CN108042797B - Application of bispecific phosphatase 9 in preparation of drugs for treating fatty liver and related diseases - Google Patents

Abstract

The invention discloses application of bispecific phosphatase 9 in preparation of a medicament for treating fatty liver and related diseases. Belongs to the new application of DUSP 9. According to the invention, a human normal liver L02 cell line and a DUSP9 overexpression L02 cell line are taken as research objects, and the function of DUSP9 gene is researched through a liver cell lipid accumulation model induced by combined stimulation of Palmitate (PA) and Oleic Acid (OA), so that the DUSP9 overexpression can obviously improve the lipid accumulation in liver cells, and the DUSP9 can protect the liver cells from lipoid degeneration. DUSP9 is an endogenous protein in the body, and therefore, it is highly safe as a drug.

Description

Application of bispecific phosphatase 9 in preparation of drugs for treating fatty liver and related diseases

Technical Field

The invention belongs to the field of gene functions and applications, and particularly relates to a function and an application of DUSP9 in fatty liver treatment, and an application of DUSP9 as a target gene in preparation of a medicament for preventing, relieving and/or treating fatty liver and related diseases.

Background

With the change of modern life style and environment, metabolic diseases such as obesity, lipid abnormality and diabetes become global serious diseases, and seriously threaten public health and quality of life. NAFLD (Non-alcoholic fatty liver disease) is a metabolic disease with no history of excessive drinking and pathological characteristics of accumulation and degeneration of fat in liver cells, and its pathogenesis includes obesity, type II diabetes, dyslipidemia and metabolic syndrome^[1-3]. NAFLD is the most common chronic liver disease, with a prevalence of approximately 25% according to a wide range of global statistics, with 10-20% of patients being the most commonThe terminal progression is nonalcoholic steatohepatitis (NASH), and about one third of NASH patients progress to cirrhosis and even liver cancer^[4][5]. In addition, along with the appearance of NAFLD and NASH, the body also generates a series of metabolic disorder syndromes, such as dyslipidemia and diabetes. However, at present, no effective medicine or treatment means exist to overcome the clinical problem, and therefore, it is very important to find a new target for preparing a medicine for preventing, relieving and/or treating fatty liver diseases.

Dual specificity phosphatase 9 (DUSP 9) belongs to the family of tyrosine protein phosphatases and was originally discovered by Muda in 1997 to inhibit the activity of the MAPK signaling pathway by dephosphorylating or inactivating mitogen-activated protein kinase (MAPK)^[6]. The literature reports about DUSP9 are also mostly focused on the regulation mechanism of the DUSP9 on MAPK signal pathway, and DUSP9 plays a role as an anti-cancer gene in a plurality of tumors such as liver cancer, stomach cancer, kidney cancer and the like^[7-11]. However, the role of DUSP9 in fatty liver has not been reported for a long time.

Reference to the literature

[1].Reccia I,Kumar J,Akladios C,Virdis F,Pai M,Habib N,et al.Non-alcoholic fatty liver disease:A sign of systemic disease.Metabolism 2017Jul；72:94-108.

[2].Machado MV,Diehl AM.Pathogenesis of Nonalcoholic Steatohepatitis.Gastroenterology 2016Jun；150(8):1769-1777.

[3].Mota M,Banini BA,Cazanave SC,Sanyal AJ.Molecular mechanisms oflipotoxicity and glucotoxicity in nonalcoholic fatty liver disease.Metabolism2016Aug；65(8):1049-1061.

[4].Dyson J,Jaques B,Chattopadyhay D,Lochan R,Graham J,Das D,etal.Hepatocellular cancer:the impact of obesity,type 2diabetes and amultidisciplinary team.J Hepatol 2014Jan；60(1):110-117.

[5].Gaggini M,Morelli M,Buzzigoli E,DeFronzo RA,Bugianesi E,Gastaldelli A.Non-alcoholic fatty liver disease(NAFLD)and its connection withinsulin resistance,dyslipidemia,atherosclerosis and coronary heartdisease.Nutrients 2013May 10；5(5):1544-1560.

[6].Muda M,Boschert U,Smith A,Antonsson B,Gillieron C,Chabert C,etal.Molecular cloning and functional characterization of a novel mitogen-activated protein kinase phosphatase,MKP-4.J Biol Chem 1997Feb 21；272(8):5141-5151.

