CN108114269B - Application of tumor necrosis factor α -induced protein 8-like molecule 2 in preparation of medicines for treating fatty liver and related diseases - Google Patents

Abstract

The invention discloses an application of a tumor necrosis factor α induced protein 8-like molecule 2 in preparation of a medicament for treating fatty liver and related diseases, belongs to a new application of TIPE2, takes a human normal liver cell line L02 and a TIPE2 over-expressed L02 cell line as research objects, stimulates and induces a liver cell lipid accumulation model through Palmitate (PA) and Oleic Acid (OA) so as to research the function of a TIPE2 gene, researches show that the TIPE2 over-expression can obviously improve the lipid accumulation in liver cells and protect the liver cells from fatty sample degeneration, the TIPE2 plays a protective role in liver lipid metabolic diseases, and the TIPE2 is an endogenous protein of an organism, so that the TIPE 6332 has high safety as the medicament.

Description

Application of tumor necrosis factor α -induced protein 8-like molecule 2 in preparation of medicines for treating fatty liver and related diseases

Technical Field

The invention belongs to the field of gene function and application, and particularly relates to a function and application of a tumor necrosis factor α induced protein 8-like molecule 2 (TIPE 2) gene in fatty liver and related diseases, particularly an application in preparation of a medicament for preventing, relieving and/or treating fatty liver and related diseases.

Background

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic disease in developed and even developing countries, mainly due to a series of clinical diseases caused by overnutrition. NAFLD is a metabolic disease without excessive drinking history and with liver cell fat accumulation and degeneration as pathological characteristics, and its pathogenesis includes obesity, type II diabetes, dyslipidemia and metabolic syndrome^1-3. NAFLD is the most common chronic liver disease, and its incidence rate is about 25% according to global statistics, of which 10-20% of patients eventually develop nonalcoholic steatohepatitis (NASH), while about one third of NASH patients gradually develop cirrhosis, 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 and treatment means exist to overcome the clinical problem, and therefore, the search for effective prevention measures and treatment means is imperative.

The tumor necrosis factor α induced protein 8 (TIPE) family genes are identified and confirmed in recent years and comprise TIPE, TIPE2, TIPE2 and TIPE3, and research shows that TIPE family molecules mainly play an important role in tumor occurrence development and immune regulation mechanisms^6-10. TIPE2 is the most studied gene in TIPE family molecules, is highly expressed in hepatocytes, and is widely involved in immune regulation through T cell receptors, Toll-like receptors, JNK signaling pathway, NF-kappa B, mTORC signaling pathway^7,11-14. TIPE2 participates in the molecular pathological mechanism of various tumors by inhibiting MAPK signal pathway and NF-kB signal pathway. No report has been made on the study of TIPE2 in liver fat accumulation and degeneration.

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 2016 Jun；150(8):1769-1777.

[3]Mota M,Banini BA,Cazanave SC,Sanyal AJ.Molecular mechanisms oflipotoxicity and glucotoxicity in nonalcoholic fatty liver disease.Metabolism2016 Aug；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 2 diabetes and amultidisciplinary team.J Hepatol 2014 Jan；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 2013 May 10；5(5):1544-1560.

[6]Freundt EC,Bidere N,Lenardo MJ.A different TIPE of immunehomeostasis.Cell 2008 May 02；133(3):401-402.

[7]Lou Y,Liu S.The TIPE(TNFAIP8)family in inflammation,immunity,andcancer.Mol Immunol 2011 Oct；49(1-2):4-7.

[8]Moniz LS,Vanhaesebroeck B.A new TIPE of phosphoinositide regulatorin cancer.Cancer cell 2014 Oct 13；26(4):443-444.

[9]Goldsmith JR,Chen YH.Regulation of inflammation and tumorigenesisby the TIPE family of phospholipid transfer proteins.Cell Mol Immunol 2017Jun；14(6):482-487.

[10]Sun H,Gong S,Carmody RJ,Hilliard A,Li L,Sun J,et al.TIPE2,anegative regulator of innate and adaptive immunity that maintains immunehomeostasis.Cell 2008 May 02；133(3):415-426.

[11]Oho M,Nakano R,Nakayama R,Sakurai W,Miyamoto A,Masuhiro Y,etal.TIPE2(Tumor Necrosis Factor alpha-induced Protein 8-like 2)Is a NovelNegative Regulator of TAK1 Signal.J Biol Chem 2016 Oct 21；291(43):22650-22660.

