Construction method and application of lung-related disease model simulating muscle-lung axis communication steady-state disorder

Abstract

The application discloses a construction method and application of a lung-related disease model simulating cross-axis homeostasis disorder of muscle-lung. The construction method comprises: specifically inhibiting expression of a target gene in muscle satellite cells of an adult non-human mammal to induce a muscle aging phenotype and a secondary lung-related disease phenotype. The lung-related disease model simulating cross-axis homeostasis disorder of muscle-lung obtained by using the construction method has at least the following advantages: precise simulation of an aging background, in-depth mechanism analysis, good model stability, and high drug screening value.

Description

Technical Field

[0001] This application relates to the field of disease modeling technology, specifically to the construction method and application of models for lung-related diseases (including lung cancer, COPD, etc.) that simulate muscle-lung axis communication homeostasis imbalance. Background Technology

[0002] Lung cancer and chronic obstructive pulmonary disease (COPD) are highly comorbid in the elderly, often presenting clinically as "lung cancer combined with COPD." Aging is the core pathological background driving this comorbid phenotype: with age, the lung tissue microenvironment undergoes irreversible remodeling, characterized by typical features including abnormal proliferation of airway goblet cells, hypersecretion of mucus, decline in alveolar epithelial barrier function, and significant downregulation of the Club cell-specific secreted protein CC16—all recognized early signs of lung cancer. CC16, an important anti-inflammatory and antioxidant barrier protein, not only weakens airway clearance and local immune surveillance capabilities but also, through a persistent inflammatory microenvironment and oxidative stress, synergistically promotes epithelial-mesenchymal transition (EMT) and the accumulation of genomic instability, making it an important early symptom of lung cancer. Chronic inflammation is one of the most important contributing factors to lung cancer, but the reasons are not yet fully understood.

[0003] Elderly individuals with lung cancer and COPD often experience progressive skeletal muscle loss, muscle weakness, and metabolic disorders, clinically termed "pulmonary-muscle comorbidity" or "senile pulmonary cachexia / sarcopenia." This is a significant cause of death from lung cancer, and currently, there is no effective treatment. A suitable model to simulate the comorbidity of lung cancer with COPD and sarcopenia is lacking, making drug development for this highly lethal comorbidity difficult.

[0004] In the field of basic research, current mainstream animal models of lung cancer mainly employ methods such as driver gene mutation activation or xenograft / syngeneic transplantation of tumor cells. These methods primarily use young adult individuals aged 6-8 weeks, which, while capable of mimicking some lung lesions relatively well, have the following significant limitations:

[0005] Ignoring the core context of aging: Both lung cancer and COPD are highly prevalent in the elderly population, and aging itself can lead to a decline in immune system function, mitochondrial dysfunction, and chronic low-grade inflammation (inflammatory aging). Existing models lack this crucial biological context of aging, making it difficult to accurately reflect the pathophysiological characteristics of elderly patients with chronic lung diseases.

[0006] Limitations of natural aging models: Some studies have attempted to construct lung cancer and COPD models using naturally aging animals (such as 18-24 month old mice), but this method has problems such as long cycle, high cost, large individual differences, and high mortality. Furthermore, it cannot distinguish the independent contributions of systemic aging and aging of specific tissues (such as muscles) to the occurrence and development of lung cancer and COPD.

[0007] The limitations of traditional gene knockout models: Although systemic gene knockout techniques (such as systematic knockout of aging-related genes such as telomerase, p53, and SIRT1) can induce premature aging phenotypes, they often lead to embryonic lethality, developmental abnormalities, or multi-organ systemic lesions. This makes it difficult to attribute lung and muscle phenotypes to the aging effects of specific tissues and to accurately analyze the independent role of muscle-derived signals in the regulation of lung function in the "muscle-lung" axis.

[0008] Furthermore, existing models have significant gaps in simulating the disruption of muscle-lung axis communication. Preclinical studies suggest that aging skeletal muscle can secrete various pro-inflammatory factors (such as IL-6, TNF-α, and myostatin), metabolites, and exosomes. These muscle-derived factors may affect the lung's immune microenvironment and tissue remodeling through blood circulation. However, the current lack of animal models that can specifically induce muscle tissue aging while preserving the normal physiological state of other tissues severely limits research into the crucial scientific question of "how aging muscle drives or exacerbates lung diseases such as lung cancer and COPD."

