S100A6 protein with extremely low endotoxin content as well as preparation method and application of S100A6 protein

Abstract

The invention relates to a preparation method of an S100A6 protein with low endotoxin content, and the preparation method comprises the following steps: connecting an S100a6 gene into a pET-28a vector to obtain a pET-28a-S100a6 plasmid; introducing into Escherichia coli, carrying out induced expression, and centrifuging to obtain thalli containing S100A6 protein; cracking, crushing and centrifugally filtering the thalli to obtain a supernatant; performing nickel column purification and Q column chromatography purification on the supernatant to obtain a protein solution 2; and purifying with a desalting column to obtain the S100A6 protein. According to the preparation method, the endotoxin content lt can be obtained; the S100A6 protein can be used for researching the S100A6 protein or gene-related pathways and mechanisms in a living body, so that an effective scheme is provided for the treatment of diseases. The S100A6 protein can be applied to the research of the S100A6 protein or gene-related pathways and mechanisms in the living body. The invention further provides a regulation and control effect of the S100A6 receptor RAGE in CVB3 viral myocarditis, and a new target spot and a new treatment scheme are provided for development of drugs for treating the CVB3 viral myocarditis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to the expression and purification of proteins and their medical uses, and specifically to an S100A6 protein with extremely low endotoxin content, a preparation method thereof, and its application in medicine. Background Art

[0002] Studies have shown that S100A6, a member of the DAMPs family, plays multiple regulatory roles in inflammatory responses. For example, progesterone regulates S100A6 expression through its receptor, subsequently activating the downstream RAGE / EGFR / ERK1 / 2 signaling pathway, modulating inflammatory responses and mediating embryo implantation. S100A6 can promote inflammatory responses after myocardial injury by regulating B cell levels, impairing cardiac function. S100A6 can also activate inflammatory responses by promoting the production of reactive oxygen species (ROS) by neutrophils. Chika Harada et al. found that after acute lung injury, the expression of S100A6, S100A8, and StefinA3 was significantly upregulated, modulating the inflammatory response and thereby exacerbating acute lung injury. Thus, S100A6 expression is upregulated in various inflammatory diseases, accelerating disease progression. However, whether increased S100A6 release is involved in the inflammatory response induced by CVB3 (Coxsackievirus group B type 3) infection has not been reported.

[0003] The S100A6 protein is known to play a key role in tumorigenesis, inflammatory responses, and cellular regulation. It is an important calcium-binding protein. Due to the increasing demand for its experimental research and clinical application, the preparation of high-purity, low-endotoxin S100A6 protein is crucial. However, existing purification methods result in high levels of endotoxins in purified S100A6 protein. Endotoxins are endogenous substances in Escherichia coli. Due to their unique structure, they remain active even after heating at 100°C for one hour. Even in extremely small amounts, they can induce a high fever in humans. During the purification of proteins expressed in E. coli, endotoxins often remain in the protein solution even after fragmentation and purification by affinity chromatography. To remove endogenous endotoxins, ion exchange column chromatography is often used. However, existing purification methods have the following problems: the supernatant obtained by centrifuging the disrupted bacterial solution is passed through a weak anion column and then a hydrophobic chromatography column to obtain the final product. This method does not consider the possibility of nonspecific adsorption in the first purification step, so a large amount of non-target protein will remain in the target protein solution, and the problem of endotoxin removal is also not considered. Alternatively, the S100A6 protein is purified using a GST tag. However, the purification of the GST-tagged protein requires the addition of reduced glutathione to the elution buffer. This substance is easily oxidized and needs to be prepared immediately before use. Therefore, this purification method is only suitable for small-scale preparation and not for large-scale production. It also does not consider the problem of endotoxin removal. Some methods also use Q columns for purification first, followed by ultra-high-resolution gel filtration chromatography columns. Although this solves the problem of endotoxin removal, ultra-high-resolution gel filtration chromatography columns are expensive and have slow flow rates, requiring extremely high sample pretreatment requirements, making large-scale preparation impossible. Therefore, the cost is very high and it is not suitable for conventional methods. From the perspective of low-cost preparation, existing methods are unable to remove endotoxins, and residual endotoxins can affect cell-based experiments: endotoxin (LPS) is a component of the cell wall of Gram-negative bacteria. Even extremely low concentrations (>0.1 EU / μg) can activate immune cells (such as macrophages) and interfere with functional studies of S100A6 (such as cell migration and signaling pathway analysis). Current endotoxin removal methods for protein samples mainly include: ultrafiltration, which only partially removes endotoxins and may cause protein loss; polymyxin B affinity chromatography, which is costly and may affect protein activity; and ion exchange chromatography (such as Q columns): studies have shown that anion exchange chromatography can bind endotoxins (negatively charged).

[0004] Furthermore, traditional purification methods are inefficient for removing endotoxins. Currently, purification of S100A6 relies on affinity chromatography (e.g., His-tag purification) or gel filtration chromatography. However, these methods are ineffective in removing endotoxins and often require additional steps (e.g., ultrafiltration, Triton X-114 treatment), resulting in complex processes and reduced protein recovery.

[0005] Currently available S100A6 is expensive. For example, Merck, a renowned reagent manufacturer, charges 148 yuan per microgram. Furthermore, the endotoxin content of commonly available S100A6 proteins is only listed as less than 1 EU / microgram of protein. This high price and excessive endotoxin content significantly hinder further research on S100A6. Rapidly preparing S100A6 at low cost and with ultra-low endotoxin levels remains a major challenge. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a S100A6 protein with low endotoxin content, a low-cost preparation method thereof, and application in medicine.

[0007] The technical solutions for achieving the above-mentioned purpose include the following.

[0008] The first invention of the present invention is to provide a method for preparing S100A6 protein with low endotoxin content, comprising the following steps:

[0009] S1 ligated the S100a6 gene into the pET-28a vector to obtain the pET-28a-S100a6 plasmid;

[0010] S2: introducing the pET-28a-S100a6 plasmid into Escherichia coli, inducing expression, and obtaining bacteria containing the S100A6 protein by centrifugation;

[0011] S3: lysing and crushing the bacterial cells, centrifuging and filtering to obtain a supernatant;

[0012] S4 purifies the supernatant through a nickel column to obtain protein solution 1;

[0013] S5 purifying the protein solution 1 through Q column chromatography to obtain protein solution 2;

[0014] S6 purifies the protein solution 2 through a desalting column to obtain S100A6 protein.

[0015] In some embodiments, in step S5, the protein solution 1 is diluted with Q-Buffer A at a volume ratio of 1:1-4 before loading, and the volume ratio is more preferably 1.5-2.5.

[0016] In some embodiments, the packing material in the Q column is FastFlow.

[0017] In some embodiments, in step S5, the sample is loaded at a flow rate of 2.5 to 10 mL / min, and the flow-through is collected after the peak appears; after the loading is completed, the Q column is flushed with Q-Buffer A at a flow rate of 2.5 to 10 mL / min until the Cd and UV280 baselines are stable.

[0018] In some of the examples, the vector was linearized with BamHI and NdeI, and the S100a6 gene was ligated to the pET-28a vector by seamless cloning to obtain the pET-28a-S100a6 plasmid.

[0019] The second aspect of the present invention is to provide a S100A6 protein with low endotoxin content obtained by the above preparation method.

[0020] In some embodiments, the endotoxin content of the S100A6 protein is less than 0.02 EU per 1 μg of S100A6 protein, preferably, the endotoxin content is between 0.0163 and 0.02 EU per 1 μg of S100A6 protein.

[0021] The third aspect of the present invention is to provide the use of the S100A6 protein with low endotoxin content in the research of drugs for treating myocarditis.

[0022] A fourth aspect of the present invention is to provide a reagent for knocking out the S100A6 gene for use in preparing a drug for myocarditis.

[0023] The S100A6 gene is an S100A6 gene in an isolated cell, or an S100A6 gene in a cell in an organism.

[0024] The organism can be a mammal, including humans, horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, sheep, and cows.

[0025] In some embodiments, the myocarditis is CVB3 viral myocarditis.

