Protein complex for protecting mitochondria from being damaged, pharmaceutical composition and application

Abstract

The invention discloses a protein complex for protecting mitochondria from being damaged, a pharmaceutical composition and application. According to the application, the influence of nitrate on mitochondrial injury in in-vivo radiation-induced salivary gland injury and in-vitro human salivary gland cell injury is evaluated, the action mechanism in the mitochondrial injury is further deeply explored, and a result shows that sialic acid transporter interacts with FKBP8, so that the mitochondrial injury can be inhibited, and the mitochondrial injury can be inhibited. In turn, particles are formed and anchored on the outer mitochondrial membrane, thereby maintaining membrane integrity. Therefore, the invention provides a brand new therapeutic target for preventing radiation-induced salivary gland injury and other mitochondrial membrane related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, in particular to a protein complex for protecting mitochondria from damage, a pharmaceutical composition and applications. BACKGROUND

[0002] Salivary glands play a key role in digestion, lubrication, maintaining the stability of the oral microenvironment, and exerting antibacterial activity, and are essential for physical health. However, as the main treatment for head and neck tumors, radiation therapy (IR) often causes irreversible dysfunction of salivary glands, and further causes symptoms such as xerostomia (i.e., dry mouth), dental caries, mucosal inflammation, fungal infection, and taste alteration. Current treatment methods can only temporarily alleviate related symptoms, but cannot restore the structure and function of the glands. Therefore, developing strategies to prevent or reverse radiation therapy-induced salivary gland damage remains a key problem to be solved.

[0003] The key mechanism of radiation (IR) induced salivary gland dysfunction mainly involves the loss of acinar cells, mitochondrial dysfunction, and the increase of oxidative stress in the tissue. Mitochondrial damage is the core pathogenic mechanism because mitochondria play a driving role in cellular energy metabolism. Mitochondrial dysfunction includes mitochondrial dynamics imbalance, ATP synthesis inhibition, calcium homeostasis disorder, and metabolic reprogramming. Mitochondrial dysfunction is not only a key mechanism of IR-induced salivary gland dysfunction, but also an important cause of salivary gland dysfunction in many other diseases (such as neurological diseases, cardiovascular diseases, and liver diseases). Specifically, radiation can damage the integrity of the mitochondrial membrane, down-regulate the expression of anti-apoptotic proteins (such as BCL-2, BCL-XL), increase membrane permeability, and further induce cytochrome c-mediated apoptosis. Subsequently, Bak / Bax oligomers cause inner membrane herniation and leakage into the cytoplasm. The continuously increasing membrane permeability promotes the influx of calcium ions and the excessive production of reactive oxygen species (ROS), thereby activating the mitochondrial permeability transition pore (mPTP). This series of cascade reactions ultimately cause mitochondrial permeability imbalance, inner membrane rupture, and pyroptosis. Damaged mitochondria have the following two outcomes: one is local repair through membrane stabilizing complexes, and the other is removal through mitophagy. Therefore, maintaining the integrity of the mitochondrial double membrane is expected to fundamentally solve the problem of mitochondrial dysfunction and significantly improve the treatment effect on IR-induced salivary gland dysfunction.

[0004] The information in the background section merely serves to explain the general context of the application and should not be regarded as an admission that this information forms part of the common general knowledge in the art. SUMMARY

[0005] To solve the technical problems in the prior art, the present application provides a protein complex for protecting mitochondria from damage, a pharmaceutical composition and applications. Specifically, the present application includes the following contents.

[0006] In a first aspect of the present application, a protein complex for protecting mitochondria from damage is provided, which comprises a sialic acid transporter and FKBP8.

[0007] In some embodiments, the protein complex for protecting mitochondria from damage according to the present application, wherein the sialic acid transporter and FKBP8 are connected or interacted through non-covalent bonds.

[0008] In some embodiments, the protein complex for protecting mitochondria from damage according to the present application, wherein the sialic acid transporter and FKBP8 are connected or interacted through covalent bonds.

[0009] In some embodiments, the protein complex for protecting mitochondria from damage according to the present application, wherein the protein complex exists in the form of a condensate.

[0010] In a second aspect of the present application, a pharmaceutical composition for reducing the degree of radiation damage is provided, which comprises the protein complex or a precursor according to the present application.

[0011] In some embodiments, the pharmaceutical composition for reducing the degree of radiation damage according to the present application, wherein the precursor comprises a nucleic acid for producing the protein complex, or a nucleic acid for producing the sialic acid transporter and / or FKBP8.

[0012] In some embodiments, the pharmaceutical composition for reducing the degree of radiation damage according to the present application, wherein the precursor comprises both a sialic acid transporter precursor and a FKBP8 precursor.

[0013] In some embodiments, the pharmaceutical composition for reducing the degree of radiation damage according to the present application, wherein the composition is designed such that the sialic acid transporter precursor and the FKBP8 precursor exist in separate forms, respectively, and the protein complex exists in the form of a condensate during use.

[0014] In a third aspect of the present application, an engineered cell is provided, wherein the cell comprises the protein complex or a precursor thereof according to the present application introduced by artificial means.

[0015] In a fourth aspect of the present application, a method for promoting the complexation of a sialic acid transporter and FKBP8 is provided, which comprises the step of aggregating the sialic acid transporter and FKBP8 by adding a nitrate to complex.

[0016] In a fifth aspect of the present application, there is provided a method for reducing the extent of radiation damage to an organism, comprising the step of contacting the organism with a protein complex as described herein.

[0017] In certain embodiments, the method for reducing the extent of radiation damage to an organism according to the present application, wherein the organism comprises a cell, a tissue, an organoid or an organ, is an in vitro method.

[0018] In a sixth aspect of the present application, there is provided a mutant sialin protein comprising a mutation of the amino acid at position 494.

[0019] In certain embodiments, the mutant sialin protein according to the present application, wherein the amino acid at position 494 is mutated from arginine to a hydrophobic amino acid.

[0020] In a seventh aspect of the present application, there is provided a method for inhibiting the complexation or aggregation of sialin and FKBP8, wherein the method comprises the step of mutating the amino acid at position 494 of sialin, or using a sialin protein with a mutation at the amino acid at position 494.

[0021] In an eighth aspect of the present application, there is provided a method for protecting mitochondria from damage, comprising the step of forming a complex, preferably an aggregate, of sialin and FKBP8 at the mitochondrial membrane.

[0022] The present application evaluates the effect of nitrate on IR-induced salivary gland damage and in vitro human salivary gland (HSG) cell damage, and further explores the underlying mechanism, and the results show that Sialin interacts with FKBP8 to form granules and anchor on the outer mitochondrial membrane, thereby maintaining the integrity of the membrane. Therefore, the present application provides a new therapeutic target for preventing IR-induced salivary gland damage and other diseases related to mitochondrial membrane. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Inorganic nitrate reduces IR-induced mitochondrial damage in vivo. (A) Representative SMG images of TOM20 and AQP5 IF staining in the control group, IR and IR + nitrate group (n = 6). (B, C) Statistical analysis of AQP5 and TOM20 protein expression in the control, IR and IR + nitrate groups. (D, E) Statistical analysis of mitochondrial structure of SMG tissue by TEM and control, IR and IR + nitrate groups. (F, G) Representative images of LC3 IF staining in the control, IR and IR + nitrate groups (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001.

[0024] Figure 2 Nitrate protects IR-induced mitochondrial damage in vitro. (A, B) JC-1 mitochondrial staining of control, IR and IR + nitrate groups (n = 6). (C, D) TMRE staining of control, IR and IR + nitrate groups (n = 6). (E, F) mPTP assay of control, IR and IR + nitrate groups (n = 6). (G, H) Representative images of OPA1 IF staining in control, IR and IR + nitrate groups (n = 6). (I, J) Representative images of TOM20 IF staining in control, IR and IR + nitrate groups (n = 6). (K, L) Mitochondrial structure of HSG by TEM and statistical analysis of control, IR and IR + nitrate groups. (M, N) Detection of mitochondrial damage-related proteins (BCL-2, BCL-XL, Cyto C) and statistical analysis of total protein in HSG of control, IR and IR + nitrate groups (N = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

[0025] Figure 3 Nitrate protects IR-induced mitochondrial damage in vivo by sialic acid. (A) Representative images of Sialin and TOM20 IF staining in control, IR and IR + nitrate groups (n = 6). Pearson’s coefficient of Sialin and TOM20 fluorescence colocalization in yellow font. (B) Statistical analysis of sialic acid protein expression in control, IR and IR + nitrate groups. (C) Overlap of Sialin and TOM20 fluorescence intensity peaks shown in micrographs of the three groups combined. (D) K14-Cre; Slc17a5 fl / + and K14-Cre; Slc17a5 fl / fl Representative images of IF staining (n = 6). Pearson’s coefficient of Sialin and TOM20 fluorescence colocalization in yellow font. (E) K14-Cre; Slc17a5 fl / + and K14-Cre; Slc17a5 fl / fl Statistical analysis of Sialin and TOM20 protein expression in control, IR and IR + nitrate groups. (F) Overlap of Sialin and TOM20 fluorescence intensity peaks shown in micrographs of the two groups combined. (G, H) K14-Cre; Slc17a5 fl / + and K14-Cre; Slc17a5 fl / fl Representative images of AQP5 IF staining and statistical analysis in control, IR and IR + nitrate groups. (I, J) K14-Cre; Slc17a5 fl / + and K14-Cre; Slc17a5 fl / fl Mitochondrial structure of MG tissue and statistical analysis in control, IR and IR + nitrate groups.