[0009] In summary, there is an urgent need in this field to develop an animal model of lung-related diseases that induces muscle aging through muscle-specific gene manipulation and thereby simulates the dysregulation of the muscle-lung axis communication homeostasis. Summary of the Invention

[0010] Based on this, this application provides at least one method for constructing and applying a model of lung-related diseases (lung cancer, COPD) that simulates the dysregulation of muscle-lung axis communication homeostasis.

[0011] In a first aspect of this application, a method for constructing a model of lung-related diseases (lung cancer, COPD) simulating muscle-lung axis communication homeostasis imbalance is provided, the method comprising:

[0012] Specifically inhibits the expression of target genes in muscle satellite cells of adult non-human mammals to induce muscle senescence phenotype and secondary lung cancer and COPD-like phenotypes.

[0013] In a second aspect of this application, a lung-related disease model simulating muscle-lung axis communication homeostasis disorder, such as a lung cancer combined with COPD model, is provided, constructed by the construction method described in the first aspect.

[0014] In a third aspect of this application, the use of the lung-related disease model described in the second aspect, which simulates the dysregulation of muscle-lung axis communication homeostasis, such as a lung cancer combined with COPD model, in screening drugs for the treatment of aging-related diseases is provided.

[0015] In a fourth aspect of this application, the use of an animal model simulating muscle-lung axis communication homeostasis dysregulation by muscle-specific knockout of the Tet1 gene is provided in screening drugs that regulate muscle-lung axis signaling pathways.

[0016] The lung-related disease model simulating muscle-lung axis communication homeostasis imbalance obtained using the construction method of this application has at least the following advantages:

[0017] (1) Precise simulation of aging background: By using genetic methods to specifically simulate aging in muscles, the problems of long cycle and high cost of using naturally aging animals are avoided, while eliminating the confounding factors of systemic aging.

[0018] (2) In-depth analysis of the mechanism: It can clearly prove that muscle aging signals are the key factors leading to increased susceptibility to lung cancer and COPD and systemic complications, and verify the existence of the "muscle-lung" axis.

[0019] (3) The model has good stability: the genotype determines the phenotype, the individual differences are small, and the experiment has high reproducibility.

[0020] (4) High value for drug screening: It is particularly suitable for screening drugs for the treatment of lung cancer and COPD and anti-aging therapies for elderly patients with sarcopenia. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0022] Figure 1 In one embodiment of this application, the model mice exhibit a muscle aging phenotype; wherein:

[0023] a: Twelve weeks after induction and knockout, qRT-PCR results showed that the level of Tet1 mRNA in muscle stem cells was significantly downregulated;

[0024] b: Appearance of mice after 12 weeks of induction and knockout;

[0025] ce: After 12 weeks of induction knockout, mice showed decreased body weight (c), increased hair loss rate (d), and increased arched back rate (e).

[0026] f: After twelve weeks of induced knockout, the running distance and running time of mice decreased;

[0027] g: After 12 weeks of induction and knockout, the muscle weight of mice decreased;

[0028] h: After twelve weeks of induction and knockout, the muscle strength of mice decreased;

[0029] i: After 12 weeks of induction and knockout, the muscle fatigue resistance of mice decreased;

[0030] j: Twelve weeks after induction and knockout, HE staining showed that the cross-sectional area of ​​mouse muscle fibers was significantly reduced.

[0031] Figure 2 In one embodiment of this application, the model mice exhibit an early lung cancer combined with COPD phenotype; wherein:

[0032] a: Twelve weeks after induction knockout, PFT pulmonary function tests showed a significant decrease in tidal volume;

[0033] b: Twelve weeks after induction knockout, PFT pulmonary function tests showed a significant decrease in dynamic lung compliance;

[0034] c: Twelve weeks after induction and knockout, PFT lung function tests showed a slight increase in respiratory rate in mice;

[0035] d: Twelve weeks after induction and knockout, HE staining showed that the airways of mice were significantly thickened;

[0036] e: Twelve weeks after induction and knockout, HE staining showed that abnormal cell proliferation and cell clusters were formed in the alveoli of mice;

[0037] f: Twelve weeks after induction and knockout, PAS staining showed abnormal proliferation of goblet cells (purple-red) in the lungs of mice;

[0038] g: Twelve weeks after induction and knockout, RNA next-generation sequencing data showed that lung Scgb1a1 / CC16 gene expression was significantly downregulated;

[0039] h: Twelve weeks after induction and knockout, SCGB1A1 / CC16 immunofluorescence staining showed that the level of CC16 secreted by mouse lung club cells was significantly reduced;

[0040] i: Twelve weeks after induction and knockout, CD45 immunohistochemistry showed that a large number of immune cells infiltrated the lungs of mice.

