Application of a targeted overexpression agent of NEU1 in hepatic stellate cells in the preparation of a drug for treating liver fibrosis

Abstract

The present invention discloses the use of a targeted overexpression agent for NEU1 in hepatic stellate cells in the preparation of a medicament for treating liver fibrosis. The present invention seeks protection for the use of a targeted overexpression agent for NEU1 in hepatic stellate cells in the preparation of a medicament for treating liver fibrosis. The present invention finds that targeted overexpression of NEU1 in hepatic stellate cells can effectively combat liver fibrosis. Therefore, this targeted overexpression agent for NEU1 in hepatic stellate cells has the potential to be developed into a medicament for treating liver fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the use of a targeted overexpression agent of hepatic stellate cells NEU1 in the preparation of a drug for treating liver fibrosis. Background Art

[0002] Hepatic fibrosis (HF) is the result of an excessive repair response caused by various chronic liver diseases, including genetic disorders, chronic viral hepatitis, alcoholic hepatitis, cholestasis, and drug-induced liver injury, resulting in abnormal deposition of various extracellular matrices (ECM). The development and progression of HF are related to hepatic stellate cells (HSCs), hepatic macrophages, endoplasmic reticulum stress (ERs), and autophagy. Liver fibrosis is a dynamic process that, without intervention, can progress to cirrhosis and even hepatocellular carcinoma. Therefore, delaying or reversing the occurrence and progression of HF is of great significance. Summary of the Invention

[0003] The purpose of the present invention is to provide an application of a targeted overexpression agent of hepatic stellate cells NEU1 in the preparation of a drug for treating liver fibrosis.

[0004] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0005] Use of a targeted overexpression agent for hepatic stellate cells NEU1 in the preparation of a drug for treating liver fibrosis.

[0006] Preferably, the drug uses a targeted overexpression agent for hepatic stellate cells NEU1 as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using a pharmaceutically acceptable carrier.

[0007] More preferably, the carrier is in solid, liquid or semi-solid form.

[0008] More preferably, the dosage forms include injections, tablets, capsules and drops.

[0009] Beneficial effects:

[0010] The present invention found that targeted overexpression of NEU1 in hepatic stellate cells can effectively resist liver fibrosis. Therefore, targeted overexpression agents of NEU1 in hepatic stellate cells have the prospect of being developed into drugs for treating liver fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1: NEU1 gene expression levels (1A) and protein expression levels (1B, 1C) during spontaneous activation of mouse primary hepatic stellate cells in vitro;

[0012] Figure 2 : NEU1 gene expression level in mouse primary astrocytes after transfection of NEU1 plasmid;

[0013] Figure 3 : Immunofluorescence staining of α-SMA in primary mouse astrocytes after NEU1 plasmid intervention (3A), gene expression levels of fibrosis indicators (Acta2, Col1, Ctgf) (3B), and protein expression level of fibrosis indicator α-SMA (3C);

[0014] Figure 4 : H&E and picrosirius red staining results of bile duct ligation (BDL) mice after AAV-NEU1 intervention (4A), protein expression levels of fibrosis markers (FN, COL1, COL3, VIMENTIN, α-SMA) (4B), and gene expression levels of fibrosis markers (Acta2, Col1, Ctgf) (4C). DETAILED DESCRIPTION

[0015] The essential contents of the present invention are described in detail below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

[0016] 1. Experimental Materials

[0017] 1. Experimental Animals

[0018] C57BL / 6J mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. SPF-grade male mice, 6-8 weeks old and weighing approximately 20-22 g, were selected. They were randomly assigned to each cage and housed 6-8 mice. After one week of adaptive feeding, they were used for experiments.

