Application of SNX16 of myeloid cells as target spot in preparation of medicine for preventing and treating heart aging

Abstract

The invention discloses application of SNX16 in myeloid cells as a target spot in preparation of a medicine for preventing and treating heart aging, and belongs to the technical field of biological medicine. According to the application disclosed by the invention, the effect of the SNX16 in the myeloid cells in heart aging is researched by utilizing a naturally aged mouse model with the myeloid cells deficient in the SNX16, and a result shows that the heart function of the naturally aged mouse is obviously reduced by the myeloid cells deficient in the SNX16, the expression level of aging-related proteins P16 and P21 in the heart tissue of the naturally aged mouse is increased, and the heart aging of the naturally aged mouse is inhibited. Macrophages in heart tissues of naturally aged mice are promoted to be polarized to M1 type; myeloid cell deficiency SNX16 regulates and controls polarization of macrophages to M1 type through a CSF1R signal channel, the expression level of CSF1R protein in the macrophages is remarkably increased through the myeloid cell deficiency SNX16, a downstream channel of the CSF1R signal channel is affected, and finally development of heart aging is aggravated. The invention finds that the SNX16 protein in the myeloid cell can be used as a new target for preparing the medicine for treating or preventing the heart aging for the first time, and provides a theoretical basis for target selection of prevention and treatment of the heart aging and research and development of related medicines.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically involving the application of SNX16 of myeloid cells as a target in the preparation of drugs for preventing and treating cardiac aging. Background Technology

[0002] Aging is a universal, time-dependent decline in physiological function. Among the body's organs, the heart is one of the most affected by aging due to the lack of proliferative capacity of cardiomyocytes. Cardiac aging refers to the changes in heart structure and the decline in heart function caused by increasing age. Cardiac aging is a significant factor inducing cardiovascular disease, and the incidence of cardiovascular diseases related to cardiac aging, such as atherosclerosis, myocardial infarction, and heart failure, is increasing dramatically. With the continuous increase in the aging population, the risks posed by cardiac aging are also growing. However, the pathogenesis of cardiac aging is not fully understood, and there are currently no safe and effective drugs to delay cardiac aging in clinical practice. Therefore, exploring new diagnostic and therapeutic targets related to cardiac aging will provide a theoretical basis for the development of drugs to delay cardiac aging in clinical practice, and has significant scientific value.

