CN117883574A - Application of malic enzyme 1 inhibitor in preparation of medicines for treating pulmonary fibrosis - Google Patents
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Abstract
The invention provides an application of a malic enzyme 1 inhibitor in preparing a medicine for treating pulmonary fibrosis, and belongs to the technical field of biological medicines. It was found that administration of ME1 inhibitors significantly improved lung function in IPF mice, including lung volume indicators such as inspiratory capacity, lung ventilation function tests such as airway resistance, dynamic compliance, and quasi-static compliance; inflammatory factors IL-1 beta and IL-6 concentration in the IPF mouse alveolar lavage fluid are reduced, and inflammatory exudation of the lung is reduced; the transcription level of the fibrosis factors FN-1 and COL-I is reduced, the collagen specific amino acid hydroxyproline is reduced, the fibrosis focus is reduced, and the lesion degree is reduced. Compared with a control group, inflammatory cell infiltration of the IPF mice with the Me1 conditional knockout is reduced, and inflammation is reduced; at the same time, the area of fibrosis is reduced, collagen is reduced, and HYP content is also reduced. Thus, targeted therapies against ME1 may be effective in ameliorating pulmonary fibrosis disease progression.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of a malic enzyme 1 inhibitor in preparation of a medicine for treating pulmonary fibrosis.
Background
Interstitial lung disease (INTERSTITIAL LUNG DISEASE, ILD) is a generic term for a group of diffuse pulmonary diseases involving the lung interstitium, alveoli and/or bronchioles. It is classified more frequently and of complex etiology, with idiopathic pulmonary fibrosis (Idiopathic pulmonary fibrosis, IPF) being the most representative disease. IPF is well developed in the middle-aged and elderly, and has an unclear etiology, characterized by progressive and irreversible pulmonary parenchyma fibrosis, resulting in progressive decline of lung function. In recent years, the incidence of IPF has been on the rise. Meanwhile, the median survival time after diagnosis of IPF patients is only 2 to 5 years, and the survival rate of 5 years is lower than 30%, which is a serious fatal pulmonary fibrosis disease, and is called "cancer not cancer". The pathogenesis of IPF is very complex and has wide factors involved, so far no specific therapeutic drug is found, and no definite therapeutic basis is available. Therefore, the pathogenesis of IPF is deeply explored, and a more effective new drug target is searched for, so that the best scientific basis can be provided for the targeted treatment of the disease.
Malate 1 (M1) is a cytoplasmic NADP dependent enzyme that can produce NADPH for fatty acid biosynthesis. The activity of this enzyme, i.e. the reversible oxidative decarboxylation of malic acid, can link glycolysis with the citric acid cycle. At the same time, ME1 can also link fatty acid synthesis to glutamine metabolic pathways via NADPH produced. Research shows that ME1 is highly expressed in various tumors, and can enhance proliferation and invasion capacity of tumor cells. In addition, ME1 is also involved in the development of various non-neoplastic diseases such as cardiovascular disease, obesity, fatty liver, and the like. Unfortunately, there is no experimental evidence to date to confirm the role of ME1 in pulmonary fibrosis disease, whether inhibition of ME1 expression can alleviate the disease progression of pulmonary fibrosis.
Disclosure of Invention
In view of the above, the invention aims to provide an application of a malic enzyme 1 inhibitor in preparing a medicament for treating pulmonary fibrosis.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of a malic enzyme 1 inhibitor in preparing a medicament for treating pulmonary fibrosis.
Preferably, the malic enzyme 1 inhibitor comprises one or both of a modulator that reduces the expression of malic enzyme 1 or a modulator that reduces the product of malic enzyme 1.
Preferably, the modulator that reduces the malic enzyme 1 product comprises a protease, a nuclease, or a malic enzyme 1 antibody that degrades the malic enzyme 1 product.
Preferably, the modulator of reduced expression of malic enzyme 1 comprises an agent or small molecule compound that knocks out or silences malic enzyme 1.
Preferably, the agent that knocks out or silences malic enzyme 1 comprises a nucleic acid molecule siRNA, shRNA or miRNA.
