the adipocytes were starved with serum for 8 hours, and recombinant murine LIF was added to the cell culture medium of the adipocytes to a final concentration of 10ng/ml, and treated for 8 hours.

CT-1 group of cells: starving adipocytes with serum for 8 hours, adding recombinant murine CT-1 to the cell culture medium of adipocytes to a final concentration of 10ng/ml, and treating for 8 hours

CNTF group cells: starving adipocytes with serum for 8 hours, adding recombinant murine CNTF to the cell culture medium of adipocytes to a final concentration of 10ng/ml, and treating for 8 hours

IL-6+ SC144 group cells: serum starved adipocytes for 8 hours, pre-treated adipocytes with 10 μ M SC144 for 30 minutes, followed by co-treatment of mature adipocytes with recombinant murine IL-6(10ng/ml) and SC144(10 μ M) for 8 hours.

Total RNAs of the above groups of cells were extracted as templates, respectively, and RT-PCR was performed using ATX primers (Table 2) (the method was as described above) to detect the expression level of ATX mRNA in the cells.

Lysates of the cells of each group are respectively prepared, and the protein levels of P-STAT3 and STAT3 in the cells of each group are detected by immunoblotting, wherein primary antibodies are antibodies against P-STAT3 and STAT3 respectively.

As shown in FIG. 2, the IL6 family cytokines IL-6, LIF, CT-1 or CNTF treated adipocytes could all be activated to increase P-STAT3 protein levels in the cells and significantly up-regulate the expression of ATXmRNA in adipocytes (FIGS. 2A-D). After 30 minutes of pretreatment of adipocytes with gp130 inhibitor SC144, the expression of ATX in IL-6 treated adipocytes was significantly inhibited (fig. 2E).

The above results indicate that IL-6 family cytokines can up-regulate the expression of ATX in adipocytes by activating the gp130-STAT3 signaling pathway.

Example 4 use of gp130 inhibitors for the intervention and treatment of disorders of glycaemic metabolism resulting from obesity

First, gp130 inhibitor SC144 inhibits the expression of ATX in adipose tissue of obese mice

The experimental mice select male C57BL/6 mice, the mice are bred according to a 12-hour day and night breeding period, and the mice are approved by the ethical examination society of experimental animals of Beijing university.

Healthy male C57BL/6 mice of 8 weeks old were selected and divided into 3 groups (the flow is as shown in the left panel of FIG. 3A):

normal (ND) group: feeding normal food, and orally administering medicinal solvent (0.9% NaCl with 40% propylene glycol) to prepare 40% propylene glycol (volume ratio, propylene glycol/H) 7 weeks after feeding₂O), a final concentration of 0.9% NaCl (mass to volume, NaCl (g)/solvent (L))) was prepared using 40% propylene glycol as a solvent; calculating the dosage of the solvent according to the weight of the mouse; the volume of the medicine is the same as that of the medicine to be taken;

obesity (HFD) group: feeding high fat diet (HFD,60 kcal% fat, Research Diets), orally administering a pharmaceutical solvent (40% propylene glycol physiological saline: formulating 40% propylene glycol (volume ratio, propylene glycol/H) 7 weeks after feeding₂O), a final concentration of 0.9% NaCl (mass to volume, NaCl (g)/solvent (L))) was prepared using 40% propylene glycol as a solvent; calculating the dosage of the solvent according to the weight of the mouse; the volume of the medicine taken by the mice in the experimental group is the same, and the blood sugar metabolic disease caused by obesity is simulated;

obesity + SC144(HFD + SC144) group: feeding a high fat diet, orally administering a p130 inhibitor SC144 drug (5mg/kg) 7 weeks after feeding; the drug is treated once daily for one week.

Mice were weighed weekly and after one week of drug treatment, mice were sacrificed and adipose tissue and plasma were isolated.

Extracting total RNA of the adipose tissues of the mice of each group respectively as a template, and performing RT-PCR (the method is described in the above) by using ATX primer, IL-6 primer or LIF primer (Table 2) respectively to detect the mRNA expression levels of ATX, IL-6 and LIF in the adipose tissues; detecting the level of ATX protein in the plasma of each group of mice by immunoblotting, wherein primary antibodies are anti-ATX antibodies respectively; the protein levels of P-STAT3 and STAT3 in the adipose tissues of each group of mice were detected by immunoblotting, and the primary antibodies were antibodies against P-STAT3 and STAT3, respectively.

The results are shown in figure 3, and compared to Normal Diet (ND) mice, both ATX mRNA levels in adipose tissue and ATX protein levels in plasma were significantly up-regulated in High Fat Diet (HFD) fed obese mice (figures 3B, 3C). After one week of oral administration of the gp130 inhibitor SC144 to HFD obese mice, the body weight of the obese mice did not significantly change, but both atxmna levels in adipose tissue and ATX protein levels in plasma were significantly down-regulated, returning essentially to levels fed to Normal Diet (ND) mice (fig. 3A-C). gp130 inhibitor SC144 treatment significantly down-regulated P-STAT3 levels in adipose tissue of HFD obese mice, but had no significant effect on STAT3 total protein levels (fig. 3C).

