Application of siRNA composition in preparation of medicine for treating liver diseases and medicine

Abstract

The invention discloses application of a siRNA composition in preparation of a medicine for treating liver diseases and the medicine. The siRNA composition at least comprises PCBP2-siRNA and PD-L1-siRNA. The invention discovers that PCBP2-siRNA and PD-L1-siRNA can effectively reduce type I collagen deposition in a mouse hepatic fibrosis model induced by CCL4 for the first time. The result shows that the two siRNAs both have a potential anti-fibrosis effect. And when the PCBP2-siRNA and the PD-L1-siRNA are used in a combined manner, the inhibition effect of the PCBP2-siRNA and the PD-L1-siRNA on the type I collagen deposition is more obvious than that of the PCBP2-siRNA which is independently used with any siRNA. The result shows that the liver fibrosis degree can be more effectively relieved through combined application of the two siRNAs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an application of a siRNA composition in the preparation of a drug for treating liver disease and the drug. Background Art

[0002] Liver cirrhosis is one of the most common causes of death worldwide. The large number of cases and deaths indicates that liver disease has become a major cause of symptoms and poor quality of life and cannot be ignored. Previous studies have shown that the development of these liver diseases is associated with fibrosis. Therefore, it is necessary to have a more comprehensive understanding of the pathological characteristics of liver fibrosis and potential treatments.

[0003] The complex pathological characteristics of liver fibrosis and the serious consequences of its continued progression mean that it is particularly important to provide effective treatments. However, most of the drugs currently used in clinical practice for specific pathological characteristics are based on their efficacy to treat diseases with similar pathological characteristics. For example, ursodeoxycholic acid, which is approved for the treatment of primary biliary cirrhosis and primary sclerosing cholangitis, is a widely used lipid-lowering drug that helps maintain bile acid homeostasis and energy metabolism. In addition, pentoxifylline, which is mainly used to treat cerebrovascular diseases, and pirfenidone, which is used to treat idiopathic pulmonary fibrosis, are also used in the treatment of liver fibrosis due to their anti-inflammatory effects. At the same time, some specific anti-fibrotic drugs are under development, and there have been many potential clinical trials for the treatment of metabolic dysfunction-associated steatohepatitis (MASH), where fat accumulation in the liver leads to progressive inflammation and fibrosis. For example, the fibroblast growth factor analog, fibroblast growth factor 21, was found to improve liver fibrosis in stages F2 or F3, prevent MASH progression for up to 24 weeks, and had acceptable good tolerability in a phase 2b clinical trial. Phase 3 clinical trials are currently being designed to further evaluate its efficacy and safety. Lanifibranor is an oral pan-peroxisome proliferator-activated receptor agonist. In a Phase 2b trial, 48% and 34% of patients treated with 1200mg and 800mg, respectively, achieved at least one point improvement in fibrosis stage without deterioration, compared with 29% in the placebo group. However, the use of lanifibranor is associated with a higher incidence of gastrointestinal events.

[0004] Therefore, developing a combination of drugs that target multiple aspects of pathogenesis may be a useful approach but will involve identifying drugs with synergistic effects. The development of safer and more effective substances, the identification of combinations with multiple targets, and the precise targeting of specific genes may represent future research directions for the treatment of liver fibrosis. Summary of the invention

[0005] The purpose of the present invention is to provide an application of a siRNA composition in the preparation of a drug for treating liver disease, as there is no combination of identification and multiple targets, as well as a drug that accurately targets a specific gene, for the treatment of liver fibrosis in the prior art.

[0006] The second object of the present invention is to provide a drug based on the siRNA composition.

[0007] The third object of the present invention is to provide a method for preparing the drug.

[0008] The technical solution adopted to achieve the purpose of the present invention is:

[0009] A use of an siRNA composition in preparing a drug for treating liver disease, wherein the siRNA composition comprises at least PCBP2-siRNA and PD-L1-siRNA.