[0026] The fifth aspect of the present invention is to provide the use of RGAE inhibitors in the preparation of a drug for improving or treating CVB3 viral myocarditis.

[0027] In some of these embodiments, the RGAE inhibitor is TTP488 and / or FPS-ZM1.

[0028] The present invention discovered that after infection with the CVB3 virus, S100A6 protein is secreted into the cell supernatant, with particularly high expression levels in macrophage supernatants. The present invention provides a novel method for preparing S100A6 protein. By developing a suitable expression and purification method, this method can produce S100A6 protein with an endotoxin content of <0.02 EU / μg, significantly lower than that of currently commercially available products. This S100A6 protein can be used to study S100A6-related pathways and mechanisms in vivo, thereby providing effective treatment options for diseases.

[0029] The present study discovered the role of S100A6 in CVB3-induced myocarditis after activating RAGE expression. Furthermore, the RAGE inhibitors TTP488 and FPS-ZM1 significantly ameliorated CVB3-induced cellular inflammatory responses. These studies demonstrate the important role of S100A6 in CVB3-induced inflammatory responses and confirm the regulatory role of the S100A6 receptor, RAGE, in CVB3-induced myocarditis, providing new targets and therapeutic options for drug development to treat this disease.

[0030] The present invention discovered that increased release of S100A6 is involved in the inflammatory response induced by CVB3 infection. By purifying and expressing S100A6, a low-endotoxin S100A6 protein was obtained. A series of molecular biology experiments revealed that S100A6 can upregulate the expression of inflammatory factors such as IL-6, IL-1β, and Ptgs2, while also increasing ROS levels, promoting macrophage inflammatory responses and oxidative stress damage, and participating in CVB3-induced inflammatory responses. This invention demonstrates that S100A6 is a key regulator of the development and progression of CVB3-induced myocarditis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 CVB3 promotes S100A6 secretion into the cell supernatant. (A) RAW264.7 cells were stimulated with CVB3 (MOI = 20) for 12 hours. (B) Neutrophils were stimulated with CVB3 (MOI = 20) for 4 hours. (C) NRVCs were infected with CVB3 (MOI = 20) for 48 hours. (D) AC16 cells were infected with CVB3 (MOI = 10) for 48 hours. (E) H9C2 cells were infected with CVB3 (MOI = 10) for 48 hours. S100A6 levels in the supernatant were measured by ELISA. Data are expressed as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0032] Figure 2 Construction of pET-28a-S100a6. (A) Map of plasmid pET-28a-S100a6. (B) PCR amplification of the S100a6 (mouse) gene fragment. M: DNA Maker; Lanes 1-3: S100a6 amplified fragments. (C) BamHI and NdeI linearized vector pET-28a. M: DNA Marker; Lanes 1-2: Linearized vector. (D) Colony PCR verification of positive transformants. M: DNA Marker; Lanes 1-4: Amplified fragments from selected transformants.

[0033] Figure 3SDS-PAGE electrophoresis analysis of S100A6 protein expression and purification. (A) SDS-PAGE analysis of the nickel column purification process for S100A6 protein. Lanes 1-4 represent the bacterial suspension at the end of induction of the S100A6 protein expression strain, lane 5 represents the total bacterial lysate sample during purification, lane 6 represents the supernatant after centrifugation of the lysate, lane 7 represents the flow-through during nickel column loading, and lanes 8-11 represent protein samples collected during nickel column elution. (B) SDS-PAGE analysis of the nickel column purification process for S100A6 protein. Lanes 1-8 represent protein samples collected during nickel column elution. (C) SDS-PAGE analysis of the Q column purification process for S100A6 protein. Lanes 1-11 represent the flow-through samples collected during Q column loading. (D) SDS-PAGE analysis of the desalting column purification process for S100A6 protein. Lanes 1-11 represent the flow-through samples collected during desalting column loading.

[0034] Figure 4 S100A6 activates the JAK2 / STAT3 signaling pathway in RAW264.7 cells. RAW264.7 cells were stimulated with S100A6 (10 μg) for 12 hours, and the expression levels of JAK2, p-JAK2 (Y1007 / 1008), STAT3, and p-STAT3 (Y705) were assessed by Western blotting. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0035] Figure 5 CVB3 activates the JAK2 / STAT3 signaling pathway in RAW264.7 cells. RAW264.7 cells were co-incubated with CVB3 (MOI = 20) for 12 hours, and the levels of JAK2, p-JAK2 (Y1007 / 1008), STAT3, and p-STAT3 (Y705) proteins were measured by Western blotting. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0036] Figure 6 S100A6 promotes the transformation of RAW264.7 cells into proinflammatory macrophages. RAW264.7 cells were stimulated with S100A6 protein (10 μg) for 12 h. (A) Cell morphology. (B) Cell viability was assessed using CCK-8 assay. (C) RT-qPCR assays were used to assess the expression of IL-1β, IL-6, Ptgs2, TNF-α, CXCL2, IL-23, and CXCL9. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0037] Figure 7CVB3 promotes the transformation of RAW264.7 cells into pro-inflammatory macrophages. RAW264.7 cells were stimulated with CVB3 (MOI = 20) for 12 h. (A) Cell morphology. (B) Cell viability was assessed using CCK-8 assay. (C) RT-qPCR was used to measure IL-1β, IL-6, and Ptgs2 mRNA levels. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0038] Figure 8 S100A6 promotes ROS production in RAW 264.7 cells. RAW 264.7 cells were stimulated with S100A6 protein (10 μg) for 12 hours, and ROS levels were measured using a DCFH-DA probe. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0039] Figure 9 CVB3 promotes ROS production in RAW264.7 cells. RAW264.7 cells were stimulated with CVB3 (MOI = 20) for 12 hours, and ROS levels were measured using a DCFH-DA probe. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0040] Figure 10 S100A6 activates RAGE expression in RAW264.7 cells. RAW264.7 cells were stimulated with S100A6 protein (10 μg) for 12 hours. (A) RT-qPCR analysis of RAGE, TLR2, TLR3, TLR4, TLR5, TLR6, and TLR9 levels. (B) Western blotting analysis of RAGE expression. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0041] Figure 11 CVB3 upregulates RAGE expression in RAW264.7 cells. CVB3 (MOI = 20) was co-incubated with RAW264.7 cells for 12 hours. (A) RT-qPCR analysis of RAGE expression. (B) Western blotting analysis of RAGE expression. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0042] Figure 12FPS-ZM1 and TTP488 do not affect the viability of RAW264.7 cells. RAW264.7 cells were treated with FPS-ZM1 (10 and 20 μM) and TTP488 (0.5 and 1 μM) for 14 hours. (A) Cell morphology. (B) Changes in cell viability were assessed using CCK-8 assay. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0043] Figure 13 FPS-ZM1 and TTP488 inhibit S100A6-induced inflammatory responses in RAW264.7 cells. RAW264.7 cells were treated with FPS-ZM1 (10 and 20 μM) and TTP488 (0.5 and 1 μM) for 2 h, followed by S100A6 (10 μg) stimulation for 12 h. (A) Cell morphology (red arrows indicate polarized cells). (B) Cell viability was assessed by CCK-8 assay. (C) RT-qPCR assays were used to assess IL-6, IL-1β, and Ptgs2 expression levels. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0044] Figure 14 FPS-ZM1 and TTP488 inhibit CVB3-induced inflammatory responses in RAW264.7 cells. RAW264.7 cells were treated with FPS-ZM1 (10 and 20 μM) and TTP488 (0.5 and 1 μM) for 2 h and then incubated with CVB3 (MOI = 20) for 12 h. (A) Cell morphology (red arrows indicate polarized cells). (B) Cell viability was assessed by CCK-8 assay. (C) RT-qPCR assay for IL-6, IL-1β, and Ptgs2 expression levels. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0045] Figure 15 FPS-ZM1 and TTP488 can reduce the increase in ROS levels in RAW264.7 cells induced by S100A6. RAW264.7 cells were treated with FPS-ZM1 (10 and 20 μM) and TTP488 (0.5 and 1 μM) for 2 hours and incubated with S100A6 (10 μg) for 12 hours. ROS levels were detected using a DCFH-DA probe. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0046] Figure 16FPS-ZM1 and TTP488 can reduce CVB3-induced increases in ROS levels in RAW264.7 cells. RAW264.7 cells were treated with FPS-ZM1 (10 and 20 μM) and TTP488 (0.5 and 1 μM) for 2 hours and then incubated with CVB3 (MOI = 20) for 12 hours. ROS levels were detected using a DCFH-DA probe. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0047] Figure 17 FPS-ZM1 and TTP488 ameliorate CVB3-induced myocarditis. Mice were intraperitoneally injected with CVB3 (1×107 pfu / mouse) and orally gavaged with FPS-ZM1 (5 mg / kg) and TTP488 (4 mg / kg) on ​​days 1 and 3 of infection (n=10). (A) Survival curves. (B) Body weight changes. (C) Cardiac morphology. (D) H&E staining (blue arrows indicate inflammatory cell infiltration; green arrows indicate myocardial rupture). Data are expressed as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0048] Figure 18 FPS-ZM1 and TTP488 reduce the expression of CVB3 and inflammatory factors in mouse heart tissue. FPS-ZM1 (5 mg / kg) and TTP488 (4 mg / kg) were administered orally four days after CVB3 infection. (A) RT-qPCR analysis of VP1 mRNA levels. (B) RT-qPCR analysis of IL-6, IL-1β, and Ptgs2 expression levels. Data are expressed as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0049] Figure 19 FPS-ZM1 and TTP488 inhibit the JAK2 / STAT3 signaling pathway to improve CVB3-induced myocarditis. Mice were intraperitoneally injected with CVB3 (1×107 pfu / mouse) and then orally gavaged with FPS-ZM1 (5 mg / kg) and TTP488 (4 mg / kg) (days 1 and 3 after infection). On day 4 of infection, heart tissue was harvested. IHC assayed the expression levels of RAGE, JAK2, p-JAK2 (Y1007 / 1008), STAT3, and p-STAT3 (Y705). Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0050] Figure 20S100A6 gene knockout inhibits IL-6 and Ptgs2 expression in mouse heart tissue. Mice (n=5) were intraperitoneally injected with CVB3 (5×106 pfu / mouse). (A) Survival curves. (B) RT-qPCR analysis of IL-6, IL-1β, and Ptgs2 expression levels. Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001.