[0003] Heart tissue contains various types of immune cells, including monocytes, macrophages, and neutrophils, which play a dual role in cardiac aging. For example, macrophages can eliminate senescent cardiomyocytes, thus delaying cardiac aging; on the other hand, they can increase the secretion of inflammatory factors and matrix metalloproteinase 9 (MMP9), leading to cardiomyocyte hypertrophy and extensive collagen fiber deposition, further exacerbating cardiac aging. Therefore, macrophages, as one of the core cells of the body's immune system, play a crucial role in the occurrence and development of cardiac aging. SNX16, a member of the sorting protein family (SNXs), is composed of 344 amino acids and contains a PX domain and a Coiled-coil (CC) domain. Its PX domain specifically binds to phospholipid components (phosphatidylinositol) on the cell membrane. Studies have reported that SNX16, as a sorting linker protein, plays an important role in tumors, neurological diseases, and infectious diseases by regulating various processes such as signaling pathways, membrane transport, cell migration, and viral infection. Previous studies have reported that the total mRNA expression level of SNX16 protein in heart tissue derived from heart failure patients is higher than that in heart tissue derived from healthy controls. However, heart tissue contains a variety of cell types, including cardiomyocytes, fibroblasts, and immune cells (monocytes, macrophages, and neutrophils derived from myeloid cells). The specific mRNA expression level of SNX16 protein in a particular cell type (such as cardiomyocytes or immune cells) within heart tissue is unclear, and no relevant research has been reported. Currently, there are no reports on the role of SNX16 in myeloid cells in cardiac aging. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide the application of SNX16 protein in myeloid cells as a target in the preparation of drugs for the prevention or treatment of cardiac aging. This invention utilizes a naturally aging mouse model with SNX16 deficiency in myeloid cells to investigate whether SNX16 in myeloid cells participates in cardiac aging by regulating macrophage polarization (M1 / M2 macrophage balance). The study found that SNX16 deficiency in myeloid cells significantly reduced cardiac function in naturally aging mice, increased the expression levels of aging-related proteins P16 and P21 in the cardiac tissue of naturally aging mice, and promoted the M1 polarization of macrophages in the cardiac tissue of naturally aging mice. We also found that SNX16 deficiency in myeloid cells can regulate macrophage M1 polarization through the CSF1R signaling pathway. SNX16 deficiency in myeloid cells significantly increased the expression level of CSF1R protein in macrophages and affected downstream pathways of the CSF1R signaling pathway, namely increasing JNK phosphorylation and inhibiting STAT3 phosphorylation, thereby promoting the polarization of cardiac macrophages towards the pro-inflammatory M1 phenotype and ultimately exacerbating the development of cardiac aging. The above experimental results indicate that SNX16 expression in myeloid cells has a negative regulatory effect on cardiac aging. Using SNX16 in myeloid cells as a target, drugs that promote high SNX16 expression in myeloid cells can be used to treat cardiac aging. This invention is the first to discover a novel function of the SNX16 protein in myeloid cells in cardiac aging research. The SNX16 protein in myeloid cells can serve as a new target for the preparation of drugs to treat or prevent cardiac aging. This invention provides a theoretical basis for target selection in the prevention and treatment of cardiac aging and the development of related drugs.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the application of SNX16 protein in myeloid cells as a target in the preparation of drugs for the prevention or treatment of cardiac aging. In myeloid cells, SNX16 can inhibit macrophage polarization to M1 type and promote macrophage polarization to M2 type through the CSF1R signaling pathway, thereby delaying the development of cardiac aging.

[0006] SNX16, a member of the sorting protein family (SNXs), is composed of 344 amino acids and contains a PX domain and a Coiled-coil (CC) domain. The PX domain specifically binds to phospholipid components (phosphatidylinositol) on the cell membrane. Studies have reported that SNX16, as a sorting linker protein, plays an important role in tumors, neurological diseases, and infectious diseases by regulating various processes such as signaling pathways, membrane transport, cell migration, and viral infection. Therefore, this invention investigates the role of SNX16 protein in myeloid cells in cardiac aging using a mouse model with a deficient SNX16 gene. The study found that SNX16 protein in myeloid cells can serve as a novel target for the preparation of drugs to treat or prevent cardiac aging, providing a theoretical basis for target selection in the prevention and treatment of cardiac aging and related drug development.

[0007] Based on the above technical solution, the drug is further described as a substance capable of specifically upregulating SNX16 expression in myeloid cells. Studies have found that SNX16 deficiency in myeloid cells significantly reduces cardiac function in naturally aging mice, increases the expression levels of aging-related proteins P16 and P21 in the cardiac tissue of naturally aging mice, and promotes the M1 polarization of macrophages in the cardiac tissue of naturally aging mice. We also found that SNX16 deficiency in myeloid cells can regulate macrophage M1 polarization through the CSF1R signaling pathway. SNX16 deficiency in myeloid cells significantly increases the expression level of CSF1R protein in macrophages and affects downstream pathways of the CSF1R signaling pathway, namely increasing JNK phosphorylation and inhibiting STAT3 phosphorylation, thereby promoting the polarization of cardiac macrophages towards the pro-inflammatory M1 phenotype and ultimately exacerbating cardiac aging. Therefore, SNX16 expression in myeloid cells has a negative regulatory effect on cardiac aging. Targeting SNX16 in myeloid cells and using drugs that promote high SNX16 expression in myeloid cells can be used to treat cardiac aging.

[0008] Based on the above technical solution, the drug further comprises a gene-based drug with an adenovirus vector that specifically targets myeloid cells and overexpresses SNX16, a commercially available liposome-encapsulated purified SNX16 protein, or a SNX16 peptide drug that specifically targets myeloid cells. Any substance that is biologically available and targets the SNX16 protein in myeloid cells is acceptable.

