CN117045791A - Application of targeted overexpression agent of hepatic stellate cells NEU1 in preparation of drugs for treating hepatic fibrosis - Google Patents

Abstract

The invention discloses an application of a target overexpression agent of hepatic stellate cells NEU1 in preparing a medicament for treating hepatic fibrosis, and discloses an application of the target overexpression agent of hepatic stellate cells NEU1 in preparing the medicament for treating hepatic fibrosis. The invention discovers that the targeted over-expression hepatic stellate cell NEU1 can effectively resist hepatic fibrosis. Therefore, the targeted overexpression agent of hepatic stellate cell NEU1 has a prospect of developing drugs for treating hepatic fibrosis.

Description

Application of targeted overexpression agent of hepatic stellate cells NEU1 in preparation of drugs for treating hepatic fibrosis

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of a targeted overexpression agent of hepatic stellate cells NEU1 in preparation of medicines for treating hepatic fibrosis.

Background

Liver fibrosis (HF) is the result of abnormal deposition of various extracellular matrices (extracellular matrix, ECM) due to excessive repair reactions caused by various chronic liver diseases such as genetically related diseases, chronic viral hepatitis, alcoholic hepatitis, cholestasis and drug-induced liver injury. The mechanism of hepatic fibrosis development is related to hepatic stellate cells (hepatic stellate cells, HSC), hepatic macrophages, endoplasmic reticulum stress (endoplasmic reticulum stress, ERs), autophagy, and the like. Liver fibrosis is a dynamic process that continues to develop cirrhosis, even hepatocellular carcinoma, without intervention. Therefore, it is important to delay or reverse the occurrence and development of liver fibrosis.

Disclosure of Invention

The invention aims to provide an application of a target overexpression agent of hepatic stellate cells NEU1 in preparation of a drug for treating hepatic fibrosis.

The above object of the present invention is achieved by the following technical scheme:

use of a targeted overexpression agent of hepatic stellate cells NEU1 in the preparation of a medicament for treating liver fibrosis.

Preferably, the drug takes the targeting overexpression agent of hepatic stellate cells NEU1 as an active ingredient, and is prepared into a pharmaceutically acceptable dosage form by using a pharmaceutically acceptable carrier.

More preferably, the carrier is in solid, liquid or semi-solid form.

More preferably, the dosage forms include injections, tablets, capsules and drops.

The beneficial effects are that:

the invention discovers that the targeted over-expression hepatic stellate cell NEU1 can effectively resist hepatic fibrosis. Therefore, the targeted overexpression agent of hepatic stellate cell NEU1 has a prospect of developing drugs for treating hepatic fibrosis.

Drawings

Fig. 1: the NEU1 gene expression level (1A) and the protein expression level (1B, 1C) of the mouse primary hepatic stellate cells in the in vitro spontaneous activation process;

fig. 2: gene expression levels of NEU1 in primary astrocytes of mice after transfection of NEU1 plasmid;

fig. 3: NEU1 plasmid intervention mice primary astrocyte α -SMA immunofluorescence staining pattern (3A), gene expression level of fibrosis index (Acta 2, col1, ctgf) (3B), protein expression level of fibrosis index α -SMA (3C);

fig. 4: bile Duct Ligation (BDL) mice AAV-NEU1 dry prognosis H & E, sirius scarlet staining results (4A), protein expression levels of fibrosis indicators (FN, COL1, COL3, VIMENTIN, α -SMA) (4B), gene expression levels of fibrosis indicators (Acta 2, col1, ctgf) (4C).

Detailed Description

The following describes the essential aspects of the present invention in detail with reference to examples, but is not intended to limit the scope of the present invention.

1. Experimental materials

1. Experimental animal

The experimental C57BL/6J mice are purchased from Jiangsu Jiyaokang biotechnology Co., ltd, SPF grade male mice of 6-8 weeks old are selected, and the weight is about 20-22g. The feed is randomly distributed according to 6-8 animals per cage, and the feed can be used for experiments after one week of adaptive feed.