[0051] Figure 21 S100A6 gene knockout inhibits activation of the RAGE / JAK2 / STAT3 signaling pathway in mouse heart tissue. Mice were intraperitoneally injected with CVB3 (5×106 pfu / mouse). Heart tissue was harvested on day 4 of infection (n=3). IHC assayed the expression of RAGE, JAK2, p-JAK2 (Y1007 / 1008), STAT3, and p-STAT3 (Y705). Data are presented as mean ± SEM. ns indicates no significant difference. *p < 0.05, **p < 0.01, ***p < 0.001. DETAILED DESCRIPTION

[0052] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0053] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0054] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0055] Definitions To facilitate understanding of this technology, certain terms and phrases are defined below.

[0056] CVB3 virus, or CVB3, is the abbreviation for Coxsackievirus group B type 3.

[0057] CVB3 viral myocarditis is a myocardial inflammatory disease caused by infection with Coxsackievirus B3 (CVB3), and its pathological characteristics are myocardial cell degeneration, necrosis and immune cell infiltration.

[0058] TTP488: Azeliragon, Azeliragon.

[0059] FPS-ZM1: N-Benzyl-4-chloro-N-cyclohexylbenzamide.

[0060] The present invention experimentally verified that after CVB3 infection, S100A6 activated RAGE expression, RAGE promoted the phosphorylation of JAK2 and STAT3, and upregulated the level of intracellular inflammatory response, emphasizing the role of a new S100A6 / RAGE / JAK2 / STAT3 signaling pathway in the inflammatory response of CVB3 viral myocarditis.

[0061] The present study's results demonstrate that CVB3, upon infection of target cells and stimulation of macrophages, promotes the secretion of S100A6 into the cell supernatant. S100A6 subsequently activates its receptor, RAGE, which in turn regulates the expression of IL-6, IL-1β, and Ptgs2, the production of ROS, and the JAK2 / STAT3 signaling pathway, modulating the inflammatory response. Inhibition of RAGE and knockout of S100A6 alleviate CVB3-induced inflammation in RAW264.7 cells and mouse heart tissue.

[0062] The present invention is further described in detail below with reference to specific embodiments.

[0063] Example 1

[0064] 1. The sources of materials of the present invention include the following:

[0065] 1 cell line

[0066] Table 1 Cell lines

[0067]

[0068] 2 viruses

[0069] The CVB3 strain (Nancy strain) was purchased from ATCC and stored in our laboratory.

[0070] 3 Animals

[0071] BALB / c mice were purchased from Jiangsu Jicui Yaokang Experimental Animal Technology Co., Ltd. (certificate number: NO.202318065, NO.410983241100024653). - / -Mice were constructed using the C57BL / 6J mouse strain genetic background at the request of Southern Model Organisms, Inc. (certificate number: 4115036633). Mice were housed in a clean laboratory animal room with free access to food and water, and a 12-hour alternating light cycle. All animal experiments conducted in this paper were approved by the Nanchang University Ethics Committee (10775) and were conducted in strict accordance with the guidelines.

[0072] 4 Antibodies

[0073] Table 2 Antibodies

[0074]

[0075] 5 Experimental reagents

[0076] Table 3 Experimental reagents

[0077]

[0078]

[0079] Preparation of other reagents: Unless otherwise specified, all were prepared according to conventional methods.

[0080] 2. Experimental methods

[0081] Plasmid construction

[0082] (1) Primer design: Upstream and downstream primers were designed based on the CDS region of the mouse S100a6 gene sequence included in NCBI. At the same time, two restriction endonucleases, BamHI and NdeⅠ, were selected based on the target gene sequence (Gene ID: 20200; NCBI Reference Sequence: NM_001417970.1; CDS: 63..332) and the multiple cloning site region of the pET-28a vector, and restriction sites were introduced at the 5' end of the upstream and downstream primers, respectively. Upstream primer: GTGCCGCGCGGCAGCCATATGatggcatgccctctggatc (SEQ ID NO: 1); downstream primer: ACGGAGCTCGAATTCGGATCCttatttcagagcttcattgtagatc (SEQ ID NO: 2).

[0083] The amino acid composition of the S100A6 protein is as follows (SEQ ID NO: 25):

[0084] macpldqaig llvaifhkys gkegdkhtls kkelkeliqk eltigsklqd aeiarlmddldrnkdqevnf qeyvaflgal aliynealk.

[0085] (2) Preparation of linearized vector: Use restriction endonucleases BamHI and NdeⅠ to linearize the pET-28a vector according to Takara's instructions. The vector linearization system is as follows: 1μg of pET-28a, 1μL of BamHI, 1μL of NdeⅠ, and 5μL of 10×QuickCut buffer, and add distilled water to make up to 50μL. After preparing the reaction system in a 200μL centrifuge tube, incubate it in a 37℃ water bath for 2h. Add 5μL of 10×loading buffer, mix well, load the sample into the agarose electrophoresis sample tank, and electrophorese at 100V for 30min. Cut the gel according to the size of the linearized vector and the position of the marker to recover the linearized vector for later use.

[0086] (3) PCR amplification of the S100a6 fragment: RNA was extracted from mouse heart tissue and reverse transcribed into cDNA using oligo dt primers. The target fragment was amplified according to the Vazyme operating instructions. The amplification system is as follows:

[0087] Table 5 PCR amplification reaction system

[0088]

[0089] The PCR amplification program is as follows:

[0090] Table 6 PCR amplification reaction program

[0091]

[0092]

[0093] After PCR amplification, the DNA fragments were separated by 1% agarose gel electrophoresis and the fragments were cut and recovered at the size of the S100a6 gene using a UV gel cutting instrument.