[0010] In the above technical solution, the nucleotide sequence of the sense strand of the PCBP2-siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand of the PCBP2-siRNA is shown in SEQ ID NO.2.

[0011] In the above technical solution, the nucleotide sequence of the sense strand of the PD-L1-siRNA is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand of the PD-L1-siRNA is shown in SEQ ID NO.4.

[0012] In the above technical solution, the liver disease is liver fibrosis or liver cirrhosis.

[0013] The second aspect of the present invention provides a drug for treating liver disease, which at least includes a targeting vector, a targeting ligand loaded on the targeting vector and the siRNA composition.

[0014] In the above technical solution, the molar ratio of PCBP2-siRNA to PD-L1-siRNA in the drug is 1:(0.1-10).

[0015] In the above technical solution, the targeting ligand is a targeting ligand of insulin-like growth factor II receptor.

[0016] In the above technical solution, the targeting carrier is a small molecule polypeptide organic substance.

[0017] In the above technical solution, the dosage of the drug is 0.5-5 mg / kg based on the total amount of siRNA.

[0018] The third aspect of the present invention provides a method for preparing the drug for treating liver disease, comprising the following steps:

[0019] respectively preparing solutions of targeting vector, targeting ligand, PCBP2-siRNA and PD-L1-siRNA;

[0020] The targeting carrier solution, the targeting ligand solution, the PCBP2-siRNA solution and the PD-L1-siRNA solution are uniformly mixed and co-incubated to obtain the drug.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention first discovered that both PCBP2-siRNA and PD-L1-siRNA can effectively reduce type I collagen deposition in a CCL4-induced mouse liver fibrosis model. This indicates that both siRNAs have potential anti-fibrotic effects. And when PCBP2-siRNA and PD-L1-siRNA are used in combination, their inhibitory effect on type I collagen deposition is more significant than when either siRNA is used alone. This indicates that the combined use of these two siRNAs can more effectively reduce the degree of liver fibrosis.

[0023] 2. The present invention proposes and constructs the Apt-PM@siRNA nano-delivery system, and applies it to the treatment of liver fibrosis for the first time. The system has faster drug delivery efficiency and accurate drug delivery targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Shown are schematic diagrams and actual pictures of the drug of the present invention, wherein A is a schematic diagram of the Apt-PM@siRNA nanocomplex; B and C are transmission electron microscopy pictures of the siRNA nanocomplex.

[0025] Figure 2 Shown are the expressions of PCBP2 and PD-L1 during fibrosis, where A is PCBP2, in the figure, blue: DAPI, green: α-SMA, red: PCBP2; B is PD-L1, in the figure, blue: DAPI, green: α-SMA, red: PD-L1.

[0026] Figure 3 Shown are the cell uptake diagrams of siRNA nanocomplexes at different time periods, where A is 2 h; B is 6 h; and C is 24 h.

[0027] Figure 4Shown are diagrams showing the silencing effects of different siRNAs in cells, wherein A is the silencing effect of PCBP2-siRNA in mouse embryonic fibroblasts (3T3); B is the silencing effect of PCBP2-siRNA in human hepatic stellate cells (LX-2); C is the silencing effect of PD-L1-siRNA in mouse embryonic fibroblasts (3T3); and D is the silencing effect of PD-L1-siRNA in human hepatic stellate cells (LX-2).

[0028] Figure 5 Shown is the detection of collagen fibers by Sirius red staining in liver specimens of mice in different groups.

[0029] Figure 6 Shown is the immunohistochemical study of collagen I in liver specimens from different groups of mice. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] In this embodiment, the siRNA sequences were purchased from Suzhou Genetron Gene Co., Ltd.