[0041] Figure 3 This application illustrates muscle-lung axis communication in a mouse model mediated by inflammatory factors in one embodiment; wherein:

[0042] a: After twelve weeks of induction and knockout, serum inflammatory factors CCL6 and CXCL11 were significantly upregulated;

[0043] b: After 12 weeks of induction and knockout, upstream inflammatory factors were upregulated in the muscles of model mice, while downstream inflammatory factors were mainly upregulated in the lungs and liver.

[0044] Figure 4 This application illustrates the lung cancer and COPD phenotypes in a small molecule Anta reversible model mouse model according to one embodiment of the present application:

[0045] a: Appearance of mice after treatment with succinic acid antagonist (Anta);

[0046] bd: After Anta treatment, the mice regained their body weight (b), the rate of hair loss decreased (c), and the rate of arched back decreased (d).

[0047] e: After Anta treatment, the running distance and running time of mice were effectively prolonged;

[0048] f: After Anta treatment, HE staining showed that the thickening of the lung airways in mice was reversed;

[0049] g: After Anta treatment, PAS staining showed a decrease in the proliferation of goblet cells (purple-red) in the mouse lungs;

[0050] h: After Anta treatment, SCGB1A1 / CC16 immunofluorescence staining showed that CC16 secretion in mouse lung club cells was restored;

[0051] i: After Anta treatment, CD45 immunohistochemistry showed a reduction in the infiltration of immune cells in the mouse lungs. Detailed Implementation

[0052] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0054] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0055] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0056] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0057] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0058] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it refers to either "with" or "without" a parallel solution. If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."

[0059] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0060] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0061] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0062] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0063] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0064] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0065] The inventors of this application have creatively simulated the natural aging process of muscles by inducing knockout of Tet1 in adult muscle stem cells. Muscle aging and lung-related disease (lung cancer, COPD) phenotypes caused by the disorder of communication in the "muscle-lung" axis can be induced in just 4 months, thus constructing a disease model that is closer to the clinical characteristics of chronic lung disease in the elderly.

[0066] This application provides an exemplary method and application for constructing a lung-related disease model that simulates the imbalance of muscle-lung axis communication homeostasis. By specifically knocking out aging regulatory genes in muscles, the aging phenotype is simulated in muscles, thereby constructing a disease model that is closer to the clinical characteristics of chronic lung disease in the elderly.

[0067] One aspect of this application provides a method for constructing a lung-related disease model simulating muscle-lung axis communication homeostasis disorder, the method comprising:

[0068] Specifically inhibiting the expression of target genes in muscle satellite cells (also known as "muscle stem cells") of adult non-human mammals to induce muscle aging phenotypes and secondary lung-related disease phenotypes.

[0069] In some embodiments, the target genes include DNA dioxygenase family genes.

[0070] For example, the DNA dioxygenase family genes include one or more of Tet1, Tet2, and Tet3.

[0071] In some embodiments, the DNA dioxygenase family gene is Tet1.

[0072] In some embodiments, the specific inhibition of target gene expression in muscle satellite cells of adult non-human mammals includes specific knockout and / or knockdown of the target gene.

[0073] For example, specific knockout and / or knockdown of the target gene is achieved using the Cre-LoxP system.

[0074] In some implementations, the construction method includes the following steps:

[0075] S100. Provide a first transgenic animal: the genome of the first transgenic animal contains an inducible Cre recombinase expression cassette regulated by a muscle satellite cell-specific promoter;

[0076] S200. Provide a second transgenic animal: the genome of the second transgenic animal contains the target gene, and the target gene is flanked by LoxP sites;

[0077] S300. The first transgenic animal is crossed with the second transgenic animal to obtain offspring animals; the genome of the offspring animals simultaneously contains the inducible Cre recombinase expression cassette regulated by the muscle satellite cell specific promoter and the target gene flanked by LoxP sites;

[0078] S400. The offspring animals are cultured to adulthood, and an inducer is applied to activate Cre recombinase activity to achieve specific knockout of the target gene in muscle satellite cells. The animals are then cultured to obtain a lung-related disease model that simulates muscle-lung axis communication homeostasis disorder.