[0026] Figure 4 Sialin maintains mitochondrial membrane integrity. (A) JC-1 mitochondrial staining of vehicle and sialate knockdown HSG after 20 min of CCCP treatment (n=6). (B) TMRE staining of vehicle and sialate knockdown HSG after 20 min of CCCP treatment (n=6). (C) mPTP assay of vehicle and sialate knockdown HSG after 20 min of CCCP treatment (n=6). (D) Mitochondrial structure of HSG and statistical analysis of vehicle and sialate knockdown HSG after 20 min of CCCP treatment (n=6).

[0027] Figure 5 Sialin promotes mitochondrial function through binding to FKBP8. (A) Schematic showing the steps for identification of sialate-binding proteins in HSG lysates using Co-IP and LC-MS / MS. (B) Co-IP of sialate and FKBP8 in HSG. (C) Representative images of IF staining of sialate and FKBP8 protein in vehicle and sialate overexpressing HSG cells (n=3). Pearson’s coefficient of sialin and FKBP8 fluorescence colocalization in yellow font. (D) JC-1 mitochondrial staining of vehicle and FKBP8 overexpressing HSG after 20 min of CCCP treatment (n=6). (E) mPTP assay of vehicle and FKBP8 overexpressing HSG after 20 min of CCCP treatment (n=6). (F) TMRE staining of vehicle and FKBP8 overexpressing HSG after 20 min of CCCP treatment (n=6). (G) Representative images of IF staining of OPA1 protein in vehicle and FKBP8 overexpressing HSG after 20 min of CCCP treatment (n=6). (H) Representative images of IF staining of TOM20 protein in vehicle and FKBP8 overexpressing HSG after 20 min of CCCP treatment (n=6).

[0028] Figure 6 Sialin reduces mitochondrial function disruption. (A) JC-1 mitochondrial staining of vehicle and sialate overexpressing HSG after 20 min of CCCP treatment (n=6). (B) mPTP assay of vehicle and sialate overexpressing HSG after 20 min of CCCP treatment (n=6). (C) TMRE staining of vehicle and sialate overexpressing HSG after 20 min of CCCP treatment (n=6). (D) Representative images of IF staining of OPA1 protein in vehicle and sialate overexpressing HSG after 20 min of CCCP treatment (n=6). (E) Representative images of IF staining of TOM20 protein in vehicle and sialate overexpressing HSG after 20 min of CCCP treatment (n=6). (F) Mitochondrial structure of HSG and statistical analysis of vehicle and sialate overexpressing HSG after 20 min of CCCP treatment (n=6).

[0029] Figure 7 Sialin binds to FKBP8 and forms granules. (A) JC-1 mitochondrial staining of vehicle and FKBP8 knockdown HSGs after 20 min of CCCP treatment (n=6). (B) mPTP assay of vehicle and FKBP8 knockdown HSGs after 20 min of CCCP treatment (n=6). (C) TMRE staining of vehicle and FKBP8 knockdown HSGs after 20 min of CCCP treatment (n=6). (D, E) Representative images of IF staining of TOM20 and FKBP8 proteins in vehicle and sialin overexpressing HSGs (n=6). Pearson’s coefficient of TOM20 and FKBP8 fluorescence colocalization in yellow font. (F) Representative time-lapse images of FKBP8-mCherry puncta before and after photobleaching in sialin overexpressing, sialin knockdown and control groups. (G) In vitro phase separation assay of purified sialin and FKBP8. (H) Representative images of sialin and FKBP8 protein IF staining in IR and IR+ nitrate groups. (I) Intracellular pH was detected using BCECF-AM probe and statistical analysis was performed in control, nitrate and IR+ nitrate groups (n=19).

[0030] Figure 8 Nitrate alleviates mitochondrial damage by promoting Sialin-FKBP8 interaction. (A) Schematic diagram showing the steps of analyzing Sialin-FKBP8 interaction by atomistic modeling analysis. (B) Artificial intelligence predicts the binding site of Sialin-FKBP8 complex. (C) Energy contribution of each amino acid at the interface of Sialin-FKBP8 interaction. (D) Co-IP of FLAG-Sialin and HA-FKBP8 in HSGs with wild type (WT) and Sialin R4949A mutant (MUT) plasmids. (E) Co-IP of FLAG-Sialin and HA-FKBP8 in HSGs with WT and MUT plasmids after addition of nitrate. (H) Mitochondrial damage related proteins (BCL-2, BCL-XL, Cyto C) detection and statistical analysis of total proteins in Sialin-knockdown cells of WT and MUT groups (n=3). (I) Mitochondrial damage related proteins (BCL-2, BCL-XL, Cyto C) detection and statistical analysis of total proteins in Sialin-knockdown cells of WT and MUT groups under IR and IR+ nitrate conditions, respectively (n=3). DETAILED DESCRIPTION

[0031] The following examples are presented to more fully demonstrate the application and should not be construed as limiting the application. Unless otherwise indicated, the techniques and conditions used in the examples are in accordance with those described in the literature (e.g., see J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001) or as described in the manufacturer's manuals. Unless otherwise indicated, the reagents and instruments used are commercially available and are conventional products.

[0032] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of the term "about" in relation to a value or a range of values is intended to include each and every value falling within the range and each and every value falling within the range as well as each and every value intermediate to any value within the range. The upper and lower limits of each range are included in the range.

[0033] Unless defined otherwise, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited.

[0034] Previous studies have shown that nitrate can reduce radiation-induced salivary gland injury by restoring mitochondrial membrane potential (ΔΨm), inhibiting reactive oxygen species (ROS), activating the PI3K-Akt signaling pathway, and inhibiting the ROS / NLRP3 / Caspase-1 / GSDMD apoptosis body formation axis. Although nitrate typically functions through the nitrate-nitrite-nitric oxide pathway, no significant changes in key nitric oxide synthases (iNOS, XOR) were observed after radiation. This suggests that nitrate may directly protect mitochondria through a mechanism independent of classical nitric oxide signaling.

[0035] Sialin, as a nitrate transporter channel on the cell membrane of mammalian cells, plays a crucial role in the physiological function of nitrate in vivo. Previous studies of the present application have confirmed that Sialin responds to nitrate stimulation, and the nitrate-Sialin cycle can protect damaged mitochondria by reducing the level of mitochondrial reactive oxygen species (ROS) in salivary glands and restoring the mitochondrial membrane potential. In addition to the nitrate transport function, the lack of Sialin can also cause damage to the epithelial barrier, stem cell function and neural integrity, which fully highlights its multifunctional role in maintaining cell homeostasis. Given that Sialin is highly expressed in salivary acinar cells and plays an important role in protecting mitochondria, it becomes a potential key protein for nitrate to play a role in protecting mitochondria in radiation-induced (IR) salivary gland injury. Considering that mitochondrial membrane repair is a process of dynamic interaction between mitochondrial membrane proteins, the present application screens and identifies a key protein (FKBP8) that binds to Sialin in the process of mitochondrial protection, which is a multifunctional protein-FK506 binding protein 8 located in the outer membrane of mitochondria.

[0036] In one aspect of the present application, a protein complex comprising Sialin and FKBP8 is provided for protecting mitochondria from damage, and in a preferred embodiment, the complex of the present application is used for mitochondrial dysfunction or mitochondrial damage, particularly radiation therapy-induced mitochondrial dysfunction or mitochondrial damage. In a preferred embodiment, the complex of the present application is used for radiation therapy-induced salivary gland damage or salivary gland dysfunction.