[0032] Mouse PCBP2-siRNA

[0033] Sense chain: 5-GUCAGUGUGGCUCUCUUAU-3

[0034] Antisense strand: 3-CAGUCACACCGAGAGAAUA-5

[0035] Human PCBP2-siRNA

[0036] SEQ ID NO.1: positive strand: 5'-GUCAGUGUGGCUCUCUCAU-3'

[0037] SEQ ID NO.2: Antisense strand: 3'-CAGUCACACCGAGAGAGUA-5'

[0038] Mouse PD-L1-siRNA

[0039] Sense strand: 5'-GAAUUCUGACGACAUGAUA-3'

[0040] Antisense strand: 3'-CUUAAGACUGCUGUACUAU-5'

[0041] Human PD-L1-siRNA

[0042] SEQ ID NO.3: Sense strand: 5'-GAUUUCUUUUGAAGAUAUA-3'

[0043] SEQ ID NO.4: Antisense strand: 3'-CUAAAGAAAACUUCUAUAU-5'

[0044] In this specific embodiment, the targeting carrier adopts a small molecule polypeptide substance, hereinafter referred to as PM, the molecular formula of which is a hydrophobic functional group -CGGFLG-HHHKKHHHKKK (issued patent number: 202410255441.X). The polypeptide substance has a relatively common delivery effect in fibrosis-related cells, and can be released at a fixed point in the liver fibrosis microenvironment. It can be recognized and hydrolyzed by the hydrolase in the body through the enzyme cleavage sequence of CGGFLG in the molecular structure, thereby releasing the substance combined with it and achieving a certain therapeutic effect. Then, the above-mentioned PM small molecule peptide that can be widely released in fibrosis-related cells is connected to PCBP2-siRNA and PD-L1-siRNA, thereby achieving a certain therapeutic effect.

[0045] IFG2R targeting ligand (IFG2R Aptamer) was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0046] Example 1 Preparation and characterization of drugs

[0047] In this example, Apt-PM@siRNA nanocomplexes were successfully prepared to load siRNA, which improved the loading efficiency of siRNA in the nanocomplexes and made them feasible in in vivo animal studies. IFG2R targeting ligands were used to enhance the delivery of nanocomplexes to activated hepatic stellate cells in fibrotic livers.

[0048] Reaction process of Apt-PM@siRNA nano-delivery system:

[0049] (1) Weigh 4 mg of the synthesized PM pure product powder, add 1 mL of ultrapure water to dissolve it, and prepare a PM mother solution with a concentration of 4 mg / mL. Ultrasonicate for 20 s to assist dissolution to form a stable dispersed phase.

[0050] (2) Take 1OD of siRNA (purchased from Suzhou Gene Gene Co., Ltd.) (1OD of both PCBP2-siRNA and PD-L1-siRNA) and add 125 μl of DEPC water to make a 20 μM stock solution.

[0051] (3) IFG2R Aptamer (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was prepared into a 20 μM stock solution.

[0052] (4) A certain volume of PM, siRNA, and APT was pipetted and mixed by blowing and incubating for 30 minutes to form an Apt-PM@siRNA nano-delivery system with a siRNA concentration of 100 nM (preparation principle as shown in Figure 1 As shown in Figure 1, the formation of Apt-PM@siRNA nanocomplexes mainly depends on the electrostatic interaction between negatively charged siRNA and positively charged PM peptide. Therefore, a large amount of siRNA in the complex can enhance condensation and thus reduce the particle size. Figure 1 As shown in B and 1C, the particle size of the nanocomplex was determined by transmission electron microscopy.

[0053] Example 2 Target Verification

[0054] CCl4-induced liver fibrosis in mice (C57)

[0055] CCL4: olive oil was mixed evenly in a ratio of 1:4 and injected intraperitoneally into mice. The dose (μL) given to each mouse was twice the body weight (g). (The mice were intraperitoneally injected with a mixture of carbon tetrachloride and olive oil (1:4, v / v) at a dose of 2μL per gram of body weight.) Induction was performed three times a week, and 30 mice were randomly divided into 5 groups, induced 3 times, 6 times, 9 times, and 12 times, respectively. Mice with different induction times were euthanized after 1 week, 2 weeks, 3 weeks, and 4 weeks of induction, and the liver was removed and cut into two halves, one half was frozen at -80°C, and the other half was stored in formalin for histological analysis.

