Application of SIRT1 activator in preparation of anti-inflammatory immune drugs

Abstract

The invention belongs to the field of biotechnology and medicine, discloses application of an SIRT1 protein activator in preparation of an anti-inflammatory immune drug, and creatively provides application of the SIRT1 protein activator in preparation of the anti-inflammatory immune drug. The SIRT1 activator is especially applied to drugs for treating autoimmune diseases with external secretory gland lymphocyte infiltration and I-type interferon (IFN) characteristic excessive activation as the core, and the SIRT1 activator inhibits I-type interferon excessive activation through targeted regulation, so that the SIRT1 activator has a good clinical application prospect in relieving and treating sicca syndrome. And the method has a wide application scene and a practical practical value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and medicine, and particularly relates to application of a SIRT1 activator in preparation of anti-inflammatory immune drugs. BACKGROUND

[0002] Type I interferon response is an important anti-viral immune defense mechanism of the body, but its excessive activation can destroy immune tolerance and induce autoimmune response, forming autoimmune diseases. Sjogren's syndrome (SS) is a typical autoimmune disease with lymphocyte infiltration of exocrine glands and characteristic overexpression of type I interferon, and is divided into primary Sjogren's syndrome and secondary Sjogren's syndrome. Patients have symptoms of dry eyes and dry mouth due to damage to the lacrimal and salivary glands. With the progression of the disease, patients also have complications such as fatigue, pain, lymphoma and interstitial lung disease, which seriously affect the quality of life of patients.

[0003] Studies have shown that there is a significant overexpression of interferon-stimulated genes (ISGs) in the peripheral blood or affected tissues of SS patients, including characteristic molecules such as MX1, IFI44, ISG15 and IFIT3, which directly reflects the persistent activation of the type I IFN signaling pathway. In addition, the level of type I IFN is positively correlated with disease activity, and when patients have systemic involvement (such as interstitial lung disease, vasculitis, etc.), the content of type I IFN in serum is significantly increased, suggesting that abnormal activation of type I IFN plays a key role in the progression of the disease.

[0004] At present, there is no ideal drug for treating Sjogren's syndrome in clinical practice. Existing therapies (such as hydroxychloroquine and glucocorticoids) can only relieve symptoms such as dry mouth and dry eyes, and cannot correct immune disorders, and long-term use can easily cause adverse reactions such as infection and osteoporosis. As the second largest autoimmune disease, Sjogren's syndrome not only seriously affects the physical and mental health of patients, but also causes a huge economic burden. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides the application of a SIRT1 activator in preparation of anti-inflammatory immune drugs, to solve the problems in the background art.

[0006] The present application solves the technical problems by adopting the following technical solutions: The present application provides the application of a SIRT1 activator in preparation of anti-inflammatory immune drugs.

[0007] Preferably, the anti-inflammatory immune drug is a drug for treating autoimmune diseases with lymphocyte infiltration of exocrine glands and excessive activation of type I interferon (IFN) as the core.

[0008] Preferably, the anti-inflammatory immunological drug is a drug for treating Sjogren's syndrome.

[0009] Preferably, the SIRT1 activator comprises SRT1720 or a pharmaceutically acceptable salt or derivative thereof, wherein the chemical structural formula of SRT1720 is as follows:

[0010] Preferably, the drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0011] Preferably, the dosage form of the drug comprises an injection, a tablet, a granule, a capsule, a dripping pill or a sustained-release preparation.

[0012] Preferably, the Sjogren's syndrome comprises primary Sjogren's syndrome or secondary Sjogren's syndrome.

[0013] Preferably, the drug further comprises a JAK inhibitor or an immunomodulator.

[0014] The application also provides an application of a gene encoding a SIRT1 protein in the preparation of a drug for treating Sjogren's syndrome.

[0015] The application also provides an application of a biological material containing a gene encoding a SIRT1 protein in the preparation of a drug for treating Sjogren's syndrome, wherein the biological material comprises an expression vector or a host cell.