[0009] Based on the above technical solution, the drug is further defined as a commercially available liposome-encapsulated traditional Chinese medicine (TCM) or natural active substances derived from TCM that specifically target myeloid cells and upregulate the expression levels of SNX16 protein or SNX16 mRNA. As exogenous drugs, TCM and natural active substances derived from TCM have different sites of action. In this invention, both TCM and natural active substances capable of upregulating the expression levels of SNX16 protein or SNX16 mRNA fall within the scope of protection of this invention.

[0010] Based on the above technical solution, the SNX16 purified protein, SNX16 polypeptide drug, traditional Chinese medicine, and natural active substances derived from traditional Chinese medicine are further encapsulated in a commercially available liposome carrier that can specifically target myeloid cells.

[0011] Based on the above technical solution, the commercially available liposome carrier capable of specifically targeting myeloid cells is further coupled with mannose on its surface, enabling it to actively target myeloid cells in the body that highly express mannose receptors.

[0012] Based on the above technical solution, the adenovirus vector that can specifically target myeloid cells and overexpress SNX16 carries the myeloid cell-specific promoter CD11b, which can restrict the expression of the SNX16 gene to myeloid cells only.

[0013] Based on the above technical solution, the drug further includes commercially available liposome-encapsulated pharmaceutically acceptable excipients capable of specifically targeting myeloid cells.

[0014] Based on the above technical solution, the pharmaceutically acceptable excipients further include fillers, diluents, binders, disintegrants, and emulsifiers.

[0015] Based on the above technical solution, the drug is further prepared into a pharmaceutically permissible dosage form, which includes one or more combinations of tablets, granules, oral liquid preparations, drops, injection preparations, and capsule preparations.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of SNX16 protein in myeloid cells as a target in the preparation of drugs for the prevention or treatment of cardiac aging. Using a naturally aging mouse model with SNX16 deficiency in myeloid cells, this invention investigates whether SNX16 in myeloid cells participates in cardiac aging by regulating macrophage polarization (M1 / M2 macrophage balance). The results show that SNX16 deficiency in myeloid cells significantly reduces cardiac function in naturally aging mice, increases the expression levels of aging-related proteins P16 and P21 in the cardiac tissue of naturally aging mice, and promotes M1 polarization of macrophages in the cardiac tissue of naturally aging mice. We also found that SNX16 deficiency in myeloid cells can affect CSF1R... Signaling pathways regulate macrophage polarization toward the M1 phenotype. The absence of SNX16 significantly increases the expression level of CSF1R protein in macrophages and affects downstream pathways of the CSF1R signaling pathway, namely increasing JNK phosphorylation and inhibiting STAT3 phosphorylation, thereby promoting the polarization of cardiac macrophages toward the pro-inflammatory M1 phenotype and ultimately exacerbating cardiac aging. This invention is the first to discover a novel function of SNX16 protein in myeloid cells in cardiac aging research. SNX16 protein in myeloid cells can serve as a new target for the preparation of drugs for the treatment or prevention of cardiac aging. This invention provides a theoretical basis for the selection of targets for the prevention and treatment of cardiac aging and the development of related drugs, and has very broad application prospects. Attached Figure Description

[0017] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0018] Figure 1 This is a flowchart illustrating the function of the SNX16 protein in myeloid cells in cardiac aging, as demonstrated in this invention.

[0019] Figure 2 The graph shows the cardiac function results of mice in the experimental and control groups. In the graph, A: left ventricular ejection fraction; B: left ventricular shortening fraction; C: systolic diameter; D: diastolic diameter; E: systolic volume; F: diastolic volume; G: left ventricular myocardial mass; H: left ventricular mass fraction; n≥8, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0020] Figure 3 Figure 1 shows the results of expression of aging-related proteins in the heart tissue of mice in the experimental and control groups. In Figure 2, A shows the expression of p16 and p21 proteins in mouse heart tissue by Western blotting; B shows the gray-scale statistical results of p16 protein expression in Figure 2; C shows the gray-scale statistical results of p21 protein expression in Figure 2; n=3, *P<0.05, **P<0.01.