[7].Wu S,Wang Y,Sun L,Zhang Z,Jiang Z,Qin Z,et al.Decreasedexpression of dual-specificity phosphatase 9is associated with poor prognosisin clear cell renal cell carcinoma.BMC cancer 2011Sep 26；11:413.

[8].Lou S,Ren L,Xiao J,Ding Q,Zhang W.Expression profiling basedgraph-clustering approach to determine renal carcinoma related pathway inresponse to kidney cancer.Eur Rev Med Pharmacol Sci 2012Jun；16(6):775-780.

[9].Liu Y,Lagowski J,Sundholm A,Sundberg A,Kulesz-MartinM.Microtubule disruption and tumor suppression by mitogen-activated proteinkinase phosphatase 4.Cancer Res 2007Nov 15；67(22):10711-10719.

[10].Xu H,Dembski M,Yang Q,Yang D,Moriarty A,Tayber O,et al.Dualspecificity mitogen-activated protein(MAP)kinase phosphatase-4plays apotential role in insulin resistance.J Biol Chem 2003Aug 08；278(32):30187-30192.

[11].Keyse SM.Dual-specificity MAP kinase phosphatases(MKPs)andcancer.Cancer Metastasis Rev 2008Jun；27(2):253-261.

Disclosure of Invention

The invention aims to solve the defects and shortcomings of the prior art, provide a correlation between DUSP9 gene expression and fatty liver, provide a new application of a target gene DUSP9 for treating fatty liver, and further apply the DUSP9 gene to the treatment of fatty liver.

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

in a first aspect of the invention, there is provided the use of a dual specificity phosphatase 9 for the manufacture of a medicament for the protection of the liver.

Preferably, the drug has a function of inhibiting liver lipid accumulation.

In a second aspect of the present invention, there is provided a use of the dual specificity phosphatase 9 for the preparation of a medicament for the prevention, alleviation and/or treatment of fatty liver and related diseases.

The invention relates to an application of dual-specificity phosphatase 9 in preparing a medicament for preventing, relieving and/or treating fatty liver and related diseases, wherein the active ingredient of the medicament is dual-specificity phosphatase 9.

The invention relates to an application of bispecific phosphatase 9 in preparing a medicament for preventing, relieving and/or treating fatty liver and related diseases, in particular to a medicament for screening and preventing, relieving and/or treating fatty liver and related diseases by using the bispecific phosphatase 9 as a medicament target, wherein the medicament is an agent for improving the expression level of the bispecific phosphatase 9.

Preferably, the agent for increasing the expression level of the dual-specificity phosphatase 9 is administered by direct naked DNA injection, liposome-encapsulated DNA direct injection, gold-encapsulated DNA gene gun bombardment, plasmid-carried DNA of reproduction-defective bacteria, DNA of replication-defective adenovirus carrier, PEG-modified protein drug injection, liposome-encapsulated protein intravenous injection, or protein microsphere preparation subcutaneous injection.

The dual specificity phosphatase 9 or DUSP9 comprises a gene or a protein. The DUSP9 gene is transcribed and translated into a bispecific phosphatase 9 protein product in a subject.

Such fatty liver and related diseases include, but are not limited to: insulin resistance, metabolic syndrome, obesity, diabetes, hyperglycemia, hyperlipidemia, simple hepatic steatosis, non-alcoholic steatohepatitis, hepatic fibrosis, liver cirrhosis, liver cancer, etc.

The invention experimentally determines the relationship between the expression of the dual specificity phosphatase 9 and fatty liver and related diseases:

according to the invention, a human normal liver L02 cell line and a DUSP9 overexpression L02 cell line are taken as research objects, and the function of DUSP9 gene is researched through a liver cell lipid accumulation model induced by combined stimulation of Palmitate (PA) and Oleic Acid (OA), so that the DUSP9 overexpression can obviously improve the lipid accumulation in liver cells, and the DUSP9 can protect the liver cells from lipoid degeneration. Therefore, DUSP9 plays a protective role in liver lipid metabolic diseases, and the treatment of liver lipid metabolic diseases by over-expressing DUSP9 gene has potential treatment and application values.

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

(1) the invention discovers a new function of DUSP9 gene, namely that DUSP9 gene has the function of protecting fatty liver diseases.

