CN108126184B

CN108126184B - Application of nuclear factor kappa B inhibitor protein 3 combined with A20 in preparation of drugs for treating fatty liver and related diseases

Info

Publication number: CN108126184B
Application number: CN201810023792.2A
Authority: CN
Inventors: 李红良; 李枫
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2018-01-10
Filing date: 2018-01-10
Publication date: 2020-06-09
Anticipated expiration: 2038-01-10
Also published as: CN108126184A

Abstract

The invention discloses application of nuclear factor kappa B arrestin 3 combined with A20 in preparing a medicament for treating fatty liver and related diseases. Belongs to a new application of ABIN 3. According to the invention, an L02 cell line, an ABIN3 overexpression L02 cell line, a mouse primary hepatocyte and an ABIN3 overexpression mouse primary hepatocyte are taken as experimental objects, a fatty liver cell model is induced by combining palmitic acid and oleic acid, and an oil red O staining result shows that the area of a red fat drop in the ABIN3 overexpression L02 cell is obviously reduced compared with that of the L02 cell under combined stimulation of the palmitic acid and the oleic acid with the same concentration. The same trend of oil red O staining results was also observed in primary hepatocytes of mice overexpressing ABIN3, indicating that ABIN3 overexpression has a significant inhibitory effect on lipid accumulation in fatty liver cells induced by palmitic acid and oleic acid in combination. Therefore, ABIN3 has the functions of inhibiting liver lipid accumulation and protecting liver.

Description

Application of nuclear factor kappa B inhibitor protein 3 combined with A20 in preparation of drugs for treating fatty liver and related diseases

Technical Field

The invention belongs to the field of functions and applications of genes, and particularly relates to a ubiquitin chain binding protein, namely a function and an application of a nuclear factor kappa B inhibitor protein 3(A20binding inhibitor of NF-kappa B activation, ABIN3) combined with A20 in fatty liver and related diseases, and an application of ABIN3 as a target gene in preparation of drugs for preventing, relieving and/or treating fatty liver and related diseases.

Background

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by the presence of pathological features of steatosis and fat accumulation in parenchymal hepatocytes, although patients have no history of excessive alcohol consumption [1 ]. Studies have shown that approximately 10% of patients with NAFLD develop nonalcoholic steatohepatitis (NASH), while approximately 20% of these NASH patients develop cirrhosis within 10 years. NAFLD is reported to be highly prevalent in developed countries, with an adult incidence of 30% and a child incidence of 13% [2 ]. In recent years, NASH has become a common cause of hepatoenzymological abnormality of physical examination people and one of the pathological changes in the early stage of cirrhosis, so that the research on NAFLD is receiving more and more attention [3 ]. Although some therapies are clinically able to improve some parameter indicators of liver function and histology, no exact treatment for NAFLD is currently established. Therefore, the discovery of specific molecules and signal transduction pathways participating in NAFLD has very important theoretical and practical significance for further systematically clarifying the generation and development mechanism of NAFLD, regulating and controlling NAFLD from the cellular molecular level and exploring a new therapeutic target for preventing and treating NAFLD.

The analysis of the human ABIN3 protein mRNA promoter shows that ABIN3 can be abundantly expressed in the pathway of Lipopolysaccharide (LPS) and TNF-induced NF- κ B activation, which reveals that ABIN3 protein expression may be involved in NF- κ B regulation similar to other family proteins of ABIN, human ABIN3 can also bind to A20 and edit ubiquitin chain through the latter, thereby achieving the inhibition of TNFR1, IL-1R and other receptor-mediated NF- κ B activation, but no effect on IKK- β -mediated NF- κ B activation is produced [4 ]. in human cells, ABIN3 can inhibit Toll-like receptor (TLR), TNF signal-induced NF- κ B activity, the regulation mechanism is that firstly, human ABIN3 protein has an AHD region capable of binding NF- κ B, which can bind to corresponding site on NF- κ B to inhibit NF- κ B activation, which can inhibit NF- κ B activation by binding to corresponding site on NF- κ B activation, which single- κ B protein can inhibit NF- κ B activation in liver disease, and further inhibit liver disease induced by ABIN 3625 protein activation, thus, ABIN 366 can inhibit liver activation induced liver disease.

Reference to the literature

1.Paschos,P.and K.Paletas,Non alcoholic fatty liver disease andmetabolic syndrome.Hippokratia,2009.13(1):p.9-19.

2.Greenfield,V.,O.Cheung,and A.J.Sanyal,Recent advances innonalcholic fatty liver disease.Curr Opin Gastroenterol,2008.24(3):p.320-7.

