Pharmaceutical compositions and uses thereof

Abstract

The present application relates to a kind of reagent in the use of preparation of medicine, pharmaceutical composition and its use and the method for promoting potential regeneration.The use of reagent for inhibiting the expression or activity of Lonp1 gene in preparation of medicine, the medicine is used to promote crypt regeneration, and the medicine is used to anti-aging or treat senile-related diseases;Pharmaceutical composition includes: reagent for inhibiting the expression or activity of Lonp1 gene;Method includes: organoid is contacted with reagent, reagent is used to inhibit the expression or activity of Lonp1 gene, and organoid has the potential of generating crypt.This pharmaceutical composition can promote crypt regeneration, effectively used in anti-aging or treat senile-related diseases;And, when culturing crypt differentiation formation organoid in vitro, the above-mentioned method can inhibit the expression or activity of Lonp1 gene, can increase the number and size of organoid, effectively promote crypt regeneration in organoid, can be used in crypt regeneration-related experimental in vitro study.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, specifically to a pharmaceutical composition and its use, and more specifically to the use of a reagent in the preparation of a drug, a pharmaceutical composition and its use, and a method for promoting potential regeneration. Background Technology

[0002] Aging is a universal law in the biological world, an inevitable natural decline that occurs over time. Oxidative damage theory is one of the main theories governing the aging mechanism. During the aging process, the gut's homeostasis is disrupted, and gut function weakens, which seriously affects the body's health.

[0003] Therefore, there is an urgent need to develop a drug that can combat aging or treat aging-related diseases. Summary of the Invention

[0004] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, the present invention provides the use of an agent for inhibiting the expression or activity of the Lonp1 gene in the preparation of a pharmaceutical, a pharmaceutical composition thereof, and a method for promoting crypt regeneration. The pharmaceutical and pharmaceutical compositions of the present invention can be used to promote crypt regeneration, anti-aging, and / or treat age-related diseases.

[0005] This invention is based on the following discoveries of the inventors:

[0006] Mitochondria, as the center of cellular energy metabolism, oxygen consumption, superoxide radical production, and apoptosis regulation, play a crucial role in cellular senescence, particularly in adult stem cell senescence and the maintenance of stemness. The accumulation of mitochondrial DNA mutations and oxidative damage is considered a significant contributor to tissue aging, and the decline in both the quality and quantity of mitochondrial DNA is closely related to cellular senescence and individual lifespan. Based on the understanding of mitochondrial regulation of small intestinal senescence, this study not only provides new insights into the mechanisms of mitochondria and aging but also offers a new theoretical basis for the development of mitochondrial-targeted drugs and the treatment of age-related diseases.

[0007] Mitochondrial ionopeptidase 1 (also known as Lonp1) is an ATP-dependent protein encoded by nuclear DNA, located in the mitochondrial matrix, and plays an important role in regulating the expression of mitochondrial genes and maintaining mitochondrial stability.

[0008] Based on this, the inventors made a surprising discovery through experiments: during the process of inducing the differentiation of small intestinal crypts into small intestinal organoids in mice, knocking down the expression or activity level of the Lonp1 gene can increase the number and size of organoids, and has the ability to promote crypt regeneration in organoids, thus resisting small intestinal aging. Therefore, by regulating the expression or activity level of the Lonp1 gene in cells, crypt regeneration capacity can be promoted, providing potential for anti-aging or the treatment of age-related diseases.

[0009] In one aspect of the invention, a reagent is proposed for use in the preparation of a drug, said reagent being used to inhibit the expression or activity of the Lonp1 gene, and said drug being used to promote crypt regeneration. The inventors have experimentally discovered that the above-mentioned reagent can inhibit the expression or activity of the Lonp1 gene, effectively promoting crypt regeneration, and can be used in in vitro studies related to crypt regeneration.

[0010] According to an embodiment of the present invention, the crypts are derived from the tonsils, large intestine and / or small intestine.