(2) Based on the role of DUSP9 in protecting fatty liver disease, it can be used for preparing a medicament for preventing, alleviating and/or treating fatty liver. DUSP9 is an endogenous protein in the body, and therefore, it is highly safe as a drug.

Drawings

FIG. 1 identification of Stable cell lines overexpressing DUSP9

FIG. 2 overexpression of DUSP9 inhibits hepatocyte fat accumulation

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.

Cells for experiments and culture

Human liver cell line L02 was purchased from the Chinese academy of sciences cell Bank (catalog No. GNHu6), and human embryonic kidney HEK293T cells were purchased from the American Type Culture Collection (ATCC). The cells were cultured in DMEM high-glucose medium (containing 10% FBS, 1% penicillin-streptomycin) in 5% CO₂The cells are cultured in a special incubator at the constant temperature of 37 ℃, the culture time of the cells for experiments is not more than three months, and the mycoplasma detection is carried out every three months. Cells were cryopreserved using FBS containing 10% DMSO.

DUSP9 overexpression plasmid construction

1) The DUSP9 gene was amplified by PCR with the primers:

forward direction: 5'-TCGGGTTTAAACGGATCCATGGAGGGTCTGGGCCGCTC-3', respectively;

and (3) reversing: 5'-GGGCCCTCTAGACTCGAGCTAGGTGGGGGCCAGCTCGAA-3', respectively;

2) the PCR products were subjected to agarose gel electrophoresis, followed by recovery of DNA fragments using a DNA gel recovery kit (Tiangen);

3) the obtained DNA product and restriction endonuclease,

buffer or

Green buffer、ddH₂O is mixed uniformly (50. mu.l system) and placed at 37 ℃ for reaction. Use of

AxyPrep^TMRecovering an enzyme digestion product by using a PCR Clean-Up Kit (Axygen);

4) use of

Performing recombination reaction by using a PCR one-step directional cloning kit (Novoprotein) according to the kit instruction;

5) preparing escherichia coli competent cells, performing a transformation experiment on the ligation product, coating a plate, placing the plate in an incubator at 37 ℃, and culturing overnight;

6) taking out the overnight cultured plate from the 37 ℃ incubator, selecting clone and shaking bacteria, and detecting colony PCR positive clone;

7) taking 5-10 mul of the bacterial liquid identified as positive by PCR, inoculating the bacterial liquid into 5ml of LB (containing resistance) culture medium, and culturing in a shaking table at 220rpm and 37 ℃ overnight;

8) taking out overnight cultured bacterial liquid, and carrying out plasmid extraction on turbid bacterial liquid (Tiangen plasmid DNA miniextraction kit);

9) the extracted plasmid can be directly used for constructing lentivirus package.

Lentiviral vector construction and packaging

1) At 200. mu.l

Adding a transfection reagent (PEI Max or Lipofectamine 2000) into the I Reduced Serum Medium, gently mixing uniformly, and centrifuging briefly; (DNA. mu.g: transfection reagent. mu.l ═ 1:0.5 to 1:5)

2) At 200. mu.l

Adding 1 μ g of target gene plasmid and packaging plasmid (0.5 μ g of pMD2.G (Addgene, 12259) and 0.75 μ g of psPAX2(Addgene, 12260) commonly used for lentivirus) into I Reduced Serum Medium, gently mixing, and centrifuging briefly;

3) gently mixing the systems 1) and 2), centrifuging briefly, and incubating at room temperature for 20 min;

4) the mixed system was added dropwise to a 6-well plate and gently mixed.

5) After 6h of transfection, fresh culture medium is replaced;

6) harvesting virus-containing supernatant 48-72h after transfection, centrifuging at 3000rpm for 10min, removing precipitate, and filtering with 0.45 μm filter membrane;

7) the filtered virus was used immediately for infection or stored at-80 ℃.

Western blot analysis

1) Glue making

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.

2) Protein extraction

Cells were lysed with an appropriate amount of RIPA (50mM Tris-HCl PH7.4,150mM NaCl, 1% Triton X-100ornP-40, 1% Sodium desoxyholate, 0.1% SDS, 1mM EDTA, protease or phosphatase inhibitor added prior to use) on ice for 10-30min, and sonication increased protein extraction efficiency; centrifuging at 4 deg.C and 12000rpm for 10min to obtain supernatant as total protein; protein quantification was performed using BCA Protein Assay Kit, and Western blot analysis was performed on 30-50. mu.g of total Protein.