Preferably, the small molecule compound has a structural formula:
The invention also provides application of the malic enzyme 1 serving as a drug action target in developing or designing a drug for treating pulmonary fibrosis.
Preferably, the pulmonary fibrosis comprises idiopathic pulmonary fibrosis, fibrosis non-specific interstitial pneumonia, chronic allergic pneumonia, connective tissue disease-related interstitial lung disease or pneumoconiosis.
Preferably, the medicament is capable of improving lung function and lung inflammation.
Preferably, the medicament further comprises a pharmaceutically acceptable carrier.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides application of a malic enzyme 1 inhibitor in preparing a medicament for treating pulmonary fibrosis. The present study found that administration of ME1 inhibitors significantly improved lung function in IPF mice, including lung volume indicators such as Inspiratory Capacity (IC), and lung ventilation function tests such as airway Resistance (RI), dynamic compliance (Cdyn), and quasi-static compliance (Cchord); inflammatory factors IL-1 beta and IL-6 concentration in the IPF mouse alveolar lavage fluid are reduced, and inflammatory exudation of the lung is reduced; the transcription level of the fibrosis factors FN-1 and COL-I is reduced, the collagen specific amino acid hydroxyproline is reduced, the fibrosis focus is reduced, and the lesion degree is reduced. Compared with a control group, inflammatory cell infiltration of the IPF mice with the conditional knocking-out of Me1 is reduced, and inflammation is reduced to a certain extent; at the same time, the area of fibrosis is reduced, collagen is reduced, and HYP content is also reduced. Thus, targeted therapies against ME1 may be effective in ameliorating pulmonary fibrosis disease progression.
Drawings
FIG. 1 shows HE staining results of Me1 -/- Sftpc-Creet 2 IPF model mice and Me1 +/+ Sftpc-Creet 2 IPF model mice, on a scale of 200 μm;
FIG. 2 is the Masson staining results of Me1 -/- Sftpc-Creet 2 IPF model mice and Me1 +/+ Sftpc-Creet 2 IPF model mice, scale 200 μm;
FIG. 3 shows Hydroxyproline (HYP) content measurements of mice model Me1 -/- Sftpc-CreERT2 IPF and mice model Me1 +/+ Sftpc-CreERT2 IPF, wherein **P＜0.01,*** P < 0.001, representing the comparison of each group to the BLM-Me1 +/+ Sftpc-CreERT2 group;
FIG. 4 is a graph showing the protective effect of ME1 inhibitors on the lung function of IPF mice, wherein A is the effect of different groups on the inhalation amount (IC) of the lung function of mice; b is the effect result of different groups on the airway Resistance (RI) of the lung function of the mice; c is the effect of different groups on the quasi-static compliance (Cchord) of mouse lung function; d is the effect of different groups on the dynamic compliance (Cdyn) of mouse lung function; *P＜0.5,**P＜0.01,***P＜0.001,### P < 0.001, wherein x represents each group compared to BLM group, # represents blm+ld group compared to blm+hd group;
FIG. 5 shows that ME1 inhibitors significantly reduce inflammation in IPF mice, wherein A is the IL-1β concentration in the bronchoalveolar lavage fluid (BALF) of mice in different groups; b is the concentration of IL-6 in the bronchoalveolar lavage fluid of mice in different groups; c is the HE staining of the lung tissue of the mice in different groups, and the scale is 200 mu m; d is the inflammation scoring results for different groups of HE staining results; *P＜0.5,**P＜0.01,***P＜0.001,#P＜0.5,## P < 0.01, wherein x represents each group compared to BLM group, # represents blm+ld group compared to blm+hd group;
FIG. 6 shows that ME1 inhibitor is effective in alleviating pulmonary fibrosis in IPF mice, wherein A is transcript levels in Col-I in lung tissue of mice of different groups; b is the transcription level of the lung tissue Fn-1 of different groups of mice; c is the content of hydroxyproline in the lung tissue of different groups of mice; d is the result of Masson staining of lung tissues of mice of different groups, and the scale is 200 mu m; e is the fibrotic lesion scoring results for the Masson staining results, for the different groups, ***P＜0.001,### P < 0.001, with x indicating each group compared to the BLM group, # indicating blm+ld group and blm+hd group.