[0056] Immunofluorescence co-localization of PCBP2, PD-L1 and α-SMA

[0057] The livers of the above five groups frozen at -80°C were subjected to immunofluorescence co-localization of PCBP2, PD-L1, and α-SMA. The expression levels of PCBP2 and PD-L1 were observed to change with the progress of liver fibrosis. The livers were frozen and sliced, washed with PBS three times for 5 minutes each time; fixed with 4% paraformaldehyde for 15 minutes, and the slides were washed with PBS three times for 5 minutes each time; 0.5% Triton X-100 (prepared in PBS) for permeabilization at room temperature for 15 minutes; wash the slides with PBS 3 times, 5 minutes each time, dry the PBS with absorbent paper, add blocking solution (containing 10% goat serum, prepared in PBS) on the slides to cover the tissue, and block at room temperature for 1 hour; absorb the blocking solution with absorbent paper, do not wash, add a sufficient amount of diluted primary antibody (1; 500, prepared with blocking solution) to each slide and put it into a wet box, incubate at 4°C overnight or at room temperature for two hours, wash with PBS 3 times, 5 minutes each time, dry the excess liquid on the slides with absorbent paper, and then add diluted fluorescent secondary antibody (1; 500, prepared with blocking solution), incubate at room temperature for 1 hour, wash with PBS 3 times, 5 minutes each time; (Starting from the addition of fluorescent secondary antibody, all subsequent steps should be performed in a darker place.) Use absorbent paper to dry the liquid on the slides, and seal the slides with anti-fluorescence quencher sealing solution containing DAPI. Observe and collect images under confocal microscope. Figure 2 A and Figure 2 As shown in B, α-SMA is a marker for activating hepatic stellate cells. The targets of the study were co-stained with it to explore the expression of PCBP2 and PD-L1 during fibrosis. The results showed that as liver fibrosis continued to develop, the expression of PCBP2 and PD-L1 was also continuously upregulated. (The more times of induction, the more yellow there is. This shows that the expression of PCBP2 and PD-L1 in hepatic stellate cells is continuously upregulated.)

[0058] Example 3

[0059] Cell culture

[0060] Subsequent experiments were performed using mouse embryonic fibroblasts (3T3) and human hepatic stellate cells (LX-2).

[0061] (1) Observe under a microscope and wait until the confluence of adherent cells reaches 80-90% before performing cell passaging in the clean bench;

[0062] (2) Discard the old culture medium in the cell culture flask, remove the remaining culture medium with a pipette, add about 1 mL of pancreatin, and gently shake to cover the bottom of the culture flask with the pancreatic digestion solution. When white mist appears on the wall of the cell culture flask and most of the cells are observed to become round and fall off under a microscope, use a pipette to absorb the pancreatic digestion solution and blow it evenly against the wall of the cell culture flask to blow off the remaining adherent cells. Then transfer the pancreatic digestion solution containing the cell suspension to a 15 mL pipette;

[0063] (3) Add about 3 mL of culture medium (the culture medium for both cells is DMEM high glucose) to a 15 mL centrifuge tube, and then centrifuge at 1000 rpm for 5 minutes;

[0064] (4) After centrifugation, discard the supernatant and add about 4 mL of culture medium to the cell pellet. Mix the cell pellet by pipetting to form a cell suspension. Divide the cell suspension into different equal parts and transfer them to new culture bottles for culture.

[0065] (5) Finally, tighten the cap of the culture flask and place it in a cell culture incubator at 37°C and 5% CO2.