[12]Wang K,Ren Y,Liu Y,Zhang J,He JJ.Tumor Necrosis Factor (TNF)-alpha-Induced Protein 8-like-2(TIPE2)Inhibits Proliferation and Tumorigenesisin Breast Cancer Cells.Oncol Res 2017Jan 02；25(1):55-63.

[13]Zhang X,Wang J,Fan C,Li H,Sun H,Gong S,et al.Crystal structure ofTIPE2provides insights into immune homeostasis.Nat Struct Mol Biol 2009Jan；16(1):89-90.

[14]Zhang Y,Shao Z,Zhang X,Jia X,Xia Y,Zhang Y,et al.TIPE2Play aNegative Role in TLR4-Mediated Autoimmune T Helper 17Cell Responses inPatients with Myasthenia Gravis.J Neuroimmune Pharmacol 2015Dec；10(4):635-644.

Disclosure of Invention

In order to solve the defects and shortcomings of the prior art, the invention aims to provide a correlation between the expression of the TIPE2 gene and fatty liver and related diseases, provide a new application of a target gene TIPE2 for treating fatty liver and related diseases, and further apply the TIPE2 gene to the treatment of fatty liver and related diseases.

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

the invention provides an application of a tumor necrosis factor α -induced protein 8-like molecule 2 in preparation of a liver-protecting drug.

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

In a second aspect of the invention, the application of the tumor necrosis factor α -induced protein 8-like molecule 2 in preparing a medicament for preventing, alleviating and/or treating fatty liver and related diseases is provided.

The invention relates to an application of a tumor necrosis factor α -induced protein 8-like molecule 2 in preparation of a medicament for preventing, relieving and/or treating fatty liver and related diseases, wherein the active component of the medicament is the tumor necrosis factor α -induced protein 8-like molecule 2.

The invention relates to an application of a tumor necrosis factor α -induced protein 8-like molecule 2 in preparation of a medicament for preventing, relieving and/or treating fatty liver and related diseases, in particular to a medicament for screening, preventing, relieving and/or treating fatty liver and related diseases by taking the tumor necrosis factor α -induced protein 8-like molecule 2 as a medicament target, wherein the medicament is a reagent for improving the expression quantity of the tumor necrosis factor α -induced protein 8-like molecule 2.

Preferably, the administration mode of the reagent for improving the expression quantity of the tumor necrosis factor α induced protein 8-like molecule 2 is direct naked DNA injection, liposome-coated DNA direct injection, gold-coated DNA gene gun bombardment, reproduction-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA, PEG modified protein drug injection, liposome-coated protein intravenous injection or protein microsphere preparation subcutaneous injection.

The RNF14 gene is transcribed and translated into a protein product of the tumor necrosis factor α -induced protein 8-like molecule 2 in a subject body.

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 determines the relation between the expression of the tumor necrosis factor α induced protein 8-like molecule 2 and fatty liver and related diseases through experiments:

the invention takes a human normal liver cell line L02 and a TIPE2 over-expressed L02 cell line as research objects, and further researches the function of the TIPE2 gene by stimulating and inducing a liver cell lipid accumulation model through Palmitate (PA) and Oleic Acid (OA). Research shows that TIPE2 overexpression can obviously improve lipid accumulation in liver cells and protect the liver cells from lipoidosis. TIPE2 plays a protective role in liver lipid metabolism disorders.

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

(1) the invention discovers a new function of the TIPE2 gene, namely the TIPE2 gene has the function of protecting fatty liver and related diseases.

(2) Based on the role of TIPE2 in protecting fatty liver and related diseases, the TIPE2 can be used for preparing medicines for preventing, relieving and/or treating fatty liver and related diseases. Since TIPE2 is an endogenous protein in the body, it is highly safe as a drug.

Drawings

FIG. 1 shows the Western Blot analysis result of protein expression of TIPE2 of L02 cell line.

FIG. 2 shows the results of oil red O staining (magnification:. times.400) of L02 cells stimulated with PA and OA.

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 experiment 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.

Cell culture

The human liver cell line L02 was purchased from the Chinese academy of sciences cell Bank (catalog No. GNHu6), and the 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 and 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.

TIPE2 overexpression plasmid construction

1) The TIPE2 gene was amplified by PCR with the primers:

forward direction: 5'-CGCGGATCCATGGAGTCCTTCAGCTCAAAG-3' the flow of the air in the air conditioner,

and (3) reversing: 5'-CCGCTCGAGTCAGAGCTTCCCTTCGTCTA-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 resulting DNA product is combined with the restriction endonucleases BamHI and XholI (thermo),

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) HEK293T cells were cultured in 6-well plates starting the day before, so that the expected cell aggregation per well on the second day after plating was 50% or more.

