Protein compositions for treating fibrosis and uses thereof

Abstract

The invention discloses a composition. The composition is prepared from at least two of nAG protein, TGF-beta 3 and IL-10; or at least two of a nucleic acid molecule for coding nAG protein, a nucleic acid molecule for coding TGF-beta3 and a nucleic acid molecule for coding IL-10; or at least two of an expression vector for expressing nAG protein, an expression vector for expressing TGF-beta3 and an expression vector for expressing IL-10. The composition containing the protein can be used for treating fibrotic diseases, such as pulmonary fibrosis and hepatic fibrosis.

Description

Technical Field

[0001] The present application belongs to the field of biopharmaceutical technology, and specifically relates to a protein composition for treating fibrosis and its use. Background Art

[0002] Pulmonary fibrosis is a lung injury caused by a variety of factors, including toxins, autoimmunity, drugs, infection, or trauma. It may also be linked to age, genetics, and environmental factors. Pulmonary histopathology demonstrates structural destruction of normal lung tissue, along with varying degrees of inflammation and collagen deposition. Due to the prevalence of respiratory viruses in recent years, the disease is becoming more prevalent among younger patients, with an increasing incidence rate. Currently, there is no effective treatment that can completely reverse pulmonary fibrosis.

[0003] Liver fibrosis is an abnormal proliferation of connective tissue in the liver caused by chronic damage from various pathogenic factors, such as viruses, alcohol, or inflammation. This leads to impaired liver function and, in severe cases, progression to cirrhosis and even liver cancer. Changing living conditions have led to a continuous increase in the incidence of liver fibrosis worldwide. Treating liver fibrosis is crucial for preventing the progression of chronic liver disease to cirrhosis and liver cancer. However, there is currently no treatment that can completely reverse liver fibrosis, making continued research into the treatment of both pulmonary and liver fibrosis crucial.

[0004] The current academic community believes that fibrosis is the result of a special mechanism of wound healing in mammals, and the accumulation of scars during wound healing is the core cause of fibrosis. Therefore, exploring and simulating "scarless healing" is a core idea for solving fibrosis. Early studies have found that some lower vertebrates and invertebrates have extraordinary tissue regeneration capabilities. The tissue regeneration ability of mammals is limited to the gestation period. Mammals in the embryonic stage can achieve "scarless healing", and this ability gradually disappears during postnatal development. The way wounds heal tends to quickly form fibrotic tissue (or scar tissue). Therefore, it is very important to develop a product that can treat fibrosis. Summary of the Invention

[0005] The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present application provides a protein composition for treating fibrosis.

[0006] In its first aspect, the present application provides a composition. According to embodiments of the present application, the composition comprises: at least two of nAG protein, TGF-β3, and IL-10; or at least two of a nucleic acid molecule encoding nAG protein, a nucleic acid molecule encoding TGF-β3, and a nucleic acid molecule encoding IL-10; or at least two of an expression vector expressing nAG protein, an expression vector expressing TGF-β3, and an expression vector expressing IL-10. The composition containing the above-mentioned proteins in the present application can treat fibrotic diseases, such as pulmonary fibrosis and hepatic fibrosis.

[0007] According to an embodiment of the present application, the composition comprises:

[0008] nAG protein, TGF-β3, IL-10; or

[0009] A nucleic acid molecule encoding nAG protein, a nucleic acid molecule encoding TGF-β3, or a nucleic acid molecule encoding IL-10; or

[0010] Expression vector expressing nAG protein, expression vector expressing TGF-β3, expression vector expressing IL-10.

[0011] According to an embodiment of the present application, the expression vector is selected from a non-pathogenic viral vector.

[0012] According to an embodiment of the present application, the expression vector is selected from a plasmid expression vector and a viral expression vector.

[0013] In a second aspect of this application, a pharmaceutical composition is provided. According to embodiments of this application, the pharmaceutical composition comprises the composition described in the first aspect, and optionally, a pharmaceutically acceptable excipient. As previously mentioned, the composition can treat fibrotic diseases. Thus, a pharmaceutical composition containing the composition can also effectively treat fibrotic diseases, such as pulmonary fibrosis and hepatic fibrosis.