2. Apparatus and device

Tail vein visual tail injection fixator (Beijing middle department life); FRESCO 17 high speed cryocentrifuge (ThermoFisher Scientific Co., U.S.A.); biosafety cabinet (us ThermoFisher Scientific company); CO ₂ Cell incubator (us Thermo Fisher Scientific company); synergy 2 multifunctional enzyme labeling apparatus (BioTek company, usa); thermomixer comfort metal bath heater (eppendorf, germany); orbital Shaker TS-100 decolorizing shaker (Kylin-Bell Lab Instraments, haimen Co., ltd.);XI Cell type vertical electrophoresis tank (BIO-RAD Co., U.S.); mini PROTEANII (BIO-RAD Co., USA); powerPac Basic electrophoresis apparatus (BIO-RAD Co., U.S.); a Tanon-520 day gel imaging system (Shanghai Techno Co., ltd.) tissue grinder (Shanghai Net letter Co.); the LightCycler96 fully automatic fluorescent quantitative PCR System (Roche diagnostics products Co., ltd.); LSM700 laser confocal microscope (Carl Zeiss company, germany); -80 ℃ ultra low temperature refrigerator (company ThermoFisher Scientific usa).

3. Materials and reagents

DMEM with/without double antibody medium was purchased from keji organisms (south tokyo, china, jiangsu province); south america brazil source bovine serum was purchased from Gibco company of the united states; type IV collagenase is available from Gibco company, usa;2000Reagent purchased from Invitrogen corporation, USA; opti-MEM medium was purchased from Gibco, inc., USA; the anti-fluorescence quenching sealing liquid is purchased from Biyundian; nycodenz cell sorting powder was purchased from America Accurate Chemical; bovine Serum Albumin (BSA) was purchased from Sigma, usa; triton X-100 was purchased from Sigma Co., USA; FITC-labeled goat anti-mouse IgG was purchased from Kaiyi organisms; the RIPA strong lysate is purchased from Biyun Tian; 30% (w/v) acrylamide/methylene bisacrylamide solution was purchased from a manufacturer; 0.5M pH=6.8 Tris-HCl buffer solution and 1.5M pH=8.8 Tris-HCl buffer solution purchased from an emerging organism; cocktail protease inhibitors and phosphatase inhibitors were purchased from Roche, usa; protein loading markers were purchased from ThermoFisher Scientific, usa; antibody PAI-1, VIMENTIN, available from proteontech; antibodies COL1, COL3, FN, α -SMA were purchased from us Cell Signaling Technology company; hiScript Q RT SuperMix for Qpcr, qRT-PCR SYBR Green Kit are available from Nanjinouzan Biotechnology Inc. Adeno-associated virus 8 (Adenoassociated virus, AAV 8) wraps control plasmid (NC plasmid) and NEU1 over-expression plasmid carries GFAP promoter to be designed and synthesized by Henry Biotechnology Co., ltd and wraps into virus liquid with virus titer of 10≡13; the mouse NEU1 plasmid was designed and synthesized by Hanheng biosciences, inc., and the mouse NEU1: NM-010893.

2. Experimental method

1. Establishment and grouping of mouse biliary tract ligation models

The bile duct ligation model of the C57BL/6 mouse is established by adopting a bile duct ligation method for 2 weeks. Divided into Sham surgery (Sham), bile duct ligation model (BDL), sham surgery + astrocyte-specific over-expressed NEU1 (AAV-neu1 + Sham) and bile duct ligation model + astrocyte-specific over-expressed NEU1 (AAV-neu1 + BDL) 4.

The tail of the mouse is intravenous injected with virus liquid, and a bile duct ligation model is established after 3 weeks: the mice were anesthetized with pentobarbital sodium, fixed on an operating table, the abdominal epidermis was cut off, the liver was gently peeled off, and after the position of the common bile duct was observed, the mice were ligated with a 5-0 suture needle, and after 2 weeks, the mice were examined.

2. Extraction and grouping of mouse primary astrocytes (HSCs)

Since the primary astrocytes of mice spontaneously activate in vitro, complete activation can be considered without the addition of additional modeling agents, typically by culturing until day 7 (D7). The groups were divided into group D1, group D7, and group D7+NEU1 plasmid 3.