[0019] 2. Instruments and equipment

[0020] Mouse tail vein visual tail injection holder (Beijing Zhongke Life Science); FRESCO 17 high-speed refrigerated centrifuge (ThermoFisher Scientific, USA); biological safety cabinet (ThermoFisher Scientific, USA); CO2 cell culture incubator (Thermo Fisher Scientific, USA); Synergy 2 multifunctional microplate reader (BioTek, USA); Thermomixer comfort metal bath heater (Eppendorf, Germany); Orbital Shaker TS-100 decolorization shaker (Kylin-Bell Lab Instruments Co., Ltd., Haimen City); XI Cell vertical electrophoresis tank (BIO-RAD, USA); Mini PROTEAN II (BIO-RAD, USA); PowerPac Basic electrophoresis instrument (BIO-RAD, USA); Tanon-520 Tianneng gel imaging system (Shanghai Tianneng Technology Co., Ltd.); tissue grinder (Shanghai Jingxin Company); LightCycler 96 fully automated fluorescence quantitative PCR system (Roche Diagnostics Products Co., Ltd.); LSM700 laser confocal microscope (Carl Zeiss, Germany); -80°C ultra-low temperature freezer (ThermoFisherScientific, USA).

[0021] 3. Materials and Reagents

[0022] DMEM with or without double-antibody culture medium was purchased from Keygen Biotechnology (Nanjing, Jiangsu, China); fetal bovine serum from Brazil was purchased from Gibco, USA; type IV collagenase was purchased from Gibco, USA; 2000 Reagent was purchased from Invitrogen, USA; Opti-MEM medium was purchased from Gibco, USA; anti-fluorescence quenching mounting fluid was purchased from Beyotime; Nycodenz cell sorting powder was purchased from Accurate Chemical, USA; bovine serum albumin (BSA) was purchased from Sigma, USA; Triton X-100 was purchased from Sigma, USA; FITC-labeled goat anti-mouse IgG was purchased from Keygen Biotechnology; RIPA strong lysis buffer was purchased from Beyotime; 30% (w / v) acrylamide / methylenebisacrylamide solution was purchased from Bioscientific; 0.5M pH = 6.8 Tris-HCl buffer solution and 1.5M pH = 8.8 Tris-HCl buffer solution were purchased from Shengxing Biotechnology; cocktail protease inhibitors and phosphatase inhibitors were purchased from Roche, USA; protein loading marker was purchased from ThermoFisher Scientific, USA; antibodies PAI-1 and VIMENTIN were purchased from Proteintech; antibodies COL1, COL3, FN, and α-SMA were purchased from Cell Signaling Technology, USA; HiScript Q RT SuperMix for qPCR and qRT-PCR SYBR Green Kit were purchased from Nanjing Novozymes Biotechnology Co., Ltd. Adenoassociated virus 8 (AAV8)-encapsulated control plasmid (NC plasmid) and a NEU1 overexpression plasmid carrying the GFAP promoter were designed and synthesized by Hanbio Biotechnology Co., Ltd. and packaged into viral fluid with a titer of 10^13. The mouse NEU1 plasmid was designed and synthesized by Hanbio Biotechnology Co., Ltd. (mouse NEU1: NM-010893).

[0023] 2. Experimental Methods

[0024] 1. Establishment and grouping of mouse bile duct ligation model

[0025] A bile duct ligation model was established in C57BL / 6 mice by bile duct ligation for 2 weeks. The mice were divided into four groups: sham operation group (Sham), bile duct ligation model group (BDL), sham operation + stellate cell-specific overexpression of NEU1 (AAV-Neu1+Sham), and bile duct ligation model + stellate cell-specific overexpression of NEU1 (AAV-Neu1+BDL).

[0026] The virus solution was injected into the tail vein of mice, and a bile duct ligation model was established 3 weeks later: the mice were anesthetized with sodium pentobarbital, fixed on the operating table, the abdominal epidermis was cut open, the liver was gently peeled open, and after observing the location of the common bile duct, it was ligated with a 5-0 suture needle. The test was performed 2 weeks later.

[0027] 2. Extraction and grouping of primary mouse stellate cells (HSCs)

[0028] Because primary mouse stellate cells can spontaneously activate in vitro, no additional modeling agent is required. Generally, cells are considered fully activated by day 7 (D7) of culture. They were divided into three groups: D1, D7, and D7 + NEU1 plasmid.

