CN114075590A

CN114075590A - Application of SIRT3 mediated macrophage Frataxin deacetylation modification in improvement of inflammatory diseases

Info

Publication number: CN114075590A
Application number: CN202010832347.8A
Authority: CN
Inventors: 沈伟利; 高晶; 魏彤; 黄程淋
Original assignee: SHANGHAI INSTITUTE OF HYPERTENSION
Current assignee: SHANGHAI INSTITUTE OF HYPERTENSION
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2022-02-22
Anticipated expiration: 2040-08-18
Also published as: CN114075590B

Abstract

The invention provides an application of SIRT3 mediated macrophage Frataxin deacetylation modification in improvement of inflammatory diseases. The invention discloses that SIRT3 and Frataxin can interact for the first time, and SIRT3 mediates deacetylation modification of Frataxin, so that polarization of macrophages is adjusted. By modulating the interaction of SIRT3 with Frataxin, M2 type polarization of macrophages can be modulated to alleviate or treat inflammation-related diseases such as hypertension. The invention has important significance for preventing and treating diseases related to mitochondrial metabolism and blood pressure disorder.

Description

Application of SIRT3 mediated macrophage Frataxin deacetylation modification in improvement of inflammatory diseases

Technical Field

The invention belongs to the field, and particularly relates to SIRT3 mediated Frataxin deacetylation modification and application thereof in inhibiting macrophage polarization and improving inflammatory diseases; preferably, the inflammatory disease may be an inflammatory disease associated with hypertension.

Background

Macrophages are representative of innate immune regulatory cells, and their mediated activation of innate immunity and release of a number of inflammatory mediators are thought to be one of the important pathogenesis of induced inflammation and inflammation-related disorders. Macrophages have the ability to polarize and deform, conferring different cellular phenotypes and functions. The concept of macrophage polarization was proposed by researchers' observations that macrophages exposed to Interferon-gamma (Interferon gamma, IFN-gamma and Interleukin-4 (Interleukin-4, IL-4) have distinct gene expression profiles, which were classified into two subtypes M1 and M2 Lipopolysaccharide (LPS), IFN-gamma, induce the differentiation of M1 macrophages, exhibiting a pro-inflammatory phenotype characterized by the secretion of high levels of pro-inflammatory factors, and M1 macrophages are important sources of many inflammatory cytokines, including Tumor cell necrosis factor (Tumor necrosis factor alpha, TNF alpha), Interleukin-1 beta (Interleukin-1 beta, IL-1 beta), Interleukin-12 (Interleukin-12, IL-12), Interleukin-18 (Interleukin-18, IL-18) and Interleukin-23 (Interleukin-23, IL-23), which have been identified as important mediators and drivers of chronic inflammatory and autoimmune diseases. IL-4 and IL-13 promote macrophage M2 type polarization (alternative activation type), and are characterized by expressing high levels of galactose, mannose receptors and anti-inflammatory factors. Has effects in promoting tissue repair, blood vessel regeneration and fibrosis. Therefore, the interconversion and existence ratio of M1/M2 macrophage can precisely regulate the change of microenvironment in tissues, plays a key role in prognosis and prognosis of inflammation-related diseases, and elucidates the reason of macrophage polarization, so as to provide a new target for treating the inflammation diseases.

Under normal physiological conditions, iron storage, absorption and release by macrophages in the reticuloendothelial system are of great importance for the maintenance of iron metabolic homeostasis in vivo. Macrophages are the bridge connecting iron metabolism and immune response, and controlling available iron in the body is a key step of immune response. The iron level of macrophages can change the polarization, M1 type macrophages have higher iron ion accumulation capacity, and the increase of the intracellular iron level enhances the proinflammatory reaction. While type M2 metabolizes and outputs iron, thereby lowering intracellular iron concentrations, low intracellular iron levels may be shown to inhibit expression of pro-inflammatory cytokines.

However, in the field, the intrinsic mechanism of how macrophages sense the change of microenvironment and start the transition of iron metabolic flux to induce macrophage polarization under the condition of inflammatory diseases is not clear, and further understanding on macrophage polarization regulation is urgently needed to develop a drug for regulating macrophage polarization for clinical application.

Disclosure of Invention

The invention aims to provide SIRT3 mediated macrophage Frataxin deacetylation modification and application thereof in improving inflammatory diseases; preferably, the inflammatory disease may be an inflammatory disease associated with hypertension.

In a first aspect of the invention, there is provided the use of a complex of SIRT3 interacting with Frataxin for: as a target for regulating macrophage polarization, preparing a medicament for regulating macrophage polarization; as a target for screening the medicament for regulating the polarization of the macrophages, screening the medicament for regulating the polarization of the macrophages; or as a target for assessing the polarization state of macrophages, and preparing an agent for assessing the polarization state of macrophages.

In another aspect of the invention, there is provided the use of a modulator that modulates the interaction of SIRT3 with Frataxin for modulating macrophage polarization; preferably, the modulator is an up-modulator that promotes the interaction of SIRT3 with Frataxin, which reduces the level of acetylation of lysine 189 in Frataxin (i.e., promotes deacetylation of lysine 189 in Frataxin), thereby promoting M2-type polarization of macrophages; or the regulator is a down regulator for inhibiting the interaction of SIRT3 and Frataxin, and the down regulator increases the acetylation level of the 189 th lysine of Frataxin (namely promotes the acetylation of the 189 th lysine of Frataxin), thereby promoting the M1 type polarization of macrophages.

In a preferred example, the SIRT3 interaction with Frataxin includes: SIRT3 regulates the acetylation modification mode of lysine 189 in Frataxin; preferably, the SIRT3 and Frataxin interaction is an interaction in mitochondria.

In another preferred example, the up-regulator comprises: substances for enhancing the activity of SIRT3 or Frataxin, substances for enhancing the expression, stability or effective acting time of SIRT3 or Frataxin; preferably comprising a compound selected from: expression constructs (including expression vectors) for recombinant expression of SIRT3 or Frataxin, polypeptides or compounds for enhancing the effect of SIRT3 on Frataxin, chemical up-regulators of SIRT3 or Frataxin, and up-regulators of the driving ability of SIRT3 or Frataxin gene promoters.

In another preferred embodiment, the down-regulating agent comprises: a substance that down-regulates SIRT3 or Frataxin activity or a substance that down-regulates SIRT3 or Frataxin expression, stability or reduces its effective duration of action; preferably comprising: an agent that knocks out or silences SIRT3 or Frataxin, a binding molecule (e.g., an antibody or ligand) that specifically binds SIRT3 or Frataxin, a small chemical molecule antagonist or inhibitor against SIRT3 or Frataxin, angiotensin II.

In another preferred embodiment, the expression construct (expression vector) comprises: viral vectors, non-viral vectors; preferably, the expression vector comprises: adeno-associated virus vectors, lentiviral vectors, and adenoviral vectors.

In another preferred embodiment, the agent that knocks out or silences SIRT3 or Frataxin includes (but is not limited to): a CRISPR gene editing reagent for SIRT3 or Frataxin, an interference molecule which specifically interferes with the expression of a coding gene of SIRT3 or Frataxin, a homologous recombination reagent or a site-directed mutation reagent for SIRT3 or Frataxin, and the homologous recombination reagent or the site-directed mutation reagent carries out loss-of-function mutation on SIRT3 or Frataxin.

In another preferred embodiment, the interfering molecule comprises an siRNA, shRNA, miRNA, antisense nucleic acid, or the like, or a construct capable of forming the siRNA, shRNA, miRNA, antisense nucleic acid, or the like.

In another preferred embodiment, the SIRT3 is selected from the following group: (a) polypeptide with amino acid sequence as shown in SEQ ID No. 1; (b) a SIRT3 derivative with the polypeptide function of (a) or (b) formed by substituting, deleting or adding one or more (such as 1-20, 1-10, 1-5, 1-3 or 1-2) amino acid residues in the amino acid sequence shown in (a), or an active fragment thereof; (c) the SIRT3 derivative with homology of more than or equal to 90 percent (such as homology of more than or equal to 92 percent, more than or equal to 94 percent, more than or equal to 96 percent, more than or equal to 98 percent or more than or equal to 99 percent) or an active fragment thereof is compared with the amino acid sequence shown in SEQ ID NO. 1.

In another preferred embodiment, Frataxin is selected from the group consisting of: (a) polypeptide with amino acid sequence as shown in SEQ ID No. 2; (b) a Frataxin derivative with polypeptide functions of (a) or (b) or an active fragment thereof, which is formed by substituting, deleting or adding one or more (such as 1-20, 1-10, 1-5, 1-3 or 1-2) amino acid residues in the amino acid sequence shown in (a); (c) the Frataxin derivative with homology of more than or equal to 90 percent (such as homology of more than or equal to 92 percent, more than or equal to 94 percent, more than or equal to 96 percent, more than or equal to 98 percent or more than or equal to 99 percent) or an active fragment thereof is compared with the amino acid sequence shown in SEQ ID NO. 2.

In another aspect of the invention, there is provided the use of an agent that specifically recognizes the complex of SIRT3 interacting with Frataxin or recognizes the acetylation modification of lysine 189 of Frataxin, for the preparation of an agent or kit for assessing the polarization of macrophages; preferably, the specifically recognized reagents include (but are not limited to): a binding molecule (e.g., an antibody or ligand) that specifically binds SIRT3 or Frataxin; more preferably, the binding molecule that specifically binds Frataxin comprises: a binding molecule that specifically recognizes or binds to Frataxin acetylated lysine at position 189.

In a preferred embodiment, the agent that specifically recognizes the complex of SIRT3 interacting with Frataxin indicates that M2-type polarization of macrophages is dominant (promoted) if an enhanced interaction of SIRT3 with Frataxin is detected; if a decrease in the interaction of SIRT3 with Frataxin is detected, this indicates that M1-type polarization of macrophages is dominant (promoted).