[0094] (4) Recombination of fragments and vectors: Thaw the Assembly Master Mix on ice and spin down for later use. Add the S100a6 DNA fragment and the pET-28a linearized fragment to the Assembly Master Mix at a ratio of 1:1 or 1:4 and mix thoroughly. Use a PCR amplification instrument at 30°C for 40 minutes. Then, place on ice for 10 minutes before use.

[0095] (5) Transformation: Use a micropipette to add the recombinant product to a 1.5 mL centrifuge tube containing 50 μL of DH-5α competent cells, gently and slowly pipette to mix, and then let it stand on ice for 30 minutes. Set the metal bath to 42°C in advance. After standing, transfer the centrifuge tube to the metal bath and heat shock it at 42°C for 90 seconds. Then place it on ice and let it stand for 3 minutes. Add 700 μL of LB medium without resistance and shake it at 200 rpm in a 37°C floor shaker for 1 hour. Then centrifuge at 3000 rpm at room temperature for 5 minutes, discard 600 μL of supernatant and resuspend the precipitate. Use glass beads to evenly spread the resuspended bacterial suspension on LB solid medium containing kanamycin sulfate and culture it in a constant temperature incubator at 37°C for 14 hours.

[0096] (6) Enzyme Digestion Verification: Select transformants and inoculate them into 5 mL of LB liquid medium containing kanamycin sulfate. Culture them in a floor shaker at 37°C overnight to extract the plasmid. The extracted plasmid is digested with BamHI and NdeⅠ, and the positive transformants that have been verified are selected for use.

[0097] (7) Sequencing verification: 10 μL of the positive transformant plasmid that was verified to be correct by enzyme digestion was aspirated, stored at low temperature, and sent to a sequencing company for first-generation sequencing using pET-28a universal sequencing primers. After the sequencing results were returned, they were aligned with the S100a6 CDS region sequence using Snapgene software. The plasmid that was sequenced correctly was stored for future use.

[0098] 2. Rubber recovery: proceed according to conventional methods.

[0099] 3S100A6 protein purification

[0100] (1) Construction of S100A6 protein expression strain: The plasmid verified by sequencing was heat-shocked into E. coli BL21 (DE3) competent cells, plated and cultured, and positive clones were screened and cultured and sent to a sequencing company for verification. The positive clones that were sequenced correctly were stored in a -80°C freezer.

[0101] (2) S100A6 seed culture: Remove the S100A6 expression strain from -80°C freezer and thaw in 4°C refrigerator. Streak the plate onto solid LB medium containing kanamycin sulfate and culture overnight in a 37°C constant temperature biochemical incubator. Pick a single colony and transfer it to 20 mL of liquid LB medium containing kanamycin sulfate. Culture at 37°C, 220 rpm for 6-7 h.

[0102] (3) Induction of expression of the S100A6 expression strain: After culturing the seed liquid, pour it into 1 L of liquid LB medium containing kanamycin sulfate and culture at 37°C and 220 rpm for 3-4 hours. When the OD reaches 0.8, cool the fermentation liquid in an ice bath and adjust the shaker temperature to 16°C. After the shaker temperature drops to 16°C, add IPTG to a final concentration of 0.5 mM to the fermentation liquid. After overnight induction, centrifuge at 8000 rpm and 15°C for 10 minutes, collect the cells and store them in a -20°C refrigerator.

[0103] (4) Nickel column purification of S100A6 protein: Remove the centrifuged bacterial cells from a -20°C freezer, add Ni-Buffer A at a ratio of 1:15 (w / v), and mix thoroughly using a homogenizer. Pour the mixture into a high-pressure crusher and crush continuously at 750 bar and 6°C for 10 min. After crushing, centrifuge at 13,000 rpm and 6°C for 15 min. Filter the supernatant through a 0.22 μm filter and place on ice until use.

[0104] The nickel column was flushed with Ni-Buffer B at a flow rate of 4 mL / min. After the Cd and UV280 baselines stabilized, the column was flushed with Ni-Buffer A at a flow rate of 4 mL / min until the Cd and UV280 baselines stabilized. The sample was then loaded at a flow rate of 2.5 mL / min. After loading was complete, the column was flushed with Ni-Buffer A at a flow rate of 2.5 mL / min until the UV280 and Cd baselines were balanced.

[0105] Elute the protein from the nickel column using a linear elution method. Increase the concentration of Ni-Buffer B linearly from 0% to 50% over 10 minutes. When the UV280 peak begins, add 5 mL of the protein flow-through solution to each tube. Stop collecting protein after the UV280 baseline stabilizes.

[0106] Rinse the nickel column with Ni-Buffer B again. After the Cd and UV280 baselines are stable, rinse with ultrapure water until the Cd and UV280 baselines are stable. Then rinse with 20% ethanol solution until the Cd and UV280 baselines are stable. Remove the column and store it in a 4°C refrigerator.

[0107] (5) Q column purification of S100A6 protein: The collected protein solution was diluted with Q-Buffer A at a ratio of 1:2 (v / v).

[0108] First, flush the Q column with Q-Buffer B at a flow rate of 4 mL / min. Once the Cd and UV280 baselines are stable, flush the Q column with Q-Buffer A until the Cd and UV280 baselines are stable. After equilibration, load the sample at a flow rate of 2.5 mL / min. After the peak appears, collect the flow-through. After loading, flush the Q column with Q-Buffer A at a flow rate of 2.5 mL / min until the Cd and UV280 baselines are stable.

[0109] Rinse the Q column with Q-Buffer B until the Cd and UV280 baselines are stable, then rinse with Q-Buffer A until the Cd and UV280 baselines are stable. After equilibration, rinse the Q column with ultrapure water until the Cd and UV280 baselines are stable, then rinse with 20% ethanol until the Cd and UV280 baselines are stable. Remove the column and store in a refrigerator at 4°C.

[0110] (6) Purification of S100A6 protein by desalting column: First, flush the desalting column with TY-Buffer at a flow rate of 5 mL / min until the Cd and UV280 baselines are stable. Then, load the sample at a flow rate of 5 mL / min. After loading 15 mL at a time, flush with desalting buffer again. After the UV280 peak appears, quickly add the protein solution. When the UV280 peak decreases to a slope of -1, stop collecting. Repeat this process until all samples have been loaded.

[0111] After the sample loading is completed, the desalting column is first flushed with TY-Buffer at a flow rate of 5 mL / min until the Cd and UV280 baselines are stable; then the desalting column is flushed with pure water at a flow rate of 5 mL / min until the Cd and UV280 baselines are stable; finally, the column is flushed with 20% ethanol solution until the Cd and UV280 baselines are stable, then the column is removed and stored in a 4°C refrigerator.

[0112] 4: Determination of endotoxin content of S100A6 protein: (1) Add Limulus Amebocyte Lysate (LRW) to the endotoxin working solution standard until the endotoxin content of the solution reaches 50EU / mL to 100EU / mL, and vortex for 15 minutes to fully dissolve.

[0113] (2) 10-fold gradient dilution of the endotoxin standard working solution was performed to prepare a standard curve.

[0114] (3) Then, 100 μL of LRW blank solution, serially diluted endotoxin standards, S100A6 protein, and positive control were added to a 96-well plate and incubated at 37°C for 10 min using a microplate reader.

[0115] (4) Prepare the horseshoe crab reagent using LRW in advance, add 100 μL of horseshoe crab reagent to the sample and use an enzyme-labeled instrument for dynamic monitoring.

[0116] (5) The measurement results showed that the endotoxin content in 1 μg of S100A6 protein was <0.02 EU, which meets the endotoxin level requirements for cell experiments. The obtained S100A6 protein was used in the following experiments.

[0117] 3. Analysis of experimental results

[0118] S100A6 protein purification

[0119] In previous experiments, we discovered and confirmed that S100A6 can be secreted extracellularly after CVB3 infection. Therefore, we purified and expressed the S100A6 protein for subsequent investigation of its molecular mechanism.