[0029] Extraction of HSCs: After anesthesia, mice were fixed on a plate, the laparotomy was performed to expose the portal vein, and the liver was digested with type IV collagenase after cannulation to obtain the digested cells. The cells were resuspended in Nycodenz density gradient sorting solution and gently covered with a layer of PBS. After centrifugation, HSCs were visible as white rings located in the middle layer between the sorting solution and PBS. Carefully aspirate the cells and centrifuge them before plating them on a plate. The cells were then placed in a 37°C, 5% CO2 cell culture incubator for incubation, and the medium was changed daily.

[0030] Cell transfection (taking the amount required for a 6-well plate as an example): ① Prepare transfection solution: Solution A: Opti-MEM medium 200 μL / well + Neu1 plasmid 2 μg / well; control group: Opti-MEM medium 200 μL / well + NC plasmid 2 μg / well; Solution B: Opti-MEM medium 150 μL / well + 20005μL / well; Mix solution A and solution B, gently invert to mix, and let it stand at room temperature for 20 minutes before use; ② Discard the cell culture medium, wash with PBS buffer solution, and add 800μL of serum-free double-antibody-free DMEM culture medium to each well; Add 200μL of transfection solution to each well, gently shake the 6-well plate and incubate in an incubator; ③ Change the medium 4 hours after transfection, wash with PBS buffer solution, and add 2mL10% FBS DMEM high-glucose culture medium containing double-antibody to each well. Incubate overnight and then carry out subsequent experiments.

[0031] 3. Detection of gene expression levels of NEU1 and fibrosis-related indicators (real-time fluorescence quantitative PCR)

[0032] (1) RNA extraction

[0033] Remove the cells, discard the culture medium, wash once with PBS, add an appropriate amount of Total RNA Extraction Reagent, cover and repeatedly pipette to lyse the cells, and let them stand at room temperature for 5 minutes to completely dissociate the ribosomes. Add chloroform to the lysate, shake vigorously, and let it stand at room temperature for 2 minutes. Centrifuge at 4°C, 12,000 rpm, for 15 minutes. Gently aspirate the supernatant and place it in a new centrifuge tube. Add an equal volume of isopropanol, invert to mix, and let it stand at room temperature for 10 minutes. Centrifuge at 4°C, 12,000 rpm, for 10 minutes, discard the supernatant, add 75% ethanol to wash thoroughly, centrifuge at 4°C, 12,000 rpm, for 3 minutes, discard the supernatant, dry at room temperature, and dissolve in an appropriate amount of DEPC water.

[0034] (2) cDNA synthesis

[0035] The experiment was performed using the 5× Super Mix reverse transcription system. The desired amount of RNA, DEPC water (16 μL per RNA volume), and 5× qRT Super Mix (4 μL) were added sequentially. After vortexing and centrifugation, the mixture was placed in an RNA reverse transcription instrument for reverse transcription. The reverse transcription program was as follows: 50°C for 15 minutes, 85°C for 2 minutes, and 4°C for 2 hours. The resulting 5× cDNA was stored at 4°C for short-term storage and at -80°C for long-term storage.

[0036] (3) Real-time quantitative PCR (RT-Q-PCR)

[0037] The RT-Q-PCR system (10 μL) consists of a mixture of primers: cDNA: luciferase in a ratio of 1:4:5. Add each mixture to the corresponding wells of the PCR plate. After completion, apply the membrane, scrape it flat, and spin for 1 minute before placing it in the Q-PCR instrument, setting the program, and performing the experiment. The Q-PCR procedure is as follows:

[0038]

[0039] (4) Primer sequence

[0040]

[0041] 4. Detection of protein expression levels of NEU1 and fibrosis-related indicators