In another preferred embodiment, the specific recognition reagent further comprises (but is not limited to): primers for specifically amplifying SIRT3 gene or Frataxin gene; a probe that specifically recognizes the SIRT3 gene or Frataxin gene; a chip for specifically recognizing SIRT3 gene or Frataxin gene.

In another preferred embodiment, using said agent for identifying acetylation modification of Frataxin lysine 189, a decrease in the level of acetylation of Frataxin lysine 189 is detected, indicating that M2-type polarization of macrophages is predominant; if an increased level of acetylation of lysine 189 in Frataxin is detected, this indicates that M1-type polarization of macrophages is dominant.

In another preferred embodiment, the polarization of the macrophages comprises M2 type polarization or M1 type polarization; preferably, said promoting M2-type polarization of macrophages comprises inhibiting a macrophage inflammatory phenotype; more specifically, the method comprises the following steps: promoting the release of macrophage iron ions, increasing the synthesis of mitochondrial iron-sulfur cluster and heme, improving the expression of inflammation-inhibiting factors, reducing the expression of inflammatory factors, reducing the accumulation of cell lipid peroxides, improving mitochondrial oxidative stress and delaying inflammatory lesions caused by hypertension; or, said promoting M1-type polarization of macrophages comprises promoting a macrophage inflammatory phenotype; more specifically, the method comprises the following steps: promoting macrophage iron ion accumulation, reducing mitochondrial iron-sulfur cluster and heme synthesis, increasing inflammatory factor expression, and increasing cell lipid peroxide accumulation.

In another preferred embodiment, the inflammatory factors include (but are not limited to): TNF- α, IL-6, Inducible Nitric Oxide Synthase (iNOS), or INF- γ; or, the anti-inflammatory factors include (but are not limited to): IL-10, YM-1, ARG-1 or IL-4.

In another aspect of the invention, there is provided a method of screening for potential agents that modulate macrophage polarization comprising: (1) treating an expression system expressing a complex of SIRT3 and Frataxin interaction with a candidate substance; and, (2) detecting the interaction of SIRT3 with Frataxin in the system; (ii) if the candidate substance statistically promotes the interaction of SIRT3 with Frataxin, then the candidate substance is a potential substance that promotes M2-type polarization of macrophages; if the candidate substance statistically inhibits the interaction of SIRT3 with Frataxin, the candidate substance is a potential substance for promoting M1 type polarization of macrophages.

In a preferred embodiment, step (1) comprises: in the test group, adding a candidate substance to the expression system; and/or, the step (2) comprises: detecting the interaction condition of SIRT3 and Frataxin in the system; and comparing the expression system with a control group, wherein the control group is an expression system without the candidate substance; (ii) if the candidate substance statistically promotes the interaction of SIRT3 with Frataxin, then the candidate substance is a potential substance that promotes M2-type polarization of macrophages; if the candidate substance statistically inhibits the interaction of SIRT3 with Frataxin, the candidate substance is a potential substance for promoting M1 type polarization of macrophages.

In another preferred example, the detecting the interaction between SIRT3 and Frataxin in the system comprises: detecting SIRT 3-mediated acetylation modification of Frataxin lysine 189, wherein if the candidate substance causes SIRT 3-mediated reduction (significant reduction, such as reduction by more than 10%, more than 20%, more than 50%, more than 80%, etc.) of the acetylation level of Frataxin lysine 189, the candidate substance is a potential substance for promoting M2 type polarization of macrophages; if the candidate substance causes SIRT 3-mediated increase (a significant increase, such as an increase of more than 10%, more than 20%, more than 50%, more than 80%, etc.) in the acetylation level of lysine 189 in Frataxin, the candidate substance is a potential substance for promoting M1 type polarization of macrophages.

In another preferred embodiment, the candidate substance includes (but is not limited to): regulatory molecules designed to SIRT3 or Frataxin, fragments or variants thereof, genes encoding the same or upstream and downstream molecules or signaling pathways thereof, or constructs thereof (e.g., shRNA, siRNA, gene editing agents, expression vectors, recombinant viral or non-viral constructs, etc.), small chemical molecules (e.g., specific inhibitors or antagonists), interactive molecules, etc.

In another preferred embodiment, the system is selected from: a cell system (e.g., a cell or cell culture expressing SIRT3 or Frataxin), a subcellular (culture) system, a solution system, a tissue system, an organ system, or an animal system.

In another preferred example, the method further comprises: the obtained potential substance is subjected to further cell experiments and/or animal experiments to further select and identify a substance useful for regulating the polarization of macrophages from the candidate substances.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, SIRT3 deletion exacerbates mitochondrial iron accumulation in macrophages, promoting macrophage polarization;

[A]after SIRT3-shRNA is transferred into M1920 cells, qPCR (quantitative polymerase chain reaction) is used for detecting SIRT3 mRNAHorizontal; compared with the normal control group, the composition has the advantages that,^*P<0.05；

[B] after inhibiting the expression of SIRT3, and stimulating by Ang II for 24 hours, M1920 cells ABCB7, Mito-ferrin, Frataxin and SIRT3 protein immunoblotting bands;

[C]inhibiting SIRT3 expression and stimulating with Ang II for 24 hours, and detecting the MDA level in the cells; compared with the normal control group, the composition has the advantages that,^*P<0.05; compared with the control group of Ang II,^#P<0.05；

[D]inhibiting SIRT3 expression and stimulating with Ang II for 24 hours, detecting mRNA level of IL-6 and iNOS by qPCR, comparing with normal control group,^*P<0.05; compared with the control group of Ang II,^#P<0.05，^##P<0.01；

[E]inhibiting SIRT3 expression and stimulating with Ang II for 24 hours, qPCR detecting Arg-1, IL-10 mRNA level, comparing with normal control group,^*P<0.05; compared with the control group of Ang II,^#P<0.05。

FIG. 2, loss of Frataxin activity promotes macrophage mitochondrial iron accumulation, increases accumulation of cellular lipid peroxides, and leads to macrophage polarization;

[A] after M1920 cells are transferred into Frataxin-shRNA and stimulated by Ang II for 24 hours, Lipoflour staining is carried out, and the fluorescence intensity is detected by flow cytometry;

[B]detecting the MDA level in the cells, compared with the normal control group,^*P<0.05; compared with the control group of Ang II,^#P<0.05；

[C]qPCR was performed to detect the mRNA levels of INF-gamma and iNOS, and compared with the normal control group,^*P<0.05; compared with the control group of Ang II,^#P<0.05；

[D]the mRNA levels of Arg-1 and YM-1 were determined by qPCR, and compared to the normal control,^*P<0.05; compared with the control group of Ang II,^#P<0.05；

[E] m1920 cells increased Frataxin expression and were stimulated with Ang II for 24 hours, stained with lipoflur, and fluorescence intensity was detected by flow cytometry;

[F] after M1920 cells increased Frataxin expression and stimulated with Ang II for 24 hours, Mito-FerroGreen stained and fluorescence intensity was detected by flow cytometry;

[G]detecting the MDA level in the cells, compared with the normal control group,^*P<0.05; compared with the control group of Ang II,^#P<0.05；

[H]the mRNA level of IL-6 and TNF-alpha is detected by qPCR, compared with a normal control group,^*P<0.05; compared with the control group of Ang II,^#P<0.05；

[I]the mRNA levels of Arg-1 and YM-1 were determined by qPCR, and compared to the normal control,^*P<0.05; compared with the control group of Ang II,^#P<0.05。

FIG. 3, SIRT3 regulates acetylation modification of lysine 189 site (Lys189) of Frataxin protein;

[A] respectively immunoprecipitating IgG and Frataxin by 293T cell lysate, and detecting endogenous Frataxin and SIRT3 protein immunoblot images;

[B] immunoprecipitating IgG and SIRT3, respectively, and detecting endogenous Frataxin and SIRT3 protein immunoblot images;

[C]293T is introduced with exogenous HA-SIRT3 and Flag-Frataxin, immunoprecipitation Flag, and detection of exogenous Frataxin and SIRT3 protein immunoblot images;

[D] immunoprecipitating HA, detecting exogenous Frataxin and SIRT3 protein immunoblot images;

[E] frataxin189 site lysine acetylation mass spectrum;

[F] human, mouse, dog, donkey, fruit fly, nematode and fungal Frataxin189 site lysine;

[G]293T cells are transferred into SIRT3-shRNA, and 293T cells are provided with Frataxin 189K-Ac, Frataxin and SIRT3 protein immunoblotting strips;

[H]293T cells are respectively transferred into Frataxin WT, Frataxin189 site lysine-arginine mutation (FXN189KR) and 189 site lysine-glutamine mutation (FXN189 KQ) plasmids, and 293T cells Frataxin, Complex I and Complex II protein immunoblotting bands;

[I] measurement of the activity of 293T cells complete I < 0.05; compared to the FXNWT group, # P < 0.05.

FIG. 4, increasing the expression of SIRT3, promoting the deacetylation of Frataxin (Lys189) sites, inhibiting the polarization of macrophages;

[A] the 293T cell is transferred into exogenous SIRT3, and 293T cells Frataxin 189K-Ac, Frataxin and SIRT3 protein immunoblotting strips;

[B]after the M1920 cell lentivirus infection increases the expression of SIRT3, the qPCR detects the SIRT3 mRNA level, compared with a normal control group,^*P<0.05；

[C] m1920 cell lentiviral infection increased SIRT3 expression and stimulated with Ang II for 24 hours, Frataxin and SIRT3 western blot bands for M1920 cells;

[D]detecting the MDA level in the cells, compared with the normal control group,^*P<0.05; compared with the control group of Ang II,^#P<0.05；

[E]increase SIRT3 expression and stimulate with Ang II for 24 hours, qPCR detects IL-6, iNOS, mRNA level, compare with normal control group,^*P<0.05; compared with the control group of Ang II,^#P<0.05；

[F]increases SIRT3 expression and stimulates with Ang II for 24 hours, qPCR detected Arg-1 and IL-10 mRNA levels, compared to normal control,^*P<0.05; compared with the control group of Ang II,^#P<0.05。

Detailed Description

Through intensive research, the inventor firstly reveals that SIRT3 and Frataxin can interact, and SIRT3 mediates deacetylation modification of Frataxin, so that polarization of macrophages is regulated. By modulating the interaction of SIRT3 and Frataxin, M2 type polarization of macrophages can be modulated to alleviate or treat inflammation-related diseases (such as hypertension).