[0021] Figure 4 Figure A shows the results of macrophage polarization towards the M1 type in the heart tissues of mice in the experimental and control groups. A: Western blotting was used to detect the protein expression levels of CD163, CD86, ARG-1, IL-10, and MCP-1 in mouse heart tissues; B and F represent the grayscale statistical results of the protein expression levels of CD163, CD86, ARG-1, IL-10, and MCP-1 in Figure A, respectively; n=3, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0022] Figure 5 To detect the mRNA expression levels of Cd206 (A), Il-10 (B), Arg-1 (C), and Tnf-α (D) in the heart tissues of mice in the experimental and control groups by RT-qPCR; n=3, *P<0.05, **P<0.01.

[0023] Figure 6 shows the experimental results of the SNX16 signaling pathway regulating CSF1R in macrophages participating in M1 polarization of macrophages in the experimental and control groups; A: Western blot detection of p16, IL-6, CSF1R protein expression and JNK and STAT3 phosphorylation levels in mouse peritoneal macrophages; B and C are the gray-scale statistical results of JNK and STAT3 protein phosphorylation levels in Figure A; n=3, *P<0.05, **P<0.01. Detailed Implementation

[0024] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0025] The materials and methods used in the embodiments are as follows: Mouse model required for the experiment: Ordered SNX16 from Cyagen Biotech. flox / flox The C57BL / 6 mice (C57BL / 6 mice with Loxp sequences inserted at both ends of exon 2 of the SNX16 gene) and the Lyz2-Cre C57BL / 6 tool mice (C57BL / 6 mice that specifically express Cre recombinase in myeloid cells). The SNX16... flox / flox After mating C57BL / 6 mice with Lyz2-Cre C57BL / 6 tool mice, experimental group SNX16 mice were obtained. CKO C57BL / 6 mice (C57BL / 6 mice with myeloid cells lacking the SNX16 gene) and control SNX16 miceLoxP / LoxP C57BL / 6 mice (SNX16 mice that do not express Lyz2-Cre recombinase) flox / flox C57BL / 6 mice); experimental group SNX16 CKO and control group SNX16 LoxP / LoxP The C57BL / 6 mice used in the experiment were divided into three groups: 2-3 month old male C57BL / 6 mice as the young mouse group, 12-14 month old male C57BL / 6 mice as the middle-aged mouse group, and 22-24 month old male C57BL / 6 mice as the old mouse group.

[0026] Cardiac echocardiography to detect diastolic and systolic function in mice: Echocardiography was performed on mice using a small animal in vivo echocardiography system (Vevo3100). Mice were anesthetized with 1.5% isoflurane. After anesthesia, hair was removed from the chest area using depilatory cream. The mice were then fixed in a supine position on a heating plate to maintain a constant body temperature. An MX400 (30MHz) probe was used to perform transthoracic echocardiography on the mice, measuring left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), systolic diameter (Diameter; s), diastolic diameter (Diameter; d), systolic volume (Volume; s), diastolic volume (Volume; d), left ventricular mass (LV Mass), and left ventricular mass fraction (LV MassCor).

[0027] Western blot experimental procedure: Left ventricular tissue was collected, and homogenized in liquid nitrogen with pre-cooled protein lysis buffer containing PMSF (1 mmol / L). The homogenate was centrifuged at 12000 rpm for 30 min to extract total protein. The supernatant was collected, and protein quantification was performed using the BCA method. 50 µg of total protein sample was subjected to 10% denaturing polyacrylamide gel electrophoresis, electroblotting onto a PVDF membrane. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with antibody against the target protein, followed by three washes with PBST buffer. Horseradish peroxidase-conjugated secondary antibody was added, and the membrane was incubated at room temperature for 1 h, followed by three washes with PBST buffer for color development. β-tubulin was used as the primary antibody control. Optical density scanning was used for semi-quantitative analysis of the developed bands, and the ratio of the target band to the β-tubulin signal area was used to assess the target protein expression level.