Detailed Description
The invention provides application of a malic enzyme 1 inhibitor in preparing a medicament for treating pulmonary fibrosis.
In the present invention, the malic enzyme 1 inhibitor preferably comprises one or both of a modulator for reducing the expression of malic enzyme 1 or a modulator for reducing the product of malic enzyme 1. The modulator that reduces the malic enzyme 1 product preferably comprises a protease, a nuclease or a malic enzyme 1 antibody that degrades the malic enzyme 1 product. The modulator of the expression of malic enzyme 1 preferably comprises an agent or small molecule compound that knocks out or silences malic enzyme 1. The agent for knocking out or silencing malic enzyme 1 preferably comprises a nucleic acid molecule siRNA, shRNA or miRNA. In the present invention, the siRNA refers to a short double-stranded RNA capable of inducing RNA interference by cleaving some mRNA, and includes a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto, and can inhibit the expression of the target gene, and can be used for gene knockdown and gene therapy. In the present invention, the shRNA (short hairpin RNA) is a single-stranded RNA that includes a stem portion and a loop portion that form a double-stranded portion by hydrogen bonding, is converted into siRNA by protein processing such as Dicer, and performs the same function as siRNA. In the present invention, miRNA refers to 21 to 23 non-coding RNAs which regulate gene expression after transcription by promoting degradation of target RNA or by inhibiting translation thereof. In the invention, the small molecule compound has the structural formula:
The small molecule compounds of the above formula are available from MedChemExpress, CAS under 522649-59-8.
The invention also provides application of the malic enzyme 1 serving as a drug action target in developing or designing a drug for treating pulmonary fibrosis.
In the present invention, the pulmonary fibrosis preferably includes idiopathic pulmonary fibrosis, fibrosis nonspecific interstitial pneumonia, chronic allergic pneumonia, connective tissue disease-related interstitial lung disease or pneumoconiosis.
In the invention, after the ME1 inhibitor is given, the lung function of the IPF mice is obviously improved, the inflammatory exudation of the lung is reduced, the fibrosis focus is reduced, the lesion degree is reduced, and the curative effect of the high-dose ME1 inhibitor is obviously better than that of the low-dose ME1 inhibitor. Since the AT2 cells are effector cells for initiating and progressing IPF diseases, the transgenic mice with the conditional knocking out of Me1 by the AT2 cells are utilized to explore the role of ME1 in pulmonary fibrosis diseases, and whether ME1 can be used as a potential treatment target to promote the treatment development of the pulmonary fibrosis diseases, and the results show that inflammatory cell infiltration of the IPF mice with the conditional knocking out of Me1 is reduced, and the inflammation is reduced to a certain extent; at the same time, the area of fibrosis is reduced, collagen is reduced, and HYP content is also reduced. Thus, ME1 inhibitors are useful as a novel regimen for the treatment of pulmonary fibrosis diseases.
In the present invention, the medicament further comprises a pharmaceutically acceptable carrier, such as one or more of a flavoring agent, an excipient, a binder, a preservative, a stabilizer and a diluent. The route of administration of the medicament of the present invention includes oral, intravenous, parenteral, intramuscular, subcutaneous, intraperitoneal, intranasal, rectal or topical administration. In the present invention, the dosage of the drug of the present invention may be determined by the type of the treatment disease, the severity of the disease, the administration route, the age, sex, health condition of the patient, etc., and for example, the dosage of the drug of the present invention may be 1 mug to 1000 mg/day per patient.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the examples below, the ME1 inhibitor is available from MedChemExpress, CAS under the formula 522649-59-8
The Me1 gene AT2 cell line knockout (Me 1 -/- Sftpc-CreERT2 mice) was from the Shanghai south mode biotechnology, inc, where Me1 +/+ Sftpc-CreERT2 mice were also littermates of the Shanghai south mode biotechnology, inc.
In the following examples, the solvent of the ME1 inhibitor solution was 10% dmso by volume and 90% corn oil by volume as solvents.
The solvent of the bleomycin solution is PBS.