[0037] The present application also provides the use of a Sialin and FKBP8 complex as a target for preventing and treating radiation damage, i.e., the use of a Sialin and FKBP8 complex in the preparation of a medicament for treating or ameliorating radiation damage.

[0038] In one aspect of the present application, a pharmaceutical composition for reducing the degree of radiation damage is provided, which comprises the protein complex or precursor of the present application, or an accelerator for promoting the formation of the complex. The present application therefore also provides the use of an agent in the preparation of a medicament for treating or ameliorating radiation damage, wherein the agent comprises an accelerator for promoting the formation of the complex of the present application.

[0039] The pharmaceutical composition of the present application also includes a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared in the form of a lyophilized preparation or an aqueous solution by mixing the protein complex or precursor having a desired purity with an optional pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used, and it can include at least one of a buffer, an antioxidant, a preservative, an isotonic agent, a stabilizer, and a surfactant. In addition, in order for the pharmaceutical composition to be useful for in vivo administration, they must be sterile. The pharmaceutical composition can be rendered sterile by filtration through a sterile filtration membrane.

[0040] In some embodiments, the pharmaceutical composition can also contain at least one additive of a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, a growth inhibitor, and an active agent required for the specific indication to be treated. The specific amount of additive to be added can be adjusted as necessary.

[0041] In the present application, the promoter is a substance for promoting the formation of a sialic acid transporter and FKBP8 complex, and includes a small molecule substance and / or a biological macromolecule, etc. Examples of the small molecule substance include, but are not limited to, a nitrate such as an alkali metal salt or an alkaline earth metal salt of nitric acid, including but not limited to a potassium salt, a sodium salt, a lithium salt, a calcium salt, a magnesium salt, etc., and also includes a crosslinking agent, in particular a bifunctional crosslinking agent; examples of the biological macromolecule include a soluble protein fragment, and examples of the biological macromolecule also include a particle such as a nanoparticle formed from a plurality of biological macromolecules.

[0042] In one embodiment, the promoter is a nitrate. In another embodiment, the promoter is a sialic acid transporter overexpression vector or an overexpression recombinant plasmid. In yet another embodiment, the promoter is an FKBP8 overexpression vector or an overexpression recombinant plasmid. The sialic acid transporter overexpression vector or the overexpression recombinant plasmid, the FKBP8 overexpression vector or the overexpression recombinant plasmid can be designed and synthesized according to known gene databases or protein databases, for which there is no particular limitation, or can be purchased from commercially available products.

[0043] In the present application, the sialic acid transporter and FKBP8 complex is a condensate, and preferably the condensate is capable of anchoring and stabilizing the outer membrane of mitochondria.

[0044] In one aspect of the present application, an engineered cell is provided, the cell comprising a protein complex or a precursor thereof introduced by artificial means. In certain embodiments, the cell is a salivary gland epithelial cell.

[0045] In one aspect of the present application, there is provided a method for promoting the complexation of sialic acid transporter and FKBP8, comprising the step of promoting the aggregation of sialic acid transporter and FKBP8 to form a complex by adding a promoting agent. In this aspect, the promoting agent is the promoting agent as mentioned above.

[0046] In one aspect of the present application, there is provided a method for reducing the extent of radiation damage in an organism, comprising the step of contacting the organism with the protein complex. Preferably, the organism comprises a cell, a tissue, an organoid or an organ, and the method is an in vitro method, and the method can be used for the study of molecular mechanism of radiation damage, drug optimization, drug structure design, drug screening, etc. In some embodiments, the method of the present application is a method for non-therapeutic purposes.

[0047] In one aspect of the present application, there is provided a sialic acid transporter mutant comprising a mutation at amino acid 494, preferably the amino acid 494 is mutated from arginine to a hydrophobic amino acid. The amino acid mutation can be generated using genetic or chemical methods known in the art. The genetic methods can include site-directed mutagenesis, PCR, whole gene synthesis, etc. The present application thus also provides a method for inhibiting the complexation or aggregation of sialic acid transporter and FKBP8, comprising the step of mutating the amino acid 494 of sialic acid transporter, or using a sialic acid transporter with a mutation at amino acid 494, and the method is an in vitro method, and the method can be used for the study of molecular mechanism of radiation damage, drug optimization, drug structure design, drug screening, etc. In some embodiments, the method of the present application is a method for non-therapeutic purposes.

[0048] In one aspect of the present application, there is provided a method for protecting mitochondria from damage, comprising the step of forming a complex of sialic acid transporter and FKBP8, preferably an aggregate, at the mitochondrial membrane, and the method is an in vitro method, and can be used for the study of molecular mechanism of radiation damage, drug optimization, drug structure design, drug screening, etc. In some embodiments, the method of the present application is a method for non-therapeutic purposes.

[0049] The present application also provides a method for screening a substance useful for preventing or treating mitochondrial radiation damage (for reducing the extent of radiation damage), comprising: (1) measuring a marker in a cell model, to obtain a first measurement value, preferably the cell model overexpresses sialic acid transporter and / or FKBP8; (2) applying the substance to be tested to the cell model; (3) measuring the marker of the cell model after applying the substance to be tested, to obtain a second measurement value; (4) comparing the first measurement value and the second measurement value, thereby screening a substance useful for preventing and treating mitochondrial radiation damage.

[0050] In the screening method of the present application, the marker includes binding or agglutination between sialic acid transporter and FKBP8.

[0051] In the screening method of the present application, when the second measurement value is greater than the first measurement value, the substance to be tested is screened as a substance useful for preventing and treating mitochondrial radiation damage.

[0052] In the screening method of the present application, further comprising the step of providing a control cell model for testing, wherein the control cell model preferably overexpresses a mutant of sialic acid transporter, particularly a protein mutated at amino acid position 494.

[0053] In the present application, the protein or complex of proteins involved can be isolated, the term "isolated protein or complex of proteins or variant or derivative thereof means a protein not in its natural environment. A recombinantly produced protein expressed in a host cell can be considered "isolated", as well as a naturally occurring or recombinant protein purified by any suitable technique.

[0054] In the present application, by the step of administering to a subject in need thereof a therapeutically effective amount of a sialic acid transporter and FKBP8 complex, or a promoter thereof, or a pharmaceutical composition. The term "subject" and "patient" used herein are used interchangeably herein to refer to any animal that can require pharmaceutical treatment as described herein. Subjects and patients thus include, but are not limited to: primates (including humans), canine, feline, murine and other mammalian subjects. Preferably, the subject is a human.

[0055] In the present application, the term "treatment" refers to therapeutic treatment and prophylactic or preventative measures, which aim to prevent or slow down (reduce) the extent of a physiological change or disorder, such as radiation damage, that is not desired to occur. Beneficial or desired results include, but are not limited to, one or more of the following, whether detectable or undetectable: alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether or not detectable to the eye. "Treatment" also means prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those in which the condition or disorder is to be prevented.

[0056] The term "effective amount" as used herein means that amount of a drug or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, or human by a researcher or clinician. Further, the term "therapeutically effective amount" means that amount of an agent that, when administered to a subject in need thereof, results in the improvement of the treatment, healing, prevention, or amelioration of a disease, disorder, or side effects, or a decrease in the rate of advancement of a disease or condition, as compared to the subject not receiving the agent. The term also includes within its scope amounts effective to enhance normal physiological function. Generally, an effective amount herein will vary according to factors such as the given pharmaceutical composition, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the subject to be treated, and the like, but can nevertheless be routinely determined by one of skill in the art. Effective amounts of the pharmaceutical compositions of the present application can be readily determined by one of skill in the art by routine methods known in the art.

[0057] Examples I. Experimental Materials and Methods 1. Animals Male C57BL / 6J mice (6 weeks old, 18-20 grams in weight) were purchased from Beijing SPF Biotechnology Co., Ltd. Animals were randomly divided into three experimental groups, 6 mice in each group: (1) control group, mice did not receive irradiation, drinking standard drinking water without added nitrate; (2) IR group, mice only received irradiation, drinking drinking water without added nitrate; (3) IR + nitrate group, mice received irradiation, drinking water with 4 mM nitrate added. Among them, sodium nitrate (NaNO3) was dissolved in drinking water, and the concentration of 4 mM was added to the nitrate one week before irradiation, and this concentration was maintained throughout the observation period. The irradiation procedure was as follows: mice were anesthetized by inhalation of isoflurane. They were placed in a prone position on the experimental platform of the Radsource 2000 X-ray machine, and the submandibular gland area received a single 15 Gy irradiation dose (dose rate: 1 Gy per minute; total irradiation time: 15 minutes).