3.Polyzos,S.A.,J.Kountouras,and C.Zavos,Nonalcoholic fatty liverdisease:the pathogenetic roles of insulin resistance and adipocytokines.CurrMol Med, 2009.9(3):p.299-314.

4.Wullaert,A.,et al.,LIND/ABIN-3is a novel lipopolysaccharide-inducible inhibitor of NF-kappaB activation.J Biol Chem,2007.282(1):p.81-90.

5.Ogushi,I.,et al.,Nuclear factor kappa B decoy oligodeoxynucleotidesprevent endotoxin-induced fatal liver failure in a murine model.Hepatology,2003. 38(2):p.335-44.

6.Freudenberg,M.A.,D.Keppler,and C.Galanos,Requirement forlipopolysaccharide-responsive macrophages in galactosamine-inducedsensitization to endotoxin.Infect Immun,1986.51(3):p.891-5.

Disclosure of Invention

In order to solve the defects and shortcomings of the prior art, the invention aims to provide a correlation between ABIN1 gene expression and fatty liver and related diseases, provide a new application of a target gene ABIN1 for treating fatty liver and related diseases, and further apply ABIN1 to the treatment of fatty liver diseases.

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 nuclear factor κ B inhibitory protein 3, associated with a20, in the preparation of a medicament for the protection of the liver.

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

In a second aspect of the invention, the application of the nuclear factor kappa B inhibitor protein 3 combined with A20 in preparing a medicament for preventing, relieving and/or treating fatty liver and related diseases is provided.

The invention relates to an application of nuclear factor kB arrestin 3 combined with A20 in preparing a medicament for preventing, relieving and/or treating fatty liver and related diseases, wherein the active component of the medicament is nuclear factor kB arrestin 3 combined with A20.

The invention relates to application of nuclear factor kappa B inhibitor protein 3 combined with A20 in preparation of drugs for preventing, relieving and/or treating fatty liver and related diseases, in particular to application of nuclear factor kappa B inhibitor protein 3 combined with A20 as a drug target in screening of drugs for preventing, relieving and/or treating fatty liver and related diseases, wherein the drugs are reagents for improving the expression level of nuclear factor kappa B inhibitor protein 3 combined with A20.

Preferably, the agent for increasing the expression level of nuclear factor kappa B inhibitor protein 3 bound to A20 is administered by direct naked DNA injection, liposome-encapsulated DNA direct injection, gold-encapsulated DNA gene gun bombardment, plasmid-carried DNA for reproduction-defective bacteria, target DNA carried by replication-defective adenovirus, PEG-modified protein drug injection, intravenous injection of liposome-encapsulated protein, or subcutaneous injection of protein microsphere formulation.

The nuclear factor kappa B inhibitor protein 3 or ABIN3 combined with A20 comprises genes or proteins. The ABIN3 gene is transcriptionally translated in a subject into a nuclear factor kappa B arrestin 3 protein product that binds to A20.

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 nuclear factor kappa B inhibitor protein 3 combined with A20 and fatty liver and related diseases through experiments:

the invention takes a normal human hepatocyte L02 cell line, an ABIN3 overexpression L02 cell line, a mouse primary hepatocyte and an ABIN3 overexpression mouse primary hepatocyte as experimental objects, and researches the function of the ABIN3 gene by using oil red O staining and WB through a Palmitic Acid (PA) and Oleic Acid (OA) combined induced fatty liver cell model. The results of oil red O staining showed a significant reduction in the area of red fat droplets in ABIN3 overexpressing L02 cells compared to normal L02 cells under the same combined stimulation of palmitic acid and oleic acid. The oil red O staining result with the same trend is also observed in primary hepatocytes of mice over-expressed by ABIN3, which shows that the ABIN3 over-expression has obvious inhibition effect on lipid accumulation induced by combination of palmitic acid and oleic acid. The research of the inventor proves that: in a fatty liver cell model induced by combination of palmitic acid and oleic acid, ABIN3 has the functions of inhibiting liver lipid accumulation and protecting liver.

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

(1) the invention discovers a new function of the ABIN3 gene, namely the ABIN3 gene has the function of inhibiting fatty liver and related diseases.

(2) Based on the effect of ABIN3 in inhibiting fatty liver and related diseases, the ABIN3 can be used for preparing medicines for preventing, relieving and/or treating fatty liver and related diseases. ABIN3 is an endogenous protein in the body, and therefore, it is highly safe as a drug.

Drawings

FIG. 1 is a graph of WB identification of a stable L02 cell line stably expressing ABIN 3;

FIG. 2 is a graph of the results of oil red O staining of L02 stable cell lines stably expressing ABIN3 following stimulation with PA and OA;

FIG. 3 is a graph of the results of WB identifying ABIN3 expression from primary hepatocytes of mice overexpressing ABIN 3;

FIG. 4 is a graph of the results of oil red O staining of primary hepatocytes of mice overexpressing ABIN3 after stimulation 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.