[0014] In the third aspect of the present application, the present application proposes use of the composition described in the first aspect or the pharmaceutical composition described in the second aspect in preparing a drug for treating fibrosis.

[0015] According to an embodiment of the present application, the fibrosis is selected from pulmonary fibrosis and liver fibrosis.

[0016] In a third aspect, the present application provides a method for reducing the expression of α-SMA and / or Col1-1a, or increasing the expression of E-cad. According to an embodiment of the present application, the method comprises: contacting a test sample with the composition of the first aspect, wherein the test sample comprises a nucleic acid molecule encoding at least one of α-SMA, Col1-1a, and E-cad.

[0017] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become obvious from the description below, or will be learned through practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0019] Figure 1 This is the change in α-SMA and E-cad mRNA levels in NRK-4F cells after strong expression of TGFβ2 and β3 in Example 1 of the present application;

[0020] Figure 2 This is the change in α-SMA and E-cad mRNA levels in NRK-4F cells after strong expression of IL-6, IL-10, and IL-11 in Example 1 of the present application;

[0021] Figure 3 This is the change in Col1-1a mRNA level in NRK-4F cells after strong expression of nAG, MMP-1, MMP-9, and MMP-13 in Example 1 of the present application;

[0022] Figure 4 The expression changes of α-SMA and Col1-1a mRNA levels in NRK-4F cells after strong expression of TGFβ3, IL-10 and nAG alone or in combination in Example 1 of the present application;

[0023] Figure 5 This is the AAV expression in the lungs of each group of mice in Example 2 of this application;

[0024] Figure 6 This is the change in lung index of each group of mice in Example 2 of this application;

[0025] Figure 7 HE and Masson staining of lung tissues of mice in each group in Example 2 of the present application;

[0026] Figure 8 The Ashcroft score of lung tissue fibrosis in each group of mice in Example 2 of the present application is shown in FIG.

[0027] Figure 9 The collagen volume fraction (CVF%) of the lung tissue of each group of mice in Example 2 of the present application is shown in FIG.

[0028] Figure 10 The changes in liver function of mice were evaluated by AST and ALT in the serum of each group of mice in Example 3 of the present application;

[0029] Figure 11 HE and picrosirius red staining of liver tissues of mice in each group in Example 3 of the present application;

[0030] Figure 12 These are the results of Col1-1a immunohistochemical staining of mice in each group in Example 3 of the present application;

[0031] Figure 13 These are the measurement area results after immunohistochemical section analysis of each group of mice in Example 3 of the present application. DETAILED DESCRIPTION

[0032] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and should not be understood as limiting the present application.

[0033] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of this application, unless otherwise specified, "plurality" means two or more.

[0034] This application details

[0035] Definitions and General Terms

[0036] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in this application, but not excluding other contents.

[0037] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0038] In this article, the term "expression vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the nucleic acid molecule contains a nucleotide sequence that can express a target protein, and the nucleic acid molecule can be transferred into a host cell and / or between host cells. The expression vector may include a vector mainly used to insert DNA or RNA into a cell, a vector mainly used to replicate DNA or RNA, and a vector mainly used for expression of transcription and / or translation of DNA or RNA. The expression vector also includes vectors with multiple of the above functions. The expression vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the expression vector can produce a desired expression product by culturing a suitable host cell containing the expression vector.

[0039] As used herein, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active composition (nucleic acid molecule, expression vector or protein) with a liquid carrier, a finely divided solid carrier, or both.

[0040] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a particular target dosage form. Except for any conventional excipients that are incompatible with the compositions (nucleic acid molecules, expression vectors or proteins) of the present application, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of this application.

[0041] In this article, the term "administration" refers to the introduction of a predetermined amount of material into a patient by a certain suitable manner. The composition (nucleic acid molecule, expression vector or protein) or pharmaceutical composition of the present application can be administered by any common approach, as long as it can reach the expected tissue. The various modes of administration are expected to include tracheal injection, oral and nasal atomization inhalation, peritoneum, intravenous injection, intramuscular injection, subcutaneous injection, etc., but the application is not limited to these exemplified modes of administration. Preferably, the composition of the present application is administered by intravenous injection or subcutaneous injection.