2) 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)

3) 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;

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

5) dripping the mixed system into a 6-well plate for culturing HEK293T cells, and gently mixing;

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

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

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

Infection of cells

(1) L02 cell plating: two wells were infected with each virus and one well was left as a blank for later screening of cells.

(2) First infection: the virus solution was mixed with the medium of L02 cells to be infected (at the same density as normal transfection) at a ratio dependent on the virus titer and cell viability (this time 500. mu.l virus solution +2ml complete medium per well) and then 2.5. mu.l polybrene (8mg/ml) was added to give a final concentration of 8. mu.g/ml.

(3) The liquid can be changed to stop infection within 2h after infection, and the liquid can be continuously infected for 24h to the maximum extent if the cell bearing capacity is strong.

(4) And (3) secondary infection: after 24h of infection, the infection was repeated once more.

Drug-added screening cells

48h after the first infection, adding complete culture medium (with a final concentration of 1 mu g/ml) containing puromycin into a six-well plate (including blank wells), and when the cells in the blank wells die completely, passaging the cells in the six-well plate to a T25 culture flask, wherein the blank cells die after 24-48 h. After the cells are full, collecting a part of cells, performing Western blot verification on overexpression, and freezing and storing a part of cells.

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-100or NP-40, 1% Sodium deoxyholate, 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 for 10min at 12000g to obtain supernatant as total protein; the protein quantification is carried out by adopting BCAProtein Assay Kit, and Western blot analysis is carried out 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. 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

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

6) Primary antibody incubation

After blocking, the protein membrane was washed 3 times with TBST for 5min each, and a primary antibody (TIPE2, Proteitech, 15940-1-AP) was added and incubated overnight at 4 ℃.

7) Incubation with secondary antibody

The anti-rabbit secondary antibody is purchased from Beijing Boolong immune technology Co., Ltd, and has the product number of BF03008/BF 03008X.

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) And (6) developing.

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.

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 TIPE2 overexpression on hepatocyte fat deposition

Constructing a lentivirus expression vector for over-expressing TIPE2, transfecting HEK-293T cells, packaging lentivirus, infecting L02 cells to construct a stable cell strain (pHAGE-TIPE2) for over-expressing TIPE2, meanwhile, taking an over-expressing empty vector as a control group (pHAGE group), and detecting whether the stable cell strain highly expresses TIPE2 or not by Western blot; the L02 cell plates with successful expression are divided into 4 groups, namely a pHAGE control group, a pHAGE-TIPE2 control group, a pHAGE experimental group and a pHAGE-TIPE2 experimental group. After the cells were attached, the experimental group was stimulated with palmitate (PA, final concentration 0.4mM) and oleic acid (OA, 0.8mM), while the control group was stained with oil Red O after 16h with the same amount of BSA.

The Western blot detection result is shown in FIG. 1, and the protein expression level of the L02 cell strain TIPE2 infected with the lentivirus over-expressing TIPE2 is obviously higher than that of the unloaded control group. The results of oil red O staining are shown in FIG. 2, where there was no significant staining of the cells in both the control and TIPE 2-overexpressing groups when BSA was used to treat the cells, and the red lipid droplets were much larger in the cells of the experimental group compared to the control group when stimulated with PA + OA. However, compared with the pHAGE experimental group, the number of cells stained by oil red O and the degree of staining of cells in the TIPE2 overexpression group are obviously reduced, which indicates that the TIPE2 overexpression can inhibit lipid deposition of liver cells caused by PA and OA stimulation

The results show that TIPE2 overexpression can inhibit lipid deposition of liver cells caused by PA and OA stimulation, and TIPE2 plays a protective role in pathological processes of fatty liver and related diseases thereof.

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

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Application of <120> tumor necrosis factor α induced protein 8-like molecule 2 in preparation of drugs for treating fatty liver and related diseases

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Claims (1)

1. The application of the tumor necrosis factor α -induced protein 8-like molecule 2 as a drug target in screening drugs for preventing, relieving and/or treating non-alcoholic fatty liver is characterized in that the drugs are drugs for improving the expression level of the tumor necrosis factor α -induced protein 8-like molecule 2, the drugs have the function of inhibiting liver lipid accumulation, and the application is non-diagnostic and non-therapeutic.

Images