[0058] purchased from Cyagen Bioscience Slc17a5 fl / fl Mice and K14-Cre-ERT mice, followed by crossbreeding to produce K14-Cre; Slc17a5 fl / fl Mice, K14-Cre epithelial cell-specific knockout was induced by a single injection of tamoxifen (Sigma-Aldrich; T5648) at 200 mg / kg body weight by intraperitoneal injection K14-Cre; Slc17a5 fl / fl Mice. K14-Cre; Slc17a5 fl / + C57BL / 6 (6 weeks old) mice were used as K14-Cre; Slc17a5 fl / flControl. Mouse tail tips were obtained at 10 days post birth for DNA extraction and PCR genotyping. Animals were housed under specific pathogen-free conditions with free access to water and food. Animals were randomly assigned to receive a single dose of 15 Gy irradiation or no irradiation. The experimental part has been approved by the Animal Care and Use Committee of Capital Medical University (AEEI-2021-025).

[0059] 2. Cell culture and transfection Human salivary gland cell line HSGs were cultured in high glucose DMEM (Gibco) containing 10% fetal bovine serum and 1% penicillin / streptomycin. Cells were cultured in a 5% CO2 humidified incubator at 37°C and were routinely tested for mycoplasma. Nitrate was dissolved in PBS and added to cells after filtration through a 0.22 pm filter. Nitrate (1 mM) was added to the culture medium after cell adhesion growth (4 hours after cell seeding). Cells were irradiated with a Radsource 2000 X-ray instrument at a dose of 5 Gy (dose rate of 1 Gy / min, irradiation time of 5 minutes). CCCP (10 mM) was added before measuring mitochondrial function, and the incubation time was 20 minutes.

[0060] Sialin and FKBP8 overexpression and knockdown lentivirus, overexpression lentivirus with HA-tag and Flag-tag, and FKBP8 plasmid with mcherry fluorescence were purchased from Shanghai Genechem Co., Ltd. Wild-type Sialin plasmid and Sialin R494A Mutant plasmid and FKBP8 plasmid with HA-tag were from Mabnus Biotech Co., Ltd. Wuhan. Plasmids were transfected into HSGs cells with Lipo3000 transfection reagent. Lentivirus was transfected into HSGs cells, and cells were selected with medium containing 1 pg / ml puromycin for 7 days. Western blot detection was used to verify the knockdown and overexpression efficiency.

[0061] 3. Immunofluorescence staining Submandibular glands were fixed with 4% paraformaldehyde, dehydrated with gradient ethanol, and then embedded in paraffin. Paraffin sections were deparaffinated and treated for antigen retrieval by boiling the slides in sodium citrate buffer (10 mmol / L, pH 6.0) for 20 min. Then, the sections were immersed in PBS containing 5% bovine serum albumin (BSA) and 0.2% Triton X-100 for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. After washing with PBS for 10 min three times, the sections were incubated with secondary antibodies for 2 h at room temperature. Subsequently, the sections were sealed with a slice containing DAPI. Images were taken using a NIS confocal microscope.

[0062] HSG cells were fixed with 4% paraformaldehyde and incubated with PBS containing 5% bovine serum albumin (BSA) and 0.2% Triton X-100 for 1 h at room temperature. Then, the antibody incubation, DAPI staining, and image acquisition were the same as for tissue sections.

[0063] The primary antibodies used were as follows: rabbit polyclonal anti-AQP5 antibody (1:100; Abeam; ab315855), rabbit polyclonal anti-TOM20 antibody (1:200; Thermo Fisher Scientific; PA5-110506), rabbit polyclonal anti-OPA1 antibody (1:200, Thermo Fisher Science; PA5-79771), rabbit polyclonal anti-LC3A / B antibody.

[0064] The secondary antibodies used were as follows: donkey polyclonal anti-mouse IgG (H+L) and Alexa Fluor 488 antibody (1:1000; Thermo Fisher Scientific; A-21202), donkey polyclonal anti-antibody IgG (H+L) Alexa Fluor 594 (1:1000).

[0065] 4. Transmission electron microscopy (TEM) Submandibular gland specimens were cut into ultrathin sections with a thickness of about 70 nm, double-stained with uranium and lead, and then observed under a transmission electron microscope (Hitachi HT7700 Exalens).

[0066] Cellular electron microscopy sample preparation: First, the cells were digested with 0.25% trypsin without EDTA, and then the cell pellet was centrifuged. Next, the cells were fixed in 2.5% glutaraldehyde at 4°C for 2 hours, and then the tissue sample was prepared.

[0067] 5. JC-1 and TMRE staining Mitochondrial membrane potential was measured in HSGs cells using JC-1 fluorescent probe (Thermo Fisher Scientific; T3168) and TMRE detection kit (Beyotime; C2001S). After digestion and resuspension, cells were seeded in 4-well confocal dishes. After incubation with nitrate for 24 hours, cells were incubated with JC-1 (10 μg / ml) and TMRE probe (10 μM) respectively for 20 minutes at 37 °C. Cells were then washed twice with detection buffer and then observed under confocal microscope.

[0068] 6. Mitochondrial permeability transition pore (mPTP) assay Cellular mitochondrial membrane permeability was detected using mPTP fluorescent detection kit (Beyotime; C2009S) according to the manufacturer’s instructions. After washing twice with PBS, cells were treated with calcein AM and calcein AM + CoCl2 respectively. Cells were then incubated at 37 °C in the dark for 45 minutes and further replaced with pre-warmed medium containing 10% FBS and incubated in the dark for another 30 minutes. Finally, cells were washed with PBS and observed under confocal microscope.

[0069] 7. Mitochondrial isolation Mitochondria were isolated using Mitochondrial Isolation Kit for Culture Cells (Thermo Fisher Scientific; 89874). Briefly, cells were seeded in culture dishes and incubated for 48 hours, then lysed with Lysis Buffer A on ice for 2 minutes. Reagent B was then added to the lysate and incubated for 5 minutes, followed by the addition of Reagent C (including protease inhibitors) and centrifugation at 700 x g for 10 minutes. The supernatant was centrifuged at 12000 x g for 15 minutes at 4 °C. The supernatant (cytosolic proteins) was then transferred to a new tube. Reagent C (including protease inhibitors) was added to the pellet and centrifuged again (12000 x g for 5 minutes at 4 °C). The pellet was the isolated mitochondria. Subsequently, Western blot analysis was employed to assess the purity of mitochondria.

[0070] 8. Blotting Cell total and mitochondrial proteins were collected using RIPA lysis buffer (Beyotime; P0013B) and protease inhibitor cocktail (Thermo Fisher Scientific; 87786). Proteins were separated by 10% SDS-PAGE (Bio-rad; 1610183) and then transferred to PVDF membranes. After blocking with 5% milk in PBS, the membranes were incubated with primary antibodies (part numbers as follows: ab109012, 12741, PA5-30517, ab129113, 15071, 2764, ab133504, ab14715, ab8245, 8570, ab205606, ab9110) overnight at 4°C. After incubation with secondary antibodies at room temperature, the membranes were treated with ECL substrate (Beyotime; P0018S) and then exposed to film and digital imaging. Image J software was used for gray-scale analysis, with 3 technical replicates per independent group to obtain the mean value as the sample.

[0071] 9. Electrophysiological measurements Standard whole-cell patch-clamp method was used to evaluate the changes of Sialin ion channel membrane current. First, the changes of Sialin ion channel current in cell membrane were detected by reducing the NO3 - concentration (50 / 30 / 10 / 1 mM, pH=4). Subsequently, the changes of Sialin ion channel response to different nitrate concentrations were detected in the extracellular electrode solution containing 1 mM NO3 - . Cells were randomly divided into 6 groups (control group and different concentrations of nitrate: 10 µM, 100 µM, 1 mM, 4 mM, 8 mM). Single cells were attached to the coverslips, and after cell adhesion, 1 mM nitrate was added to the culture medium (4 hours after cell inoculation). In addition, under the condition that the extracellular electrode solution was 1 mM NO3 - and pH was 4, the cells were divided into the following six groups: (1) control group without nitrate; (2) 1 mM nitrate group; (3) Sialin overexpression group (OE-Sialin) with 1 mM nitrate; (4) OE vector group containing 1 mM nitrate; (5) Sialin knockout group (sh-Sialin) with 1 mM nitrate; (6) sh vector group containing 1 mM nitrate. Digital data were analyzed using pClamp 10.6 and OriginPro 2024, and further statistical analysis was performed using Graphpad prism 9.0.