[0042] As used herein, the term "treatment" refers to a method for obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the development of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a composition (nucleic acid molecule, expression vector or protein) or a pharmaceutical composition to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a composition (nucleic acid molecule, expression vector or protein) described herein to an individual in need.

[0043] Detailed description of the protein composition for treating fibrosis and its use

[0044] The present application proposes a composition, a pharmaceutical composition, and a method for reducing the expression of α-SMA and / or Col1-1a, or increasing the expression of E-cad, which will be described in detail below.

[0045] Composition

[0046] In a first aspect of the present application, the present application provides a composition. According to an embodiment of the present application, the composition comprises:

[0047] At least two of nAG protein, TGF-β3, and IL-10; or

[0048] At least two of a nucleic acid molecule encoding nAG protein, a nucleic acid molecule encoding TGF-β3, and a nucleic acid molecule encoding IL-10; or

[0049] At least two of an expression vector expressing nAG protein, an expression vector expressing TGF-β3, and an expression vector expressing IL-10.

[0050] The present application found that nAG protein, TGF-β3, and IL-10 can reduce the levels of α-SMA and Col1-1a in cells. Therefore, a composition containing the above proteins can treat fibrotic diseases, such as pulmonary fibrosis and liver fibrosis.

[0051] In this article, "nAG protein" refers to anterior gradient protein 2, and its amino acid sequence can be found in GenBank: ABS01492.1.

[0052] According to an embodiment of the present application, the composition includes: nAG protein, TGF-β3, and IL-10.

[0053] According to an embodiment of the present application, the composition includes: a nucleic acid molecule encoding nAG protein, a nucleic acid molecule encoding TGF-β3, and a nucleic acid molecule encoding IL-10.

[0054] According to an embodiment of the present application, the composition includes: an expression vector expressing nAG protein, an expression vector expressing TGF-β3, and an expression vector expressing IL-10.

[0055] In this article, "an expression vector expressing nAG protein, an expression vector expressing TGF-β3, and an expression vector expressing IL-10" can be one expression vector that can simultaneously express nAG protein, TGF-β3, and IL-10, or two expression vectors that jointly express nAG protein, TGF-β3, and IL-10, or three expression vectors that separately express nAG protein, TGF-β3, and IL-10. The specific types are not limited and are all within the scope of protection of this application.

[0056] In an optional embodiment of the present application, the expression vector for expressing nAG protein carries a nucleic acid molecule encoding nAG protein.

[0057] In an optional embodiment of the present application, the expression vector for expressing TGF-β3 carries a nucleic acid molecule encoding TGF-β3.

[0058] In an optional embodiment of the present application, the expression vector for expressing IL-10 carries a nucleic acid molecule encoding IL-10.

[0059] According to an embodiment of the present application, the expression vector is selected from a non-pathogenic viral vector.

[0060] According to an embodiment of the present application, the expression vector is selected from a plasmid expression vector and a viral expression vector.

[0061] In an optional embodiment of the present application, the expression vector is an adeno-associated virus, such as AVV6 or AVV8.

[0062] Pharmaceutical composition

[0063] In a second aspect of this application, a pharmaceutical composition is provided. According to embodiments of this application, the pharmaceutical composition comprises the composition described in the first aspect, and optionally, a pharmaceutically acceptable excipient. As previously mentioned, the composition can treat fibrotic diseases. Thus, a pharmaceutical composition containing the composition can also effectively treat fibrotic diseases, such as pulmonary fibrosis and hepatic fibrosis.

[0064] The administration of the pharmaceutical composition of the present application can be carried out by any acceptable mode of administration. The pharmaceutical composition of the present application can be formulated into preparations in solid, semisolid, liquid or gaseous form, such as injections, lyophilized powders, and the current methods for preparing these dosage forms are known or obvious to those skilled in the art. Typical routes of administering such pharmaceutical compositions include, but are not limited to, tracheal injection, oral and nasal aerosol inhalation, subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection or infusion techniques. The pharmaceutical composition of the present application is formulated so as to allow the biologically active ingredients contained therein to be bioavailable after the composition is administered to the patient.

[0065] use

[0066] In the third aspect of the present application, the present application proposes use of the composition described in the first aspect or the pharmaceutical composition described in the second aspect in preparing a drug for treating fibrosis.