SIRT3 and Frataxin and interaction thereof

The sirtuins (sirts) family is a group of nicotinamide adenine dinucleotide coenzyme (NAD +) dependent deacetylases. The member SIRT3 in the SIRTs family is located mainly in mitochondria, SIRT3 can activate succinate dehydrogenase, acetyl CoA synthetase 2 and isocitrate dehydrogenase 2 through its deacetylation activity; subunit NDUF9A of electron transport chain complex I, NADH dehydrogenase and ATP synthase; the long-chain acyl coenzyme A dehydrogenase and other key energy metabolism related enzymes in the fatty acid oxidase beta-oxidation process enhance the oxidation respiration of mitochondria, maintain the stable energy metabolism of the mitochondria and reduce the generation of active oxygen (ROS), but the regulation of SIRT3 through which way is carried out is not clear in the field. The present inventors focused on the study of SIRT3 in macrophages and found that in obese patients, an increase in inflammatory factors in peripheral blood mononuclear macrophages was accompanied by a decrease in SIRT 3; in a mouse acute hyperlipidemia model induced by Pox407, the reduction of macrophage SIRT3 can promote the secretion of inflammatory factors TNF-alpha and Il-1 beta and reduce the inflammation of vascular intima; in a hypertension mouse model established by angiotensin II (AngII) perfusion, the decrease of macrophage SIRT3 in perivascular adipose tissues is found to be inversely related to the secretion of inflammatory factor Il-1 beta, and the overexpression of SIRT3 can reduce the secretion of Il-1 beta and repair angioremodeling induced by Ang II. Thus, SIRT3 was shown to be involved in regulating macrophage inflammatory factor secretion. However, the mechanism by which SIRT3 promotes macrophage polarization by affecting macrophage iron metabolism is not clear.

Mitochondria are the center of cellular free radical metabolism and iron metabolism, and maintenance of mitochondrial iron homeostasis involves a dynamic balance between iron uptake, iron storage, and iron utilization. Mitochondrial iron uptake is primarily responsible for transport by the mitochondrial iron transporter 1/2(Mitoferrin1/2) and the ATP-binding transporter ABCB10(ATP-binding cassette B10); mitochondrial iron export is responsible for ABCB6 and ABCB 7; mitochondrial iron storage is associated with Mitochondrial ferritin (mitochondral ferritin, MtFt); frataxin is located in a mitochondrial matrix and participates in the synthesis of heme and iron-sulfur clusters in mitochondria, wherein the former is a prosthetic group of hemoglobin, myoglobin, cytochrome, peroxidase, catalase and the like; the latter is involved in the composition of ferredoxin, cytochrome b and electron transport chain complexes I, II, III. Therefore, Frataxin functions as an iron chaperone, an iron-sensitive negative regulator, and a regulatory metabolic switch, and the iron metabolism-related proteins cooperate to precisely regulate the level of mitochondrial iron.

Since the synthesis of iron-sulfur clusters is central to both mitochondrial iron metabolism and the operation of whole cellular iron, Frataxin is considered to be the pivotal hub of mitochondrial iron metabolism. The FRDA (Friedreich ataxia, FRDA) gene encoding human Frataxin, located distal to the long arm of chromosome 9, is a 95kb gene consisting of 7 exons. The mutation of the gene leading to Friedreich's ataxia syndrome is the most common hereditary syndrome abroad. Research reports that the Frataxin defect can cause iron-sulfur cluster and heme biosynthesis disorder and respiratory chain electron transporter complex I-III defect; mitochondrial iron overload, overriding MtFt iron storage function, further mediates increased variable iron pools, oxidative stress and free radical accumulation in mitochondria. However, it is not clear in the art how Frataxin is regulated.

After intensive research, the inventor finds that SIRT3 interacts with Frataxin, and SIRT3 does not affect the expression of macrophage Frataxin on the level of transcription and protein, but has posttranslational modification on Frataxin and influences the activity of Frataxin. Based on the localization of SIRT3 in mitochondria, the inventors applied acetylation quantitative proteomics analysis technology to compare the results from Wild Type (WT) and SIRT3 Knockout (SIRT 3)^-/-) According to the difference of the acetylation levels of mouse mitochondrial proteins, 36 mitochondrial proteins regulated and controlled by SIRT3 are screened, 116 acetylation sites are identified, and the acetylation level of Frataxin protein, a key molecule for regulating and controlling mitochondrial iron metabolism, is found to be obviously increased, wherein K189 has the most obvious acetylation regulation effect. By comparing the amino acid sequences of Frataxin of multiple species, the inventors found that the acetylation modification of lysine at position 189, which is highly conserved from invertebrates to humans, can affect the function of Frataxin. Whether the acetylation modification of the site can reduce the activity of Frataxin to a greater extent or not and promote mitochondrial iron overload and lipid peroxide accumulation is worthy of being deeply researched.

From the above, the present inventors further believe that: cell SIRT3 is down regulated to promote the Frataxin to be highly acetylated, and the Frataxin acetylation modification mediates mitochondrial iron accumulation by reducing the synthesis of mitochondrial iron-sulfur clusters and heme, so that mitochondrial oxidative stress is caused, macrophage polarization is promoted, and the Frataxin is involved in the generation of inflammatory diseases. In addition, the inventor uses angiotensin II (AngII) to simulate a cell model of macrophage polarization in a hypertension pathological state, takes mitochondrial iron metabolism as an entry point, and researches 1) the influence of SIRT3 on mitochondrial iron homeostasis by regulating acetylation modification of macrophage Frataxin; 2) mitochondrial iron overload mediates oxidative stress of macrophages; 3) the oxidative stress of the macrophage promotes macrophage polarization, and the macrophage secretes inflammatory factors, thereby further clarifying the relationship between SIRT 3/Frataxin-macrophage polarization and opening up a new means for diagnosing and treating inflammatory diseases.

As used herein, promoting M2-type polarization of macrophages includes inhibiting the macrophage inflammatory phenotype; more specifically, the method comprises the following steps: promoting the release of macrophage iron ions, increasing the synthesis of mitochondrial iron-sulfur cluster and heme, improving the expression of inflammation-inhibiting factors, reducing the expression of inflammatory factors, reducing the accumulation of cell lipid peroxides, improving the oxidative stress of mitochondria and delaying inflammatory lesions caused by hypertension.

As used herein, promoting M1-type polarization of macrophages includes promoting a macrophage inflammatory phenotype; more specifically, the method comprises the following steps: promoting macrophage iron ion accumulation, reducing mitochondrial iron-sulfur cluster and heme synthesis, increasing inflammatory factor expression, and increasing cell lipid peroxide accumulation.

When applied to the present invention, the SIRT3 or Frataxin may be naturally occurring, such as it may be isolated or purified from a mammal. In addition, the SIRT3 or Frataxin can also be artificially prepared, for example, recombinant SIRT3 or Frataxin can be produced according to the conventional genetic engineering recombination technology for application in experiments or clinics. The SIRT3 or Frataxin comprises a full-length protein or a bioactive fragment thereof. Preferably, the amino acid sequence of the SIRT3 may be substantially identical to the sequence shown in SEQ ID NO. 1 or homologues thereof, including for example human SIRT 3; the amino acid sequence of Frataxin can be substantially identical to the sequence shown in SEQ ID No. 2 or homologues thereof, including, for example, human Frataxin having homology thereto. The corresponding nucleotide coding sequence can be conveniently derived from the amino acid sequence of SIRT3 or Frataxin.

Amino acid sequences of SIRT3 or Frataxin formed by substitution, deletion or addition of one or more amino acid residues are also included in the invention. SIRT3 or Frataxin, or a biologically active fragment thereof, includes a replacement sequence of a portion of conserved amino acids, which does not affect its activity or retains some of its activity. Appropriate substitutions of amino acids are well known in the art and can be readily made and ensure that the biological activity of the resulting molecule is not altered. These techniques allow one of skill in the art to recognize that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. See Watson et al, Molecular Biology of The Gene, fourth edition, 1987, The Benjamin/Cummings Pub. Co. P224.

Any biologically active fragment of SIRT3 or Frataxin may be used in the present invention. Herein, a biologically active fragment of SIRT3 or Frataxin is meant to be a polypeptide that still retains all or part of the function of full-length SIRT3 or Frataxin. Typically, the biologically active fragment retains at least 50% of the activity of full-length SIRT3 or Frataxin. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of full-length SIRT3 or Frataxin.

The invention may also employ modified or improved SIRT3 or Frataxin, for example, SIRT3 or Frataxin modified or improved to promote its half-life, effectiveness, metabolism, and/or potency of the protein. That is, any variant that does not affect the biological activity of SIRT3 or Frataxin may be used in the present invention.

The SIRT3 and Frataxin can form a complex (protein complex) for: as a target for regulating macrophage polarization, preparing a medicament for regulating macrophage polarization; as a target for screening the medicament for regulating the polarization of the macrophages, screening the medicament for regulating the polarization of the macrophages; or as a target for assessing the polarization state of macrophages, and preparing an agent for assessing the polarization state of macrophages.