Extraction of HSCs: fixing the mice on a plate after anesthesia, opening the abdomen to expose hepatic portal vein, digesting the liver with IV type collagenase after intubation to obtain digested cells, preparing density gradient sorting solution by Nyodenz, suspending the cells, lightly covering a layer of PBS, centrifuging to see that HSCs are positioned in the middle layer of the sorting solution and PBS, forming a white ring, carefully sucking, centrifuging to perform plating, and placing at 37 ℃ and 5% CO ₂ Incubation in cell incubator, daily changing.

Cell transfection (in the example of 6 well plate required): (1) preparing a transfection solution, namely: 200. Mu.L/well of Opti-MEM medium+Neu1 plasmid 2. Mu.g/well; the control group was 200. Mu.L/well of Opti-MEM medium+2. Mu.g/well of NC plasmid; and (2) liquid B: 150. Mu.L/well of Opti-MEM Medium+20005 μl/well; mixing the solution A and the solution B, gently reversing and uniformly mixing, and standing at room temperature for 20min for use; (2) discarding the cell culture medium, washing with PBS buffer solution, and adding 800 mu L of serum-free double-antibody-free DMEM medium into each well; 200 mu L of transfection solution is added to each hole, and the 6-hole plate is gently shaken and then placed in an incubator for incubation; (3) after 4h of transfection, the solution is changed, after the solution is washed by PBS buffer solution, 2mL of DMEM high-sugar double-antibody-containing medium with 10% FBS is added into each hole, and the subsequent experiment can be carried out after the culture is performed overnight.

3. NEU1 and fibrosis related index gene expression level detection (real-time fluorescent quantitative PCR)

(1) Extraction of RNA

Taking out the cells, discarding the culture solution, washing with PBS once, adding a proper amount of Total RNAExtraction Reagent, covering and repeatedly blowing the lysed cells, and standing at room temperature for 5min to completely dissociate the nucleoprotein. Chloroform was added to the lysate, and after vigorous shaking, the mixture was allowed to stand at room temperature for 2min. Centrifuge at 12000rpm for 15min at 4 ℃. The supernatant was gently aspirated and placed in a fresh centrifuge tube, an equal volume of isopropanol was added, and after inversion mixing, the tube was left at room temperature for 10min. Centrifuging at 4deg.C and 12000rpm for 10min, removing supernatant, adding 75% ethanol, washing thoroughly, centrifuging at 4deg.C and 12000rpm for 3min, removing supernatant, drying at room temperature, and dissolving with appropriate amount of DEPC water.

(2) cDNA Synthesis

Experiments were performed using a 5 XSuperMix reverse transcription system, in which the amount of RNA required, DEPC water (16. Mu.L-RNA amount) and 5 XqRT SuperMix (4. Mu.L) were added sequentially, and after vortexing and mixing, the mixture was put into an RNA reverse transcription apparatus for reverse transcription, and the reverse transcription procedure was as follows: 15min at 50 ℃, 2min at 85 ℃ and 2h at 4 ℃. The reverse transcription is completed to obtain 5 XcDNA, and the cDNA is stored in refrigerator at 4 deg.C for short term and-80 deg.C for long term.

(3) Real-time fluorescence quantitative PCR (RT-Q-PCR)

The system (10. Mu.L) required for RT-Q-PCR was: primer: cDNA: luciferase = 1:4: and 5, respectively adding the materials into holes corresponding to the PCR white board, pasting a film after finishing, scraping, and immediately separating for 1min, and then putting the materials into a Q-PCR instrument for setting a program to perform experiments. The Q-PCR procedure was as follows:

(4) Primer sequences

4. Protein expression level detection of NEU1 and fibrosis related indexes

4.1 extraction and detection of tissue proteins

(1) Protein extraction: an appropriate amount of liver tissue (about 20-25 mg) was cut into a 2mL centrifuge tube. 3 steel balls and 1mLRIPA strong lysate were added to each tube, and the mixture was lysed in a tissue mill at 75Hz for 45s. The tissue homogenate was transferred to a 1.5mL centrifuge tube and lysed on ice for 30min. Centrifugation was performed at 13000rpm for 15min at 4℃to allow tissue fragments to be thoroughly centrifuged to the bottom of the tube. Measuring protein concentration, taking 200 mu L of protein supernatant for experiment, and storing 200 mu L of protein supernatant in a refrigerator at-80 ℃ for standby;