[0120] First, S100a6 was constructed into the pET-28a vector for subsequent protein purification and expression. We analyzed the pET-28a vector sequence and the S100a6 CDS region sequence, selected the BamHI and NdeⅠ nuclease endonuclease sites, and inserted S100a6 into the pET-28a vector to form the pET-28a-S100a6 plasmid for subsequent experiments. The plasmid map is shown below. Figure 2 As shown in A. Mouse heart tissue RNA was extracted and reverse transcribed into cDNA as a template for amplifying the S100a6 gene fragment. The S100a6 fragment was amplified by PCR ( Figure 2 B). At the same time, the vector was linearized using BamHI and NdeⅠ ( Figure 2 C), and then the two fragments were connected by seamless cloning. The next day, transformants were selected and verified by colony PCR ( Figure 2 C) The correct transformants were sent for first-generation sequencing. The sequencing results were compared with the S100a6 sequence in the NCBI library, indicating that the pET-28a-S100a6 recombinant plasmid was successfully constructed.

[0121] Next, the plasmid with the correct sequencing results was selected for subsequent S100A6 protein purification and expression. The BL21 (ED3) Escherichia coli containing the pET-28a-S100a6 plasmid was induced to express overnight at 16°C with 0.5mM IPTG, and the bacteria containing the S100A6 protein were obtained by centrifugation. After lysis using a high-pressure cell disruptor, they were used for the next step of purification and expression. SDS-PAGE was used to detect the total bacterial solution after E. coli disruption, the supernatant after centrifugation and filtration, the flow-through during nickel column purification, the flow-through during nickel column elution, the flow-through during Q column purification, and the pure enzyme solution obtained after salt exchange. The results of SDS-PAGE electrophoresis showed that the S100A6 protein was soluble in E. coli BL21 (DE3), and a clear target protein band was visible at 10kDa ( Figure 3 This result indicates that the soluble expression and high-purity preparation of S100A6 protein were successfully achieved through the purification strategy.

[0122] The purification process described herein tested the endotoxin content of a nickel column sample after passing through a Cytiva Q(FF) at different dilution ratios. The nickel column-purified sample was diluted with Q-A Buffer at a ratio of 1:1 and 1:2, then purified using Cytiva Q(FF), and the flowthrough was collected. The collected flowthrough sample was then passed through a desalting column (HiPrep26 / 10), and a portion was tested with Limulus Amebocyte Lysate (LAL).

[0123] 1:1 dilution 1:2 dilution sample >1EU / mL >1EU / mL Dilute the sample 10 times >1EU / mL 0.49 EU / mL Dilute the sample 100 times >1EU / mL 0.06EU / mL

[0124] Note: 1:1 dilution method: 1 mL of sample purified from nickel column is mixed with 1 mL of Q-A Buffer solution.

[0125] The 1:2 dilution method is as follows: 1 mL of the sample purified by nickel column is mixed with 2 mL of Q-A Buffer solution.

[0126] As shown in the table above, after nickel column purification, a 1:2 dilution ratio for Cytiva Q(FF) effectively removes endotoxin contamination. The protein concentration after desalting is approximately 0.3 mg / mL, so the endotoxin content per mg of protein is approximately 20 EU. After multiple experiments, it was found that at a 1:2 dilution ratio, the endotoxin content per μg of protein was consistently between 0.0163 and 0.02 EU, ultimately determining the optimal dilution ratio to be 1:2.

[0127] Furthermore, during the purification process, we hope to reduce the amount of filler while also allowing the filler itself to bind more endotoxins. DEAE filler is a weak anionic adsorbent with a limited ability to adsorb endotoxins. Therefore, when developing industrial purification methods, a large amount of DEAE needs to be loaded, which increases production costs. Furthermore, an increase in the amount of filler loaded also increases pressure during the purification process, inevitably leading to a decrease in flow rate and increased production time. Currently, there are two types of fillers for the strong anion column Q, a strong adsorption type: High Performance (HP) filler with higher resolution, and Fast Flow, which emphasizes flow rate and stronger binding. The Q(HP) type sacrifices its high flow rate due to its high resolution, making it difficult to apply in industrial production. Q(FF), on the other hand, combines the properties of being usable at high flow rates and having stronger binding to negatively charged substances. Therefore, after repeated adjustments and comparisons during multiple experiments, the preparation method described herein optimized and selected the Q(FF) type filler for endotoxin removal.

[0128] This indicates that after nickel column purification, a 1:2 dilution ratio through Cytiva Q(FF) effectively removes endotoxin contamination, reducing the endotoxin content in the purified S100A6 protein to less than 0.02 EU / μg. Therefore, the optimal dilution ratio was determined to be 1:2.

[0129] Example 2

[0130] The S100A6 protein used in this example was prepared by the method described in Example 1.

[0131] Cell culture

[0132] (1) Cell recovery: 45 mL of DMEM high glucose medium, 5 mL of FBS and 500 μL of PBS were mixed and prepared into complete cell culture medium and stored at 4 °C for later use. The cell room environment and the clean bench were sterilized by ultraviolet irradiation for 30 minutes, and the complete cell culture medium was rewarmed to 37 °C in a water bath. The frozen cells were removed from the liquid nitrogen tank and incubated in a 37 °C water bath for 4 minutes until the cells were completely thawed. The cells in the cryopreserved tube were transferred to a 15 mL centrifuge tube containing 2 mL of complete cell culture medium and centrifuged at 1200 rpm for 4 minutes. The supernatant was discarded, 6 mL of complete culture medium was added to resuspend the cell pellet, and then the cell pellet was transferred to a T25 cell culture flask and cultured in a cell culture incubator at 37 °C with 5% CO2.

[0133] (2) Cell passaging and culture: When the cell confluence in the 6 cm cell culture dish reaches more than 90%, discard the old culture medium. Add 2 mL of new complete culture medium and gently pipette until the RAW264.7 cells are completely resuspended. The cells are then passaged in a new 6 cm cell culture dish at a ratio of 1:3.

[0134] (3) Cell cryopreservation: After the cells cultured in the T25 cell culture flask are completely digested, add 2 mL of complete medium and gently pipette the cells. Then transfer them to a 15 mL centrifuge tube and centrifuge at 1200 rpm for 4 minutes. Discard the supernatant and add 900 μL of serum to resuspend the cells. Then transfer them to a cryopreservation tube. At the same time, add 100 μL of DMSO, mix well, and place the cell cryopreservation tube in a cell freezing box. Freeze in a -80°C ultra-low temperature freezer overnight and then store the cells in liquid nitrogen.

[0135] Neutrophil extraction

[0136] Neutrophils were isolated from mouse bone marrow using conventional methods for bone marrow cell and neutrophil isolation. After harvesting, cells were resuspended in 1 mL of complete culture medium. 10 μL was aspirated and counted using a bovine cell counter. Another 10 μL was aspirated and tested for cell viability using 4% trypan blue. The remaining cells were stored at 4°C until further use.

[0137] Viral infection

[0138] (1) After the confluence of RAW264.7 cells reached 90%, they were digested, counted, and plated into 3.5 cm cell culture dishes, with 1×10 cells per well. 6 After the cells were cultured overnight, CVB3 (MOI=20) was co-incubated with RAW264.7 cells for 12 h for subsequent experiments.

[0139] (2) Neutrophils were extracted and incubated with CVB3 (MOI=20) for 4 h, and the cell supernatant was collected for subsequent experiments.

[0140] (3) AC16 cells and H9C2 cells were infected according to conventional methods.

[0141] S100A6 protein stimulates cells

[0142] (1) When the confluence of RAW264.7 cells reached 90%, the cells were gently blown down in the old culture medium using a micropipette, and then transferred to a 15 mL centrifuge tube. After centrifugation at 1200 rpm for 4 min, the supernatant was discarded and the cells were resuspended in 10 mL complete cell culture medium and counted using a cell counter. The cells were evenly distributed into 3.5 cm cell culture dishes, with 1 × 10 cells per dish. 6 After overnight cell culture, the cells were stimulated with 10 μg of S100A6 protein for 12 h for subsequent experiments.