[0067] According to an embodiment of the present application, the fibrosis is selected from pulmonary fibrosis and liver fibrosis.

[0068] method

[0069] In a fourth aspect, the present application provides a method for reducing the expression of α-SMA and / or Col1-1a, or increasing the expression of E-cad. According to an embodiment of the present application, the method comprises: contacting a test sample with the composition of the first aspect, wherein the test sample comprises a nucleic acid molecule encoding at least one of α-SMA, Col1-1a, and E-cad.

[0070] According to an embodiment of the present application, the composition includes nAG protein, TGF-β3, and IL-10.

[0071] In a fifth aspect of the present application, a method for treating fibrotic diseases is provided. According to an embodiment of the present application, the method comprises: administering a pharmaceutically acceptable dose of the composition of the first aspect or the pharmaceutical composition of the second aspect to a subject.

[0072] The effective amount of the compositions and pharmaceutical compositions described herein may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, and the like. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0073] The compositions and pharmaceutical compositions of the present application can be incorporated into medicines suitable for parenteral administration (e.g., tracheal injection, intravenous, subcutaneous, intraperitoneal, intramuscular). These medicines can be prepared into various forms. For example, liquid, semisolid and solid dosage forms, etc., include but are not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical medicines are in the form of injection solutions or infusion solutions. The aforementioned compositions and pharmaceutical compositions can be administered by tracheal injection, oral and nasal aerosol inhalation, intravenous infusion or injection, or intramuscular or subcutaneous injection.

[0074] As used herein, the term "subject" refers to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, athletic animals, and pets. Tissues, cells, and progeny of biological entities obtained in vivo or cultured in vitro are also included.

[0075] According to an embodiment of the present application, the administration route of the method is tracheal injection or intravenous injection.

[0076] According to an embodiment of the present application, the fibrotic disease is selected from pulmonary fibrosis and liver fibrosis.

[0077] In an optional embodiment of the present application, the fibrotic disease is selected from pulmonary fibrosis, and the administration route of the method is intratracheal injection and intravenous injection.

[0078] In an optional embodiment of the present application, the fibrotic disease is selected from liver fibrosis, and the administration route of the method is intravenous injection.

[0079] Below in conjunction with embodiment, the scheme of the application will be explained. Those skilled in the art will appreciate that the following examples are merely for illustration of the application and should not be considered as limiting the scope of the application. Where specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents used or instruments not indicated by the manufacturer are conventional products that can be obtained commercially.

[0080] The NCBI reference sequences corresponding to the amino acid sequences of the therapeutic drug proteins in the following examples are as follows:

[0081] TGF-β2:NM_001329107.1;

[0082] TGF-β3:NM_009368;

[0083] IL-6:NM_001314054.1;

[0084] IL-10:NM_010548.2;

[0085] IL-11:NM_001290423.2;

[0086] nAG:ABS01492.1;

[0087] MMP-1:NM_001134530;

[0088] MMP-9:NM_013599.5;

[0089] MMP-13:NM_008607.2.

[0090] Example 1: Protein screening

[0091] 1. In vitro cell model: Select rat kidney fibroblasts (NRK-49F, CRL-1570) were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Invitrogen), 100 U / ml penicillin, and 100 μg / ml streptomycin (Invitrogen). TGF-β1 (5 ng / ml, Pepro Tech) was added to the complete culture medium and incubated for 24 hours to establish an in vitro cell model.

[0092] 2. Based on the construction method of the in vitro cell model in step 1 above, conventionally cultured NRK-49F cells (ie, without adding TGF-β1) were used as a blank control group; NRK-49F cells with TGF-β1 added in step 1 above were used as a model group; an expression vector AAV6 carrying the target protein (ie, therapeutic drug) was constructed, and each expression vector was introduced into NRK-49F cells to obtain NRK-49F cells that strongly expressed each therapeutic drug, and the NRK-49F cells that strongly expressed each therapeutic drug were cultured using the method in step 1 above as treatment groups. Then, experimental group cells were constructed separately, and the blank control group, model group, and treatment group cells were incubated for 24 hours for subsequent detection. Among them, the NRK-49F cells in each treatment group are as follows:

[0093] Group 1:

[0094] 1) NRK-49F cells that strongly express TGF-β2;

[0095] 2) NRK-49F cells that strongly express TGF-β3;

[0096] Group 2:

[0097] 1) NRK-49F cells that strongly express IL-6;

[0098] 2) NRK-49F cells that strongly express IL-10;

[0099] 3) NRK-49F cells that strongly express IL-11;

[0100] Group 3:

[0101] 1) NRK-49F cells that strongly express nAG;

[0102] 2) NRK-49F cells that strongly express MMP-1;

[0103] 3) NRK-49F cells that strongly express MMP-9;

[0104] 4) NRK-49F cells that strongly express MMP-13.