Modulators of SIRT3 interaction with Frataxin and uses thereof

The inventor finds that SIRT3 interacts with Frataxin, and Lys189 site of mitochondrial Frataxin protein can generate acetylation modification and regulate the activity of the mitochondrial Frataxin protein; meanwhile, the NAD dependent deacetylase SIRT3 in mitochondria can regulate deacetylation modification of Frataxin, so that the polarization process of macrophages in which Frataxin participates is regulated. Signal regulation of the SIRT3/Frataxin axis has important significance on mitochondrial iron metabolism, macrophage polarization and influence related to hypertension inflammation, and on prevention and treatment of diseases related to mitochondrial metabolism and blood pressure disorder (such as hypertension).

Based on the new findings of the present inventors, the present invention provides the use of modulators that modulate the interaction of SIRT3 with Frataxin for modulating macrophage polarization.

Preferably, the modulator is an up-modulator that promotes the interaction of SIRT3 with Frataxin, which reduces the acetylation level of lysine 189 of Frataxin (i.e., promotes the deacetylation of lysine 189 of Frataxin), thereby promoting M2-type polarization of macrophages.

As used herein, the up-regulating agents include enhancers, agonists, and the like. Any substance that can increase the activity of a target protein (e.g., SIRT3 or Frataxin in the present invention), maintain the stability of a target protein, promote the expression of a target protein, promote the secretion of a target protein, prolong the effective action time of a target protein, or promote the transcription and translation of a target protein can be used in the present invention as an effective substance having an up-regulating function.

In a preferred mode of the invention, the SIRT3 or Frataxin up-regulator may comprise: an expression vector or expression construct that expresses (preferably overexpresses) SIRT3 or Frataxin after transfer into a cell. Typically, the expression vector comprises a gene cassette comprising a gene encoding SIRT3 or Frataxin operably linked to expression control sequences. The term "operably linked" or "operably linked" refers to the condition wherein certain portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

In the present invention, a polynucleotide sequence encoding SIRT3 or Frataxin may be inserted into a recombinant expression vector, such that it may be transferred into a cell and overexpressed to produce SIRT3 or Frataxin. Any plasmid and vector can be used in the present invention as long as they can replicate and are stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements. For example, the expression vector includes: viral vectors, non-viral vectors; preferably, the expression vector includes (but is not limited to): lentiviral vectors, adenoviral vectors, and the like. Methods well known to those skilled in the art can be used to construct expression vectors containing DNA sequences of SIRT3 or Frataxin and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

The SIRT3 or Frataxin up-regulator can also comprise: polypeptides or compounds for enhancing the effect of SIRT3 on Frataxin, chemical up-regulators of SIRT3 or Frataxin, up-regulators for promoting the driving capability of SIRT3 or Frataxin gene promoters, and the like.

According to the disclosure of the invention, the up-regulator is preferably an up-regulator which promotes the interaction of SIRT3 and Frataxin, thereby promoting the deacetylation of lysine 189 in Frataxin.

According to the present disclosure, and the effects of M2 type polarization of macrophages known in the art on disease states. The up-regulator for promoting the interaction of SIRT3 and Frataxin can inhibit macrophage inflammatory phenotype by promoting M2 type polarization of macrophages; more particularly, the compound can promote the release of macrophage iron ions, increase the synthesis of mitochondrial iron-sulfur cluster and heme, improve the expression of inflammation-inhibiting factors, reduce the expression of inflammation factors, reduce the accumulation of cell lipid peroxides, improve the oxidative stress of mitochondria and delay inflammatory lesions caused by hypertension.

As another preferred mode, the modulator is a down-regulator that inhibits the interaction of SIRT3 with Frataxin, and increases the level of acetylation of lysine 189 in Frataxin (i.e., promotes acetylation of lysine 189 in Frataxin), thereby promoting M1-type polarization of macrophages.

As used herein, the downregulator of the interaction of SIRT3 with Frataxin includes inhibitors, antagonists, blockers, and the like, and these terms are used interchangeably.

The down-regulator refers to any substance that can reduce the activity of a target protein (such as SIRT3 or Frataxin in the present invention), reduce the stability of the target protein or its encoding gene, down-regulate the expression of the target protein, reduce the effective action time of the target protein, or inhibit the transcription and translation of the target protein encoding gene, and these substances can be used in the present invention as substances useful for down-regulating the target protein. For example, the down-regulating agent is: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of SIRT3 or Frataxin genes; an antibody or ligand that specifically binds to a protein encoded by SIRT3 or Frataxin gene, and the like.

As an alternative of the invention, the down-regulator may be a small molecule compound directed to SIRT3 or Frataxin. Screening of such small molecule compounds can be performed by one skilled in the art using routine screening methods in the art. For example, in the examples of the present invention, several alternative screening methods are provided in conjunction with the regulatory mechanisms disclosed herein.

As an alternative mode of the invention, the down regulator can be an interfering RNA molecule (such as siRNA, shRNA, miRNA and the like) which specifically interferes with the genes of SIRT3 or Frataxin, and it can be understood that the interfering RNA molecule can be prepared and obtained according to the information provided by the invention. The method for preparing the interfering RNA molecule is not particularly limited, and includes, but is not limited to: chemical synthesis, in vitro transcription, and the like. The interfering RNA may be delivered into the cell by using an appropriate transfection reagent, or may also be delivered into the cell using a variety of techniques known in the art.

As an alternative mode of the invention, CRISPR/Cas (such as Cas9) system can be adopted for targeted gene editing, so that targeted knockout of target genes can be carried out. Common knockout methods include: co-transferring the sgRNA or a nucleic acid capable of forming the sgRNA, Cas9 mRNA or a nucleic acid capable of forming the Cas9 mRNA into a targeted region or targeted cell. After the target site is determined, known methods can be employed to cause the sgRNA and Cas9 to be introduced into the cell. The nucleic acid capable of forming the sgRNA is a nucleic acid construct or an expression vector, or the nucleic acid capable of forming the Cas9 mRNA is a nucleic acid construct or an expression vector, and these expression vectors are introduced into cells, so that active sgrnas and Cas9 mrnas are formed in the cells.

The above are some representative ways to down-regulate SIRT3 or Frataxin, and it is understood that other methods known in the art to modulate SIRT3 or Frataxin may be adopted after the general scheme of the present invention is understood by those skilled in the art, and these methods are also included in the present invention.

According to the present disclosure, and the effects of M1 type polarization of macrophages known in the art on disease states. The down-regulator for down-regulating the interaction between SIRT3 and Frataxin can promote the accumulation of macrophage iron ions by promoting the M1 type polarization of macrophages, reduce the synthesis of mitochondrial iron-sulfur clusters and heme, increase the expression of inflammatory factors, increase the accumulation of cell lipid peroxides and promote inflammatory lesions related to hypertension.

SIRT3 and Frataxin as drug screening targets

After the functions and action mechanisms of the SIRT3 and Frataxin are known, substances for regulating the interaction of the SIRT3 and Frataxin can be screened on the basis of the characteristics, and the substances are used for regulating the polarization of macrophages and further regulating diseases related to the polarization of the macrophages.

Accordingly, the present invention provides a method of screening for potential agents that modulate macrophage polarization, the method comprising: (1) treating an expression system expressing a complex of SIRT3 and Frataxin interaction with a candidate substance; and (2) detecting the interaction of SIRT3 with Frataxin in the system; (ii) if the candidate substance statistically promotes the interaction of SIRT3 with Frataxin, then the candidate substance is a potential substance that promotes M2-type polarization of macrophages; if the candidate substance statistically inhibits the interaction of SIRT3 with Frataxin, the candidate substance is a potential substance for promoting M1 type polarization of macrophages.

As a preferred mode of the invention, when SIRT3 interacts with Frataxin, the SIRT3 mediated acetylation modification situation of the 189 th lysine of Frataxin is detected, if the candidate substance reduces the SIRT3 mediated acetylation level of the 189 th lysine of Frataxin, then the candidate substance is a potential substance for promoting M2 type polarization of macrophages; if the candidate substance results in an SIRT 3-mediated increase in the acetylation level of lysine 189 in Frataxin, the candidate substance is a potential substance for promoting M1-type polarization of macrophages.

In a preferred mode of the invention, in order to make it easier to observe the change of the interaction between SIRT3 and Frataxin during screening, a control group can be provided, and the control group can be a system which does not add the candidate substance and expresses SIRT3 and Frataxin.

The system for expressing SIRT3 and Frataxin can be a cell (or cell culture) system, and the cell can be a cell which endogenously expresses SIRT3 and Frataxin; or may be a cell that recombinantly expresses SIRT3 and Frataxin. The system for expressing the SIRT3 and Frataxin can also be (but is not limited to) a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model) and the like.

As a preferred embodiment of the present invention, the method further comprises: the potential substances obtained are subjected to further cell experiments and/or animal experiments to further select and identify substances that are truly useful for modulating macrophage polarization.

The method for detecting expression, activity, amount of existing, interaction or acetylation of SIRT3 and Frataxin in the present invention is not particularly limited. Conventional protein quantitative or semi-quantitative detection techniques may be employed, such as (but not limited to): co-immunoprecipitation, SDS-PAGE method, Western-Blot method, ELISA, etc.

In another aspect, the invention also provides compounds, compositions or medicaments, or potential substances, obtained by the screening method. Some of the preliminarily screened substances may constitute a screening library so that one may finally screen substances therefrom which are truly useful for regulating macrophage polarization and the like, thereby being used clinically.