[0079] In some embodiments, in step S100, the muscle satellite cell-specific promoter includes the Pax7 promoter.

[0080] In some embodiments, in step S100, the inducible Cre recombinase comprises the Cre-ERT2 fusion protein; exemplaryly, the Cre-ERT2 fusion protein comprises the sequence shown in SEQ ID NO: 1.

[0081] The Cre-ERT2 fusion protein is an inducible site-specific recombinase created by fusing a Cre recombinase derived from bacteriophage P1 with a mutant estrogen receptor ligand-binding domain (ERT2). Its key characteristic is that the ERT2 mutant loses the ability to bind natural estrogen but exhibits high affinity for tamoxifen. In the absence of an inducer, this fusion protein binds to heat shock protein 90 (Hsp90) and is anchored in the cytoplasm. Upon administration of tamoxifen, the drug binds to ERT2, causing Hsp90 to dissociate, and the fusion protein enters the nucleus, catalyzing DNA recombination between two co-directional loxP sites, thereby achieving spatiotemporally specific knockout or activation of the target gene.

[0082] In some embodiments, in step S400, the inducing agent includes tamoxifen.

[0083] Non-human mammals used in this application include, but are not limited to, rodents.

[0084] For example, the rodent is selected from mice, rats and hamsters.

[0085] In some implementations, the construction method includes the following steps:

[0086] S100'. Provides the first transgenic mouse: whose genome contains an inducible Cre recombinase expression cassette regulated by a muscle satellite cell-specific promoter;

[0087] S200'. Provide a second transgenic mouse whose genome contains the target gene and is flanked by LoxP sites;

[0088] S300'. The first transgenic mouse is crossed with the second transgenic mouse to obtain offspring animals, which are double transgenic mice, containing the inducible Cre recombinase expression cassette regulated by the muscle satellite cell specific promoter and the target gene flanked by LoxP sites;

[0089] S400'. After culturing the double transgenic mice for 4 to 8 weeks until they reach adulthood, an inducer is applied to activate Cre recombinase activity, thereby achieving specific knockout of the target gene in muscle satellite cells; after culturing for another 4 to 12 weeks, a lung-related disease model simulating muscle-lung axis communication homeostasis disorder is obtained.

[0090] For example, in step S400', the double transgenic mice are cultured for any duration of 4, 5, 6, 7, 8 weeks or between 4 and 8 weeks.

[0091] In some embodiments, the method of administering the inducer includes intraperitoneal injection of tamoxifen into adult double transgenic mice for five consecutive days at a dose of 75–100 mg / kg body weight / day. Exemplary examples include 75 mg / kg body weight / day, 80 mg / kg body weight / day, 85 mg / kg body weight / day, 90 mg / kg body weight / day, 95 mg / kg body weight / day, 100 mg / kg body weight / day, etc.

[0092] In another aspect of this application, the use of gene knockout specifically targeting adult induced muscle stem cells in constructing animal models of muscle aging that mimic lung-related diseases is provided.

[0093] Another aspect of this application provides a lung-related disease model (e.g., a lung cancer combined with COPD model) that simulates muscle-lung axis communication homeostasis disorder using the construction method described above.

[0094] Another aspect of this application provides the use of lung-related disease models (e.g., lung cancer combined with COPD models) that simulate muscle-lung axis communication homeostasis imbalance as described above in screening drugs for the treatment of aging-related diseases.

[0095] In some embodiments, the method of screening drugs for treating age-related diseases using the aforementioned lung-related disease model simulating muscle-lung axis communication homeostasis disorder includes:

[0096] S100''. The test drug was administered to a lung-related disease model simulating muscle-lung axis communication homeostasis disorder, and compared with a lung-related disease model that was not administered the test drug;

[0097] S200''. Drugs that can improve the symptoms of age-related diseases after administration are candidate drugs for the treatment of aging and related diseases.

[0098] In some implementations, age-related diseases include, but are not limited to, lung cancer, chronic obstructive pulmonary disease (COPD), sarcopenia, etc.

[0099] In some implementations, symptoms of age-related diseases include, but are not limited to, hair loss, hunchback, weight loss, muscle fiber atrophy, decreased motor function, decreased respiratory function, thickening of the lung airways, goblet cell hyperplasia, decreased secretion of CCl6 by Club cells, or infiltration of lung immune cells.

[0100] In some embodiments, the drug to be tested includes anti-inflammatory small molecules.