Meanwhile, mRNA expression levels of IL-6 and LIF in adipocytes of mice in each group were detected by RT-PCR, and compared with mice in ND group, the mRNA expression levels of IL-6 and LIF in adipose tissues of HFD obese mice were significantly upregulated, but the mRNA expression levels of IL-6 and LIF in adipose tissues of HFD obese mice were not significantly affected by oral administration of gp130 inhibitor SC144 (FIGS. 3D, 3E).

Plasma samples of each group of mice were subjected to lipid extraction, and the plasma LPA content was determined by ESI LC MS/MS method (see the detailed examples). The detection result shows that the plasma LPA concentration of the HFD obese mice is remarkably up-regulated compared with that of the ND mice, and the oral administration of the gp130 inhibitor SC144 can restore the total LPA content of the plasma of the HFD obese mice to the level of the ND mice (figure 4A). Further detection analysis of various LPA subtypes gave the same results (fig. 4C-H). In addition, the plasma concentration of the other active lipid S1P, which was not regulated by ATX, was not significantly different between ND mice and HFD obese mice, and oral gp130 inhibitor SC144 treatment had no significant effect on the concentration of S1P in the plasma of HFD obese mice (fig. 4B).

The results of the above studies indicate that activation of gp130 signaling pathway in adipose tissue of HFD obese mice results in upregulation of adipocyte ATX expression, resulting in increased plasma ATX and LPA levels; the oral administration of the gp130 inhibitor SC144 can down-regulate the expression of ATX in adipose tissues of HFD obese mice, and the ATX and LPA content in plasma is recovered to be normal.

II, detecting insulin tolerance test and glucose tolerance test

Healthy male C57BL/6 mice of 8 weeks of age were selected and divided into 4 groups (the flow is shown in the left panel of FIG. 4A):

obesity (HFD) group: feeding high fat diet (HFD, 60% fat content, produced by Research Diets), orally administering a pharmaceutical solvent (40% propylene glycol physiological saline: 40% propylene glycol (volume ratio, propylene glycol/H) formulated) 7 weeks after feeding₂O), a final concentration of 0.9% NaCl (mass to volume, NaCl (g)/solvent (L))) was prepared using 40% propylene glycol as a solvent; calculating the dosage of the solvent according to the weight of the mouse; the volume of the medicine taken by the mice in the experimental group is the same;

Obesity + Ki16425(HFD + Ki16425) group: feeding high fat diet, injecting LPA receptor 1/3(LPAR1/3) inhibitor Ki16425(5mg/kg) intraperitoneally after 7 weeks of feeding; the drug is treated once daily for one week.

The body weight of the mice was weighed weekly, and after one week of drug treatment, glucose tolerance (GTT) and Insulin tolerance (ITT) experiments were performed on each group of mice (see the detailed implementation method).

As a result, as shown in fig. 5, oral administration of the gp130 inhibitor SC144 did not significantly affect the body weight of HFD obese mice (fig. 5A), but significantly improved insulin antagonism and impaired glucose tolerance symptoms of HFD obese mice (fig. 5B-C). Ki16425 treatment, an inhibitor of LPA receptor 1/3, also had similar effects (fig. 5B-C).

The research results show that gp130 is a new target for inhibiting the expression of adipocyte ATX and interfering in obesity-related blood glucose metabolic diseases, blocking the gp130 signal pathway can become a new strategy for treating obesity-related blood glucose metabolic diseases, and gp130 inhibitors (such as SC144) can be applied to treatment of obesity-related blood glucose metabolic diseases.

Sequence listing

<110> university of Beijing teachers

Application of <120> gp130 inhibitor in preparation of products for intervening and treating blood sugar metabolism diseases caused by obesity

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223>

<400> 1

acgacuuagg acuaucuuaa a 21

Claims

1. Use of a substance that inhibits gp130 expression and/or activity or a substance that blocks gp 130-mediated JAK-STAT3 signaling pathway in at least one of 1) or 2) as follows:

1) preparing a product for inhibiting the expression of ATX in adipose tissue or adipocytes;

2) preparing a product for inhibiting plasma LPA and/or ATX levels.

2. Use according to claim 1, characterized in that: the inhibition of ATX expression in adipose tissue or adipocytes is achieved by inhibiting gp 130-mediated activation of STAT3 in the JAK-STAT3 signaling pathway.

3. Use according to claim 1 or 2, characterized in that: the substance for inhibiting gp130 expression or the substance for blocking gp 130-mediated JAK-STAT3 signal pathway is a gp130 inhibitor or siRNA for interfering gp130 expression.

4. Use according to claim 3, characterized in that: the gp130 inhibitor is SC 144;

Use of gp130 or a ligand thereof for the preparation of a product for increasing the expression of ATX in adipocytes.

6. Use according to claim 5, characterized in that: the gp130 ligand is an IL-6 family cytokine.

7. Use according to claim 6, characterized in that: the IL6 family cytokine is IL-6, LIF, CT-1 or CNT.

8. Use according to claim 7, characterized in that: the IL-6 family cytokines enhance the expression of ATX in adipocytes by activating gp130-STAT3 signaling pathway in adipocytes.

9. Use according to claim 1, characterized in that: the product is a medicament.

10. The application of a substance for inhibiting STAT3 activation in preparing products for inhibiting the expression of ATX in adipocytes;

the substance for inhibiting STAT3 activation is STAT3siRNA or STAT3 inhibitor S3I-201;

the nucleotide sequence of the STAT3siRNA is 5 ¢ -CUGGAUAACUUCAUUAGCA-3 ¢.