[0143] (2) After RAW264.7 cells have grown to full size, use complete medium to blow off the cells, then transfer them to a 15 mL centrifuge tube and centrifuge at 1200 rpm for 4 min. Discard the supernatant and use new complete medium to gently blow the cell pellet until single cells are present. After counting the cells, use 1×10 6 Plate cells in 3.5 cm cell culture dishes and culture overnight. Discard the old cell culture medium and dilute the stock solution of the receptor of advanced glycation endproducts (RAGE) inhibitor TTP488 in serum-free DMEM to 0.5 and 1 μM concentrations before adding to the cells and incubating for 2 hours. Discard the inhibitor-containing medium, wash once with PSB, and then add 1 mL of complete culture medium and 10 μg of S100A6 protein. Culture for 12 hours before use in subsequent experiments.

[0144] (3) When the RAW264.7 confluency reaches 90% or more, discard the old culture medium, blow off the cells with new complete medium, transfer to a 15 mL centrifuge tube, centrifuge at 1200 rpm for 4 min, discard the supernatant, and add new complete medium to resuspend the cells. After counting 10 μL of cell suspension, add 1×10 6After overnight culture, the culture medium was discarded and the stock solution of the RAGE inhibitor FPS-ZM1 was diluted to 10 and 20 μM in serum-free DMEM medium and then added to the cells for 2 hours. The cells were then washed once with PBS and 1 mL of complete culture medium and 10 μg of S100A6 protein were added. The cells were cultured for 12 hours before use in subsequent experiments.

[0145] CCK-8 cell activity assay

[0146] (1) When the RAW264.7 cell confluency reaches 90%, discard some of the old culture medium until the culture dish contains about 2 mL of culture medium. Use a pipette to gently blow the cells until the cells are completely detached. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 1200 rpm for 4 minutes. Resuspend the cells in 10 mL of complete culture medium and count them. Cells are plated at 1 × 10 cells per well. 5 The cells were evenly plated in a 96-well plate and cultured in a cell culture incubator containing 5% CO2 for 12 h.

[0147] (2) CVB3 stimulates RAW264.7 cells or S100A6 protein stimulates RAW264.7 cells, and the steps are the same as 2.2.7(1) and 4.2.13.

[0148] (3) Discard the old culture medium and prepare CCK-8 working solution by mixing CCK-8 storage solution and DMEM complete medium at a volume ratio of 1:9. Add 100 μL of CCK-8 working solution to each well of a 96-well plate and incubate for 1 h.

[0149] (3) Measure the OD value of each well at 450 nm using a microplate reader and calculate the cell viability. The formula for calculating cell viability is (OD value of the experimental group - OD value of the blank group) / (OD value of the MOCK group - OD value of the blank group) × 100%.

[0150] (4) After the cell activity calculation is completed, Graphad 7.0 is used to draw graphs and perform statistical analysis.

[0151] Total cell RNA extraction and reverse transcription

[0152] (1) Sample collection. Cell sample processing: After RAW264.7 cells are cultured in a 6-well cell culture plate for the specified time, the cell culture medium is discarded, the cells are washed twice with PBS, 1 mL of Trizol is added to lyse the cells, and the lysate is collected into a 1.5 mL nuclease-free centrifuge tube. Tissue sample processing: The tissue stored in liquid nitrogen is taken out and placed in a mortar. An appropriate amount of liquid nitrogen is added and repeatedly ground until the tissue becomes powdery. An appropriate amount of Trizol is added to lyse the tissue cells, and the lysate is collected into a 1.5 mL nuclease-free centrifuge tube.

[0153] (2) Centrifuge the collected cell or tissue lysate samples at 12,000 × g for 10 min at 4°C, collect the supernatant, and transfer it to a new 1.5 mL nuclease-free centrifuge tube.

[0154] (3) Add 200 μL of RNA extraction solution, mix well, and let stand at room temperature for 10 minutes.

[0155] (4) Centrifuge at 12,000 × g for 10 min at 4°C. Transfer the supernatant to a new 1.5 mL nuclease-free centrifuge tube.

[0156] (5) Add isopropanol equal to the volume of the supernatant, mix well, and let stand at room temperature for 10 minutes.

[0157] (6) Centrifuge at 12,000 × g for 10 min at 4°C. Discard the supernatant and retain the white precipitate.

[0158] (7) Add 1 mL of 75% ethanol to wash the precipitate and centrifuge at 8000 × g for 5 min at 4°C.

[0159] (8) Repeat the wash cycle once. Discard the supernatant and air-dry the precipitate. Add nuclease-free water to dissolve the RNA for later use.

[0160] (9) RNA Reverse Transcription: Extracted RNA was reverse transcribed into cDNA according to the instructions of the Takara reverse transcription kit. The reverse transcription system was as follows: 500 ng RNA; 2 μL of 5× PrimeScript RT Master Mix (Perfect Real Time) and enzyme-free sterile water to 10 μL. Reverse transcription conditions were 37°C for 15 min and 85°C for 15 s.

[0161] RT-qPCR

[0162] (1) After RNA is reverse transcribed into cDNA, it is diluted 5-fold with nuclease-free water and used as a template.

[0163] (2) Prepare the experimental system: Prepare the system according to 1 μL of cDNA, 5 μL of TB Green mix, 0.2 μL of upstream primer, 0.2 μL of downstream primer, and 3.6 μL of nuclease-free water, and add it to the eight-tube strip.

[0164] (3) After sample addition, centrifuge using a handheld centrifuge. Amplify using a Bio-Rad qPCR instrument using the following procedure:

[0165] Table 2.1 RT-qPCR reaction procedure

[0166]

[0167] (4) Data analysis: Use △△The results were quantitatively analyzed by the Ct method, and Graphpad 7.0 was used for data analysis and presentation.

[0168]

[0169] Western blotting: performed according to conventional methods.

[0170] Reactive oxygen species (ROS) detection

[0171] (1) When the RAW264.7 cell confluency reaches 90%, discard the old culture medium. Use 3 mL of new DMEM complete culture medium to blow down the cells. Transfer the resulting cell suspension to a 15 mL centrifuge tube and centrifuge at 1200 rpm for 4 minutes. Resuspend the cells in complete culture medium and count them. Count the cells at 5 × 10 cells per well. 6 The cells were seeded into 3.5 cm cell culture dishes and cultured overnight.

[0172] (2) Cells were stimulated with CVB3 or S100A6 protein.

[0173] (3) After 12 h of cell stimulation, load the cells with DCFH-DA probes. Dilute the probes with serum-free DMEM at a volume ratio of 1:1000 to a final concentration of 10 μM. Discard the old medium and add 1 mL of DCFH-DA working solution to each 3.5 cm cell culture dish. Incubate at 37°C in the dark for 30 min, shaking every 5 min.

[0174] (3) After incubation, discard the working solution. Wash the cells three times with PBS and add 1 mL of serum-free DMEM medium.

[0175] (4) Use an inverted fluorescence microscope to directly observe the green fluorescence expression in cells and calculate the positive rate of 200 cells.

[0176] (5) Graphpadprism 7.0 was used for data analysis and graphing.

[0177] Animal experiment protocol

[0178] To investigate whether the RGAE inhibitors TTP488 and FPS-ZM1 can improve CVB3 viral myocarditis:

[0179] (1) Three-week-old male BALB / c mice were acclimated for one week and randomly divided into groups with 10 mice per group. The experimental groups were as follows: MOCK group; CVB3 group; CVB3 + TTP488 (4 mg / kg) group; CVB3 + FPS-ZM1 (5 mg / kg) group; TTP488 group; and FPS-ZM1 group.

[0180] (2) TTP488 and FPS-ZM1 were dissolved in 0.5% CMCNa and given by gavage 2 h before CVB3 injection.

[0181] (3) 1×10 7 The CVB3 infection model was established by intraperitoneal injection of pfu of CVB3 into mice; mice in the MOCK group were injected with an equal volume of serum-free DMEM medium.

[0182] (4) On days 1 and 3 after CVB3 infection, mice were gavaged with TTP488 and FPS-ZM1, respectively. The mental state, weight changes, and survival rate of the mice were recorded daily.

[0183] (5) On the 4th day after CVB3 infection, the mice were euthanized and samples were collected for H&E staining, RT-qPCR, and IHC experiments.