[0011] According to an embodiment of the present invention, the crypts originate from the duodenum, jejunum, and / or ileum of the small intestine.

[0012] According to an embodiment of the present invention, the reagent comprises siRNA for knocking down the Lonp1 gene.

[0013] According to embodiments of the present invention, the siRNA has a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0014] GGAACACTATCGGGACATA (SEQ ID NO: 1).

[0015] GGGATATCATCGCCTTGAA (SEQ ID NO: 2).

[0016] In another aspect of the invention, the invention proposes the use of a reagent in the preparation of a drug, said reagent being used to inhibit the expression or activity of the Lonp1 gene, said drug being used for anti-aging and / or treatment or prevention of age-related diseases. The inventors have experimentally discovered that using the above-mentioned reagent to enhance the self-renewal and regeneration of crypt functional units of stem cells can be effectively used for anti-aging or treatment or prevention of age-related diseases.

[0017] According to an embodiment of the present invention, the aging is caused by the accumulation of mitochondrial DNA mutations.

[0018] According to an embodiment of the present invention, the reagent comprises siRNA for knocking down the Lonp1 gene.

[0019] According to embodiments of the present invention, the siRNA has a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0020] In another aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: a reagent that inhibits the expression or activity of the Lonp1 gene. The inventors have experimentally discovered that the above-mentioned pharmaceutical composition can promote crypt regeneration, effectively treating aging or treating or preventing age-related diseases. Furthermore, when culturing crypt-differentiated organoids in vitro, the above-mentioned pharmaceutical composition can promote crypt regeneration and can be used for in vitro studies related to crypt regeneration.

[0021] According to an embodiment of the present invention, the pharmaceutical composition further comprises: pharmaceutically acceptable excipients.

[0022] According to an embodiment of the present invention, the reagent includes shRNA or siRNA for knocking down the Lonp1 gene.

[0023] According to an embodiment of the present invention, the shRNA has a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0024] In another aspect, the present invention provides the use of the aforementioned pharmaceutical composition in the preparation of a medicament for anti-aging or treatment or prevention of age-related diseases. The inventors have experimentally discovered that the above-mentioned pharmaceutical composition can promote crypt regeneration and is effectively used for anti-aging or treatment or prevention of age-related diseases.

[0025] According to an embodiment of the present invention, the aging is caused by the accumulation of mitochondrial DNA mutations.

[0026] In another aspect of the invention, the present invention provides the use of the aforementioned pharmaceutical composition in the preparation of a drug for promoting crypt regeneration. The inventors have experimentally discovered that the above-mentioned reagent can inhibit the expression or activity of the Lonp1 gene, effectively promoting crypt regeneration, and can be used in in vitro studies related to crypt regeneration.

[0027] In another aspect, the present invention proposes a method for promoting crypt regeneration. According to an embodiment of the invention, the method includes: contacting organoids with a reagent used to inhibit the expression or activity of the Lonp1 gene, wherein the organoids have the potential to generate crypts. The inventors have experimentally discovered that, when crypt-differentiated organoids are cultured in vitro, contacting the organoids with the aforementioned reagent can inhibit the expression or activity of the Lonp1 gene, increase the number and size of organoids, and effectively promote crypt regeneration within the organoids. This method can be used for in vitro studies of organoid research and experiments related to crypt regeneration.

[0028] According to an embodiment of the present invention, the organoid is a large intestine organoid and / or a small intestine organoid.

[0029] According to an embodiment of the present invention, the reagent comprises siRNA for knocking down the Lonp1 gene.

[0030] According to embodiments of the present invention, the siRNA has a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0031] According to an embodiment of the present invention, the organoid is obtained by culturing or purchasing stem cells.

[0032] According to an embodiment of the present invention, the stem cells are small intestinal stem cells or stem cell lines.