[0072] 10. Total nitric oxide detection The intracellular concentration of total nitric oxide was detected in the following six groups of cells (control group \ 1 mM nitrate \ OE Sialin + 1 mM nitrate \ OE vector + 1 mM nitrate \ sh Sialin + 1 mM nitrate \ sh vector + 1 mM nitrate) respectively. Briefly, according to the Griess method, the NO level in the cell lysate was directly detected using the total nitric oxide assay kit (Beyotime, S0024). The optical density at 540 nm was recorded using SpectraMax i3x, and the NO concentration was calculated according to the standard curve. There were six biological replicates in each group, and each sample also had three technical replicates.

[0073] 11. Intracellular NO release Intracellular NO release was detected using the NO fluorescent probe (DAF-FM DA, S0019, Beyotime). Briefly, after digestion and resuspension, HSGs cells were seeded into 4-cell confocal culture dishes. After incubation with nitrate for 24 hours, the cells were incubated with DAF-FM DA probe (1:1000) at 37°C for 20 minutes. Then the cells were washed with detection buffer three times, and then observed under a confocal microscope.

[0074] 12. Saliva collection The mice were anesthetized and injected intraperitoneally with pilocarpine (2 mg / kg), and then placed on a 30° inclined plate with their heads slightly tilted downward. After 10 minutes of pilocarpine injection, one end of a capillary pipette was placed at the bottom of the oral cavity, and after the remaining saliva in the mouse's oral cavity flowed out, the other end was placed in a pre-weighed ice-cold plastic tube. Collect saliva for 30 minutes, and calculate the total saliva flow under pilocarpine stimulation.

[0075] 13. Nitrate level detection The total nitric oxide and nitrate / nitrite parameter assay kit (KGE001) was used to determine the concentration of nitrate in saliva and blood. According to the manufacturer's protocol, the saliva sample was filtered using an ultrafiltration tube, and the supernatant was collected at 3000 x g for 20 minutes at room temperature. The optical density data at 540 nm were recorded using SpectraMax i3x, and the nitrate concentration was calculated according to the standard curve.

[0076] 14. Mitochondrial metabolite detection SMG tissue metabolite analysis was analyzed by Novogene (Beijing).

[0077] 15. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis of whole cell proteins Whole cell proteins were obtained from control, IR, and IR + nitrate groups. Samples were prepared for LC-MS / MS analysis using EasyPep Mini MS SampePrep Kit (Thermo Fisher Scientific; 40006). After sample processing, they were analyzed using a mass spectrometer (Q Exactive Plus Mass Spectrometer; Thermo Fisher Scientific) and Agilent 1260 LC (Agilent). ProteinPilot software was used to analyze raw data. A 1% false discovery rate (FDR) threshold was applied to peptide and protein levels in mass spectrometry data. Proteins were annotated using UniProt and MitoMiner 4.0 databases.

[0078] 16. Immunoprecipitation mass spectrometry (IP-MS) Lentivirus of HA-tag overexpressing Sialin was transfected into cells and expanded. HA-binding protein complexes were obtained using a magnetic HA-tag IP / Co-IP kit (Thermo Fisher Scientific; 88838) and subjected to SDS-PAGE. The gel was then digested with trypsin and detected with an Agilent 1260 LC (Agilent) tandem mass spectrometer (Q Exactive Plus Mass Spectrometer; Thermo Fisher Scientific). ProteinPilot software was used to analyze raw data. A 1% false discovery rate (FDR) threshold was applied to peptide and protein levels in mass spectrometry data. Proteins were annotated using UniProt and MitoCarta 2.0 databases.

[0079] 17. Immunoprecipitation and co-immunoprecipitation (Co-IP) Cells were lysed using IP lysis buffer (Thermo Fisher Scientific; 87787) and protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific; 78440) and centrifuged at 13000 x g for 15 minutes to obtain total protein lysate. The lysate was then incubated with protein A / G agarose beads (Beyotime; P2055) at 4°C for 2 hours. Then, the corresponding primary antibody and IgG antibody (as a control) were added to the lysate overnight at 4°C, respectively. The samples were washed twice with PBS and treated with 5 x buffer at 95°C for 10 minutes. Finally, the proteins were detected by immunoblotting.

[0080] 18. Fluorescence recovery after photobleaching (FRAP) assay For FKBP8 droplets in vitro, FRAP data were acquired using the FRAP module of the NIS confocal microscope. Briefly, droplets containing mCherry-tagged proteins were bleached using a laser beam. After bleaching, images were captured continuously over time (1 image every 1.53 seconds).

[0081] 19. Purification of Sialin and FKBP8 proteins Overexpressed HA-tagged Sialin lentivirus and overexpressed Flag-tagged FKBP8 lentivirus were transfected into HSGs cells, respectively. Transfected cells were selected with puromycin for 7 days and expanded. Sialin protein was purified by using anti-HA magnetic beads (ThermoFisher Scientific; 88836) and HA peptide (Thermo Fisher Scientific; 26184). FKBP8 protein was purified by using anti-DYKDDDDK magnetic agarose (Thermo Fisher Scientific; 36797) and DYKDDDDK-peptide (Thermo Fisher Scientific; 36805). Both HA-tagged and Flag-tagged proteins were eluted using a mild elution method according to the manufacturer’s instructions. The concentration of purified proteins was determined by BCA assay (Beyotime; P0010).

[0082] 20. Measurement of intracellular pH 2’,7’-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxy methyl ester (BCECF AM, Thermo Fisher Scientific; B1170) was used to detect intracellular pH. A fixed number of cells were seeded in a clear-bottom black 96-well plate and incubated in an overnight incubator in CO2 to allow cell attachment. The next day, cells were incubated with 1 mM BCECF-AM for 60 minutes. Subsequently, the plate was washed with Hank’s Balanced Salt Solution (HBSS). Then, BCECF fluorescence was measured using a fluorescent microplate reader and pH was calculated. Fluorescence ratios were obtained using a SpectraMax i3X plate reader (excitation wavelengths: 490 and 440 nm; emission wavelength: 535 nm).

[0083] 21. Atomic modeling analysis The structure of the SLC17A5-FKBP8 complex was predicted using the AlphaFold3 multimer program. The highest scoring conformation was used for dynamic simulation or analysis. The GROMACS software package (version 2023.03) was used to run the conventional MD simulation to study the changes in the conformation of the SLC17A5-FKBP8 complex. The force field amber14sb was used to parameterize the protein, respectively. The SLC17A5-FKBP8 complex was solvated in an octahedral water box, and then the charge of the system was neutralized by adding 0.150 M chloride and sodium ions. First, the energy of the system was minimized using the steepest descent minimization method for 50000 steps. In the next step, the position of the heavy atoms was restricted, and both NVT equilibration and NPT equilibration were performed for 50000 steps. The system temperature was maintained at 300 K, and the system pressure was maintained at 1 bar. After completing the two equilibration stages, the system was now well equilibrated at the desired temperature and pressure. A 100 ns unrestrained simulation was performed. Every 20 ps. In the simulation trajectory, ChimeraX was used to map the interactive mode and animate the dynamics trajectory. The MM-GBSA method has been widely used in drug research for the estimation of binding free energy, and gmx_MPBSA was used for MM-GBSA calculation.

[0084] II. Experimental Results 1. Nitrate alleviates IR-induced mitochondrial damage in a mouse model of IR-induced salivary gland injury First, this effect of nitrate was verified using a mouse model of salivary gland radiation injury, and the supplementation of nitrate significantly increased the salivary flow rate of irradiated mice. To explore the role of nitrate in alleviating IR-induced mitochondrial damage, the expression of TOM20 (a mitochondrial marker protein) and aquaporin 5 (AQP5, a salivary gland function protein) in the IR-induced salivary gland injury model was first detected. Immunofluorescence (IF) analysis showed that the protein levels of TOM20 and AQP5 in the salivary epithelial cells of the IR group were significantly lower than those of the unirradiated control group, while the protein levels of the IR+ nitrate group were significantly higher than those of the IR group Figure 1 A-C).