[0105] Total RNA was then isolated from NRK-49F cells in each experimental group using Trizol reagent. The isolated RNA was reverse transcribed using a qPCR RT kit (Takara) to generate a cDNA template. The generated cDNA was analyzed by RT-PCR using SYBR Green Realtime PCR Master Mix Plus (Takara). Specific primers for E-cadherin, Col1-a1, and α-SMA were synthesized, and the β-actin gene was used as an endogenous standard gene. All samples were assayed in triplicate and quantified using ΔΔCT.

[0106] turn out:

[0107] 1) If Figure 1 As shown in the figure, compared with the model group, the α-SMA level of NRK-4F cells that strongly expressed TGF-β3 was significantly reduced, while the E-cad level was significantly increased. TGF-β3 was superior to TGFβ2 as a therapeutic drug, so TGF-β3 was selected for subsequent experiments.

[0108] 2) If Figure 2 As shown in the results, the α-SMA level of NRK-4F cells that strongly expressed IL-6 and IL-11 was significantly increased in the group with only TGF-β1 addition, and the E-cad level was further reduced. However, the α-SMA level of the experimental group that strongly expressed IL-10 was significantly reduced, and the E-cad level was significantly increased. Therefore, IL-10 was selected for subsequent experiments.

[0109] 3) If Figure 3 As shown, NRK-4F cells that strongly expressed nAG had significantly lower α-SMA levels than cells that strongly expressed MMP-1, MMP-9, and MMP-13, so nAG was selected for subsequent experiments.

[0110] 3. Prepare a blank control group, a model group, and a treatment group according to the method of step 2 above, wherein at least two therapeutic drugs are added to the treatment group. When the treatment group contains two or more therapeutic drugs, construct an expression vector carrying a nucleic acid molecule encoding its protein for each therapeutic drug, and then simultaneously introduce it into NRK-49F cells. The NRK-49F cells in each treatment group are as follows:

[0111] 1) NRK-49F cells that strongly express TGF-β3;

[0112] 2) NRK-49F cells that strongly express IL-10;

[0113] 3) NRK-49F cells that strongly express nAG;

[0114] 4) NRK-49F cells that strongly express TGF-β3 + IL-10;

[0115] 5) NRK-49F cells that strongly express IL-10 + nAG;

[0116] 6) NRK-49F cells that strongly express TGF-β3+nAG;

[0117] 7) NRK-49F cells that strongly express TGF-β3+IL-10+nAG.

[0118] Total RNA was then isolated from NRK-49F cells in each experimental group using Trizol reagent. The isolated RNA was reverse transcribed using a qPCR RT kit (Takara) to generate a cDNA template. The generated cDNA was analyzed by RT-PCR using SYBR Green Realtime PCR Master Mix Plus (Takara). Specific primers for E-cadherin, Col1-a1, and α-SMA were synthesized, and the β-actin gene was used as an endogenous standard gene. All samples were assayed in triplicate and quantified using ΔΔCT.

[0119] The results showed that the α-SMA level of NRK-4F cells that strongly expressed IL-10, TGF-β3, and nAG was significantly lower than that of the experimental group that strongly expressed one or two proteins. Figure 4 .

[0120] Example 2: Treatment of pulmonary fibrosis mouse model with protein combination

[0121] 1. Construction of animal model: 8-week-old adult male C57 / BL6 mice were injected intratracheally with 1.5 mg / kg bleomycin to establish a stable animal model of pulmonary fibrosis.