[0016] Compared with the prior art, the application has the following beneficial effects: The application creatively proposes an application of a SIRT1 activator in the preparation of an anti-inflammatory immunological drug, in particular, an application of the SIRT1 activator in the preparation of a drug for treating an autoimmune disease with lymphocyte infiltration of exocrine glands and excessive activation of type I interferon (IFN) as the core, mainly an application of the SIRT1 activator in the preparation of a drug for treating Sjogren's syndrome, wherein the most typical SIRT1 activator SRT1720 can effectively increase the expression of SIRT1 in submandibular gland tissue, and then reduce the protein level of IRF5 and the transcription level of Mx1, Ifi44, Isg15 and Ifit3, so as to effectively restore body weight, salivary flow rate and organ index. The application has a good clinical application prospect for treating Sjogren's syndrome, can achieve remarkable social benefits, has a wide application scene and practical value.

[0017] Other outstanding substantial features and significant progress of the application relative to the prior art are further described in detail in the embodiment part. BRIEF DESCRIPTION OF DRAWINGS

[0018] Other features, objects and advantages of the application will become more apparent from the following detailed description of non-limiting embodiments with reference to the following drawings: Figure 1 Histopathology of submandibular gland tissue of normal group, model group, SRT1720 group, and Prednisone acetate (Pre) group of mice in Example 1; Figure 2 Body weight of normal group, model group, SRT1720 group, and Prednisone acetate (Pre) group of mice in Example 1; Figure 3 Saliva volume of normal group, model group, SRT1720 group, and Prednisone acetate (Pre) group of mice in Example 1; Figure 4 Organ (submandibular gland, thymus, spleen) index of normal group, model group, SRT1720 group, and Prednisone acetate (Pre) group of mice in Example 1; Figure 5 Expression of SIRT1, TRIM21, and IRF5 in submandibular gland tissue of normal group, model group, SRT1720 group, and Prednisone acetate (Pre) group of mice in Example 1, detected by immunohistochemistry; Figure 6 Expression of SIRT1, TRIM21, IRF5, and α-Amylase in submandibular gland tissue of normal group, model group, SRT1720 group, and Prednisone acetate (Pre) group of mice in Example 1, detected by immunofluorescence; Figure 7 Expression of SIRT1, TRIM21, acetylated TRIM21, and IRF5 in submandibular gland tissue of normal group, model group, SRT1720 group, and Prednisone acetate (Pre) group of mice in Example 1, detected by immunoblotting; Figure 8 Expression of Mx1, Ifi44, Isg15, and Ifit3 in submandibular gland tissue of normal group, model group, SRT1720 group, and Prednisone acetate (Pre) group of mice in Example 1, detected by qPCR; Figure 9 Expression of IFN-α in serum of normal group, model group, SRT1720 group, and Prednisone acetate (Pre) group of mice in Example 1, detected by ELISA. DETAILED DESCRIPTION

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0020] This embodiment provides the application of SIRT1 activator in the preparation of anti-inflammatory immunotherapeutic drugs, which are autoimmune diseases with exocrine gland lymphocyte infiltration and type I interferon (IFN) overactivation as the core, mainly drugs for treating Sjögren's syndrome. Therefore, this embodiment takes drugs for treating Sjögren's syndrome as the main experimental verification object.

[0021] In this embodiment, SRT1720 was selected as the SIRT1 activator. Of course, its pharmaceutically acceptable salt or other SIRT1 activators can also be used, which will not be elaborated here. The molecular weight of SRT1720 is: C 25 H 23 N7OS has the following chemical structural formula:

[0022] The drugs in this embodiment also include pharmaceutically acceptable carriers and / or excipients, and the dosage forms of these drugs include injections, tablets, granules, capsules, pellets, or sustained-release formulations, which will not be elaborated here.

[0023] The drug in this embodiment also includes a JAK inhibitor or an immunomodulator.

[0024] In this embodiment, the SIRT1 activator SRT1720 can specifically activate SIRT1, promote TRIM21 deacetylation, inhibit IRF5 expression and transcription of interferon-stimulated genes, and at the same time reduce the level of IFN-α in the serum of model mice.

[0025] In this embodiment, anti-inflammatory immunity specifically refers to the treatment of Sjögren's syndrome, including primary Sjögren's syndrome and secondary Sjögren's syndrome. Primary Sjögren's syndrome is Sjögren's syndrome that exists independently without other known autoimmune diseases. Secondary Sjögren's syndrome is secondary to other diagnosed autoimmune diseases, with dryness symptoms appearing as a complication.