[0085] To determine whether nitrate improves mitochondrial dysfunction by modulating tissue metabolism during the repair process of salivary gland radiation damage, a non-targeted gas chromatography-mass spectrometry (GC-MS) metabolomics analysis was performed. The data showed significant metabolic perturbations between the control and IR groups, and between the IR and IR+ nitrate groups. Among these differentially expressed metabolites, N6 succinyl adenosine was found to be closely related to mitochondrial nucleotide synthesis. Subsequently, transmission electron microscopy (TEM) was used to evaluate mitochondrial morphological changes. In the control group, salivary epithelial cells contained mitochondria with normal round or rod-shaped morphology, showing clearly visible cristae arranged perpendicular to the long axis of the mitochondria. In contrast, the IR group exhibited disorganized salivary epithelial cell structures, with swollen mitochondria, reduced number of cristae, and indistinct cristae morphology. In addition, ruptured outer mitochondrial membranes and exposed inner membranes to the cytoplasm were observed. Notably, the mitochondrial morphology and integrity in the IR+ nitrate group were essentially restored to normal levels Figure 1 D, E).

[0086] During the process of apoptosis, the mitochondrial network remodels after an increase in mitochondrial membrane permeability, triggering mitophagy. Therefore, the expression of LC3, an autophagy marker protein, in salivary glands was detected. IF results showed that the upregulation of LC3 protein levels in salivary epithelial cells induced by IR was reversed by the supplementation of nitrate Figure 1 F, G). Overall, these findings suggest that nitrate effectively prevents IR-induced mitochondrial membrane damage.

[0087] 2. Nitrate protects mitochondria from IR-induced HSG damage To further verify whether nitrate can alleviate IR-induced cell damage in vitro, a radiation damage model of HSGs cells was used. The results showed that IR exposure significantly impaired cell proliferation capacity, while nitrate supplementation in the IR+ nitrate group significantly rescued this proliferation defect compared to the IR group. Changes in mitochondrial membrane potential of IR-exposed HSG cells were evaluated using JC-1 and TMRE fluorescent probes. JC-1 showed that IR exposure led to a significant decrease in mitochondrial membrane potential compared to the control group, while nitrate supplementation significantly restored mitochondrial membrane potential in the IR+ nitrate group compared to the IR group Figure 2 A, B). Consistent with these findings, TMRE staining, which specifically accumulates in metabolically active mitochondria, showed that the number of active mitochondria in the IR+ nitrate group was significantly higher than in the IR group Figure 2 C, D). Subsequently, the mitochondrial membrane permeability was evaluated using a mitochondrial permeability transition pore (mPTP) probe. IR exposure led to a significant increase in mitochondrial membrane permeability compared to the control group, while the IR-induced increase in permeability was significantly attenuated in the IR+ nitrate group Figure 2E, F). Next, the expression levels of TOM20 and OPA1 (mitochondrial inner membrane markers) were examined. IF analysis showed that, compared with the control group, IR exposure reduced the expression of TOM20 and OPA1 (E, F). Figure 2 G, I), while nitrate supplementation significantly reversed these reductions in the IR+ nitrate group ( Figure 2 GJ). Transmission electron microscopy was used to assess changes in mitochondrial ultrastructure. Cells exposed to IR exhibited characteristic mitochondrial damage, including swelling, membrane rupture, and cristae breakage. In contrast, control cells and cells treated with IR + nitrate maintained normal mitochondrial morphology, with intact membranes, well-organized tubular structures, and clearly defined cristae. Figure 2 K, L).

[0088] To further explore the nitrate-mediated mitochondrial protection mechanism, mitochondrial damage-related proteins were investigated: compared with the unexposed control group, IR exposure significantly reduced the expression of anti-apoptotic proteins BCL2 and BCL-XL, while nitrate supplementation in the IR+nitrate group reversed this downregulation. Conversely, compared with the IR group, the IR+nitrate group showed a significant reduction in IR-induced cytochrome c release. Figure 2 (M, N). In summary, these results indicate that nitrates play a crucial role in maintaining mitochondrial structural integrity and functional homeostasis after radiation exposure.

[0089] 3. Nitrates protect against IR-induced mitochondrial damage via sialic acid. To further investigate the specific molecular mechanisms of nitrate-mediated mitochondrial function protection, whole-cell proteomics analysis was performed using mass spectrometry (MS) on proteins from the control, IR, and IR+ nitrate groups. Quantitative analysis and differential expression screening identified 244 differentially expressed proteins (DEPs) in the IR group compared to the control group (146 upregulated, 98 downregulated). Similarly, a comparison between the IR+ nitrate group and the IR group revealed 278 DEPs (187 upregulated, 91 downregulated). A total of 39 proteins were identified across both comparisons. Subsequent screening of these 39 shared DEPs by cell localization identified 5 mitochondrial proteins and 5 membrane localization proteins. Within a subset of these 10 DEPs, the nitrate-associated protein sialicin was significantly downregulated in the IR group compared to the control group, but its expression was restored in the IR+ nitrate group compared to the IR group. This example first investigated whether different concentrations of nitrate could regulate the expression of sialic acid proteins. HSG cells were pre-incubated with nitrate at concentrations of 10 µM, 100 µM, 1 mM, 4 mM, and 8 mM. Specifically, Western blot analysis showed that nitrate concentrations of 10 µM and 100 µM had no significant effect on sialic acid expression. The results indicate that nitrate concentrations exceeding 1 mM can promote sialic acid protein expression, and this promoting effect plateaus at 4 mM.

[0090] Given that sialin is a nitrate transporter, further investigation was conducted to verify whether the observed changes in sialin expression corresponded to changes in nitrate transport activity. To this end, whole-cell patch-clamp electrophysiological recordings were used to quantify nitrate influx. To more accurately simulate physiological conditions, the pH of the extracellular solution was set to 4, and NO3 was set... - The concentrations were 50 mM, 30 mM, 10 mM, and 1 mM, respectively. The results showed that when NO3... - At an extracellular solution concentration of 1 mM, sialic acid ion channels on the cell membrane can be activated. Based on this, the pH of the extracellular solution was set to 4, and NO3- was... - The concentration was 1 mM. Subsequently, the cells were pre-incubated with different concentrations of nitrate (10 µM, 100 µM, 1 mM, 4 mM, 8 mM), and changes in sialic acid current across the cell membrane were measured. The results showed that the changes in current were positively correlated with the expression level of sialic acid.

[0091] To confirm that nitrate regulates mitochondrial function and maintains mitochondrial membrane stability through sialic acid, the in vitro expression of sialic acid was first assessed among the groups. qPCR and Western blotting analyses showed that nitrate upregulated sialic acid expression at both the mRNA and protein levels. Furthermore, Western blotting was used to verify the purity of mitochondrial proteins and to analyze the expression levels of sialic acid proteins isolated from the three groups (control, IR, and IR+nitrate). Data showed that sialic acid expression was increased in the IR+nitrate group compared to the IR group. IF staining revealed that sialic acid expression was significantly decreased in vivo in the IR group compared to the control group, but significantly increased in vivo in vivo in the IR+nitrate group compared to the IR group. Figure 3 (A, B). Notably, co-localization analysis based on immunofluorescence showed that Sialin and TOM20 co-localized in normal acinar cells of the control group, with most Sialin localized in mitochondria. In the IR group, the expression of both Sialin and TOM20 was significantly reduced, along with decreased co-localization. Conversely, compared to the IR group, the co-localization of these two proteins was significantly enhanced in the IR + nitrate group. Figure 3 C).

[0092] To explore the role of sialic acid in maintaining mitochondrial function, a K14-specific sialic acid knockout (CKO) mouse model was generated using the Cre / LoxP system. Further IF analysis showed that TOM20 expression was decreased in Sialin CKO mice, and co-localization between TOM20 and Sialin was reduced. Figure 3 D, E, F). Furthermore, Sialin-CKO downregulates AQP5 expression (D, E, F). Figure 3G, H). TEM showed morphological abnormalities of submandibular gland mitochondria in Sialin CKO mice, including swelling, rounding, membrane rupture and cristae disruption Figure 3 I), while the number of damaged mitochondria increased Figure 3 J). IF analysis showed that IR significantly reduced the expression of AQP5 and TOM20 in salivary glands of Sialin CKO mice, and nitrate supplementation failed to alleviate salivary gland damage.