[0042] 4.1 Tissue protein extraction and detection

[0043] (1) Protein extraction: Cut an appropriate amount of liver tissue (about 20-25 mg) into a 2 mL centrifuge tube. Add 3 steel beads and 1 mL of RIPA strong lysis buffer to each tube and lyse at 75 Hz for 45 seconds in a tissue grinder. Transfer the tissue homogenate to a 1.5 mL centrifuge tube and place it on ice for 30 minutes. Centrifuge at 13000 rpm at 4°C for 15 minutes to allow the tissue fragments to be fully centrifuged to the bottom of the tube. Determine the protein concentration, take 200 μL of protein supernatant for the experiment, and take another 200 μL of protein supernatant and store it in a -80°C refrigerator for later use;

[0044] (2) Determine protein concentration and calculate protein loading amount according to the BCA method:

[0045] ① Prepare the standard protein solution: Add 1.2 mL of protein standard solution to the protein standard, vortex mix, and prepare a 25 mg / mL standard protein stock solution. Store in a -20°C refrigerator for future use. Before use, remove the standard protein stock solution and dilute it with PBS buffer to a 0.5 mg / mL standard protein working solution.

[0046] ② Prepare the working solution: Mix Solution A and Solution B in the BCA kit at a volume ratio of 50:1 to prepare the working solution. Prepare the required amount on demand.

[0047] ③ Prepare a standard curve: Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the prepared standard protein working solution to a 96-well plate, and fill up to 20 μL with PBS buffer. Repeat to prepare two standard curves;

[0048] ④ Add 1 μL protein sample solution and 19 μL PBS buffer solution to each well, and set up two replicates for each protein sample;

[0049] ⑤ Add 200 μL of BCA working solution to the standard solution well and protein sample well respectively, and incubate in a 37°C incubator for 30 minutes;

[0050] ⑥ After incubation, place the sample in a microplate reader and measure the absorbance at 562 nm. Calculate the sample concentration of each protein based on the protein standard curve and calculate the sample volume based on a 75 μg sample load.

[0051] ⑦ Add 6× loading buffer to the protein solution (e.g., add 40 μL of 6× loading buffer to 200 μL of protein solution). Vortex and centrifuge, then heat in a 99°C metal bath for 10 minutes to denature the protein. Cool to room temperature and store at -20°C until ready for use.

[0052] (3) Western blotting

[0053] ① Fix a clean thick and thin glass plate on the gel preparation stand. Prepare separation gel of different concentrations according to the molecular weight of the target protein. Use a pipette to evenly add it between the two plates and quickly add ddH2O sealing fluid to level it. After the separation gel solidifies, pour off the ddH2O on the top layer and absorb the excess water with flat paper. Add the prepared stacking gel to the top layer of the separation gel and quickly insert the sample comb into the wells. After the stacking gel solidifies, remove it and store it in a refrigerator at 4°C for future use.

[0054] ② Remove the prepared gel and carefully remove the sample comb. Place the gel in the electrophoresis tank and add 1× electrophoresis buffer. Thaw the sample, vortex, and centrifuge before use. Add the sample to the comb in the order of loading volume and add 2μL of protein marker to each side of each plate.

[0055] ③ After turning on the power, set the voltage to 80V. After about 30 minutes, the protein sample will be flattened into a straight line. Switch the voltage to 120V and continue electrophoresis until the protein sample reaches the bottom of the gel plate;

[0056] ④ Prepare 1× transfer buffer in advance using a ratio of ddH₂O: methanol: 10× transfer buffer = 7:2:1. After electrophoresis is complete, turn off the power supply. Pre-fill a square trough with 1× transfer buffer. Soak the filter paper and sponge in the transfer cassette and lay them out, with the white side facing up. Pry the thin plates apart and immerse the thick plates, along with the gel, in 1× transfer buffer. Carefully remove the gel by spatula. Cut off the concentrated gel and lay the front of the gel onto the transfer cassette. Use a spatula to flatten the gel, removing any bubbles. Place the nitrocellulose (NC) membrane and filter paper, using a roller to remove any bubbles. Place the transfer cassette in the transfer cassette, add 1× transfer buffer, place an ice pack in the cassette, and secure the lid. Move the transfer cassette to a basin filled with cold water and add an ice pack to keep it cool. Turn on the power supply, adjust the voltage to a maximum of 300V, and set a constant current of 350mA for 90 minutes. The transfer is complete.