[0026] The Sjögren's syndrome mouse model in this embodiment is an antigen-induced Sjögren's syndrome mouse model. The SIRT1 activator SRT1720 also has a therapeutic effect on Sjögren's syndrome mouse models induced by other methods, which will not be listed here.

[0027] To verify the effectiveness of the present invention, a verification experiment was conducted in this embodiment. The materials used in the experiment included animals, drugs and reagents, and instruments, wherein: The animals were female C57BL / 6 mice, SPF grade, weighing 18g±2g; Purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; Laboratory animal production license: SCXK (Su) 2023-0009; The experimental procedure was approved by the Ethics Review Committee of the Institute of Clinical Pharmacology, Anhui Medical University. The animals were housed in the laboratory of the Institute of Clinical Pharmacology, Anhui Medical University, under conditions of 23±2℃ and 40%~70% humidity, with free access to food and water. After purchase, the mice underwent a week of acclimatization training.

[0028] Drugs and reagents include: (1) Telotamine hydrochloride and zoprazepam hydrochloride for injection (Zoletil50): Telotamine 125mg + zoprazepam 125mg, 5mL sterile water for injection (2020 Veterinary Drug Certificate No. 06), purchased from Virbac (France). The drug was dissolved in sterile water for injection and diluted 20 times with physiological saline to a concentration of 2.5mg / mL, and administered to mice via intramuscular injection. 60μL of the 2.5mg / mL concentration was administered intramuscularly to mice weighing 20g.

[0029] (2) Pilocarpine: Purchased from Abcam (USA). White powder. Weigh 10 mg and dissolve it in 10 mL of double-distilled water to prepare 1 mg / mL. When using, dilute to the required concentration (0.025 mg / mL) and administer intraperitoneally (0.125 mg / kg). For mice, inject 100 μL per 20 g of mice.

[0030] (3) Immunohistochemistry universal two-step kit (PV-9000): purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (China).

[0031] (4) SIRT1 activator SRT1720: purchased from GlpBio (USA), dissolved in DMSO to a stock solution of 100 mg / mL, diluted with physiological saline to the required concentration and dispensed for use. Each mouse was given an intraperitoneal injection of 50 mg / kg.

[0032] (5) Anti-β-actin antibody (66009-1-Ig): purchased from Proteintech (China).

[0033] (6) Anti-IRF5 antibody (ab181553): purchased from Abcam (USA) (7) Anti-TRIM21 antibody (12108-1-AP) and Anti-SIRT1 antibody (13161-1-AP): purchased from Proteintech (China) (8) Mouse IFN-α ELISA Kit: purchased from Solarbio (China) (9) EDTA Antigen Retrieval Solution: China Zhongshan Jinqiao (10) Anti-mouse secondary antibody (SA00001-1) and anti-rabbit secondary antibody (SA00001-2): purchased from Proteintech (China) (11) AceQ qPCR SYBR Green Master Mix: purchased from Novizan (China) (12) Bovine serum albumin (BSA), complete Freund's adjuvant, and incomplete Freund's adjuvant: purchased from Sigma (USA). (13) Skim milk powder: purchased from Yili (China) The main instruments include: Multiskan Go full-wavelength microplate reader: Thermo Scientific, USA -80℃ ultra-low temperature freezer: Sanyo Corporation; RM2135 Paraffin Slicer: Leica GmbH, Germany; Laser confocal microscopy: Leica GmbH, Germany.