[0184] To investigate whether S100A6 knockout can alleviate CVB3 viral myocarditis:

[0185] (1)S100A6 - / - Mice (obtained by gene knockout mice constructed by Southern Model Company using the Cas9 method) and littermate WT mice (4-6 weeks) were separated and labeled, with 5 mice in each group. The experimental groups were: WT+MOCK group; WT+CVB3 group; S100A6 - / - +MOCK group; S100A6 - / - +CVB3 group.

[0186] (2)CVB3(4.5×10 6 pfu / mouse) were injected intraperitoneally into mice, and the survival status, mental state, hair condition, and weight changes of the mice were recorded.

[0187] (3) On the fourth day of CVB3 infection, mice were euthanized and samples were collected for subsequent verification experiments.

[0188] ELISA detection of protein concentration

[0189] (1) Sample preparation: Collect the cell supernatant into a 2 mL sterile centrifuge tube without nuclease, centrifuge at 1,000 rpm for 20 min, aspirate the supernatant into a new 2 mL centrifuge tube, and store at -80°C until use.

[0190] (2) Take the S100A6 ELISA test kit out of the refrigerator 10 minutes in advance and return it to room temperature.

[0191] (3) Preparation of S100A6 standard: Add 1 mL of universal diluent to the lyophilized tube of standard and let it stand for 15 min until the standard is completely dissolved. Serially dilute the standard using universal diluent to the following concentrations: 50 ng / mL, 25 ng / mL, 12.5 ng / mL, 6.25 ng / mL, 3.12 ng / mL, 1.56 ng / mL, 0.78 ng / mL, and 0 ng / mL.

[0192] (4) Sample addition: Add 100 μL of sample or standard of different concentrations to the corresponding wells. Add 100 μL of universal diluent to the blank wells. Cover with sealing film and incubate in a 37°C incubator for 1 h.

[0193] (5) Add biotinylated antibody: Centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute 15 minutes in advance. Dilute the 100× concentrated biotinylated antibody to a 1× working solution using universal diluent. Prepare and use immediately. Remove the ELISA plate from the 37°C incubator, remove the liquid, and add 100 μL of biotinylated antibody working solution to each well. Cover with a sealing film and place in a 37°C incubator for 1 hour.

[0194] (6) Washing: Return the 20× concentrated washing solution to room temperature. Dilute the concentrated washing solution to 1× using universal diluent and set aside. Discard the liquid in the ELISA plate, add 300 μL of washing solution to each well, and let it stand for 1 min. Discard the washing solution and repeat the washing process three times.

[0195] (7) Add enzyme conjugate working solution: 15 minutes in advance, centrifuge the 100× concentrated enzyme conjugate at 1,000×g for 1 minute. Dilute it to a 1× working solution using universal diluent. Prepare it immediately before use. Add 100 μL of enzyme conjugate working solution to each well of the ELISA plate, cover with a sealing film, and incubate at 37°C for 30 minutes.

[0196] (8) Washing the plate: Wash the plate 5 times according to the steps in step (6).

[0197] (9) Add substrate: Add 90 μL of substrate to each well of the ELISA plate, cover with a sealing film, and then cover with tin foil. Incubate in a 37°C incubator in the dark for 15 min.

[0198] (10) Add stop solution: Take out the ELISA plate and add 50 μL of stop solution directly to each well.

[0199] (11) Result determination: Preheat the microplate reader in advance, measure the OD value of each well at 450 nm, and calculate the protein concentration of each well based on the standard curve.

[0200] H&E staining

[0201] After euthanasia, heart tissue was obtained from mice. Hematoxylin and eosin (H&E) staining was performed according to standard procedures. Finally, the heart tissue sections were mounted with neutral gum, allowed to dry, and observed under an inverted microscope. Cell nuclei appeared blue, and the cytoplasm was pink.

[0202] Immunohistochemistry

[0203] (1) Euthanize mice, extract heart tissue, wash twice with PBS, and then perform immunohistochemistry according to conventional methods.

[0204] (2) Observe the color changes on the tissue sections using a microscope and perform quantitative analysis using Image Pro Plus software.

[0205] Data statistical analysis

[0206] After data collection, data were analyzed and processed using Graphpad Prism 7.0 and SPSS 22.0. For data requiring comparison between two groups, if the data were normally distributed and had homogeneous variances, the independent sample t-test was used; if not, the Mann-Whitney U test was used. For data requiring comparison between multiple groups, if the data were normally distributed and had homogeneous variances, one-way analysis of variance (ANOVA) with post hoc testing was used; if the data were not normally distributed, the Kruskal-Wallis test was used. A P value < 0.05 was considered statistically significant.

[0207] Second result

[0208] 1. CVB3 infection promotes cell secretion of S100A6

[0209] As a member of the DAMPs, S100A6 plays a role in regulating the development of inflammatory responses. To investigate whether S100A6 functions as a DAMP during CVB3 infection, a series of experiments were conducted.

[0210] First, the present invention established a variety of CVB3-infected cell models and detected the level of S100A6 in the cell supernatant by ELISA. The experimental results showed that the content of S100A6 in the supernatant of RAW264.7 cells stimulated by CVB3 was significantly increased (p < 0.05) ( Figure 1 A). After 4 hours of CVB3 stimulation of neutrophils, the content of S100A6 in the supernatant also increased significantly (p<0.05) ( Figure 1 B). In addition, the levels of S100A6 in the cell supernatants of different cardiomyocyte cell lines (including NRVCs, AC16, and H9C2 cells) were detected after CVB3 infection. The results showed that CVB3 infection significantly promoted the secretion of S100A6 in these cardiomyocyte cell lines compared with the MOCK group ( Figure 1 C- Figure 1 E). The above experimental results collectively indicate that CVB3 infection can induce multiple cell types (including immune cells and cardiomyocytes) to secrete S100A6 into the extracellular environment.

[0211] 3. CVB3 activates the JAK2 / STAT3 signaling pathway in RAW 264.7 cells via S100A6

[0212] Next, we further explored whether S100A6 regulates inflammatory responses through specific signaling pathways. We found that CVB3 infection upregulates IL-6 expression in RAW264.7 cells. Previous studies have shown that upregulation of IL-6 expression in avian infectious bronchitis virus infection can further activate the JAK2 / STAT3 signaling pathway, promoting bronchitis. Other studies have shown that the JAK2 / STAT3 signaling pathway further promotes IL-6 expression, forming a cascade reaction that amplifies the inflammatory response. Therefore, we hypothesize that CVB3 infection may promote IL-6 expression through S100A6, activate the JAK2 / STAT3 signaling pathway, and regulate the inflammatory response.

[0213] To verify this hypothesis, we first examined the changes in the JAK2 / STAT3 signaling pathway after S100A6 stimulation of RAW264.7 cells. WB results showed that S100A6 stimulation significantly promoted the phosphorylation of JAK2 (Y1007 / 1008) and STAT3 (Y705). Figure 4 ), indicating that S100A6 can activate the JAK2 / STAT3 signaling pathway. Subsequently, this finding was also verified in the CVB3 infection model. Figure 5 As shown in the figure, CVB3 stimulation significantly upregulated the expression levels of JAK2, p-JAK2, STAT3, and p-STAT3 in RAW264.7 cells. In summary, CVB3 stimulation of RAW264.7 cells activates the JAK2 / STAT3 signaling pathway through S100A6, forming an IL-6 / JAK2 / STAT3 positive feedback loop and amplifying the inflammatory response.