[0122] 2. Experimental groups and intervention methods:

[0123] AAV6 was used as a vector to carry the protein combination into the living body for sustained expression. The mice were divided into the following five groups, each containing 10 mice:

[0124] 1) Control group: no treatment, continuous observation;

[0125] 2) Model group: a single intratracheal injection of 1.5 mg / kg bleomycin was administered to establish the model and the model was continuously observed;

[0126] 3) Tail vein injection group: 1.5 mg / kg bleomycin was injected into the trachea once to establish the model, and 5 days later, 5×10 AAV6 was injected into the tail vein. 11 vg / mice, continuous observation;

[0127] 4) Tracheal injection (Low) group: 1.5 mg / Kg bleomycin was injected into the trachea once to establish the model, and 5 days later, a low-dose AAV6 5×10 10 vg / mice, continuous observation;

[0128] 5) Tracheal injection (High) group: 1.5 mg / Kg bleomycin was injected into the trachea once to establish the model, and 5 days later, a low-dose AAV6 5×10 11 vg / mice, continue to observe.

[0129] 3. During the observation period, the expression of AAV in mice was observed by in vivo imaging. At the end of the observation period, samples were collected from each group of mice and subjected to lung index analysis, HE staining, Masson staining, Ashcroft score, collagen volume fraction (CVF%) and other tests.

[0130] 4. The test results are as follows:

[0131] 1) If Figure 5 As shown in the figure, 10 days after AAV6 injection, in vivo imaging was used to observe AAV expression in mice. It was observed that the intravenous injection group had a relatively obvious expression in the mice, but the expression level was highest at the tail vein injection site. This proves that the tail vein injection of AAV carrying the protein combination has some loss, but it can also be stably expressed in mice. In contrast, high expression was observed in the lungs of both high-dose and low-dose mice injected through the trachea. The expression was limited to the trachea and lungs, with no significant expression in other locations. This suggests that AAV carrying the protein combination has better targeting through intratracheal injection.

[0132] 2) Pulmonary index = mouse lung weight (g) / mouse body weight (g) × 100%. It is one of the important indicators for evaluating the severity of pulmonary fibrosis. After pulmonary fibrosis occurs, the increase in collagen fibers leads to an increase in lung weight (increased lung weight needs to be combined with other tests to exclude diseases such as pulmonary congestion and tumors). The pulmonary index is positively correlated with the severity of pulmonary fibrosis. Figure 6 As shown in the figure, it can be observed that the lung index of mice in the model group was significantly higher than that in the blank group, while the lung index of the three treatment groups was significantly decreased.

[0133] 3) HE staining: Figure 7 The first three rows are HE staining, in which the alveolar structure of the lung tissue of mice in the model group was obviously destroyed and inflammatory cells infiltrated, while the lung tissue structure of mice in the three treatment groups was alleviated to varying degrees, close to that of normal mouse lung tissue.

[0134] Masson staining: Figure 7The last two rows are Masson staining, in which the blue collagen fibers were obviously proliferated in the lung tissue structure of the mice in the model group, while the collagen fibers in the lung tissue of the mice in the three treatment groups were reduced, close to the normal lung tissue.

[0135] 4) Fibrosis score (Ashcroft score): Figure 8 As shown, at least three sections were selected from each experimental group and statistically analyzed after scoring by three pathology experts. It can be observed that the fibrosis score of mice in the model group was significantly increased, while that in the three treatment groups was significantly decreased.

[0136] 5) Collagen volume fraction (CVF%): Figure 9 As shown, the CVF% of mice in the model group was significantly higher than that in the normal group, while that in the three treatment groups was significantly lower than that in the model group and close to that in the normal group.

[0137] Example 3: Treatment of liver fibrosis mouse model with protein combination

[0138] 1. Construction of animal model: 9-week-old male C57 / BL6 mice were intraperitoneally injected with 20% CCL4 2uL / g once every three days to establish a stable animal model of liver fibrosis.