[0034] Chemiluminescence imaging system (Las4000mini): GE, USA Electronic balance (YP402N): Shanghai Jingtian Instrument Factory Slide scanner: 3DHISTECH, Hungary Pure water and ultrapure water systems: Merck, Germany QuantStudio 3 Real-Time PCR System: Thermo Scientific, USA In this embodiment, the establishment and grouping of an antigen-induced Sjögren's syndrome model mouse were first performed, including: (1) Antigen preparation Healthy female C57BL / 6 mice, approximately 6-8 weeks old, were selected. After euthanasia by cervical dislocation, the mice were immersed in benzalkonium chloride solution for 15 min. The necks of the mice were dissected in a clean bench, and bilateral submandibular gland tissue was removed. Excess water was absorbed on clean filter paper, and the tissue was weighed. The weighed submandibular gland tissue was placed in a mortar, and sterile PBS solution was added at 0.1 g / mL. The tissue was homogenized on ice until a homogenate was formed. The submandibular gland protein homogenate was transferred to a 15 mL centrifuge tube and centrifuged (4 ℃, 3000 rpm, 15 min), discarding the precipitate. The supernatant was used for protein concentration determination using the BCA method. The protein was diluted with PBS to a concentration of 5 mg / mL, and equal volumes of Freund's complete adjuvant (Day 0, Day 7, Day 14) and Freund's incomplete adjuvant (Day 21) were added. The mixture was vortexed until the antigen concentration was adjusted to an emulsifier of 2.5 mg / mL.

[0035] (2) Antigen injection The day of the first antigen injection was designated as Day 0 of the modeling process. On Day 0, Day 7, and Day 14, the submandibular gland antigen was injected into mice via multiple intradermal injections on the back, with 0.1 mL of antigen injected into 20 g mice. On Day 21, a booster immunization was administered in the same manner (the antigen was prepared in equal volumes with Freund's incomplete adjuvant).

[0036] (3) Grouping and administration Starting at week 6 (6 weeks) of modeling, mice that had successfully developed the model were selected and grouped based on saliva production and physical signs (no ear scratching, paw licking, or changes in coat color). The mice were divided into a normal group, a model group, a SIRT1 activator (SRT1720) group (50 mg / kg), and a Pre group (5 mg / kg), with six mice in each group. Except for the normal and model groups, the other two groups began receiving medication at week 7: the SIRT1 activator group received the medication via intraperitoneal injection, and the Pre group received it via gavage. After two weeks of continuous administration, the mice were sacrificed.

[0037] The indicator detection in this embodiment includes: (1) Observation of the overall condition, specifically: Observe the mouse's skin for scratching or damage, and the condition of its lips, etc.

[0038] (2) Weight measurement, specifically: Starting from the first day of modeling, the weight (g) of the mice was measured once a week to observe the changes in weight of the mice in each group.

[0039] (3) Saliva volume detection, specifically: From week 0 of modeling until sacrifice, the saliva volume of mice was measured weekly. Before saliva measurement, mice were injected intramuscularly with Salmonella 50 (100 μL per 20 g mouse) to achieve complete anesthesia, with stable breathing, loss of corneal reflex, and relaxation of limb muscles. After anesthesia, mice were injected intraperitoneally with pilocarpine (0.025 mg / mL, 100 μL per 20 g mouse). Saliva collection began 5 minutes after injection and continued for 10 minutes. The weights of cotton balls before and after saliva collection were measured (M1 and M2), and the weight difference between M2 and M1 was taken as the saliva volume.

[0040] (4) Organ index detection, specifically: Mice were weighed before sacrifice at week 9. After sacrifice, the thymus, spleen, and submandibular gland were removed and weighed. The organ index (mg / g) is the ratio of the weight of the organ (mg) to the body weight (g) of the mouse.

[0041] (5) The expression of SIRT1, TRIM21, and IRF5 and the transcription levels of Mx1, Ifi44, Isg15, and Ifit3 in the submandibular gland tissue of mice in each group were measured.

[0042] (6) Measure the IFN-α level in the serum of mice in each group.