3) Sample loading and electrophoresis

Ensuring that the sample loading quantity and the sample loading volume are consistent, performing constant-pressure electrophoresis, wherein the upper layer of glue is 80-90V, and the lower layer of glue is 100V.

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. (Note: the glue should be spread evenly and not stretched; the glue should be kept in the buffer solution during the film transfer process; no air bubble should be left between the glue and the film.)

5) Sealing of

5% skimmed milk powder (in TBST) was sealed for 1h at room temperature on a shaking table.

6) Primary antibody incubation

Incubate at 4 ℃ overnight.

7) Secondary antibody incubation (anti-rabbit secondary antibody source, from Beijing Boolong immuno-technology Co., Ltd., anti-rabbit secondary antibody with the product number BF03008/BF03008X)

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. (selection of whether to dilute the secondary antibody with 5% skim milk powder (in TBST) based on the specificity of the antibody)

8) Development

9) Bands of interest were detected using a Bio-Rad Chemi Doc XRS + gel imaging system.

Oil red O dyeing

1) Cell fixation: washing the cells with PBS for 2-3 times, removing dead cells, and adding 300 μ l of 4% paraformaldehyde for fixation for 20 min;

2) washing with 1 × PBS for 2 times, adding 60% isopropanol, rinsing for 10s, removing isopropanol, and air drying;

3) adding 500 μ l of oil red O into each well, and dyeing for 1 min;

4) PBS was added for washing 2-3 times, the background red color was removed, observed under a microscope, and photographed.

[ example 1 ] Effect of DUSP9 overexpression on hepatocyte fat deposition

Constructing a lentivirus expression vector for over-expressing DUSP9, transfecting HEK-293T cells, packaging lentivirus, infecting L02 cells to construct a stable cell strain for over-expressing DUSP9, simultaneously using an over-expressed empty vector as a control (Con), detecting whether the stable cell strain expresses DUSP9 or not by Western blot, wherein the detection result is shown in figure 1, DUSP9 in cells over-expressing DUSP9 is highly expressed, which indicates that DUSP9 achieves the function in the stable cell strain; after the cells are attached to the plates of the successfully expressed L02 cells, the cells are stimulated by adding palmitate (PA, final concentration 0.4mM) and oleic acid (OA, final concentration 0.8mM), and a drug-added control group (the same amount of BSA is added) is arranged at the same time, and oil red O staining is carried out after 16 h. Oil red O staining results as shown in fig. 2, when BSA treated cells, none of the cells in the Con group and DUSP9 overexpression group were significantly stained, and when PA + OA stimulation was added, none of the cells in the Con group and DUSP9 overexpression group were significantly stained, but the number of cells stained with oil red O and the degree of staining were significantly reduced in the cells in the DUSP9 overexpression group compared to the Con group.

The results show that DUSP9 overexpression can inhibit lipid deposition of liver cells caused by PA and OA stimulation, DUSP9 plays a protective role in the pathological process of liver steatosis, and DUSP9 as a target point can provide a new treatment idea for diseases such as fatty liver injury.

In conclusion, DUSP9 overexpression can remarkably inhibit liver fat accumulation in vitro, and DUSP9 realizes the protection effect on liver steatosis by inhibiting MAPK signal channels, and a new idea and strategy are possibly provided for clinical treatment of liver fat metabolic diseases by taking DUSP9 as a target point.

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.

Sequence listing

<110> Wuhan university

Application of <120> bispecific phosphatase 9 in preparation of drugs for treating fatty liver and related diseases

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<170>SIPOSequenceListing 1.0

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<213> Artificial Sequence (Artificial Sequence)

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tcgggtttaa acggatccat ggagggtctg ggccgctc 38

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<213> Artificial Sequence (Artificial Sequence)

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gggccctcta gactcgagct aggtgggggc cagctcgaa 39

Claims (1)

1. The application of the dual-specificity phosphatase 9 as a drug target in screening drugs for preventing, relieving and/or treating nonalcoholic fatty liver diseases, is characterized in that the drugs are drugs for improving the expression level of the dual-specificity phosphatase 9, have the function of inhibiting liver lipid accumulation, and are non-diagnostic and non-therapeutic.

Images