(2) Protein concentration was determined according to BCA method and protein loading was calculated:

(1) preparing a standard protein solution: 1.2mL of protein standard preparation solution is added into the protein standard substance, and 25mg/mL of standard protein mother solution is prepared after vortex mixing, and the protein standard substance is stored in a refrigerator at the temperature of minus 20 ℃ for standby. Taking out the standard protein mother solution before use, and diluting the standard protein mother solution into 0.5mg/mL of standard protein working solution by using PBS buffer solution;

(2) preparing a working solution: the volume ratio of A solution to B solution in the BCA kit is 50:1 preparing working fluid according to the required quantity;

(3) preparing a standard curve: 0,1,2,4,8, 12, 16 and 20. Mu.L of the prepared standard protein working solution were added to 96-well plates, respectively, and made up to 20. Mu.L with PBS buffer. Repeatedly preparing two standard curves;

(4) 1. Mu.L of protein sample solution and 19. Mu.L of LPBS buffer solution were added to each well, and two replicates were set for each protein sample;

(5) respectively adding 200 mu LBCA working solution into the standard solution hole and the protein sample hole, and incubating for 30min in an incubator at 37 ℃;

(6) after the incubation, the absorbance was measured at 562nm in a microplate reader. Calculating the sample concentration of each protein according to a protein standard curve, and calculating the loading volume according to the loading amount of 75 mug;

(7) to the protein solution, 6 Xloadingbuffer (e.g., 200. Mu.L protein solution, 40. Mu.L 6 Xloadingbuffer) was added. Vortex centrifugation and heating in a metal bath at 99deg.C for 10min to denature proteins. Cooling to room temperature, and storing at-20deg.C.

(3) Western blotting experiment

(1) And fixing the clean thick glass plate and the clean thin glass plate on a glue dispensing bracket, preparing separating glue with different concentrations according to the required target protein molecular weight, uniformly adding the separating glue between the two plates by using a liquid-transferring gun, and rapidly adding ddH2O sealing liquid to flatten. After the separating gel is solidified, ddH of the upper layer is added ₂ The O was poured off and the excess water was blotted with a piece of paper. Adding the prepared concentrated gel into the upper layer of the separating gel, and rapidly inserting into sample application hole comb. After the concentrated glue is solidified, taking out and storing in a refrigerator at 4 ℃ for standby;

(2) the prepared gel was removed and carefully the sample application well comb was pulled out, placed in the electrophoresis tank and 1 Xrunning buffer was added. The samples were thawed and centrifuged by vortexing for use. Sequentially adding samples into the comb holes according to the sample loading volume, and respectively adding 2 mu L of protein markers at the left side and the right side of each plate;

(3) after the power was turned on, the voltage was set at 80V and after about 30 minutes the protein sample was flattened into a straight line. After the voltage is switched to 120V, continuing electrophoresis until the protein sample runs to the bottommost part of the gel plate, and stopping electrophoresis;

(4) 1 Xwet transfer solution is prepared in advance, and the preparation proportion is ddH2O: methanol: 10 x wet transfer = 7:2:1. after electrophoresis, the power supply is turned off, a proper amount of 1X wet transfer liquid is added in the square groove in advance, filter paper and sponge in the transfer film clamp are soaked, the white clamp is paved, and the side with the white clamp faces upwards. The thin plate is pried and separated, the thick plate and the glue are immersed in 1X wet transfer liquid together, the glue is shoveled down carefully, the concentrated glue part is cut off and then is paved on a transfer film clamp on the front surface, the glue is paved by a special shovel to remove bubbles, a Nitrocellulose (NC) film and filter paper are paved in sequence, and the bubbles are removed by a roller each time. After the membrane is clamped, the membrane is placed in a membrane transferring groove, 1X wet transfer liquid is poured into the groove, an ice bag is placed in the groove, and a groove cover is covered. The film transferring groove is moved into a basin filled with cold water, and an ice bag is added into the basin for refrigeration. Switching on a power supply, regulating the voltage to 300V at maximum, setting constant current 350mA for 90min, and finishing film transfer;