[0028] RT-qPCR experimental procedure: Total RNA was extracted from mouse left ventricular tissue, and cDNA was obtained after reverse transcription. The transcription level of the target gene was detected by RT-qPCR.

[0029] Example 1: Myeloid cell loss of SNX16 significantly reduced cardiac function in aged mice. To investigate the effects of SNX16 in myeloid cells on cardiac function in aged mice, we separately compared the experimental group (SNX16...) with the control group (SNX16...). CKO ()) and control group (SNX16) LoxP / LoxP Echocardiography was performed on young mice (male C57BL / 6 mice aged 2-3 months), middle-aged mice (male C57BL / 6 mice aged 12-14 months), and old mice (male C57BL / 6 mice aged 22-24 months).

[0030] Experimental results are as follows Figure 2 As shown, the experimental results indicate that cardiac function in mice declines significantly with age, and the loss of SNX16 in myeloid cells significantly accelerates this decline. Particularly in aged mice, compared to normal aged mice, the loss of SNX16 in myeloid cells significantly reduces cardiac function, specifically decreasing left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), while significantly increasing left ventricular systolic diameter (Diameter s) and left ventricular diastolic diameter (Diameter d). In conclusion, the loss of SNX16 in myeloid cells significantly reduces cardiac function in aged mice.

[0031] Example 2: Myeloid cell loss of SNX16 significantly increased the expression levels of aging-related proteins in the heart tissue of aged mice. To further investigate the effects of SNX16 in myeloid cells on cardiac function in aged mice, we used Western blotting to detect the effects of SNX16 in the experimental group. CKO ) and control group (SNX16) LoxP / LoxP The expression levels of aging-related proteins p16 and p21 in the heart tissue of young and old mice were measured.

[0032] Experimental results are as follows Figure 3 As shown, the experimental results indicate that, compared with the young group, the expression levels of aging-related proteins p16 and p21 in the heart tissue of aged mice were significantly increased in both the experimental and control groups. Furthermore, the loss of SNX16 in myeloid cells promoted the upregulation of the expression levels of aging-related proteins p16 and p21 in the heart tissue of aged mice. In conclusion, the loss of SNX16 in myeloid cells significantly increases the expression levels of aging-related proteins p16 and p21 in the heart tissue of aged mice.

[0033] Example 3: Myeloid cell loss of SNX16 promotes M1 polarization of macrophages in cardiac tissue of aged mice. To investigate the effect of SNX16 deletion in myeloid cells on macrophage polarization in cardiac tissue, we first used Western blotting to detect the effect of SNX16 deletion in the experimental group. CKO()) and control group (SNX16) LoxP / LoxP The expression levels of CD163, ARG-1, and IL-10, the hallmark proteins of M2 macrophages, and CD86 and MCP-1, the hallmark proteins of M1 macrophages, in mouse heart tissue were determined.

[0034] Experimental results are as follows Figure 4 As shown, the experimental results indicate that in the aged mouse group, after myeloid cells lost SNX16, the expression levels of CD163, ARG-1 and IL-10, the hallmark proteins of M2 macrophages in the heart tissue, were significantly lower than those in the non-deleted group, while the expression levels of CD86 and MCP-1, the hallmark proteins of M1 macrophages, were significantly higher than those in the non-deleted group.

[0035] Secondly, we used RT-qPCR to detect the experimental group (SNX16). CKO ()) and control group (SNX16) LoxP / LoxP The mRNA expression levels of Cd206, Arg-1, Il-10, and Tnf-α in mouse heart tissue were analyzed, and the results are as follows: Figure 5 As shown, the experimental results indicate that the loss of SNX16 in myeloid cells led to the downregulation of Cd206, Arg-1, and Il-10 mRNA levels and the upregulation of Tnf-α mRNA levels in the heart tissue of aged mice. In conclusion, the loss of SNX16 in myeloid cells significantly promotes the M1 polarization of macrophages in the heart tissue of aged mice.