[0043] The result tracking in this embodiment includes: (1) Observation results of the overall condition, specifically: Starting from the third week of modeling, mice in the model group successively exhibited behaviors such as licking their tongues and scratching their lips, while the normal control group did not show any obvious behavior. (2) Changes in body weight of mice in each group at different time points, such as Figure 2 As shown, specifically, compared with the normal control group, the body weight of mice in the model group gradually decreased, and the body weight of mice gradually recovered after SRT1720 administration. (3) Changes in saliva volume in each group of mice at different time points, such as Figure 3 As shown, specifically, compared with the normal control group, the amount of saliva in the model mice gradually decreased, and the amount of saliva in the mice gradually recovered after SRT1720 administration; (4) Organ index of mice in each group, such as Figure 4 As shown, specifically, compared with the normal control group, the submandibular gland, thymus and spleen indices of mice in the model group increased to varying degrees, and SRT1720 could restore the submandibular gland and thymus indices of mice. (5) such as Figure 5 , 6As shown in Figures 7 and 8, the expression of SIRT1, TRIM21, acetylated TRIM21, and IRF5 in the submandibular gland tissue of mice in each group was determined by immunohistochemistry, immunofluorescence, and Western blotting. Specifically, compared with the normal control group, the expression of SIRT1 in the submandibular gland tissue of the model group mice was decreased, while the expression of TRIM21, acetylated TRIM21, and IRF5 was increased. SRT1720 can increase SIRT1 expression, promote TRIM21 deacetylation, and decrease IRF5 levels. (6) For example Figure 8 As shown, qPCR was used to detect the transcriptional levels of Mx1, Ifi44, Isg15 and Ifit3 in each group of mice. Specifically, compared with the normal group, the transcriptional level of interferon-stimulated genes in the submandibular gland tissue of model mice was significantly increased, and SRT1720 could reduce the transcriptional level of interferon-stimulated genes in the submandibular gland tissue of model mice. (7) For example Figure 9 As shown, ELISA was used to detect the expression of IFN-α in the serum of mice in each group. Specifically, compared with the normal control group, the expression of IFN-α in the serum of model mice was significantly increased, and SRT1720 could reduce the level of IFN-α in the serum of model mice.

[0044] Combining the graph and the analysis of the above results, it is clear that: The antigen-induced Sjögren's syndrome mouse model was successfully established in this embodiment. Compared with the model group, SRT1720 restored the body weight of the model mice and improved salivary flow rate. SRT1720 inhibited the transcription of type I IFN-stimulated genes by increasing SIRT1 expression, promoting TRIM21 deacetylation, and reducing IRF5 levels. The decrease in serum IFN-α levels also indicates that SRT1720 inhibited the sustained activation of type I IFN. Simultaneously, SRT1720 increased α-Amylase expression in the submandibular gland tissue of the model mice, restoring the secretory function of the salivary glands. The SIRT1 activator has a significant therapeutic effect on the antigen-induced Sjögren's syndrome model in C57BL / 6 mice.

[0045] Based on this, the present invention also provides the application of the gene encoding the SIRT1 protein in the preparation of drugs for treating Sjögren's syndrome, and the application of biomaterials containing the SIRT1 protein encoding gene in the preparation of drugs for treating Sjögren's syndrome, wherein the biomaterials include expression vectors or host cells.

[0046] While the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications and improvements can be made to it. Clearly, the present invention is not limited to the details of the exemplary embodiments described above, and can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description, and thus all changes falling within the meaning and scope of the equivalents of the claims are intended to be included within the present invention.

[0047] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Application of SIRT1 activators in the preparation of anti-inflammatory and immunomodulatory drugs.

2. The application according to claim 1, characterized in that, The anti-inflammatory and immunomodulatory drugs mentioned are for the treatment of autoimmune diseases characterized by exocrine gland lymphocyte infiltration and type I interferon (IFN) hyperactivation.

3. The application according to claim 2, characterized in that, The anti-inflammatory and immunomodulatory drug is used to treat Sjögren's syndrome.

4. The application according to claim 3, characterized in that, The SIRT1 activator includes SRT1720 or a pharmaceutically acceptable salt or derivative.

5. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or excipients.

6. The application according to claim 1, characterized in that, The dosage forms of the drug include injections, tablets, granules, capsules, pellets, or sustained-release formulations.

7. The application according to claim 3, characterized in that, The Sjögren's syndrome includes primary Sjögren's syndrome or secondary Sjögren's syndrome.

8. The application according to claim 4, characterized in that, The drug may also contain JAK inhibitors or immunomodulators.

9. Application of the gene encoding SIRT1 protein in the preparation of drugs for treating Sjögren's syndrome.

10. The use of biomaterials containing the SIRT1 protein-encoding gene in the preparation of drugs for treating Sjögren's syndrome, wherein the biomaterials include expression vectors or host cells.

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