[0101] Unless otherwise specified, the term "anti-inflammatory small molecule" in this application refers to a class of non-peptide organic compounds with a molecular weight typically less than 10 Daltons, which can reduce or alleviate local inflammatory responses in the body by modulating inflammation-related signaling pathways (e.g., inhibiting the production of pro-inflammatory cytokines, blocking the activity of inflammatory mediators, or intervening in inflammation-related metabolites).

[0102] In another aspect of this application, a method for constructing an animal model of muscle aging simulating lung-related diseases is provided, the method comprising the following steps:

[0103] 1. Constructing experimental animals carrying the inducible Cre recombinase system: Select transgenic mice carrying tissue-specific inducible Cre recombinase (Cre-ERT2);

[0104] The inducible Cre recombinase may be a Cre-ERT2 fusion protein, and the inducer may be tamoxifen.

[0105] The tissue-specific promoter of the inducible Cre recombinase may be a muscle stem cell-specific promoter (Pax7).

[0106] 2. Constructing conditional knockout mice of the target gene: Select conditional knockout (Flox) mice with LoxP sites flanking the target gene;

[0107] The target gene may be a DNA dioxygenase family / DNA active demethylase, Tet1, Tet2, or Tet3.

[0108] 3. Hybridization to obtain double transgenic mice: The mice from step 1 and step 2 are hybridized to obtain offspring mice that carry both the Pax7-Cre-ERT2 and Tet1-Flox target genes;

[0109] 4. Adult-inducible knockout: When offspring mice reach adulthood (4-8 weeks old), they are treated with an inducer to activate Cre recombinase activity and achieve specific knockout of the target gene;

[0110] For example, the specific protocol for adult induction is as follows: 4-8 week old mice are intraperitoneally injected with tamoxifen for 5 consecutive days at a dose of 75-100 mg / kg body weight / day. For instance, 6-week-old mice are intraperitoneally injected with tamoxifen for 5 consecutive days at a dose of 80 mg / kg body weight / day.

[0111] 5. Model maintenance and validation: After induction, animals were fed for 4-12 weeks to obtain an animal model of muscle aging simulating lung-related diseases (lung cancer, COPD).

[0112] In some implementations, the rearing period after the induction is 12 weeks.

[0113] In some implementations, the criteria for determining the success of the model are exemplarily as follows: (1) high Tet1 knockout efficiency in muscle stem cells; (2) mice exhibiting aging phenotypes such as hair loss, hunchback, and weight loss; (3) mice exhibiting muscle aging phenotypes such as thinning / atrophy of muscle fibers and decreased motor function; (4) mice exhibiting lung cancer combined with COPD phenotypes such as decreased respiratory function, thickened airways, abnormal proliferation of goblet cells, abnormal downregulation of CC16 expression, reduced secretion of CC16 by club cells, and immune cell infiltration; (5) significantly elevated levels of pro-inflammatory factors in peripheral blood and tissues such as skeletal muscle, lungs, and liver, with an increase of more than 2 times.

[0114] The following are some examples.

[0115] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0116] Example 1: Constructing a muscle aging model simulating lung-related diseases based on Tet1 knockout of adult induced muscular stem cells

[0117] (1) Animal strains:

[0118] Tet1 flox Mice (target gene flanked by LoxP sites);

[0119] Pax7 Cre-ERT2 Mouse (muscle stem cell specific, tamoxifen-induced Cre).

[0120] (2) Breeding:

[0121] Tet1 was obtained through hybridization flox / Tet1 flox Pax7 Cre-ERT2 / Pax7 CreERT2 ;

[0122] All mice were normally fed until 6 weeks of age before induction.

[0123] (3) Induced knockout:

[0124] Mice were injected intraperitoneally with oil (control group) or tamoxifen (model group) for 5 consecutive days. Tamoxifen was dissolved in corn oil at a dose of 80 mg / kg / day.

[0125] After the injection, medication was discontinued and the animals were observed, then fed a normal diet.

[0126] (4) Model validation (12 weeks after induction, i.e., 18 weeks of age):

[0127] • Tet1 knockout efficiency: Muscle stem cells were sorted, and total RNA was extracted after adding Trizol. The RNA was then reverse-transcribed into cDNA using NEB MuLV reverse transcriptase. The cDNA was used as a template, and qPCR was performed to detect the expression of the target gene using the forward and reverse primers listed below. GAPDH was used as an internal control, and the primer sequences used were as follows:

[0128] mGapdh Forward, 5'-GCCAGCCTCGTCCCGTAGACA-3' (SEQ ID NO: 2);

[0129] mGapdh Reverse, 5'-CAACAATCTCCACTTTGCCACTGC-3' (SEQ ID NO: 3).