Example 1
1.1 Construction of IPF mouse model
Male Me1 +/+ Sftpc-CreERT2 and Me1 -/- Sftpc-CreERT2 mice (8 weeks old, 25-30 g) are selected and fed into SPF-class experimental animal houses, and a one-time tracheal instillation Bleomycin (BLM) method is adopted to carry out Idiopathic Pulmonary Fibrosis (IPF) molding, so that the mice are divided into 4 groups, and 9 mice in each group are respectively:
①PBS-Me1+/+ Sftpc-CreERT2 group: tracheal instillation was given to PBS solution (volume consistent with subsequent BLM solution instillation volume);
②PBS-Me1-/- Sftpc-CreERT2 group: tracheal instillation was given to PBS solution (volume consistent with subsequent BLM solution instillation volume);
③BLM-Me1+/+ Sftpc-CreERT2 group: administering a BLM solution (2U/kg) tracheal instillation;
④BLM-Me1-/- Sftpc-CreERT2 group: administering a BLM solution (2U/kg) tracheal instillation;
all mice were harvested for lung tissue at day 21 post-molding for examination.
1.2 Pathological staining
The left lung was fixed in 4% paraformaldehyde for 72h, dehydrated, and paraffin-embedded sections (5 μm) were subjected to HE staining and Masson staining, respectively. HE staining was scored for inflammation according to Szapiel's score, which included no inflammation (grade 0), mild (grade 1), moderate (grade 2) and severe (grade 3); the Masson staining score was first subjected to a raster scan type screenshot, a 20X screenshot, 30 pictures were taken, and the average of all visual field scores was taken as the degree of fibrosis score for the sample.
1.3HYP content detection
Mouse lung tissue was taken at 30mg and assayed using the HYP kit (NBP 2-59747,Novus Biologicals,Littleton,CO,USA).
1.4 Analysis of results
As shown in FIGS. 1-3, the BLM-Me1 -/- Sftpc-CreERT2 group was effective in alleviating the progression of pulmonary fibrosis. After ME1 deficiency, HE staining showed a reduction in pulmonary inflammatory exudation (fig. 1); masson staining also showed reduced fibrotic lesions (fig. 2), with reduced lesion levels. Collagen-specific amino acid hydroxyproline HYP content was reduced (fig. 3).
Example 2
The ME1 inhibitor can effectively improve lung function, inflammation and fibrosis of a pulmonary fibrosis mouse.
2.1 Construction of IPF mouse model and treatment with ME1 inhibitor administration
Male C57BL/6J mice (8 weeks old, 25-30 g) are selected and raised in SPF-class laboratory animal houses, and a one-time tracheal instillation Bleomycin (BLM) method is adopted to perform Idiopathic Pulmonary Fibrosis (IPF) molding, and the mice are divided into 4 groups, namely:
① PBS group (control group): tracheal instillation with PBS (volume consistent with the volume of subsequent BLM group instillation), the next day with the same volume of solvent (solvent consisting of 10% dmso by volume and 90% corn oil by volume) as the injection of ME1 inhibitor solution, 1 time per day, 6 days per week for 3 weeks;
② BLM group (model group): the BLM solution (2U/kg) was given by tracheal instillation, the next day the same volume of solvent (solvent consisting of volume percent 10% dmso and volume percent 90% corn oil) as the ME1 inhibitor solution was injected, 1 time per day, 6 days per week for 3 weeks;
③ Blm+ld group (low dose ME1 inhibitor group): the administration of BLM solution (2U/kg) by tracheal instillation was followed by intraperitoneal injection of 50mg/kg of ME1 inhibitor solution at a low dose on the next day, 1 time per day for 6 days per week for 3 weeks;
④ Blm+hd group (high dose ME1 inhibitor group): the BLM solution (2U/kg) was given by tracheal instillation, and the ME1 inhibitor was given a high dose of 100mg/kg for intraperitoneal injection on the next day, 1 time per day, 6 days per week for 3 weeks;
all mice were sacrificed on day 21 post-dose, and corresponding samples were collected for detection.