[0066] Cellular uptake of siRNA nanocomplexes—confocal laser fluorescence imaging

[0067] The cellular uptake of nanocomplexes by LX-2 (human hepatic stellate cells) was evaluated using confocal microscopy. The cells were incubated with nanocomplexes labeled with cy'5 siRNA at 37°C for different times (2h, 6h, 24h), and each experiment was divided into 3 groups for the same time, namely: control group, PM@siRNA group, and Apt-PM@siRNA group. After the cells were transfected, they were first washed with PBS, then fixed with 10% formalin for 15 minutes, stained with LysoTracker (green lysosomal dye) for 20-30 minutes, and finally added with dapi-containing mounting medium. The cell uptake and final lysosomal release at different time points were observed under a confocal microscope. Figure 3 As shown in A, B and C, when the cells take up for 2 hours, it can be seen that the siRNA group alone will not take up, while the group with APT takes up more than the PM group due to the targeting effect, or the overall cell uptake is faster. (The yellow fluorescence area and intensity increase from left to right); when the cells take up for 6 hours, the cells have basically completed the uptake, and the non-specific uptake increases, so the difference between the PM group and the group with APT is smaller than that of 2 hours. (The difference in the yellow fluorescence area and intensity of the second and third groups is very small); when the cells take up for 24 hours, siRNA has begun to be released. As with siRNA uptake, its release will also have an order. The group with APT (siRNA+PM+Aptamer) always precedes the PM (siRNA+PM) group, so it can be seen that the area and intensity of red fluorescence in the APT group are much greater than that in the PM group. The green fluorescence gradually disappears. Because the lysosomes rupture, siRNA is released into the cytoplasm to play a role.

[0068] Silencing activity of Apt-PM@siRNA nanocomplexes

[0069] Apt-PM@siRNA nanocomplexes were prepared as described above and incubated with cells at a final concentration of 100 nM siRNA. After 6 h of incubation, DMEM medium containing 10% fetal bovine serum was supplemented and transfected for 24 or 48 h. The expression products were then analyzed by real-time fluorescence quantitative PCR and protein immunoblotting. Comparison of western blot bands showed that the drug-treated group had a certain gene silencing effect compared with the control group, which was also verified in real-time fluorescence quantitative PCR. The results are shown in Figure 4 The PCBP2-siRNA silencing effect in mouse embryonic fibroblasts (3T3) was about 77% ( Figure 4 A), the silencing effect in human hepatic stellate cells (LX-2) is about 70% ( Figure 4 B). The silencing effect of PD-L1-siRNA in mouse embryonic fibroblasts (3T3) was about 76% ( Figure 4 C), the silencing effect in human hepatic stellate cells (LX-2) is about 60% ( Figure 4 D).

[0070] Example 4

[0071] Animal experiment - Study on the anti-fibrotic activity of CCl4-induced liver fibrosis in mice

[0072] As described above, liver fibrosis was induced in mice and rats. Thirty mice were randomly divided into five groups: healthy control group; CCl4 induced; CCl4 induced plus PCBP2-siRNA nanocomplex treatment group (siRNA dose was 1 mg / kg); CCl4 induced plus PD-L1-siRNA nanocomplex treatment group (1 mg / kg); CCl4 induced plus PCBP2-siRNA nanocomplex treatment group (0.5 mg / kg) and PD-L1-siRNA nanocomplex treatment group (0.5 mg / kg). Nanocomplexes were prepared with PM and PCBP2-siRNA or PD-L1-siRNA and Aptamer at a molar ratio of 1:1:0.1, or PM, PCBP2-siRNA, PD-L1-siRNA and Aptamer at a molar ratio of 1:0.5:0.5:0.1. On days 14, 18, 23, and 27 of CCl4 induction, siRNA nanocomplexes were prepared in a final volume of 80 μL and injected via the tail vein. On day 31, mice were euthanized. The liver of each group was cut into two halves, one half was frozen at -80°C, and the other was preserved in 4% phosphate-buffered formalin.

[0073] Histological studies

[0074] The liver specimens in each group were also stained with Sirius red to detect collagen fibers, which is one of the most commonly used staining methods for type I, III, and IV collagen.