[0130] Simultaneously, the expression of the inflammatory gene Tet1 was detected. The primer sequences used were:

[0131] mTet1 Forward, 5'-ACAACATGCACAACGGAAGC-3' (SEQ ID NO: 4);

[0132] mTet1 Reverse, 5'-TTGGCCTTCATCCCTTCCAC-3' (SEQ ID NO: 5).

[0133] The results are as follows Figure 1 As shown in Figure a, Tet1 mRNA levels decreased by 81%.

[0134] • Mouse appearance and weight examination: The appearance and weight of the mice were observed, photographed, and measured. Results are as follows: Figure 1 As shown in the figure, mice exhibited premature aging phenotypes such as hair loss, weight loss, and arched back 12 weeks after induction.

[0135] • Muscle aging verification:

[0136] Muscle strength and motor ability testing: Mice were dissected in situ to expose the hind leg muscles, and muscle strength was measured in situ using a 1300A 3-in-1 whole animal system (Aurora Scientific, Canada). Simultaneously, mice were placed on a treadmill and run at 10 m / min for 10 minutes, followed by 20 m / min. The running distance and time to exhaustion were recorded. Results are as follows: Figure 1 As shown in Figure 1, compared with the control group, the model group showed a significant decrease in skeletal muscle strength and a decrease in running ability of approximately 50%.

[0137] Muscle fiber diameter measurement: Mouse TA (transferase) sections were prepared into frozen sections. Hematoxylin-eosin (HE) staining was used to visualize the muscle fiber outline and compare changes in muscle fiber diameter. The staining method was as follows: Frozen sections were washed twice with PBS, 5 minutes each time; hematoxylin staining for 1 minute, followed by rinsing with tap water for 20 minutes; incubation in 30% ethanol for 5 minutes; incubation in 50% ethanol for 5 minutes; incubation in 70% ethanol for 5 minutes; incubation in 95% ethanol for 5 minutes; staining with eosin for 10 seconds; incubation in 95% ethanol for 5 minutes; incubation in 100% ethanol for 5 minutes; incubation in xylene:ethanol = 1:1 solution for 10 minutes; incubation in xylene for 10 minutes; after drying, the sections were mounted with resin. Microscopic images were taken, and the results are shown below. Figure 1 As shown in Figure j, the muscle fiber diameter in the model group was significantly smaller than that in the control group.

[0138] The results above demonstrate that the model mice successfully simulated muscle aging / sarcopenia.

[0139] • Mouse lung function testing: Mice were anesthetized and lung function, such as tidal volume, dynamic compliance, and respiratory rate, was measured using the Buxco Pulmonary Function Testing (PFT) system (a brand of Data Sciences International, USA). Data were recorded and analyzed. Results are as follows: Figure 2 As shown in the results, compared with the control group, the model group showed a significant decrease in tidal volume and lung dynamic compliance, while the respiratory rate increased slightly.

[0140] • Detection of Scgb1a1 / CC16 expression in lung tissue: RNA transcriptome sequencing and data analysis were performed on lung tissue. Methods: Anatomical model and control lungs were dissected, and RNA was extracted using Trizol. Library construction was performed using the Illumina TruSeq RNA Library Preparation Kit. Sequencing was performed using an Illumina HiSeq 2000 instrument. FastQC was used for quality control of the sequenced data. Trimmomatic_0.39 was used to remove adapters. Hisat2 v.2.1.0 was used to align the data to the GRCm39 reference genome. FeatureCounts v.2.0.1 was used to count gene reads. DESeq2 was used for normalization and differential expression analysis. Prism 10 was used to plot the normalized Scgb1a1 gene reads. Results are as follows: Figure 2 As shown in g, the expression level of Scgb1a1 in the model group was significantly downregulated.