SIRT3 and Frataxin as diagnostic targets

Based on the above new findings of the present inventors, SIRT3 and Frataxin can be used as markers for diagnosing pathological states (diseases) associated with macrophage polarization: (i) performing a typing, differential diagnosis, and/or susceptibility analysis of the associated disease; (ii) evaluating the treatment medicines, the curative effects and the prognosis of the medicines of the relevant diseases of relevant people, and selecting a proper treatment method; (iii) early evaluating the related disease risk of related population, and early monitoring the early prevention and treatment. For example, the people with related diseases caused by abnormal expression of SIRT3 and Frataxin or abnormal acetylation of Frataxin can be separated, so that the diseases can be treated more specifically.

As a preferred mode of the present invention, evaluation was carried out by observing the acetylation modification of lysine at position 189 of Frataxin. Using said agent for identifying acetylation modification of Frataxin lysine 189, detecting a decrease in acetylation level of Frataxin lysine 189 indicates that M2 type polarization of macrophages is predominant; if an increased level of acetylation of lysine 189 in Frataxin is detected, this indicates that M1-type polarization of macrophages is dominant.

The invention also provides application of SIRT3 and Frataxin in preparing a reagent or a kit for diagnosing (or detecting) pathological states (diseases) related to macrophage polarization.

The presence, expression level or activity of SIRT3 and Frataxin or the genes encoding them can be determined by a variety of techniques known in the art and are encompassed by the present invention. For example, the conventional techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR and the like can be used, and these methods can be used in combination.

The invention also provides reagents for detecting the presence or absence and expression of a SIRT3 or Frataxin gene in an analyte. Preferably, when performing gene level detection, primers that specifically amplify SIRT3 or Frataxin; or a probe that specifically recognizes SIRT3 or Frataxin to determine the presence or absence of a SIRT3 or Frataxin gene; when performing protein level detection, antibodies or ligands that specifically bind to SIRT3 or Frataxin encoded proteins, including their acetylated states, can be used to determine the expression of SIRT3 or Frataxin proteins. In a preferred embodiment of the invention, the reagent is a primer, which can specifically amplify SIRT3 or Frataxin gene or gene fragment. The design of a specific probe for SIRT3 or Frataxin gene is well known to those skilled in the art, for example, by preparing a probe that specifically binds to a specific site on SIRT3 or Frataxin gene, but not to genes other than SIRT3 or Frataxin gene, and which carries a detectable signal. In addition, methods for detecting the expression of SIRT3 or Frataxin proteins in an analyte using antibodies that specifically bind SIRT3 or Frataxin proteins, including their acetylated states, are also well known to those of skill in the art.

The invention also provides a kit for detecting the presence or absence and expression of a SIRT3 or Frataxin gene in an analyte, the kit comprising: primers for specifically amplifying SIRT3 or Frataxin genes; a probe that specifically recognizes SIRT3 or Frataxin genes; or an antibody or ligand that specifically binds to SIRT3 or Frataxin proteins, including their acetylated states.

The kit may further comprise various reagents required for DNA extraction, PCR, hybridization, color development, and the like, including but not limited to: an extraction solution, an amplification solution, a hybridization solution, an enzyme, a control solution, a color development solution, a washing solution, and the like. In addition, the kit may further comprise instructions for use and/or nucleic acid sequence analysis software, and the like.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Materials and methods

1. Materials and reagents

In the examples of the present invention, the materials or reagents used are as follows:

angiotensin II: millipore, USA;

Anti-SIRT3 antibody: cell Signaling Technology (CST) corporation, usa;

Anti-Frataxin antibody: abcam corporation, USA;

Anti-HA antibody: shanghai Biyuntian biotechnologies;

Anti-Flag beads: sigma, USA;

HRP-labeled rabbit anti-mouse IgG: life Technologies, USA;

HRP-labeled goat anti-rabbit IgG: life Technologies, USA;

SIRT3-shRNA lentivirus: shanghai Jikai Gene technology, Inc.;

SIRT3 overexpresses lentiviruses: shanghai Jikai Gene technology, Inc.;

Frataxin-shRNA lentivirus: shanghai Jikai Gene technology, Inc.;

frataxin overexpressing lentiviruses: shanghai Jikai Gene technology, Inc.;

SIRT3 overexpression plasmid: shanghai Jikai Gene technology, Inc.;

frataxin overexpression plasmid: shanghai Jikai Gene technology, Inc.;

frataxin189 KQ (protein amino acid sequence 189 is mutated from K to Q) mutant plasmid: shanghai Jikai Gene technology, Inc.;

frataxin189 KR (protein amino acid sequence 189, K is mutated into R) mutant plasmid Shanghai Jikai Gene technology GmbH;

SYBR Green real: TOYOBO Biotech, Inc., Japan;

PCR Master Mix: TOYOBO Biotech, Inc., Japan;

AMV Reverse Transcriptase: promega corporation, USA;

(AMV RT) catalyzes: promega corporation, USA;

liperfluor: east dong renche chemical technology, ltd, japan;

Mito-FerroGreen: east dong renche chemical technology, ltd, japan;

primer: shanghai Saiban Gene technology, Inc.;

erastin: selleck Chemicals, USA;

ferrostatin-1: selleck Chemicals, USA.

2. Cell culture

M1920 mouse macrophages, purchased from Bayer Biotechnology Ltd, were cultured in RPMI1640 medium containing 10% fetal bovine serum and cultured in an incubator with 5% CO2 at 37 ℃. 293T cells were purchased from Bayer Biotechnology Ltd and were human embryonic kidney cells transfected with adenovirus E1A gene. The cells were cultured in DMEM medium containing 10% fetal bovine serum and incubated in an incubator with 5% CO2 at a constant temperature of 37 ℃.

3. Plasmid transfection

3.1 plasmid construction

(1) Carrier information

Frataxin vector element sequence: CMV-MCS-3FLAG-SV 40-Neomycin;

SIRT3 vector element sequence: CMV-MCS-HA-SV 40-Neomycin;

(2) designing a primer to obtain a target gene fragment through PCR;

(3) recovering PCR products and measuring the concentration;

(4) exchanging the PCR product with a carrier;

(5) and (3) transformation: add 10. mu.l of the exchange reaction product to 100. mu.l of competent cells, flick the tube wall down and mix well, and leave on ice for 30 minutes. Heat shock at 42 ℃ for 90 seconds and incubation in an ice water bath for 2 minutes. Add 500. mu.l LB medium and shake-incubate it at 37 ℃ for 1 hour. Taking a proper amount of bacterial liquid, uniformly coating the bacterial liquid on a flat plate containing corresponding antibiotics, and carrying out inverted culture in a constant-temperature incubator for 12-16 hours;

(6) carrying out colony PCR identification;

(7) sequencing: inoculating the identified positive clone transformant into a proper amount of LB liquid culture medium containing corresponding antibiotics, culturing for 12-16 hours at 37 ℃, and taking a proper amount of bacterial liquid for sequencing. And (4) carrying out comparison analysis on the sequence of the gene with no target in the sequencing result.

3.2 plasmid extraction and transfection

And (4) carrying out plasmid extraction by using the small-extraction medium-amount kit for the Tiangen endotoxin-free plasmid, and allowing the extracted qualified plasmid to enter a downstream process. Using Lipofectamine^TM3000 reagents were used for transfection.

4. Mouse macrophage (M1920) transfected lentivirus

4.1 Lentiviral construction

(1) Construction of recombinant plasmids

SIRT3-shRNA target sequence: 5'-CTGTACTGGCGTTGTGAAA-3' (SEQ ID NO: 3); the carrier element sequence: hU 6-MCS-CMV-EGFP;

SIRT3 target sequence: 5'-GAGGATCCCCGGGTACCGGTCGCCACCATGGTGGGGGCCGGCATCAG-3' (SEQ ID NO: 4); the vector element sequence Ubi-MCS-3 FLAG-CMV-EGFP;

Frataxin-shRNA target sequence 5'-gaGTTCTTTGAAGACCTCGCA-3' (SEQ ID NO: 5); the vector element sequence U6-MCS-Ubiquitin-Cherry-IRES-puromycin;

sequence 5'-ACGGGCCCTCTAGACTCGAGCGCCACCATGTGGACTCTCGGGCGCCG-3' of interest for Frataxin (SEQ ID NO:6), vector element sequence CMV-MCS-3FLAG-SV 40-Neomycin;

(2) extracting and purifying the recombinant plasmid with correct sequencing to obtain high-quality recombinant plasmid without endotoxin;

(3) co-transfecting 293T cells by using a high-efficiency recombinant vector and a virus packaging plasmid, performing virus cytoplasm and production, and collecting virus liquid;

(4) concentrating and purifying the virus liquid.

4.2 transfection of lentiviruses

(1) Counting M1920 cells, paving the cells into a 6-well plate, and continuously culturing for 24 hours;

(2) observing the growth condition of the cells after 24 hours until the cells grow to 50 percent;

(3) discarding the medium, adding 1ml of complete medium containing 40. mu.l/ml HitransG P infection enhancing fluid, adding appropriate amounts of lentivirus encoding SIRT3-shRNA, FXN-shRNA, or SIRT3, FXN cDNA, and empty-loading virus as control (MOI 20);

(4) incubation is carried out for 48h at 37 ℃, the fresh culture medium is replaced, and the culture is continued. GFP and mCherry fluorescence were observed under a fluorescence microscope, and transfection efficiency was checked by Western blot.

5. Preparation of acetylated antibody of lysine at position 189 of Frataxin

The Frataxin 189-lysine acetylated antibody is prepared by Hangzhou Huaan biotechnology limited;

frataxin 185-197 amino acid immunity polypeptide sequence: RELTK (Ac) ALNTKLDLC.

5.1 immunopeptide coupled Keyhole Limpet Hemocyanin (KLH)

(1)20mg KLH was completely dissolved in 2mL of 5mM EDTA in water;

(2) after 8mg of Sulfo-SMCC was completely dissolved in 50. mu.l of DMSO, 150. mu.l of 1 XPBS was added and mixed well;

(3) dropwise adding the Sulfo-SMCC solution into KLH, slightly shaking while adding, and standing for 1 hour at room temperature;

(4) the activated KLH solution was placed in a dialysis bag, which was clamped in a dialysis clamp, and then placed in 2L of 1 XPBS, and placed in a refrigerator at 4 ℃ for dialysis for 1 hour under magnetic stirring.