[0036] Example 4: SNX16 in myeloid cells regulates macrophage polarization towards the M1 type through the CSF1R signaling pathway, thereby accelerating cardiac aging in aged mice. CSF1R (colony-stimulating factor 1 receptor) is a transmembrane tyrosine kinase receptor highly expressed in monocytes, macrophages, dendritic cells, and osteoclasts. The CSF1R signaling pathway plays a crucial role in macrophage differentiation, survival, proliferation, adhesion, and migration; excessive activation of CSF1R leads to abnormal expression of pro-inflammatory cytokines such as TNF-α and IL-6. Previous studies have reported that the CSF1R signaling pathway is involved in macrophage polarization. To investigate whether SNX16 can regulate the CSF1R signaling pathway in macrophages, we extracted CSF1R from young mice (including SNX16 receptor agonists). LoxP / LoxP Control group and SNX16 CKO Experimental group) and aged group mice (including SNX16) LoxP / LoxP Control group and SNX16 CKO Western blot analysis was performed on peritoneal macrophages from the experimental group.

[0037] Experimental results are as follows Figure 6As shown, the experimental results indicate that in the aged group, the absence of SNX16 in myeloid cells significantly increased the expression level of CSF1R protein in macrophages, while also affecting downstream pathways of the CSF1R signaling pathway, namely, a significant increase in JNK phosphorylation, an inhibition of STAT3 phosphorylation, and a significant increase in the expression level of the M1 macrophage-related cytokine IL-6. In conclusion, SNX16 in myeloid cells can regulate macrophage polarization towards the M1 type through the CSF1R signaling pathway, thereby accelerating cardiac aging in aged mice.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of SNX16 protein in myeloid cells as a target in the preparation of drugs for the prevention or treatment of cardiac aging, characterized in that, In the myeloid cells, SNX16 can inhibit macrophage polarization to M1 type and promote macrophage polarization to M2 type through the CSF1R signaling pathway, thereby delaying the development of cardiac aging.

2. The application according to claim 1, characterized in that, The drug is a substance that can specifically upregulate the expression of SNX16 in myeloid cells.

3. The application according to claim 2, characterized in that, The drug comprises a gene-based drug that specifically targets myeloid cells and overexpresses SNX16 via an adenovirus vector, or a commercially available liposome-encapsulated purified SNX16 protein or SNX16 peptide drug that specifically targets myeloid cells.

4. The application according to claim 2, characterized in that, The drug is a commercially available liposome-encapsulated traditional Chinese medicine that can specifically target myeloid cells and upregulate the expression level of SNX16 protein or SNX16 mRNA, as well as natural active substances derived from traditional Chinese medicine.

5. The application according to claim 3 or 4, characterized in that, The SNX16 purified protein, SNX16 polypeptide drugs, traditional Chinese medicine, and natural active substances derived from traditional Chinese medicine are encapsulated in commercially available liposome carriers that can specifically target myeloid cells.

6. The application according to claim 5, characterized in that, The commercially available liposome carrier that specifically targets myeloid cells is coupled with mannose on its surface, enabling it to actively target myeloid cells that highly express mannose receptors in the body.

7. The application according to claim 3, characterized in that, The adenovirus vector that specifically targets myeloid cells and overexpresses SNX16 carries the myeloid cell-specific promoter CD11b, which can restrict the expression of the SNX16 gene to myeloid cells only.

8. The application according to claim 2, characterized in that, The drug also includes commercially available, pharmaceutically acceptable liposome-encapsulated excipients capable of specifically targeting myeloid cells.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients include fillers, diluents, binders, disintegrants, and emulsifiers.

10. The application according to claim 2, characterized in that, The drug is prepared into a pharmaceutically permissible dosage form, which includes one or more combinations of tablets, granules, oral liquid preparations, drops, injectable preparations, and capsule preparations.

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