[0141] • Lung histological analysis: Mouse lungs were collected and prepared into frozen or paraffin sections. Hematoxylin-eosin (HE) staining was used to visualize the lung tissue outline and compare airway changes. The staining method was as follows: Frozen sections were washed twice with PBS, 5 minutes each time; hematoxylin staining for 1 minute, followed by rinsing with tap water for 20 minutes; incubation in 30% ethanol for 5 minutes; incubation in 50% ethanol for 5 minutes; incubation in 70% ethanol for 5 minutes; incubation in 95% ethanol for 5 minutes; staining with eosin for 10 seconds; incubation in 95% ethanol for 5 minutes; incubation in 100% ethanol for 5 minutes; incubation in xylene:ethanol = 1:1 solution for 10 minutes; incubation in xylene for 10 minutes; after drying, the sections were mounted with resin. Microscopic images were taken, and the results are shown below. Figure 2 As shown in the figure, the airways of the model group mice were thickened and there was obvious abnormal cell proliferation in the alveoli.

[0142] Paraffin sections of mouse lung tissue were stained with PAS using Servicebio G1285. The staining method was as follows: dewaxing to water, washing with distilled water for 2 min; staining with Alcian blue for 10-20 min; washing with distilled water 3 times, 1-2 min each time; oxidizing in periodic acid solution for 5 min; immersion in Schiff Reagent for 10-20 min; discarding Schiff Reagent, rinsing with running water for 10 min; staining the nuclei with hematoxylin for 1-2 min; differentiating with acidic differentiation solution for 2-5 s, washing with water; re-blueing with Scott's blue solution, washing with water for 3 min; dehydrating with ethanol stepwise, clearing with xylene, and mounting with neutral resin. The images were taken under a microscope, and the results are shown below. Figure 2 As shown in f, abnormal proliferation of goblet cells was observed in the lungs of the model group.

[0143] Frozen sections of mouse lung tissue were subjected to immunofluorescence / histochemical analysis for SCGB1A1 or CD45. The specific method was as follows: fixation with 4% paraformaldehyde for 10 minutes; washing three times with PBS, 5 minutes each time; drawing water loops; blocking with 5% BSA + 5% goat serum at room temperature for 60 minutes; drying the blocking solution; adding SCGB1A1 or CD45 primary antibody working solution, incubating overnight at 4°C in a humidified chamber; washing three times with PBS, 5 minutes each time; adding secondary antibody working solution, incubating at room temperature for 1 hour; washing three times with PBS, 5 minutes each time; DAB staining; counterstaining cell nuclei with hematoxylin for 5 minutes; rinsing with running water; dehydration with graded ethanol; clearing with xylene; mounting with neutral resin (Note: from DAB staining onwards, SCGB1A1 fluorescence staining does not require subsequent steps). Images were taken under a microscope, and the number of SCGB1A1 or CD45 positive cells was counted. Results are as follows. Figure 2 As shown in the figure, compared with the control group, the model mice showed significantly reduced secretion of lung club cells SCGB1A1 / CC16 and increased immune cell infiltration.

[0144] The results above indicate that, compared with control mice, the model mice exhibit typical pathological features of lung cancer combined with COPD.

[0145] • Axis communication validation: Serum was tested by ELISA. The method was as follows: Abcam products ab277715 and ab204519 were used, and the detection was performed according to the instructions. RNA transcriptome sequencing and data analysis were performed on multiple organs. The method was as follows: RNA was extracted from the dissected model group and control muscle, lung, and liver using Trizol; library construction was performed using the Illumina TruSeq RNALibrary Preparation Kit; sequencing was performed using an Illumina HiSeq 2000 instrument; FastQC was used for quality control of the data; adapters were removed using Trimmomatic_0.39; data were aligned to the GRCm39 reference genome using Hisat2 v.2.1.0; gene counts were performed using FeatureCounts v.2.0.1; normalization and differential expression analysis were performed using DESeq2; gene function enrichment analysis was performed using R(4.4.2) ggplot2 and Phyper.

[0146] The results are as follows Figure 3 As shown in ab. Figure 3 In the model group, compared with the control group, the serum levels of inflammatory factors CCL6 increased by 2 times and CXCL11 increased by approximately 2.8 times. Figure 3 In the model group compared with the control group, upstream inflammatory factors in muscle were upregulated, while downstream inflammatory factors in lung and liver were mainly upregulated, indicating that the inflammatory factors originated from muscle tissue and acted on tissues such as lung and liver through secretion.

[0147] These results demonstrate that the model mice simulated the aging background through muscle-specific gene knockout and proved that muscle aging exacerbates lung diseases such as lung cancer and COPD, simulating the imbalance of the "muscle-lung" axis homeostasis.