(5) Dialysis was performed with fresh 1 × PBS and repeated for 2 hours. Activated dialyzed KLH was placed in a 15ml centrifuge tube and labeled on the tube and stored at 4 ℃ until needed.

(6)4mg of immunopeptide was dissolved in 50. mu.l of DMSO, added 200. mu.l of 1 XPBS, mixed rapidly and according to the polypeptide: KLH ═ 1 mg: mu.g of KLH was added immediately and the reaction was carried out overnight at 4 ℃.

(7) The crosslinked KLH-peptide complex was placed in a dialysis bag, which was then placed in a dialysis holder, and the resulting mixture was placed in 4L1 XPBS and then placed in a refrigerator at 4 ℃ and dialyzed overnight under magnetic stirring.

(8) Taking out the KLH-peptide after dialysis, putting the KLH-peptide into a clean 1.5ml centrifuge tube, subpackaging according to the immune dose, and storing at the temperature of minus 20 ℃ for later use.

5.2 immunization protocol

(1) Animal selection: new Zealand white rabbits are selected, the weight of the New Zealand white rabbits is about 2.5kg, and the New Zealand white rabbits are young and strong. The fur color is bright, and the movement is free. After 2 weeks of preculture, animals showing healthy status were selected for immunization;

(2) and thawing the KLH coupled antigen polypeptide, and fully and uniformly mixing. Extracting 0.5ml of antigen per mouse, wherein the concentration of the first-immunity antigen is 1mg/ml, the concentration of the second-immunity antigen is halved, and the dose is not changed;

(3) mixing the adjuvant completely, extracting the adjuvant, and mixing the adjuvants according to the ratio of the adjuvant: antigen 1:1 volumetric ratio draw. The first-time immunization adopts a complete adjuvant, and the second-time immunization adopts an incomplete adjuvant;

(4) the two syringes are butted by a syringe connecting pipe for complete emulsification. Emulsification standard: the emulsified immunogen is not dispersed when dropped into water with 37 degrees;

(5) injecting emulsified antigen subcutaneously at multiple points, wherein each point is injected with 0.2 ml;

(6) the immunization time is as follows: second immunization was performed 14 days after first immunization, and third immunization was performed 7 days later. And (3) collecting a small sample of serum from the middle ear artery on the 7 th day after the third immunization, detecting the small sample to be qualified, adding the immunization after the 7 th day, and collecting the whole blood after the adding of the immunization for the 7 th day.

5.3 antibody purification

(1) Fully washing the affinity chromatographic column by 20mL of pure water and 1 multiplied by PBS (pH7.4) in sequence at the flow rate of 70 mL/h;

(2) taking 10ml of serum to be purified, putting the serum into a 50ml centrifugal tube, and carrying out suction filtration by using a microporous filter membrane with the pore diameter of 0.45 mu m;

(3) the suction-filtered serum sample is loaded at a flow rate of 40ml/h and repeated once;

(4) washing affinity chromatography column with 20mL 1 × PBS (pH7.4) at flow rate of 70mL/h, connecting with protein detector after 10min, and adjusting light transmittance (T grade) of the instrument to 100 during washing;

(5) adjusting the light absorption rate (1A grade) of the protein detector to 0, opening an HD-A computer collector on a computer desktop, adjusting the full-screen range to 5, eluting the antibody at the speed of 40ml/h by using a glycine solution (pH 2.7, 0.2M), pressing a green elution record button to start elution at the moment, and starting to collect the antibody when the reading of the instrument is increased;

(6) adjusting the pH value of the antibody to about 7 in time by using 1M sodium bicarbonate in the antibody collection process, and recording the highest peak value of an elution peak;

(7) after the antibody is collected, adjusting the pH value to about 7, and recording the volume of the eluted antibody;

(8) and (4) subpackaging and storing the purified antibody at-20 ℃ for later use.

6. Extraction of cellular protein and determination of concentration

Extraction of cellular proteins by conventional methods protein concentration was determined using a Bradford protein concentration assay kit (purchased from shanghai bi yunnan biotechnology).

7. Co-immunoprecipitation

(1) And (3) detection: the interaction between FXN and SIRT3 was detected using the Thermo Scientific co-immunoprecipitation kit.

(2) Sample preparation: adding 1ml of precooled IP lysate into 293T cells in a petri dish, incubating for 20min on ice, scraping by using a cell scraper, collecting into a 1.6ml Ep tube, centrifuging for 10min at 10000g at 4 ℃, collecting supernatant, performing protein concentration determination, and taking 500 mu g of protein for detection.

(3) And (3) antibody solidification: adding the reinforced coupling resin into a Pierce centrifugal column, washing with 1 × crosslinking buffer solution and centrifuging; adding the mixed solution containing 10 mu l of SIRT3 antibody into a centrifugal column, adding 3 mu l of sodium cyanoborohydride solution, carrying out rotary incubation at room temperature for 90min, and centrifuging and retaining the flow-through solution to detect the antibody binding efficiency; washing with 1 × crosslinking buffer and quenching buffer for 1 time and centrifuging; adding 200 μ l quenching buffer and 3 μ l sodium cyanoborohydride solution, rotary incubating at room temperature for 15min, washing with 1 × crosslinking buffer and washing buffer respectively, and centrifuging for 3 times;

(4) co-immunoprecipitation: washing with IP lysis/washing buffer solution for 2 times, adding the sample into a centrifugal column, covering a bottom cover, and rotating and shaking uniformly at 4 ℃ overnight;

(5) and (3) elution: taking out the centrifugal column, centrifuging, and keeping the efficiency of detecting coprecipitation by using the cross flow liquid; washing 3 times with IP lysis/washing buffer and centrifuging; placing the centrifuge tube into a new collection tube, adding 10 μ l of elution buffer solution, centrifuging, adding 40 μ l of elution buffer solution, standing at room temperature for 5min, centrifuging, and retaining the transudate; the steps can be repeated once;

(6) preparing a sample: adding sample buffer solution, heating at 95 deg.C for 5min, and storing at-20 deg.C.

(7) And detecting the result of the co-immunoprecipitation by Western blot.

8. Western blotting test (Western blot)

Western blot assays were performed by conventional methods.

9. Fluorescent real-time quantitative PCR

9.1RNA extraction and reverse transcription

Conventional methods perform RNA extraction followed by reverse transcription into cDNA.

9.2 real-time fluorescent quantitative PCR

(1) Primer sequences are shown in the following table:

IFN-γ Forward 5′-TGGAATCCTGTGGCATCCATGAAAG-3′(SEQ ID NO:7)；

Reverse 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′(SEQ ID NO:8)；

IL-6 Forward 5′-CACAAGTCCGGAGAGGAGAC-3′(SEQ ID NO:9)；

Reverse 5′-CAGAATTGCCATTGCACAAC-3′(SEQ ID NO:10)；

TNF-α Forward 5′-GAACTGGCAGAAGAGGCACT-3′(SEQ ID NO:11)；

Reverse 5′-AGGGTCTGGGCCATAGAACT-3′(SEQ ID NO:12)；

iNOS Forward 5′-TGGCTCGCTTTGCCACGGACGAGACGGA-3′(SEQ ID NO:13)；

Reverse 5′-GGAGCTGCGACAGCAGGAAGGCAGCGGG-3′(SEQ ID NO:14)；

IL-4 Forward 5′-CCTCACAGCAACGAAGAACA-3′(SEQ ID NO:15)；

Reverse 5′-CTGCAGCTCCATGAGAACAC-3′(SEQ ID NO:16)；

IL-10 Forward 5′-CAGCCGGGAAGACAATAACT-3′(SEQ ID NO:17)；

Reverse 5′-GCATTAAGGAGTCGGTTAGCA-3′(SEQ ID NO:18)；

YM-1 Forward 5′-TTATCCTGAGTGACCCTTCTAAG-3′(SEQ ID NO:19)；

Reverse 5′-TCATTACCCTGATAGGCATAGG-3′(SEQ ID NO:20)；

ARG-1 Forward 5′-AGCTGGCTGGTGTGGTGGCAGAGGTCCA-3′(SEQ ID NO:21)；

Reverse 5′-GGGTGGACCCTGGCGTGGCCAGAGATGCT-3′(SEQ ID NO:22)；

18S rRNA Forward 5′-CATTCGAACGTCTGCCCTATC-3′(SEQ ID NO:23)；

Reverse 5′-CCTGCTGCCTTCCTTGGA-3′(SEQ ID NO:24)。

(2) each primer was formulated to 10. mu. mol/L depending on the concentration and placed on ice for use.

(3) A reaction system is prepared according to the product specification, and is added into an ABI fluorescent quantitative PCR 96 pore plate, and the process is carried out on ice to avoid illumination.

Reagent	Dosage form
		Plus solution	1μl
10μmol/L Primer Forward	0.4μl
		10μmol/L Primer Reverse	0.4μl
PCR grade water	2.2μl
		Template DNA	1μl
SYBR Green Realtime PCR Master Mix	5μl
		Total volume	10 l

(4) The cycle conditions were set according to the product specifications: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 15 seconds, extension at 60 ℃ for 1 minute, and 40 cycles; melting curve analysis was performed.

9.3 statistical data

(1) Taking CT average values of 2 auxiliary holes in each sample;

(2) obtaining delta CT by taking 18sRNA as an internal reference;

(3) further calculation 2^-△△CTAnd carrying out data statistics.