(5) the 5% defatted high protein milk was prepared with 1 XTBST solution and prepared on-line. Taking out NC membrane after membrane transfer, placing in 5% skimmed milk powder, and sealing on a shaker for 2h;

(6) a5% Bovine Serum Albumin (BSA) solution was prepared from a 1 XTBST solution and used as a primary antibody dilution, and the primary antibody was diluted according to different antibody dilution ratios for use. The corresponding bands were cut according to the molecular weight of the desired protein, 2mL of diluted primary antibody was added to each band, and each band was pressed with a laminator. Placing the pressed strips into a rotary drum and rotating in a refrigerator at 4 ℃ for incubation overnight;

(7) the primary antibody was recovered (typically 3-5 times for reuse), the strip was placed in a 1 XTBST solution and washed on a shaker for 10min, and repeated 3 times. 5% of defatted high protein milk dilution secondary antibody. After the film pressing is completed, the strip is placed at room temperature for incubation for 2 hours;

(8) the strips were taken out and placed in a 1 XTBST solution and washed on a shaker for 10min, and the washing was repeated 3 times for development. Liquid A and liquid B in the Tanon developing solution are prepared according to the following ratio of 1:1, strips were removed from the 1 XTBST solution and placed in a developing apparatus, 500. Mu.L of developing solution was added uniformly to each strip, the photographs were taken by exposure, and the photographs were saved and analyzed by Image J analysis software.

4.2 cell protein extraction and detection

(1) Cell total protein extraction: the medium was discarded. The cells were washed by adding 1mL of buffer solution to the wall, sucking the washing solution by a pipette, adding 100. Mu.L of 1 Xloadingbuffer solution to each well, scraping the cells thoroughly with a cell scraper, and transferring the cells to a 1.5mL centrifuge tube.

(2) Western blotting experiments were performed at 5.1.

5. Immunofluorescence alpha-SMA expression level detection

(1) Spreading primary hepatic stellate cells or LX2 cells of a mouse on a 35mm confocal small dish, controlling the cell density of the spread plate to be about 50%, culturing overnight, carrying out transfection treatment after cell adhesion is unfolded, and molding;

(2) discarding the culture medium, washing with PBS once, adding 500 μl of 4% paraformaldehyde solution, and fixing at room temperature for 25min;

(3) removing paraformaldehyde, adding PBS buffer solution, placing on a shaking table, cleaning for 5min, and repeating the cleaning for 3 times;

(4) 300mg BSA was weighed into 10mL PBS buffer and 20L Triton X-100 was added to prepare a Triton-containing BSA solution, 1mL BSA solution was added to each dish, and the mixture was blocked at room temperature for 2 hours;

(5) removing BSA solution, adding PBS buffer solution, placing on a shaking table, cleaning for 5min, and repeating the cleaning for 3 times;

(6) diluting the alpha-SMA antibody with BSA solution containing Triton (dilution ratio is 1:200), adding 500L of diluted alpha-SMA antibody into each dish, and incubating overnight at 4 ℃;

(7) discarding the primary antibody, adding PBS buffer solution, placing on a shaking table, cleaning for 5min, and repeatedly cleaning for 3 times;

(8) 300mg BSA is weighed and prepared into 3% BSA solution in 10mLPBS, the BSA solution is used for diluting FITC-labeled goat anti-rabbit IgG (the dilution ratio is 1:200), 500L of diluted secondary antibody is added into each dish, and the mixture is placed in a 37 ℃ incubator for light-proof incubation for 1h;

(9) discarding the secondary antibody, adding PBS buffer solution, placing on a shaking table, cleaning for 5min, and repeatedly cleaning for 3 times;

adding 500LDAPI staining solution into each dish, and incubating for 10min at 37 ℃ in a incubator in the dark;

recovering DAPI staining solution, adding PBS buffer solution, placing on a shaking table, cleaning for 5min, and repeating the cleaning for 3 times;

the buffer solution was buffered with PBS at 1:9 proportion of diluting anti-fluorescence quenching sealing liquid, adding 500L of sealing liquid into each dish, observing under a laser confocal microscope and photographing.