[0093] 4. Sialic acid deficiency leads to mitochondrial membrane damage in vitro To further explore the important role of sialic acid in maintaining mitochondrial function. Next, sialic acid knockdown cells were used to detect their effects on mitochondrial function. JC-1 and TMRE results showed that under normal conditions, knockdown of Sialin led to a decrease in mitochondrial membrane potential Figure 4 A, B). In addition, compared with the vector control group, the mitochondrial membrane potential of the Sialin knockdown group was significantly reduced after treatment with carbonyl cyanide m-chloro phenyl hydrazone (CCCP), a drug that induces mitochondrial membrane damage Figure 4 A, B). Subsequently, mitochondrial membrane permeability was evaluated between groups. mPTP assay results showed that under normal conditions, Sialin knockdown increased mitochondrial membrane permeability. After CCCP treatment, although the mitochondrial membrane permeability of the vector group increased, the mitochondrial membrane permeability of the Sialin knockdown group was significantly higher than that of the vector group Figure 4 C). These results indicate that knockdown of Sialin exacerbates CCCP-induced mitochondrial membrane damage.

[0094] TEM observations showed that under normal conditions, mitochondria in the vector group exhibited normal rod-shaped morphology with intact inner and outer membranes and distinct cristae. In contrast, mitochondria in the Sialin knockdown group showed swelling, rounding, rupture of inner and outer membranes, and disruption or loss of cristae. After CCCP treatment, the vector group showed partial mitochondrial swelling, membrane rupture, and extensive fragmentation or loss of cristae, with an increase in the number of damaged mitochondria. Notably, the Sialin knockdown group had more severe mitochondrial damage, with more damaged mitochondria, increased membrane rupture, and extensive fragmentation or loss of cristae Figure 4 D).

[0095] When CCCP dissipates mitochondrial membrane potential and activates Drp1, it also acts as a fission inducer. To determine whether Sialin knockdown induces mitochondrial fission, the expression of Drp1, a marker of mitochondrial fission, was measured. WB and IF analysis showed that there was no significant difference in Drp1 expression between the Sialin knockdown group and the vector group. Comparison of mitochondrial function between the CCCP treatment group and the CCCP + nitrate treatment group showed that even after fission induction, nitrate still maintained its protective effect. Then, a specific mitochondrial fission inhibitor, Mdivi-1, was applied to Sialin knockdown cells. The results showed that inhibiting fission did not significantly change the mitochondrial function of the sialic acid knockdown group. Overall, these results indicate that sialic acid is a key protein for maintaining mitochondrial functional stability and plays a direct protective role in maintaining mitochondrial function.

[0096] 5. Sialic acid maintains mitochondrial membrane integrity by directly interacting with FKBP8 Under basal conditions, sialic acid mediates pH-driven nitrate transport. Notably, pre-incubation with nitrate for 24 hours significantly increased sialic acid ion channel current compared to the control group. Previous studies have shown that sialic acid transports nitrate into cells, which exerts a protective effect through the nitrate-nitrite-nitric oxide (NO) pathway. Therefore, this example measured the total NO level in cells: under basal conditions, the total NO content in cells was about 0.65 µmol / L, while incubation with 1 mM nitrate increased it to about 14.12 µmol / L. To further confirm whether NO acts on mitochondria, cells were co-stained with DAF-FM DA (a non-fluorescent probe) and MitoTracker (a mitochondrial-targeting dye). The results showed that, compared with the control group, supplementation with nitrate increased mitochondrial NO content. However, no significant difference in intracellular cyclic guanosine monophosphate (cGMP) levels was detected. Subsequently, these parameters were evaluated in HSG cells with Sialin knockdown or overexpression. Even with nitrate supplementation, Sialin knockdown significantly reduced Sialin ion channel current compared to the vector control group. At the same time, the total NO and mitochondrial NO content in the Sialin knockdown group were significantly lower than those in the vector control group. In contrast, Sialin overexpression significantly increased Sialin ion channel current, as well as intracellular total NO and mitochondrial NO content, compared to the vector control group. Overall, these findings indicate that sialic acid-mediated nitrate transport is crucial for the protective effect of nitrate on mitochondrial damage. In addition, sialic acid itself may exert additional protection on mitochondria beyond its role in nitrate transport.

[0097] Given that sialic acid is a membrane protein, it can contribute to mitochondrial protection by interacting with other proteins. To verify this, the present embodiment prepared HSG cells overexpressing HA-tagged sialic acid and used immunoprecipitation combined with mass spectrometry (IP-MS) to identify potential sialic acid interacting proteins. IP-MS analysis identified 409 and 260 potential interactors in the sialic acid overexpression (OE) and vector control groups, respectively, with 205 proteins showing significant differential expression in the OE group. Among these differentially expressed proteins, there were 35 associated with mitochondria, including 10 mitochondrial membrane-associated proteins: VDAC2, AGK, OXA1L, SFXN1, TIMM50, TIMM23, SLC25A18, TOMM70A, FKBP8, and COQ5 Figure 5 A). Immunoprecipitation experiments confirmed that sialic acid and FKBP8 co-precipitated Figure 5 B). Then, the subcellular localization of Sialin and FKBP8 was analyzed using a cell line overexpressing Sialin. Co-localization analysis showed that Sialin overexpression promoted the formation of FKBP8 puncta and enhanced the co-localization of FKBP8 with Sialin Figure 5 C).

[0098] To investigate the role of the FKBP8 and Sialin complex (aggregates) in maintaining mitochondrial function, on the one hand, the present embodiment constructed a cell line overexpressing FKBP8 and evaluated mitochondrial function. JC-1 staining showed that under basal conditions, the supplementation of the FKBP8 and Sialin complex slightly reduced the mitochondrial membrane potential; however, after CCCP treatment, the supplementation of the FKBP8 and Sialin complex resulted in a significantly higher mitochondrial membrane potential than the vector control group Figure 5 D). mPTP assay results showed that under basal conditions, the mitochondrial membrane permeability of the FKBP8 and Sialin complex supplementation experimental group was higher than that of the vector control group, but after CCCP treatment, the permeability of the FKBP8 and Sialin complex supplementation experimental group was significantly reduced Figure 5 E). TMRE staining showed that under basal conditions, there was no significant difference in the number of active mitochondria between the FKBP8 and Sialin complex supplementation experimental group and the vector control group; after CCCP treatment, the number of active mitochondria in the vector control group decreased to 47.58% of the baseline, while the number of active mitochondria in the FKBP8 and Sialin complex supplementation experimental group remained at 75.29% Figure 5F) With these findings, the supplementation of FKBP8 with Sialin complex decreased the basal expression of OPA1 and TOM20, but after CCCP treatment, the expression of both proteins was significantly higher in the FKBP8 with Sialin complex supplementation group than in the vector control group Figure 5 G, H) Overall, these results show that the supplementation of FKBP8 with Sialin complex alleviates CCCP-induced mitochondrial dysfunction.

[0099] To investigate the role of FKBP8 with Sialin complex (aggregates) in maintaining mitochondrial function, on the other hand, the present embodiment also constructed a stable cell line overexpressing sialic acid. Under normal conditions, overexpression of sialic acid did not change the mitochondrial membrane potential. JC-1 staining results showed that after treatment with CCCP, the mitochondrial membrane potential of the vector control group decreased, and the supplementation of FKBP8 with Sialin complex significantly alleviated this decrease Figure 6 A). mPTP assay results showed that under untreated conditions, there was no significant difference in mitochondrial membrane permeability between the FKBP8 with Sialin complex experimental group and the vector control group. After CCCP treatment, the mitochondrial membrane permeability of the vector control group increased, but the mitochondrial membrane permeability of the FKBP8 with Sialin complex group decreased significantly Figure 6 B). Consistently, TMRE staining results showed that after CCCP exposure, the number of metabolically active mitochondria in the FKBP8 with Sialin complex experimental group was significantly higher than that in the vector control group Figure 6 C). Subsequently, the protein expression of TOM20 and OPA1 was examined, and under normal conditions, there was no difference in the expression levels of these two proteins between the FKBP8 with Sialin complex group and the vector group. After CCCP treatment, the expression of OPA1 in the vector group decreased by 49.96%, and the expression of TOM20 decreased by 47.07%, and the expression of both proteins in the FKBP8 with Sialin complex group was higher than that in the vector group Figure 6 D, E) TEM observation showed that under normal conditions, the mitochondria of the vector control group and the FKBP8 with Sialin complex group exhibited intact membranes and clear cristae. After CCCP treatment, the vector control group showed obvious mitochondrial swelling, cristae rupture or loss, and an increased number of damaged mitochondria. In contrast, the FKBP8 with Sialin complex group showed a decrease in the number of damaged mitochondria and a relatively normal morphology of cristae Figure 6 F) Overall, the supplementation of FKBP8 with Sialin complex is beneficial to the protection of mitochondrial function.