10. Flow cytometry

(1) M1920 cells were plated in 12-well plates and the next day was debranned with 0.5% BSA-1640 medium for 12 h;

(2) adding corresponding stimulating medicine, and acting for 24 hr;

(3) discarding the culture medium, and washing the cells for three times by using RPMI-1640 culture medium without serum;

(4) the Liperfluor or Mito-FerroGreen probes were added proportionally to serum-free RPMI-1640 medium, 200. mu.l per well, and incubated at 37 ℃ for 30 min.

(5) The cells were collected into a flow tube and washed 3 times with 1 × PBS.

(6) Finally, 300. mu.l of 1 XPBS was added and tested on the machine.

11. Lipid oxidation (MDA) assay

The kit comprises: lipid oxidation test kit, purchased from Shanghai Biyuntian biotech company;

11.1 sample preparation

(2) adding corresponding stimulating medicine, and acting for 24 hr;

(3) adding Western and IP cell lysate (Biyun day) 50 μ l into cells, lysing for 20min on ice, scraping with cell scraper, and collecting into centrifuge tube;

(4) centrifuging at 4 deg.C for 10min at 10000g, collecting supernatant, and storing at-20 deg.C.

11.2MDA detection

(1) Determining the protein concentration;

(2) preparing a TSA storage solution, mixing 25mg of TSA with 6.76ml of TSA preparation solution, heating at 70 ℃ for 10min, and storing at room temperature for later use;

(3) preparing a TSA working solution, and mixing the TSA storage solution: TSA diluent: preparing the TSA working solution by the antioxidant according to the proportion of 150:50:3, and preparing the TSA working solution for use at present;

(4) mixing 100 μ l sample with 200 μ l TSA working solution, heating at 100 deg.C for 15 min;

(5) 200. mu.l of the solution was taken and absorbance at 532nm was measured.

12. Complexx I Activity assays

12.1 isolation of mitochondria

(1) The kit comprises: the mitochondria isolation kit is purchased from Shanghai Biyuntian biotechnology company;

(2) to 5X 10⁶Adding 0.5ml of mitochondrion separating reagent into each cell, and incubating for 15min on ice;

(3) transferring the cell suspension into a glass homogenizer with proper size, and homogenizing for about 10-30 times;

(4) centrifuging the cell homogenate at 600g and 4 ℃ for 10min, and collecting the supernatant;

(5) centrifuging at 11000g at 4 deg.C for 10min, and removing supernatant;

(6) the mitochondria were resuspended in 50. mu.l mitochondrial storage solution.

Complex I Activity assay

(1) The kit comprises: the complete I activity detection kit is purchased from Shanghai Jimei gene medicine science and technology limited;

(2) quickly freezing and thawing the mitochondrial suspension for 3 times;

(3) determining mitochondrial protein concentration;

(4) preparing a reaction system according to the specification:

reagent	Blank control	Total Activity assay	Non-specific Activity assay
				Reagent A	195μl	195μl	190μl
Reagent B	25μl	25μl	25μl
				Reagent E	0μl	0μl	5μl
Reagent D	5μl	5μl	5μl
				Reagent C	25μl	0μl	0μl
Sample (10 ug)	0μl	25μl	25l

(5) Measuring absorbance at 340nm for 7 times at intervals of 30s for three minutes;

(6) calculate complete I activity:

[ (sample reading-background reading) × 0.25 (system volume ml) × sample dilution ] ÷ [0.025 (sample volume ml) × 6.2 (millimolar absorption coefficient) × 1 (reaction time min) ] ÷ sample protein concentration ═ μ M NADH/min/μg protein;

total activity of the sample-sample nonspecific activity-sample specific activity.

13. Statistical analysis

Data from all experiments are expressed as Mean ± SEM. The Test between the two groups was analyzed for statistical differences using the t-Test (Student's Test) Test, and the statistical differences between the groups were analyzed using the one-way ANOVA Test. P <0.05 indicates that there is a statistical difference.

14. Protein sequences

The amino acid sequence of SIRT3 (SEQ ID NO: 1; Mus musculus) referred to in the examples:

the amino acid sequences of Frataxin referred to in the examples (SEQ ID NO: 2; Mus musculus (house mouse)):

examples

Example 1 SIRT3 deletion exacerbates mitochondrial iron accumulation in macrophages, promotes macrophage polarization

The inventor utilizes lentivirus to transfer SIRT3-shRNA into M1920 cells, and utilizes qPCR and Western immunoblotting experiments to detect the inhibition efficiency of SIRT 3. As shown in FIGS. 1A and B, SIRT3 expression was significantly inhibited at the mRNA and protein levels.

Angiotensin ii (ang ii) has blood pressure increasing effect on the body; at the cellular level, the cell is stimulated, and a cell model simulating macrophage polarization under a hypertension pathological state can be obtained.

The inventor stimulates M1920 cells inhibited by SIRT3 with angiotensin II (Ang II) for 24 hours and finds that there are expression changes of some proteins related to mitochondrial iron metabolism. Mitochondrial iron metabolism-related proteins detected by the inventors include Mito-ferrin, ABCB7 and Frataxin. Mito-ferrin mediates the uptake of mitochondrial iron ions, while ABCB7 is a pathway for iron discharge from mitochondria. In the case of Ang II stimulation and SIRT3 deletion, the expression of ABCB7 and Frataxin protein has no obvious change. However, the expression of Mito-ferrin was significantly increased following the deletion of SIRT3 (FIG. 1B). This result suggests that following SIRT3 deletion, mitochondrial iron uptake is increased, exacerbating iron accumulation in macrophage mitochondria. Malondialdehyde (MDA) is a lipid peroxidation product, and its intracellular content may reflect intracellular levels of lipid peroxidation. A significant increase in MDA was observed after 24 hours of Ang II action, while the intracellular MDA content was further increased after SIRT3 deletion (fig. 1C).

Tissue macrophages are stimulated by a particular tissue environment to undergo transformation, a process known as polarization. Macrophages are classified into two categories according to their phenotype and function: m1 macrophage mainly secretes proinflammatory cytokines such as TNF-alpha, IL-6, Inducible Nitric Oxide Synthase (iNOS), INF-gamma and the like; m2 macrophages mainly express IL-10, YM-1, ARG-1, IL-4, etc. The mRNA levels of the above cytokines were measured by qPCR, and the secretion of Il-6 and iNOS by M1920 was increased and Arg-1 and IL-10 were decreased under the action of angiotensin II, indicating that macrophages were differentiated towards M1, which was more pronounced after deletion of SIRT3 (FIGS. 1D and 1E).

The above results indicate that SIRT3 deletion leads to iron accumulation within mitochondria and can promote macrophage polarization to M1.

Example 2 loss of Frataxin Activity promotes macrophage mitochondrial iron accumulation, increases accumulation of cellular lipid peroxides, and results in macrophage polarization

The synthesis of iron-sulfur clusters is central to mitochondrial iron metabolism. Frataxin is the key to the synthesis of iron and sulfur clusters and is involved in multiple processes in mitochondrial iron metabolism. Therefore, to further explore the mechanism of action of mitochondrial iron metabolism in macrophage polarization, the inventors infected M1920 cells with lentivirus, inhibited Frataxin expression (Frataxin-shRNA), and observed changes in cell function and phenotype. Lipoflour is a fluorescent probe for the detection of intracellular lipid peroxides. Lipoflur was stained and flow-measured, and Ang II induced increase in lipid peroxides was more pronounced after inhibition of Frataxin expression compared to the control group, which was caused by Ang II increase induced by flow-measurement (fig. 2A). Similar results were obtained for the detection of intracellular MDA (fig. 2B).

Furthermore, the detection of macrophage polarization-associated cytokines by qPCR revealed that the deletion of Frataxin resulted in a more pronounced increase in Ang II-induced INF- γ and iNOS (FIG. 2C), while Arg-1 and YM-1 were further reduced (FIG. 2D).

To clarify the effect of Frataxin, the present inventors transferred exogenous Frataxin into M1920 cells using lentivirus, and increased the expression of Frataxin in macrophages. Further analysis of lipid peroxidation in macrophages showed that Frataxin can mitigate Ang II-induced increase in lipoflur fluorescence intensity (fig. 2E). Mito-FerroGreen is a fluorescent probe and can detect ferrous ions (Fe) in mitochondria²⁺). As shown in FIG. 2F, after 24 hours of Ang II action, Mito-FerroGreen staining and flow cytometry observed that Ang II-acted M1920 cells exhibited intramitochondrial Fe compared to the control group²⁺This phenomenon is alleviated by aggregation, which increases the expression of Frataxin.

Similar results were obtained when the level of MDA in the cells was measured (fig. 2G). In addition, an increase in Frataxin expression may also alleviate Ang II-induced macrophage polarization to M1, as shown by reversing Ang II-induced increases in IL-6 and TNF- α and decreases in Arg-1 and YM-1 (FIG. 2H-I).

Example 3 modulation of acetylation at lysine 189 site (Lys189) of Frataxin protein by SIRT3 influences mitochondrial iron metabolism, and Frataxin (K189) -mediated iron metabolism patterns influence the inflammatory phenotype of macrophages

To explore the mechanism of interaction between SIRT3 and Frataxin, the inventors used a human embryonic kidney 293T cell line transfected with adenovirus E1A gene by tool cells, precipitated SIRT3 and Frataxin, respectively, and then examined by western blot experiments, it was observed that endogenous SIRT3 forms a complex with Frataxin, suggesting that there is interaction between SIRT3 and Frataxin (fig. 3A, B). The inventors further identified the interaction of exogenous SIRT3 with Frataxin. HA-SIRT3 and Flag-Frataxin are introduced into 293T cells by using plasmids, and expression of SIRT3 protein can be detected by immunoprecipitating Flag (figure 3C), and expression of Frataxin can be detected after immunoprecipitating HA (figure 3D).