[0075] 1. Embed the formalin-fixed liver tissue with paraffin and slice it into 4 μm thick sections. Dewax the paraffin sections to water: sequentially place the paraffin sections in xylene I for 20 min-xylene II for 20 min-xylene III for 20 min-anhydrous ethanol I for 3 min-90% alcohol for 3 min-80% alcohol for 3 min-70% alcohol for 3 min, and finally wash with distilled water.

[0076] 2. Staining with Sirius red solution: Immerse the washed sections in Sirius red solution for 1 hour.

[0077] 3. Dehydration and transparent sealing: Put the slices into 70% alcohol for 3 min-80% alcohol for 3 min-90% alcohol for 3 min-anhydrous ethanol for 10 min-xylene for 1 5 min to dehydrate and make them transparent. Take the slices out of xylene and dry them slightly, and seal them with neutral gum.

[0078] After Sirius Red forms a complex with collagen, the complex can show red in an acidic environment. Figure 5 The Sirius red staining of mice in the CCL4-induced group showed portal vein-portal vein and portal vein-central vein bridging, and obvious collagen nodules, indicating severe liver fibrosis. However, the PCBP2-siRNA treatment group and the PCBP2-siRNA treatment group significantly reduced the increase in collagen in CCl4-treated mice, and the Sirius red staining effect of the combined group was less than that of the single siRNA treatment group. In the CCL4-induced group, the collagen-positive area accounted for about 3% of the total tissue area, the collagen staining of fibrotic mice treated with PCBP2-siRNA was about 1.7%, the collagen staining of fibrotic mice treated with PD-L1-siRNA was about 1.5%, and the collagen staining of fibrotic mice in the PCBP2-siRNA and PD-L1-siRNA combination group was about 0.8%. These results show that the effect of the combined group will be more obvious than the treatment effect of the single treatment, and can better improve liver fibrosis.

[0079] Immunohistochemistry of Type I Collagen

[0080] Immunohistochemistry of collagen I was performed on liver specimens of each group:

[0081] 1. Embed the formalin-fixed liver tissue in paraffin and slice it into 4 μm thick sections. Dewax the paraffin sections to water: sequentially place the paraffin sections in xylene I for 20 min-xylene II for 20 min-xylene III for 20 min-anhydrous ethanol I for 3 min-90% alcohol for 3 min-80% alcohol for 3 min-70% alcohol for 3 min and then wash with distilled water.

[0082] 2. Antigen repair: Place the tissue sections in a pressure cooker filled with citric acid antigen repair buffer (PH6.0) for antigen repair. When the pressure in the pressure cooker rises to the point where steam begins to come out, count for 3 minutes. During this process, prevent the buffer from evaporating excessively and do not dry the slides. After natural cooling, place the slides in PBS (PH7.4) and wash and soak for 3 times, 3 minutes each time.

[0083] 3. Anionic blocking of endogenous peroxidase: Place the slices in an endogenous peroxidase anionic blocking agent and incubate at room temperature in the dark for 10 minutes. Place the slides in PBS (PH7.4) and wash and soak for 3 times, each time for 3 minutes.

[0084] 4. Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in a certain proportion on the slice, and place the slice flat in a humidified box and incubate overnight at 4°C. (Add a small amount of water in the humidified box to prevent antibody radical irradiation) Add reaction enhancement solution: Place the slice in PBS (PH7.4) and wash and soak for 3 times, 3 minutes each time. Shake the slice slightly dry.

[0085] 5. Then, add the reaction enhancement solution to the circle to cover the tissue and incubate at 37°C for 20 minutes.

[0086] 6. Enhanced enzyme-labeled polymer: Place the slices in PBST (PH7.4) and wash and soak twice, 3 minutes each time. Shake and wash and soak in PBS once, 3 minutes. After the slices are slightly dried, add enhanced enzyme-labeled polymer to the circle to cover the tissue and incubate at 37°C for 20 minutes.