[0100] 6. Sialic acid binds to FKBP8 and forms granules In this embodiment, FKBP8 knockdown HSG cells were constructed to investigate whether nitrate maintains mitochondrial functional stability by promoting FKBP8 expression or sialic acid-FKBP8 binding. JC-1 staining showed that after CCCP exposure, the mitochondrial membrane potential of both the FKBP8 knockdown group and the vector control group decreased. Notably, after nitrate supplementation, the membrane potential of the FKBP8 knockdown group was significantly lower than that of the vector control group. Figure 7 A). mPTP assay results showed no significant difference in mitochondrial membrane permeability between the FKBP8 knockdown group and the vector control group after CCCP treatment. However, nitrate supplementation reduced mitochondrial membrane permeability in the vector control group, but did not have a significant effect in the FKBP8 knockdown group. Figure 7 B). TMRE staining results showed a consistent trend: after CCCP treatment, there was no significant difference in the number of active mitochondria between the FKBP8 knockdown group and the vector control group, while nitrate supplementation increased the number of active mitochondria in the vector control group, but had no significant effect on FKBP8 knockdown. Figure 7 C). These findings suggest that nitrate-mediated protection against mitochondrial damage depends on FKBP8 expression.

[0101] To investigate whether FKBP8 plays a role in alleviating nitrate-mediated mitochondrial dysfunction through sialic acid-FKBP8 interaction and direct mitochondrial targeting, this study examined the mitochondrial localization of FKBP8 and TOM20 in sialic acid-overexpressing cells. IF analysis revealed significant differences in FKBP8 localization among the groups: in sialic acid-overexpressing cells, co-localization of FKBP8 and TOM20 indicated that enhanced binding of FKBP8 to sialicin led to enhanced mitochondrial targeting of FKBP8. Figure 7 (D, E). Therefore, Sialin overexpression promotes FKBP8 aggregation on mitochondria, and Sialin-FKBP8 binding enhances the formation of condensates to repair mitochondrial membrane damage.

[0102] To validate Sialin-FKBP8, this example prepared the mCherry-FKBP8 fusion protein (showing red fluorescence), while the vector control expressed only mCherry. IF results showed that FKBP8 formed red dot-like lesions, which aggregated and fused into larger components. Further validation was performed using live-cell time-lapse imaging combined with fluorescence recovery after photobleaching (FRAP). The mCherry-FKBP8 overexpression plasmid was transfected into Sialin-overexpressing, Sialin-knockdown, and control cells. Notably, in cells overexpressing sialic acid, the mCherry-FKBP8 lesions showed fluorescence recovery at the bleached site within approximately 30 seconds, exhibiting characteristics of phase-separated structures. Figure 7F). In contrast, no fluorescence recovery was observed in the other two groups. Moreover, increased sialic acid expression and acidification (pH ~ 6.5, induced by hydrochloric acid addition) promoted FKBP8 phase separation droplet formation Figure 7 G). Then, the sialic acid-FKBP8 interaction during the nitrate-mediated sialadenitis radiation damage mitigation process was examined using immunofluorescence. The IF results showed that Sialin-FKBP8 binding was enhanced in the sialadenitis of the IR+ nitrate group compared to the IR group Figure 7 H). Intracellular pH was measured using BCECF-AM probe: under basal conditions, the intracellular pH was 7.04; after nitrate supplementation, it decreased to 6.85; in the IR+ nitrate group, the pH was ~ 6.67 Figure 7 I). Moreover, it was also observed that, at 1 mM nitrate, the co-localization of sialic acid and FKBP8 was enhanced. Overall, these data suggest that nitrate promotes sialic acid-FKBP8 formation of aggregated condensates or complexes on mitochondria to mitigate mitochondrial function impairment.

[0103] 7. The sialic acid-FKBP8 interaction is crucial for nitrate protection against mitochondrial impairment To determine whether the Sialin-FKBP8 interaction is crucial for the protective effect of nitrate, atomistic modeling was performed to simulate their binding interface. Briefly, the complex structures of SLC17A5 (encoding sialic acid) and FKBP8 were predicted from their amino acid sequences using AlphaFold3 Figure 8 A). The predicted complex structures were further refined and stable trajectories were extracted from molecular dynamics simulations for energy and binding mode analysis. Based on the binding energy contribution of individual amino acids on the interaction interface, potential Sialin-FKBP8 binding sites were determined Figure 8 B). Among them, sialic acid residues Thr360 and Arg494 and FKBP8 residue Arg225 exhibited the most significant binding energy contribution Figure 8 C). Therefore, Arg494 of the sialic acid protein with the largest binding contribution was selected for site-directed mutagenesis. After constructing the Sialin R494A mutant (MUT) plasmid for cell transfection, immunoprecipitation was initially employed to verify the change in binding between Sialin and FKBP8. The results showed that the binding between sialic acid and FKBP8 in the MUT was significantly reduced compared to the wild type (WT) Figure 8 D). Nitrate supplementation significantly enhanced the Sialin-FKBP8 interaction in the WT group; however, this interaction was not enhanced in the MUT group even with nitrate supplementation Figure 8E) The role of Sialin-FKBP8 binding in the response to radiation damage was further explored. Under normal conditions, there was no significant change in the expression of anti-apoptotic proteins BCL2 and BCL-XL in the MUT group compared to the WT group, but the level of cytochrome c was increased Figure 8 F) After IR exposure, the MUT group showed a significant downregulation of BCL2 and BCL-XL expression and a significant upregulation of cytochrome c expression compared to the WT group Figure 8 G) In addition, Sialin knockdown cells were transfected with WT or MUT Sialin plasmids and the proteins related to mitochondrial damage were evaluated under basal and IR conditions. Under non-IR conditions, no significant differences in these proteins were observed between the WT and MUT groups. However, in the IR and IR+nitrate groups, the MUT group showed a significant decrease in BCL2 and BCL-XL expression and a significant increase in cytochrome c expression compared to the WT group Figure 8 I) These results indicate that mutation of the Sialin-FKBP8 binding site significantly eliminates the protective effect of nitrate on radiation-induced mitochondrial damage. Therefore, Sialin-FKBP8 interaction represents a key pathway for the mitigation of mitochondrial radiation damage by nitrate.

[0104] While the application has been described with reference to the example embodiments, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the application. The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the principles, and the scope of the application as set forth in the claims.

Claims

1. A protein complex for protecting mitochondria from damage, characterized in that, This includes sialic acid transporter and FKBP8.

2. The protein complex for protecting mitochondria from damage according to claim 1, wherein, The sialic acid transporter and FKBP8 interact or connect via non-covalent or covalent bonds.

3. The protein complex of claim 1, wherein, The protein complex exists in the form of an aggregate.

4. A pharmaceutical composition for reducing the degree of radiation damage, characterized by, Includes the protein complex or precursor according to any one of claims 1-3, or a promoter for promoting the formation of said complex; Preferably, the precursor comprises nucleic acids for generating protein complexes, or nucleic acids for generating sialic acid transporter protein and / or FKBP8; Preferably, the precursor includes both a sialic acid transporter precursor and an FKBP8 precursor; Preferably, the composition is designed such that the sialic acid transporter precursor and the FKBP8 precursor each exist in an independent form, and that the protein complex exists in aggregate form during use.

5. An engineered cell, characterized by, The cells contain the protein complex or precursor thereof as described in any one of claims 1-3, introduced by artificial means.

6. A method for facilitating complexation of a sialic acid transporter and FKBP8, comprising, This includes steps such as adding promoters to cause sialic acid transporter protein and FKBP8 to aggregate and then complex.

7. A method for reducing the extent of radiation damage in a living organism, comprising administering to the organism a therapeutically effective amount of a compound of claim 1. Includes the step of contacting the organism with the protein complex according to any one of claims 1-3; Preferably, the organism includes cells, tissues, organoids, or organs, and the method is an in vitro method.

8. A mutant sialic acid transporter characterized in that, It contains a mutation at amino acid position 494; Preferably, the 494th amino acid is mutated from arginine to a hydrophobic amino acid.

9. A method for inhibiting complexing or aggregation of sialic acid transporter and FKBP8, characterized by, The steps to mutate the 494th amino acid of the sialic acid transporter, or the steps to use the sialic acid transporter with the 494th amino acid mutated.

10. A method for protecting mitochondria from damage, characterized in that, This includes the step of forming a sialic acid transporter and FKBP8 complex, preferably a condensate, within the mitochondrial membrane.

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