[0057] ⑤ Prepare 5% skim high-protein milk with 1×TBST solution, and prepare it immediately before use. After the transfer is completed, remove the NC membrane, place it in 5% skim milk powder, and block it on a shaker for 2 hours;

[0058] ⑥ Prepare a 5% bovine serum albumin (BSA) solution in 1× TBST solution as the primary antibody diluent. Dilute the primary antibody according to the desired antibody dilution ratio. Cut the corresponding band according to the molecular weight of the desired protein. Add 2 mL of the diluted primary antibody to each band and press each band with a laminator. Place the pressed bands in a rotating drum and incubate in a refrigerator at 4°C overnight.

[0059] ⑦ Recover the primary antibody (usable 3-5 times), place the strips in 1×TBST solution, wash on a shaker for 10 minutes, and repeat the wash three times. Dilute the secondary antibody in 5% skim high-protein milk. After lamination, incubate the strips at room temperature for 2 hours.

[0060] ⑧ Remove the strips and place them in 1× TBST solution. Wash on a shaker for 10 minutes. Repeat this wash three times before developing. Prepare the strips by mixing Tanon developer solution A and solution B in a 1:1 ratio. Remove the strips from the 1× TBST solution and place them in a developer. Evenly apply 500 μL of developer solution to each strip. Expose and photograph, save the image, and analyze using Image J software.

[0061] 4.2 Cell protein extraction and detection

[0062] (1) Total cell protein extraction: Discard the culture medium. Add 1 mL of PBS buffer solution to the adherent cells for washing. Aspirate the washing solution with a pipette. Add 100 μL of 1× loading buffer to each well. Scrape the cells thoroughly with a cell scraper and transfer to a 1.5 mL centrifuge tube.

[0063] (2) Protein immunoblotting experiments, see 5.1.

[0064] 5. Immunofluorescence detection of α-SMA expression level

[0065] ① Primary mouse hepatic stellate cells or LX2 cells were plated on a 35 mm confocal microplate. The cell density was controlled at about 50%. After culturing overnight, the cells were allowed to adhere to the wall and expanded, and then transfected and treated to establish the model.

[0066] ②Discard the culture medium, wash once with PBS, add 500 μL of 4% paraformaldehyde solution and fix at room temperature for 25 minutes;

[0067] ③ Discard the paraformaldehyde, add PBS buffer solution and wash on a shaker for 5 minutes, repeat the washing three times;

[0068] ④ Weigh 300 mg of BSA into 10 mL of PBS buffer and add 20 L of Triton X-100 to prepare a BSA solution containing Triton. Add 1 mL of BSA solution to each dish and block at room temperature for 2 hours.

[0069] ⑤ Discard the BSA solution, add PBS buffer solution and wash on a shaker for 5 minutes, repeat the washing three times;

[0070] ⑥ Dilute α-SMA antibody with BSA solution containing Triton (dilution ratio is 1:200), add 500 μL of diluted α-SMA antibody to each dish, and incubate in a 4°C refrigerator overnight;

[0071] ⑦ Discard the primary antibody, add PBS buffer solution and wash on a shaker for 5 minutes, repeat the washing three times;

[0072] ⑧ Weigh 300 mg of BSA and dissolve it in 10 mL of PBS to prepare a 3% BSA solution. Use it to dilute FITC-labeled goat anti-rabbit IgG (dilution ratio is 1:200). Add 500 μL of the diluted secondary antibody to each dish and incubate in a 37°C incubator in the dark for 1 hour.