[0087] 7. DAB color development: Place the slices in PBST (PH7.4) and wash and soak twice, 3 minutes each time. Wash and soak in PBS once, 3 minutes. After the slices are slightly dried, add freshly prepared DAB color development solution in the circle. Control the color development time under a microscope. The positive color is brown-yellow. Rinse the slices with tap water to stop the color development.

[0088] 8. Re-stain the cell nucleus: Re-stain with hematoxylin for about 1 minute, place in distilled water, and change the water 3 times.

[0089] 9. Dehydration and transparent sealing: Put the slices into 70% alcohol for 3 min-80% alcohol for 3 min-90% alcohol for 3 min-anhydrous ethanol for 10 min-xylene for 1 5 min to dehydrate and make them transparent. Take the slices out of xylene and dry them slightly, and seal them with neutral gum.

[0090] 10. Microscopic examination and photography: Microscopic examination, image acquisition and analysis.

[0091] Results: Hematoxylin stains the cell nucleus in blue, and DAB stains the positive expression in brown. Figure 6 As shown, compared with the PCBP2-siRNA nanocomplex treatment group alone and the PD-L1-siRNA nanocomplex treatment group alone and the CCL4-induced group, the type I collagen staining was significantly reduced, while the type I collagen staining in the PCBP2-siRNA nanocomplex (0.5 mg / kg) and PD-L1-siRNA nanocomplex (0.5 mg / kg) treatment group was lower than that of the two groups used alone. In the CCL4-induced group, the type I collagen-positive area accounted for about 5% of the total tissue area, the collagen staining of the PCBP2-siRNA-treated fibrotic mice was about 2.5%, the collagen staining of the PD-L1-siRNA-treated fibrotic mice was about 1.6%, and the collagen staining of the PCBP2-siRNA and PD-L1-siRNA combined group of fibrotic mice was about 0.6%. These results show that the effect of the combined group will be more obvious than the treatment effect of the single treatment, and can better improve liver fibrosis.

[0092] The above is only a preferred embodiment of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of an siRNA composition in the preparation of a drug for treating liver disease, characterized in that: The siRNA composition includes at least PCBP2-siRNA and PD-L1-siRNA.

2. Use of the siRNA composition according to claim 1 in preparing a drug for treating liver disease, characterized in that: The nucleotide sequence of the sense strand of the PCBP2-siRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the antisense strand of the PCBP2-siRNA is shown in SEQ ID NO.

2.

3. Use of the siRNA composition according to claim 1 in preparing a drug for treating liver disease, characterized in that: The nucleotide sequence of the sense strand of the PD-L1-siRNA is shown in SEQ ID NO.3, and the nucleotide sequence of the antisense strand of the PD-L1-siRNA is shown in SEQ ID NO.

4.

4. Use of the siRNA composition according to claim 1 in preparing a drug for treating liver disease, characterized in that: The liver disease is liver fibrosis or liver cirrhosis.

5. A drug for treating liver disease, characterized in that: The method comprises at least a targeting vector, a targeting ligand loaded on the targeting vector, and the siRNA composition according to any one of claims 1 to 4.

6. The drug for treating liver disease according to claim 5, characterized in that: The molar ratio of PCBP2-siRNA to PD-L1-siRNA in the drug is 1:(0.1-10).

7. The drug for treating liver disease according to claim 5, characterized in that: The targeting ligand is a targeting ligand of insulin-like growth factor II receptor.

8. The drug for treating liver disease according to claim 5, characterized in that: The targeting carrier is a small molecule polypeptide organic substance.

9. The drug for treating liver disease according to claim 5, characterized in that: The dosage of the drug is 0.5-5 mg / kg based on the total amount of siRNA.

10. A method for preparing a drug for treating liver disease according to any one of claims 5 to 9, characterized in that: The following steps are involved: respectively preparing solutions of targeting vector, targeting ligand, PCBP2-siRNA and PD-L1-siRNA; The targeting carrier solution, the targeting ligand solution, the PCBP2-siRNA solution and the PD-L1-siRNA solution are uniformly mixed and co-incubated to obtain the drug.

Images