[0033] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0034] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0035] Figure 1 This is the verification result of the siLonp1 knockdown efficiency in Embodiment 1 of the present invention;

[0036] Figure 2 This is the verification result of the ability of knocking down Lonp1 to promote crypt regeneration in Example 2 of the present invention. Detailed Implementation

[0037] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0038] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0039] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0040] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0041] In this document, "pharmaceutical composition" can refer to a drug for the treatment of a disease or for use in in vitro cell culture experiments. When used for the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients that constitute one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining the active compound with liquid excipients, finely chopped solid excipients, or both.

[0042] In this document, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0043] In this document, the terms "pharmaceuticalally acceptable excipient" or "pharmaceuticalally acceptable carrier" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0044] In this document, the term "treatment" means used to refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0045] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0046] Example 1: Design of siRNA to knock down Lonp1

[0047] The inventors reduced the expression of LONP1 protein in mouse fibroblasts using siRNA (siLonp1#1: GGAACACTATCGGGACATA (SEQ ID NO:1) and siLonp1#2: GGGATATCATCGCCTTGAA (SEQ ID NO:2)). The control group used the siRNA sequence siNC: TTCTCCGAACGTGTCACGT (SEQ ID NO:3). The specific experimental steps are as follows: Results are shown... Figure 1 As shown, the knockdown efficiency of LONP1 protein by siLonp1 (siLonp1#1 and siLonp1#2) was verified, with siNC serving as the control group (n=5); *P<0.05, **P<0.01. The results showed that siLonp1, as shown in SEQ ID NO:1, effectively reduced the expression of LONP1 protein in cells, with significant and specific effects. Figure 1 In the bar chart for relative protein level detection, LONP1 and HSP60, from left to right, represent siNC, siLonp1#1, and siLonp1#2 groups, respectively; n represents the number of biological replicate experiments, as in Example 2. The specific steps are as follows:

[0048] Mouse fibroblasts were first cultured in DMEM complete medium (DMEM high-glucose medium + 10% NTC fetal bovine serum + 100×MEM Non-Essential Amino Acids Solution + 100×GlutMax additive). When the required cell number was reached, the cells were digested, passaged, counted, and seeded into 6-well plates at 300,000 cells per well. The cells were then cultured at 37°C in a 5% CO2 incubator. After cell adhesion (6-12 hours later), the procedure was performed according to the Lipofectamine™ RNAiMAX transfection kit (Lot #13778075). First, each siRNA was diluted to 10 μM with DEPC-treated and autoclaved ultrapure water. 18 μL of the solution was then added to the solution. RNAiMAX Reagent was added to a 150 μL container. Add 3 μL of siRNA (total amount 30 pmol) to another 1.5 mL EP tube containing medium to a separate tube containing 150 μL of medium. The mixture was transferred to a 1.5 mL EP tube of medium. Then, 150 μL of the liquid from each of the two uniformly mixed EP tubes was added to a new EP tube and incubated at room temperature for 5 minutes. 250 μL of the mixture was then evenly added dropwise to a 6-well plate containing cells. After 48 hours, the DMEM complete medium was changed daily, and the cells were cultured for another day. Protein was collected, and the expression of LONP1 protein was detected by Western blotting. Actin was used as an internal control.

[0049] Example 2: Knockdown of LONP1 protein promotes crypt regeneration

[0050] The inventors isolated crypts from the small intestine of mice with premature aging caused by accumulated mitochondrial DNA mutations and differentiated them into organoids. In this process, siLonp1#1 (SEQ ID NO:1), siLonp1#2 (SEQ ID NO:2), and the control siNC (SEQ ID NO:3) were transfected, with the only difference being the added siRNA. See the following steps for details:

[0051] (1) Small intestinal crypt isolation. Mice were dissected to obtain a segment of the small intestine and jejunum. The chyme in the intestinal segment was rinsed with pre-cooled DPBS. The small intestine was then cut into 0.5-1 cm long tissue fragments and placed in a centrifuge tube containing 10 mL of 2 mM EDTA-PBS solution. The tube was rapidly vortexed at 4°C for 20 minutes. The small intestinal crypts were then separated by vortexing for 2 minutes. The suspension in the centrifuge tube was then passed through a 70 μm filter. The collected liquid was centrifuged twice, at 110 x g and 84 x g, to obtain crypt tissue pellets, completing the small intestinal crypt isolation. The pellets were then separated using 5 mL of Gibco solution. TM Resuspended in DMEM / F-12 medium.