6. Statistical analysis

Data are expressed as mean ± SEM. Two samples were analyzed for statistical differences by independent sample t-test, and multiple samples were analyzed for statistical differences using one-way anova.

The differences were statistically significant with p <0.05 and p <0.01.

3. Experimental results

In FIG. 1, 1A is a representation of the change in gene level of Neu1 during spontaneous activation of mouse liver primary astrocytes in vitro; 1B and 1C are respectively protein level change and statistical graphs of primary astrocytes NEU1 of mice in a western blotting experiment, and the results show that NEU1 expression is obviously reduced in the process of spontaneous simulated fibrosis in vitro; the results indicate that NEU1 is significantly reduced in both gene and protein levels when cells undergo a fibrosis-like change.

FIG. 2 is a graph showing the promotion of NEU1 gene expression in primary astrocytes in mice by NEU1 plasmid, demonstrating that NEU1 plasmid can significantly increase NEU1 expression at the gene level.

In fig. 3, 3A is the immunofluorescence result of the mouse primary astrocyte fibrosis index, from which it can be seen that the α -SMA expression is increased on day 7 of activation with a significant change in cell morphology, the cell volume is enlarged, the nuclei are enlarged, the cells appear pseudopodia, the fibrosis stretch, indicating that the cells are in a spontaneous activated state, all activation characteristics are improved under the interference of NEU1 plasmid, indicating that cell activation is inhibited, compared to the unactivated state; 3B and 3C are detection results of related fibrosis indexes on gene and protein levels, and all indexes are obviously reduced, so that the inhibiting effect of NEU1 on fibrosis is demonstrated; the results show that over-expression of NEU1 can reduce the expression of fibrosis indexes, which indicates that NEU1 has good positive intervention effect on fibrosis.

In fig. 4, 4A is a graph of H & E staining and sirius scarlet staining results of mice, and it can be seen from the staining results that compared with Sham group, liver cell morphology of BDL group mice is damaged, fibrosis positive area is significantly increased, obvious lesions appear around liver sinus, which indicates that liver function of BDL group mice is significantly damaged, and molding is successful; compared with BDL group, the liver cell morphology of group mice is recovered, the fibrosis positive area is reduced, and the tissue lesions taking liver sinus as the center are obviously reduced; 4B is a decrease in the fibrosis index (FN, COL1, COL3, VIMENTIN, α -SMA) at the protein level; 4C is the measurement result of fibrosis indexes (Acta 2, col1 and Ctgf) on the gene level, and the result shows that the expression of the fibrosis indexes on the protein and gene levels is obviously reduced, which indicates that liver fibrosis of mice in AAV-Neu1+BDL groups is obviously improved, and liver fibrosis lesions of BDL mice can be improved by increasing Neu1 expression.

The above experiments demonstrate that targeting overexpressed hepatic stellate cells NEU1 can be effective against liver fibrosis. Therefore, the targeted overexpression agent of hepatic stellate cell NEU1 has a prospect of developing drugs for treating hepatic fibrosis.

The above-described embodiments serve to describe the substance of the present invention in detail, but those skilled in the art should understand that the scope of the present invention should not be limited to this specific embodiment.

Claims (4)

1. Use of a targeted overexpression agent of hepatic stellate cells NEU1 in the preparation of a medicament for treating liver fibrosis.

2. The use according to claim 1, characterized in that: the medicine takes a targeted overexpression agent of hepatic stellate cells NEU1 as an active ingredient, and is prepared into a pharmaceutically acceptable dosage form by using a pharmaceutically acceptable carrier.

3. The use according to claim 2, characterized in that: the carrier is in a solid, liquid or semi-solid form.

4. The use according to claim 2, characterized in that: the dosage forms include injections, tablets, capsules and drops.