Cells for experiments and culture:

the human normal liver cell line L02 was purchased from the Chinese academy of sciences cell Bank (catalog No. GNHu6), and the human embryonic kidney epithelial cells HEK293T were purchased from the American Type Culture Collection (ATCC). The cells are cultured in a DMEM high-sugar medium (containing 10% FBS and 1% penicillin-streptomycin) in a special 37 ℃ constant-temperature cell culture box containing 5% CO2, the culture time of the cells for experiments is not more than three months, and mycoplasma detection is performed every three months. Cells were cryopreserved using FBS containing 10% DMSO.

Separating and culturing primary hepatocytes:

and (3) separating the primary mouse hepatocytes by adopting a type IV collagenase digestion method. The mice are anesthetized by ether inhalation, the portal vein is punctured by a straight needle, the liver is perfused in situ (SC-1 and SC-2 are perfused in turn) until the liver is completely digested, the liver is taken down, the liver is repeatedly blown and filtered, and the hepatocyte suspension is collected. Centrifuging to collect liver cells in a culture dish coated with the rat tail gum, adding a complete culture medium for culture, changing the culture medium after 6h to remove dead cells, and continuing the culture.

1) Culture dish coating (taking six-hole plate as an example)

① mixing with appropriate amount of 1 × carnosol (prepared as before):

diluting absolute ethanol with sterilized ultrapure water to 30% ethanol, filtering with 0.22 μm filter, and diluting 100 × carnosol to 1 ×.

② mu.l of 1 Xcarnosic acid was added to the dish, shaken and spread evenly (to ensure that the wells were each accessible to any place at the bottom).

③ uncapped and blown overnight at a clean bench.

2) Cell plating

① the culture dish coated with the carnosic gum is opened and irradiated with ultraviolet light for 30 min.

② Trypan blue staining was performed to count cells, and cell plating was performed while adjusting the cell density according to the purpose of the experiment.

③ the liquid is changed 2h after the board is paved.

④ cells adhere for about 6-8h, and the subsequent operation can be carried out after the adhesion.

Construction of ABIN3 lentivirus overexpression plasmid:

1) ABIN3 gene was amplified by PCR with primers:

forward direction: 5'-TCGGGTTTAAACGGATCC ATGGCACATTTTGTACAGGG-3'

And (3) reversing: 5'-GGGCCCTCTAGACTCGAGCTACGGATGGACTTTCTTTAC-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 restriction endonucleases Fastdigest restriction enzymes (Thermo),

buffer or

Green buffer and ddH2O were mixed well (50. mu.l system) and reacted at 37 ℃ under conditions. 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/min 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 ABIN3 transient transfection or construction of a lentivirus stable transfection cell line.

Lentivirus vector construction and packaging:

1) the 293T cells were trypsinized and counted at 1X 10⁶293T/well into 6-well plate;

2) on the next day transfection was started when the cell confluence reached 80%;

3) 1.5ml of sterile EP tubing was taken and 2 packaging plasmids pSpax (Addgene, 12260) and pMD2G (Addgene, 12259) and overexpression or interference plasmids were added, each at 1. mu.g in 100. mu.l of serum free medium. Gently mix well and incubate for 5min at room temperature.

4) A1.5 ml sterile EP tube was taken and 3. mu.l PEI (1.6. mu.g/. mu.l) was dissolved in 100. mu.l serum free medium. Gently mix well and incubate for 5min at room temperature.

5) Mixing the DNA solution and the PEI solution gently and uniformly, and incubating for 15min at room temperature;

6) dropwise adding the DNA-PEI mixed solution into a 6-hole plate;

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

8) the virus-containing supernatant was harvested 48-72h after transfection, centrifuged at 3000rpm for 10min, the pellet removed and filtered through a 0.45 μm filter, and the filtered virus was immediately available for infection.

Cell infection:

(1) 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 the cells to be infected (at the same density as the normal transfection), at a ratio depending on the virus titer and the cell bearing capacity (500. mu.l virus solution +2mL complete medium per well in this case), and then 2.5. mu.l polybrene (8mg/mL) was added to give a final concentration of 8 ug/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 a blank well), and when the blank well cells completely die, passaging the cells in the six-well plate to a T25 culture flask, wherein the blank cells generally die after 24-48 h. After the cells are full, collecting a part of cells to carry out western verification over-expression, and freezing and storing a part of cells.