SIRT3 is an important deacetylase, and to confirm that Frataxin is one of the substrates of SIRT3, the present inventors applied acetylation quantitative proteomic analysis technique by comparing the results from Wild Type (WT) and SIRT3^-/-(SIRT3, Knockout) mice showed a marked increase in acetylation level of Frataxin proteins, with K189 being most significantly regulated by acetylation (FIG. 3E).

By aligning the amino acid sequences of Frataxin from multiple species, the inventors found that the modification of lysine acetylation at position 189, highly conserved from invertebrates to humans, may affect the function of Frataxin (fig. 3F). To determine whether SIRT3 modulates the effects of Frataxin by deacetylating lysine at position 189, the inventors used plasmids to transfer SIRT3-shRNA into 293T cells, with SIRT3 protein levels suppressed and Frataxin protein levels not significantly changed, but with significantly increased acetylation of lysine at position 189 of Frataxin (fig. 3G). Indicating that SIRT3 can regulate the acetylation level of lysine at Frataxin189 site.

Further, Frataxin plasmid with KR mutation and KQ mutation at position 189 is transferred into 293T cells, the deacetylation and acetylation state of Frataxin is simulated, and the protein level of Complex I and Complex II in which Frataxin participates in synthesis is detected. As shown in FIG. 3H, the expression of the KR-mutated 293T cell was significantly increased compared to the cells Complex I and Complex II transformed with WT Frataxin, while the expression of the KQ-mutated 293T cell Complex I and Complex II was suppressed.

Furthermore, the inventors further examined the activity of Complex I, which was significantly increased in KR-mutated 293T cells, whereas KQ mutation resulted in decreased activity of Complex I (fig. 3I). It is shown that the level of acetylation of lysine at position 189 can affect the activity of Frataxin.

Example 4 increasing the expression of SIRT3, promoting deacetylation of Frataxin (Lys189) sites, inhibiting M1 polarization of macrophages

The inventors transferred exogenous SIRT3 into 293T cells by using a plasmid, and observed that the expression of SIRT3 is obviously increased, and the lysine acetylation level at the Frataxin189 site is reduced, as shown in FIG. 4A.

To further explore the effect of changes in lysine acetylation level at Frataxin189 site on macrophage polarization, the inventors used lentiviruses to transfer exogenous SIRT3(LV-SIRT 3). A significant increase in SIRT3 mRNA and protein levels was observed in qPCR and western blot experiments (fig. 4B and 4C). While increasing the expression of SIRT3, the change in Frataxin was not significant (shown in 4C). The MDA assay found that SIRT3 overexpression inhibited Ang II-induced lipid peroxide increase (fig. 4D). And Ang II-induced polarization of macrophage M1 was also inhibited, as evidenced by inhibition of IL-6 and iNOS expression, while ARG-1 and IL-10 expression were increased (FIG. 4E).

Example 5 screening method

Cell: 293T cells recombinantly expressing SIRT3 and Frataxin.

Test group: culturing the 293T cell for recombinant expression of SIRT3 and Frataxin, and administering a candidate substance;

control group: culturing the 293T cell which expresses the SIRT3 and Frataxin in a recombination way, and not administering a candidate substance.

The interaction of SIRT3 and Frataxin in the test group and the control group, particularly the deacetylation of lysine 189 of Frataxin by SIRT3, was detected and compared. If the interaction between SIRT3 and Frataxin in the test group is significantly stronger than that in the control group, and particularly if the acetylation level of lysine at position 189 of Frataxin is significantly lower than that in the control group, it indicates that the candidate is a substance that modulates the M2 type polarization of macrophages, which is a potential substance for lowering blood pressure.

Conclusion

1. Ang II inhibits the expression of mitochondrial SIRT3, mediates macrophage mitochondrial oxidative stress and dysfunction, and participates in macrophage differentiation.

2. SIRT3 modulates deacetylation modification of Frataxin (Lys189), whereas deletion of SIRT3 exacerbates Ang II-mediated hyperacetylation of Frataxin; acetylated Frataxin exacerbates mitochondrial iron accumulation, increases accumulation of cell lipid peroxides, and promotes macrophage inflammatory phenotype differentiation.

3. The expression of SIRT3 is improved, Frataxin is deacetylated, mitochondrial oxidative stress can be improved, inflammatory factors secreted by macrophages are inhibited, and inflammatory lesions caused by hypertension are delayed.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims

Use of a complex of SIRT3 interacting with Frataxin for:

as a target for regulating macrophage polarization, preparing a medicament for regulating macrophage polarization;

as a target for screening the medicament for regulating the polarization of the macrophages, screening the medicament for regulating the polarization of the macrophages; or

As a target for evaluating the polarization state of macrophages, an agent for evaluating the polarization state of macrophages was prepared.
2. The use of claim 1, wherein the SIRT3 interacting with Frataxin comprises: SIRT3 regulates the acetylation modification mode of lysine 189 in Frataxin; preferably, the SIRT3 and Frataxin interaction is an interaction in mitochondria.
3. Use of a modulator that modulates the interaction of SIRT3 with Frataxin for modulating macrophage polarization; preferably, the first and second liquid crystal films are made of a polymer,

the modulator is an up-regulator promoting the interaction of SIRT3 and Frataxin, and the up-regulator reduces the acetylation level of lysine at position 189 of Frataxin, thereby promoting M2 type polarization of macrophages; or

The modulator is a down-regulator which inhibits the interaction of SIRT3 and Frataxin, and increases the acetylation level of lysine 189 in Frataxin, thereby promoting M1 type polarization of macrophages.
4. The use of claim 3, wherein the up-regulator comprises: substances for enhancing the activity of SIRT3 or Frataxin, substances for enhancing the expression, stability or effective acting time of SIRT3 or Frataxin; preferably comprising a compound selected from: the expression construct for recombinant expression of SIRT3 or Frataxin, the polypeptide or compound for enhancing the effect of SIRT3 on Frataxin, the chemical up-regulator of SIRT3 or Frataxin, and the up-regulator for promoting the driving capability of SIRT3 or Frataxin gene promoter; or

The down-regulating agents include: a substance that down-regulates SIRT3 or Frataxin activity or a substance that down-regulates SIRT3 or Frataxin expression, stability or reduces its effective duration of action; preferably comprising: an agent that knocks out or silences SIRT3 or Frataxin, a binding molecule that specifically binds SIRT3 or Frataxin, a small chemical molecule antagonist or inhibitor against SIRT3 or Frataxin, angiotensin II.
5. Use of an agent that specifically recognizes the complex of SIRT3 interacting with Frataxin or recognizes the acetylation modification of lysine 189 of Frataxin, for the preparation of an agent or kit for assessing the polarization of macrophages; preferably, the specifically recognizing reagent comprises: a binding molecule that specifically binds SIRT3 or Frataxin; more preferably, the binding molecule that specifically binds Frataxin comprises: a binding molecule that specifically recognizes or binds to Frataxin acetylated lysine at position 189.
6. The use according to any one of claims 1 to 5, wherein the polarization of macrophages comprises M2 type polarization or M1 type polarization; preferably, the first and second liquid crystal films are made of a polymer,

said promoting M2-type polarization of macrophages comprises inhibiting a macrophage inflammatory phenotype; more specifically, the method comprises the following steps: promoting the release of macrophage iron ions, increasing the synthesis of mitochondrial iron-sulfur cluster and heme, improving the expression of inflammation-inhibiting factors, reducing the expression of inflammatory factors, reducing the accumulation of cell lipid peroxides, improving mitochondrial oxidative stress and delaying inflammatory lesions caused by hypertension; or

Said promoting M1-type polarization of macrophages comprises promoting a macrophage inflammatory phenotype; more specifically, the method comprises the following steps: promoting macrophage iron ion accumulation, reducing mitochondrial iron-sulfur cluster and heme synthesis, increasing inflammatory factor expression, and increasing cell lipid peroxide accumulation.
7. The use of claim 6, wherein the inflammatory factor comprises: TNF-alpha, IL-6, inducible nitric oxide synthase or INF-gamma; or

The inflammation-inhibiting factors include: IL-10, YM-1, ARG-1 or IL-4.
8. A method of screening for potential agents that modulate macrophage polarization, the method comprising:

(1) treating an expression system expressing a complex of SIRT3 and Frataxin interaction with a candidate substance; and

(2) detecting the interaction condition of SIRT3 and Frataxin in the system; (ii) if the candidate substance statistically promotes the interaction of SIRT3 with Frataxin, then the candidate substance is a potential substance that promotes M2-type polarization of macrophages; if the candidate substance statistically inhibits the interaction of SIRT3 with Frataxin, the candidate substance is a potential substance for promoting M1 type polarization of macrophages.
9. The method of claim 8, wherein step (1) comprises: in the test group, adding a candidate substance to the expression system; and/or

The step (2) comprises the following steps: detecting the interaction condition of SIRT3 and Frataxin in the system; and comparing the expression system with a control group, wherein the control group is an expression system without the candidate substance; (ii) if the candidate substance statistically promotes the interaction of SIRT3 with Frataxin, then the candidate substance is a potential substance that promotes M2-type polarization of macrophages; if the candidate substance statistically inhibits the interaction of SIRT3 with Frataxin, the candidate substance is a potential substance for promoting M1 type polarization of macrophages.
10. The method of claim 8 or 9, wherein said detecting the interaction of SIRT3 with Frataxin in the system comprises: detecting SIRT 3-mediated acetylation modification of Frataxin lysine 189, wherein the candidate substance is a potential substance for promoting M2 type polarization of macrophages if the candidate substance causes SIRT 3-mediated reduction of the acetylation level of Frataxin lysine 189; if the candidate substance results in an SIRT 3-mediated increase in the acetylation level of lysine 189 in Frataxin, the candidate substance is a potential substance for promoting M1-type polarization of macrophages.