[0139] 2. Experimental groups and intervention methods:

[0140] AAV8 was used as a vector to carry the protein combination into the living body for sustained expression. The mice were divided into the following 4 groups, each containing 10 mice:

[0141] 1) Control group: 6×10 11 vg / mice control AAV8 (not carrying protein combination), but without CCL4 injection;

[0142] 2) Model group: 20% CCL4 2uL / g was injected intraperitoneally once every three days to establish the model;

[0143] 3) 2OE (two plasmids, plasmid 1 carries the nucleic acid encoding TGFβ3, and plasmid 2 carries the nucleic acid encoding nAG+IL-10): once every three days, intraperitoneal injection of 20% CCL4 2uL / g for modeling and tail vein injection of 6×10 11 vg / mice two plasmids AAV8;

[0144] 4) 3OE (three plasmids, each carrying nucleic acid encoding TGFβ, IL-10, and nAG): once every three days, intraperitoneal injection of 20% CCL4 2uL / g for modeling and tail vein injection of 6×10 11 vg / mice three-plasmid AAV8.

[0145] 3. At the end of the observation period, liver function tests and histopathological tests (HE staining, picrosirius red staining, and immunohistochemical staining) were performed on the mice in each group.

[0146] 4. The test results are as follows:

[0147] 1) Detect AST and ALT in mouse serum to evaluate changes in mouse liver function. Figure 10 As shown in the figure, the AST and ALT levels of the model group mice were significantly higher than those of the control group, but were significantly lower in the 2OE and 3OE groups than in the model group, indicating that the liver function of the mice gradually recovered after treatment.

[0148] 2) HE staining results are as follows Figure 11 As shown, the liver lobule structure of the control mice was intact and clear, with hepatocytes arranged radially around the central vein. There was no degeneration or necrosis, and no inflammatory cell infiltration. The livers of the model group mice showed partial hepatocyte necrosis, destruction of the normal lobular structure, disorganized hepatocyte cords, enlarged central veins or portal areas, and extensive inflammatory cell infiltration. Compared with the model group, these conditions were improved in the 2OE and 3OE groups.

[0149] The results of picrosirius red staining were as follows Figure 11 As shown, there was no collagen fiber deposition in the liver lobules of mice in the blank group; a large amount of collagen fiber deposition was observed in the liver sinusoids of mice in the model group, which separated the liver parenchyma and formed pseudolobules; while the collagen fiber deposition in mice in the 2OE and 3OE groups was significantly reduced.

[0150] 3) Col1-1a immunohistochemical staining results Figure 12 As shown in the figure, the liver tissues of mice in the model group showed obvious expression of Col1-1a, while the expression in the 2OE and 3OE groups decreased.

[0151] 4) Use Image J software to analyze the above immunohistochemical sections and measure the area. Figure 13 As shown in the figure, the collagen area of the model group mice was significantly higher than that of the control group, and the collagen deposition of the mice in the 2OE and 3OE treatment groups was significantly reduced.

[0152] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0153] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A composition, characterized in that include: At least two of nAG protein, TGF-β3, and IL-10; or At least two of a nucleic acid molecule encoding nAG protein, a nucleic acid molecule encoding TGF-β3, and a nucleic acid molecule encoding IL-10; or At least two of an expression vector expressing nAG protein, an expression vector expressing TGF-β3, and an expression vector expressing IL-10.

2. The composition according to claim 1, characterized in that The composition comprises: nAG protein, TGF-β3, and IL-10.

3. The composition according to claim 1, characterized in that The composition comprises: a nucleic acid molecule encoding nAG protein, a nucleic acid molecule encoding TGF-β3, and a nucleic acid molecule encoding IL-10.

4. The composition according to claim 1, characterized in that The composition comprises: an expression vector for expressing nAG protein, an expression vector for expressing TGF-β3, and an expression vector for expressing IL-10.

5. The composition according to claim 1 or 4, characterized in that The expression vector is selected from non-pathogenic viral vectors.

6. The composition according to claim 5, characterized in that The expression vector is selected from a plasmid expression vector and a viral expression vector.

7. A pharmaceutical composition, characterized in that The invention comprises the composition according to any one of items 1 to 6, and optionally pharmaceutically acceptable excipients.

8. Use of the composition according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating fibrosis.

9. The use according to claim 8, characterized in that The fibrosis is selected from pulmonary fibrosis and liver fibrosis.

10. A method for reducing the expression of α-SMA and / or Col1-1a, or increasing the expression of E-cad, characterized in that: include: The test sample is contacted with the composition according to any one of claims 1 to 6, wherein the test sample comprises a nucleic acid molecule encoding at least one of α-SMA, Col1-1a, and E-cad.

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