[0073] ⑨ Discard the secondary antibody, add PBS buffer solution and wash on a shaker for 5 minutes, repeat the washing three times;

[0074] ⑩ Add 500 μL of LDAPI staining solution to each dish and incubate in a dark incubator at 37°C for 10 minutes;

[0075] Recover the DAPI staining solution, add PBS buffer solution and wash on a shaker for 5 minutes, repeat the washing three times;

[0076] Dilute the anti-fluorescence quenching sealing solution with PBS buffer solution at a ratio of 1:9, add 500L to each dish, and observe and photograph under a laser confocal microscope.

[0077] 6. Statistical analysis

[0078] Data are expressed as mean ± SEM. Statistical differences between two samples were analyzed using independent sample t-test, and statistical differences between multiple samples were analyzed using one-way ANOVA.

[0079] The differences were statistically significant *p<0.05, **p<0.01.

[0080] 3. Experimental Results

[0081] Figure 1 In the figure, 1A is a representation of the changes in Neu1 at the gene level during spontaneous activation of primary mouse liver stellate cells in vitro; 1B and 1C are the changes and statistical graphs of NEU1 protein levels in primary mouse stellate cells in protein immunoblotting experiments, respectively. The results show that NEU1 expression is significantly reduced during spontaneous simulation of fibrosis in vitro; the results indicate that when cells undergo fibrous changes, NEU1 is significantly reduced at both the gene and protein levels.

[0082] Figure 2 The results show that NEU1 plasmid promotes the expression of NEU1 gene in primary mouse stellate cells, indicating that NEU1 plasmid can significantly increase the expression of NEU1 at the gene level.

[0083] Figure 3In the figure, 3A is the immunofluorescence result of fibrosis indicators of primary mouse astrocytes. The results show that compared with the unactivated state, the expression of α-SMA increased on the 7th day of activation, accompanied by obvious changes in cell morphology, cell volume increased, cell nucleus enlarged, pseudopodia appeared in the cells, and they stretched like fibers, indicating that the cells were in a spontaneously activated state. Under the interference of NEU1 plasmid, all activation characteristics were improved, indicating that cell activation was inhibited; 3B and 3C are the detection results of related fibrosis indicators at the gene and protein levels. All indicators were significantly reduced, indicating the inhibitory effect of NEU1 on fibrosis; the results show that overexpression of NEU1 can reduce the expression of fibrosis indicators, indicating that NEU1 has a good positive intervention effect on fibrosis.

[0084] Figure 4 Figure 4A shows the H&E staining and picrosirius red staining results of mice. From the staining results, it can be seen that compared with the Sham group, the liver cell morphology of mice in the BDL group was damaged, the fibrosis-positive area was significantly increased, and obvious lesions appeared around the hepatic sinusoids, indicating that the liver function of mice in the BDL group was significantly damaged and the model was successful; compared with the BDL group, the liver cell morphology of mice in the group was restored, the fibrosis-positive area was reduced, and the tissue lesions centered on the hepatic sinusoids were significantly reduced; 4B shows the decrease in fibrosis indicators (FN, COL1, COL3, VIMENTIN, α-SMA) at the protein level; 4C shows the measurement results of fibrosis indicators (Acta2, Col1, Ctgf) at the gene level. The results showed that the expression of fibrosis indicators at both the protein and gene levels was significantly reduced, indicating that liver fibrosis in mice in the AAV-Neu1+BDL group was significantly improved, and increasing Neu1 expression can improve liver fibrosis lesions in BDL mice.

[0085] These experiments demonstrate that targeted overexpression of NEU1 in hepatic stellate cells can effectively combat liver fibrosis. Therefore, agents that target overexpression of NEU1 in hepatic stellate cells have the potential to be developed into drugs for the treatment of liver fibrosis.

[0086] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. Use of a targeted overexpression agent for hepatic stellate cells NEU1 in the preparation of a drug for treating liver fibrosis, wherein the targeted overexpression agent is a NEU1 overexpression plasmid that promotes NEU1 overexpression.

2. The use according to claim 1, characterized in that: The drug uses a targeted over-expression agent for hepatic stellate cells NEU1 as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using a pharmaceutically acceptable carrier.

3. The use according to claim 2, characterized in that: The carrier is in 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.

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