[0148] Example 2: Application of the model in anti-aging drug screening

[0149] Using the aging animal model prepared in Example 1 as the test subjects, the morphological, functional, and histological changes of mice before and after the administration of the candidate drug were detected, so as to determine whether the candidate drug is effective in treating sarcopenia, lung cancer, and COPD.

[0150] Test group: Models given the candidate drug;

[0151] Control group: Models that did not receive the candidate drug.

[0152] If, compared with the control group, mice in the test group show reduced hair loss, reduced arching of the back, increased weight, increased running distance and time, reduced thickening of the lung airways, reduced goblet cell proliferation, and increased secretion of club cell CC16, it indicates that the drug can inhibit the progression of age-related sarcopenia, lung cancer, and COPD.

[0153] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0154] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing a lung-related disease model simulating muscle-lung axis communication homeostasis imbalance, characterized in that, The construction method includes: Specifically inhibiting the expression of target genes in muscle satellite cells of adult non-human mammals to induce muscle aging phenotypes and secondary lung-related disease phenotypes.

2. The construction method as described in claim 1, characterized in that, The target genes include DNA dioxygenase family genes; Optionally, the DNA dioxygenase family genes include one or more of Tet1, Tet2, and Tet3.

3. The construction method as described in claim 1 or 2, characterized in that, The specific inhibition of target gene expression in muscle satellite cells of adult non-human mammals includes specific knockout and / or knockdown of the target gene. Optionally, specific knockout and / or knockdown of the target gene is achieved using the Cre-LoxP system.

4. The construction method as described in claim 3, characterized in that, It includes the following steps: Provide a first transgenic animal: The genome of the first transgenic animal contains an inducible Cre recombinase expression cassette regulated by a muscle satellite cell-specific promoter; Provide a second transgenic animal: the genome of the second transgenic animal contains the target gene, and the target gene is flanked by LoxP sites; The first transgenic animal is crossed with the second transgenic animal to obtain offspring animals; the genome of the offspring animals simultaneously contains the inducible Cre recombinase expression cassette regulated by the muscle satellite cell specific promoter and the target gene flanked by LoxP sites; The offspring animals were cultured to adulthood, and an inducer was applied to activate Cre recombinase activity, thereby achieving specific knockout of the target gene in muscle satellite cells. The animals were then cultured to obtain a lung-related disease model that simulates muscle-lung axis communication homeostasis disorder.

5. The construction method as described in claim 4, characterized in that, In the process of providing the first transgenic animal: The muscle satellite cell-specific promoters include the Pax7 promoter; and / or, The inducible Cre recombinase includes the Cre-ERT2 fusion protein; optionally, the Cre-ERT2 fusion protein includes the sequence shown in SEQ ID NO:

1.

6. The construction method as described in claim 4 or 5, characterized in that, In the step of obtaining a lung-related disease model simulating muscle-lung axis communication homeostasis disorder, the inducing agent includes tamoxifen.

7. The construction method according to any one of claims 1 to 6, characterized in that, The non-human mammals referred to are rodents; Optionally, the rodent is selected from mice, rats, and hamsters; Further optionally, the continued culture period is 4 to 12 weeks; wherein the method of administering the inducer may optionally include: intraperitoneal injection of tamoxifen into offspring animals aged 4 to 8 weeks for 5 consecutive days at a dose of 75 to 100 mg / kg body weight / day.

8. The use of the lung-related disease model simulating muscle-lung axis communication homeostasis disorder constructed by the construction method of any one of claims 1 to 7 in screening drugs for the treatment of age-related diseases.

9. The use as described in claim 8, characterized in that, The age-related diseases include lung cancer, COPD, and / or sarcopenia; and / or, The uses include: The test drug was administered to a lung-related disease model simulating muscle-lung axis communication homeostasis dysregulation and compared with a lung-related disease model that was not administered the test drug. Drugs that can improve the symptoms of age-related diseases after administration are candidate drugs for the treatment of aging and related diseases; Optionally, the symptoms of the age-related diseases include one or more of the following: hair loss, hunchback, weight loss, muscle fiber atrophy, decreased motor function, decreased respiratory function, thickening of the lung airways, proliferation of lung goblet cells, downregulation of lung C16 expression, decreased secretion of lung Club cells C16, and infiltration of lung immune cells. Optionally, the drug to be tested includes anti-inflammatory small molecules.

10. The use of animal models that mimic muscle-lung axis communication homeostasis dysregulation by muscle-specific knockout of the Tet1 gene in screening drugs that regulate muscle-lung axis signaling pathways.

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