Western Blot (WB) detection:

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 on ice for 10-30min with appropriate amounts 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 inhibitors added prior to use); 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. 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 incubated overnight at 4 ℃ with primary antibody (anti-ABIN3(5303, ProSci); anti-GAPDH (2118, CST)).

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 (BF 03008/BF03008X, Beijing Boolong immune technology Co., Ltd.) (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 operation:

1) the sample group and the control group were washed 2 times with 1 × PBS, and fixed for 20min by adding 300 μ l of 3% paraformaldehyde;

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

3) washing with 1 × PBS for 2 times, and drying in a fume hood;

4) adding oil red O into 500 mul of each hole for dyeing for 1 h;

5) washing with 1 × PBS for 2 times, sorting with 60% isopropanol, and washing with 1 × PBS for 2 times; and (6) microscopic examination and photographing.

Detailed Description

EXAMPLE 1 Effect of ABIN3 overexpression on L02 cell fat deposition

Constructing a lentivirus expression vector for over-expressing ABIN3, transfecting HEK-293T cells, packaging lentivirus, infecting L02 cells to construct a stable cell strain for over-expressing ABIN3, and detecting whether the stable cell strain expresses ABIN3 by Western blot by using an over-expressed empty vector as a control.

After the detection, the cells were divided into 4 groups, L02 no-load control group, L02 stable cell line control group overexpressed by ABIN3, L02-no-load test group, and L02 stable cell line test group overexpressed by ABIN 3. After the cells were attached and cultured to 50% healing, both experimental groups were stimulated by adding Palmitate (PA) and Oleate (OA) (PA 0.2mM + OA 0.4mM), the same amount of BSA was added to the control group, and the cell samples of each group were collected after 12h for oil red O staining.

The Western blot detection result is shown in FIG. 1, and the protein expression level of the L02 cell strain ABIN3 infected with the lentivirus over-expressing ABIN3 is obviously higher than that of the unloaded control group. The results of oil red O staining are shown in fig. 2, where the control cells had no distinct red color, and when stimulated by PA + OA, the cells stained red with oil red O were significantly increased compared to the control. Under the same combined stimulation of PA + OA, the area of a red fat drop in an experiment group of an L02 stable cell line overexpressed by ABIN3 is obviously lower than that in an experiment group of an L02-no-load experiment group, which indicates that the overexpression of ABIN3 can effectively inhibit the lipid deposition of L02 cells induced by the stimulation of PA + OA.

Example 2 Effect of ABIN3 overexpression on fat accumulation in mouse Primary hepatocytes

Constructing a lentivirus expression vector for over-expressing ABIN3, transfecting HEK-293T cells, packaging lentivirus, infecting primary hepatocytes of mice to construct a cell model for over-expressing ABIN3, and detecting whether ABIN3 is over-expressed in ABIN3 infected cells by Western blot by using an over-expressed empty vector as a control (Con).

After completion of the assay, the cells were divided into 4 groups, namely: an unloaded control group, a cell control group with ABIN3 overexpressed, a L02-unloaded experimental group, and a cell experimental group with ABIN3 overexpressed. After the cells were adherent and plated to 50% union, both experimental groups were stimulated with Palmitate (PA) and Oleate (OA) (PA 0.2mM + OA 0.4mM), the control group was treated with the same amount of BSA, and the cells were stained 12h later with an oil red O stain.

The Western blot detection result is shown in FIG. 3, and the protein expression level of ABIN3 in primary hepatocytes infected with the ABIN3 overexpression lentivirus is significantly higher than that in the unloaded control group. The results of oil red O staining are shown in fig. 4, where the control cells had no distinct red color, and when stimulated by PA + OA, the cells stained red with oil red O were significantly increased compared to the control. Under the same combined stimulation of PA and OA, the area of red fat drops in the experiment group of the mouse primary hepatocytes over-expressed by ABIN3 is obviously lower than that in the experiment group of the mouse primary hepatocytes without load, which shows that the over-expression of ABIN3 can effectively inhibit lipid deposition of the mouse primary hepatocytes induced by PA and OA stimulation

The results show that the ABIN3 gene overexpression can obviously inhibit the accumulation of liver cell lipid and the occurrence and development of fatty liver and related diseases, and the ABIN3 gene has obvious inhibition effect on the fatty liver and the related diseases.

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

<120> application of nuclear factor kappa B inhibitor protein 3 combined with A20 in preparation of drugs for treating fatty liver and related diseases

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Claims

1. The application of the nuclear factor kappa B inhibitor protein 3 combined with A20 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 nuclear factor kappa B inhibitor protein 3 combined with A20, the drugs have the function of inhibiting liver lipid accumulation, and the application is non-diagnostic and non-therapeutic.