2.2 Pulmonary function detection
Anesthetized mice were immobilized on a laboratory bench and tested for pulmonary function using a pulmonary function test system (DSI Buxco, USA). Before the experiment, the mice were anesthetized by intraperitoneal injection of 0.4mL/100g of 2% pentobarbital, then the trachea was cut open, and the trachea cannula was inserted and connected to a respirator. Next, PFT systems automatically detect FRC, PV, FV and RC tests. Metrics closely related to changes in pulmonary fibrosis lung function are selected for statistical analysis, including lung volume metrics such as Inspiratory Capacity (IC), and lung ventilation function measurements such as airway Resistance (RI), dynamic compliance (Cdyn), and quasi-static compliance (Cchord).
2.3 Pathological staining
The left lung was fixed in 4% paraformaldehyde for 72h, dehydrated, and paraffin-embedded sections (5 μm) were subjected to HE staining and Masson staining, respectively. HE staining was scored for inflammation according to Szapiel's score, which included no inflammation (grade 0), mild (grade 1), moderate (grade 2) and severe (grade 3). The Masson staining score was first subjected to a raster scan type screenshot, a 20X screenshot, 30 pictures were taken, and the average of all visual field scores was taken as the degree of fibrosis score for the sample.
2.4ELISA experiments
The ELISA kit is used for detecting the concentration of IL-1 beta and IL-6 inflammatory factors in the BALF of the mice.
2.5QPCR experiments
All mouse lung tissue RNA was extracted, cDNA was obtained using reverse transcription kit (KR 103, tiangen Biotechnology), QPCR experiments were performed using SYBR Green I Q-PCR kit (TransGen Biotech), and data collection and analysis were performed by Bio-RadIQ5 system.
TABLE 1 primer sequences (5 '-3')
2.6HYP content detection
Mouse lung tissue was taken at 30mg and assayed using the HYP kit (NBP 2-59747,Novus Biologicals,Littleton,CO,USA).
2.7 Analysis of results
As shown in fig. 4-6, administration of the ME1 inhibitor to IPF mice was effective in alleviating the progression of pulmonary fibrosis. The administration of ME1 inhibitors, the lung function of IPF mice was significantly improved, including lung volume indicators such as inspiratory capacity (a in fig. 4), and lung ventilation function tests such as airway resistance (B in fig. 4), quasi-static compliance (C in fig. 4) and dynamic compliance (D in fig. 4); the IPF mice showed decreased concentrations of inflammatory factors IL-1 beta (a in fig. 5) and IL-6 (B in fig. 5) in alveolar lavage fluid, and HE staining also showed decreased inflammatory exudation in the lungs (C and D in fig. 5); the reduction of the transcription levels of fibrosis factors FN-1 (FIG. 6A) and COL-I (FIG. 6B), the reduction of collagen-specific amino acid hydroxyproline (FIG. 6C), and the reduction of fibrosis lesions (FIGS. 6D and E) by Masson staining were also shown to reduce the extent of lesions.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The application of a malic enzyme 1 inhibitor in preparing medicaments for treating pulmonary fibrosis.
2. The use of claim 1, wherein the malic enzyme 1 inhibitor comprises one or both of a modulator that reduces expression of malic enzyme 1 or a modulator that reduces a product of malic enzyme 1.
3. The use of claim 2, wherein the modulator that reduces the malic enzyme 1 product comprises a protease, a nuclease, or a malic enzyme 1 antibody that degrades the malic enzyme 1 product.
4. The use of claim 2, wherein the modulator of the expression of malic enzyme 1 comprises an agent or a small molecule compound that knocks out or silences malic enzyme 1.
5. The use according to claim 4, wherein the agent for knocking out or silencing malic enzyme 1 comprises a nucleic acid molecule siRNA, shRNA or miRNA.
6. The use according to claim 4, wherein the small molecule compound has the structural formula:
7. the application of the malic enzyme 1 as a drug action target in developing or designing a drug for treating pulmonary fibrosis.
8. The use according to claim 1 or 7, wherein the pulmonary fibrosis comprises idiopathic pulmonary fibrosis, fibrosis non-specific interstitial pneumonia, chronic allergic pneumonia, connective tissue disease-related interstitial lung disease or pneumoconiosis.
9. The use according to claim 1, wherein the medicament is capable of improving lung function and lung inflammation.
10. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier.
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