[0052] (2) Crypt implantation. 10 μL of the crypt suspension from step (1) was added to 50 μL of Matrigel and gently mixed. Then, the Matrigel-crypt solution was injected into the center of one well of a 24-well plate. The 24-well plate was then placed in a cell culture incubator and incubated for 10 minutes. After the Matirgel crypt solution forms gel-like droplets, add 1 mL of small intestinal organoid differentiation medium (DMEM / F12 (Hyclone) + 1% L-glutamine (Invitrogen) + 1% penicillin / strep (Invitrogen) + 10 μM HEPES (Invitrogen) + 1% N2 supplement (R&D Systems) + 2% B27 supplement (Invitrogen) + 0.5 μg / ml Rspo1 (R&D Systems) + 0.1 μg / ml Noggin (R&D Systems) + 0.1 μg / ml EGF (R&D Systems) + 0.1 μg / ml Wnt3a (R&D Systems)) to the culture wells.

[0053] (3) siRNA treatment. According to Lipofectamine... TM The experimental procedures for the RNAiMAX transfection kit (Lot #13778075) were performed as follows: First, dilute each siRNA to a concentration of 10 μM with DEPC water, and then add 18 μL of... RNAiMAX Reagent was added to a 150 μL container. Add 6 μL of siRNA (total amount 60 pmol) to another 1.5 mL EP tube containing medium to a separate tube containing 150 μL of medium. The mixture was transferred to a 1.5 mL EP tube of medium. Next, 150 μL of the liquid from each of the two uniformly mixed EP tubes was added to a new EP tube and incubated at room temperature for 5 minutes. Then, 250 μL of the mixture was evenly added dropwise to the culture wells containing Matrigel crypts obtained in step (2). After 48 hours, 500 μL of fresh small intestinal organoid differentiation medium was replaced daily. After seven days of culture following the addition of siRNA treatment, the organoid morphology was observed and recorded using an inverted microscope.

[0054] The results are as follows Figure 2As shown, **P < 0.01. The results showed that, compared to the control siNC, knocking down LONP1 protein with siLonp1 (siLonp1#1 and siLonp1#2) increased the size and number of organoids on day 8, indicating that knocking down LONP1 protein effectively salvaged the differentiation degree of organoids, improved the stemness of small intestinal stem cells in the crypts, and thus promoted crypt regeneration. Therefore, knocking down LONP1 expression or activity has anti-aging and age-related disease-treating effects.

[0055] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0056] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of the reagent in the preparation of a drug for inhibiting small intestinal aging, said reagent being used to inhibit... Lonp1 Gene expression or activity, the drug is used to promote crypt regeneration; the reagent is used to knock down the gene. Lonp1 The siRNA of the gene; the nucleotide sequence of the siRNA is shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. A pharmaceutical composition, characterized in that, include: inhibition LONP1 Reagents for gene expression or activity; The reagent is used for knockdown. Lonp1 The siRNA of the gene; the nucleotide sequence of the siRNA is shown in SEQ ID NO:1 or SEQ ID NO:

2.

3. The pharmaceutical composition according to claim 2, characterized in that, Further includes: Pharmaceutically acceptable excipients.

4. Use of the pharmaceutical composition of claim 2 or 3 in the preparation of a medicament for combating small intestinal aging, wherein the aging is caused by the accumulation of mitochondrial DNA mutations.

5. The use according to claim 4, characterized in that, The drug is used to promote crypt regeneration.

6. A method for promoting the regeneration of small intestinal crypts in vitro, characterized in that, include: The small intestinal crypts are brought into contact with a reagent, wherein the reagent is siRNA with a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2.

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