[0214] 4. CVB3 stimulates the expression of inflammatory factors IL-1β, IL-6, and Ptgs2 in RAW 264.7 cells through S100A6

[0215] Studies have shown that macrophages promote the occurrence and development of viral myocarditis by promoting inflammatory responses and damaging cardiac tissue. The present invention found that the expression of S100A6 changed most significantly after CVB3 stimulated the macrophage RAW 264.7. Therefore, subsequent experiments used the mouse-derived RAW264.7 macrophage cell line to further explore the role of S100A6 in the inflammatory response induced by CVB3 infection. After S100A6 protein stimulated RAW264.7 cells, it was observed that RAW264.7 cells grew tentacles and transformed into pro-inflammatory macrophage morphology, as indicated by the red arrow ( Figure 6 A). CCK-8 results showed that the activity of Raw26.7 cells was significantly increased after stimulation with S100A6 protein (p<0.05) ( Figure 6 B). Subsequently, RT-qPCR detection revealed that S100A6 protein stimulation significantly upregulated the expression levels of IL-1β, IL-6, and Ptgs2 (p<0.05), TNF-α and CXCL2 showed a slight upward trend, and the expression levels of IL-23 and CXCL9 did not change significantly ( Figure 6 C). The above results indicate that the S100A6 protein can promote the activation of RAW264.7 cells and induce the transformation of RAW264.7 cells into pro-inflammatory macrophages by specifically upregulating the expression of key inflammatory factors such as IL-1β, IL-6, and Ptgs2.

[0216] Based on the above experiments, it was confirmed that S100A6 can damage RAW264.7 cells. Next, a CVB3-stimulated RAW264.7 cell model was established to explore whether CVB3 mediates the pro-inflammatory macrophage transformation of macrophages through S100A6. After CVB3 stimulation, RAW264.7 cells will be transformed into pro-inflammatory macrophages ( Figure 7 A). CCK-8 results showed that CVB3 stimulation could significantly increase the activity of RAW264.7 cells (p<0.05) ( Figure 7 B). To further elucidate the regulatory effect of CVB3 on the expression of inflammatory factors, the mRNA levels of key inflammatory factors were detected. The results showed that CVB3 stimulation significantly upregulated the expression of IL-1β, IL-6, and Ptgs2 (p<0.05) ( Figure 7 C), this trend is highly consistent with the results of the S100A6 protein stimulation experiment. These results indicate that CVB3 stimulation of RAW264.7 cells promotes S100A6 secretion, which in turn promotes the expression of inflammatory factors IL-1β, IL-6, and Ptgs2, thereby mediating the generation of pro-inflammatory macrophages.

[0217] 5. CVB3 induces elevated ROS levels in RAW 264.7 cells via S100A6

[0218] ROS levels are key signaling molecules that regulate cellular physiological functions. Literature reports indicate that elevated ROS levels during myocarditis can modulate myocardial injury, apoptosis, and inflammation. Furthermore, immune cells contribute significantly to ROS production in myocarditis compared to cardiomyocytes. Furthermore, studies have shown that S100A6, a member of the same family as S100A9, plays a crucial role in regulating isoproterenol-induced cardiomyocyte injury through positive feedback activation of the ROS / p53 / S100A9 signaling pathway. Based on these findings, we hypothesize that in CVB3-induced myocarditis, S100A6 may exacerbate the development of the disease by regulating ROS production.

[0219] To verify this hypothesis, we first examined the effect of S100A6 on ROS levels in RAW264.7 cells. The DCFH-DA fluorescent probe method was used to detect ROS levels. Figure 8 As shown in the figure, after S100A6 stimulated RAW264.7 cells, the intracellular green fluorescence increased. In other words, compared with the MOCK group, the cellular ROS level after S100A6 protein stimulation increased significantly (p<0.05). Similarly, we detected the ROS level of RAW264.7 cells stimulated by CVB3 and found that the intracellular ROS level also increased ( Figure 9 The above results indicate that CVB3 can promote the production of ROS in RAW264.7 cells through S100A6 after stimulating cells, thereby inducing cellular oxidative stress damage.

[0220] 6. CVB3 activates RAGE receptor expression in RAW 264.7 cells via S100A6

[0221] As an important member of DAMPs, S100A6 must bind to specific receptors to exert its biological functions. Next, we conducted a screening experiment for S100A6-related receptors.

[0222] Potential receptors of S100A6 were screened, including pattern recognition receptors (TLR2, TLR3, TLR4, TLR5, TLR6, and TLR9) and non-pattern recognition receptor RAGE. RT-qPCR results showed that after S100A6 stimulation, RAGE expression was significantly increased (p<0.05), TLR4 expression was slightly increased, while TLR2, TLR3, TLR4, TLR5, TLR6, and TLR8 expression did not change significantly ( Figure 10 A). WB results showed that after S100A6 protein stimulated cells, the expression level of RAGE was significantly upregulated (p<0.05) ( Figure 10 B).

[0223] Next, a CVB3-stimulated RAW264.7 cell model was established. RT-qPCR and WB assays revealed that RAGE expression was significantly increased after CVB3 stimulation (p<0.05) ( Figure 11 A, Figure 11 B). In summary, co-incubation of CVB3 with RAW264.7 cells stimulated the secretion of S100A6, which then upregulated the expression of its specific receptor RAGE, exerting a regulatory effect.

[0224] 7. RAGE inhibitors FPS-ZM1 and TTP488 reduce CVB3-induced S100A6-mediated secretion of IL-1β, IL-6, and Ptgs2 in RAW 264.7 cells

[0225] It has been preliminarily demonstrated that CVB3 activates its receptor RAGE through S100A6, promoting the expression of IL-1β, IL-6, and Ptgs2, activating the JAK2 / STAT3 signaling pathway, and increasing ROS levels, thereby regulating inflammatory responses and oxidative stress in RAW264.7 cells. To further validate this mechanism, we conducted reverse validation experiments using RAGE-specific inhibitors. In this study, two RAGE inhibitors, TTP488 and FPS-ZM1, were selected for subsequent experiments. TTP488, also known as Azeliragon, is a bioavailable RAGE inhibitor. FPS-ZM1 binds to sRAGE, including S100B and amphotericin, and is a high-affinity RAGE inhibitor. Based on literature searches and preliminary experimental results, we determined 10 μM and 20 μM FPS-ZM1 and 0.5 μM and 1 μM TTP488 as working concentrations for subsequent experiments.

[0226] The effects of the above inhibitor concentrations on RAW264.7 cells were evaluated. Cell morphology ( Figure 12 A) and CCK-8 ( Figure 12 B) The results showed that the selected concentrations of inhibitors alone had no significant effect on cell morphology and activity, indicating that these concentrations were suitable for subsequent experiments.

[0227] We evaluated whether the inhibitors could reverse the pro-inflammatory polarization of RAW264.7 cells induced by S100A6. The results showed that the number of pro-inflammatory polarized cells in the FPS-ZM1 and TTP488 pretreatment groups was significantly reduced compared with the S100A6 group ( Figure 13 A). CCK-8 results showed that the cell activity in the RAGE inhibitor intervention group was lower than that in the S100A6 group, and the inhibitory effect of FPS-ZM1 was better than that of TTP488 ( Figure 13B). To further evaluate the effects of RAGE inhibitors, we detected the mRNA levels of key inflammatory factors by RT-qPCR. Figure 13 As shown in Figure C, pretreatment with RAGE inhibitors significantly reduced the expression levels of IL-6, IL-1β, and Ptgs2 in a dose-dependent manner. In other words, RAGE inhibitors TTP488 and FPS-ZM1 can effectively inhibit the secretion of inflammatory factors IL-6, IL-1β, and Ptgs2 in RAW264.7 cells induced by S100A6, and partially reverse the S100A6-induced cell polarization shift toward pro-inflammatory phenotypes.

[0228] Next, the FPS-ZM1 and TTP488 inhibitors were used to verify the CVB3-stimulated RAW264.7 cell model to see if the experimental results were consistent with those of the S100A6 protein. Cell morphology results showed that the RAGE inhibitors FPS-ZM1 and TTP488 could indeed rescue the CVB3-induced pro-inflammatory polarization of cells ( Figure 14 A). CCK-8 results showed that the cell activity of the RAGE inhibitor-treated group decreased in a dose-dependent manner and was significantly lower than that of the CVB3-stimulated group (p<0.05) ( Figure 14 B). Next, the expression levels of IL-6, IL-1β, and Ptgs2 were detected. The results showed that pretreatment with RAGE inhibitors significantly reduced the expression levels of these three inflammatory factors (p<0.05) in a dose-dependent manner ( Figure 14 C).

[0229] These results are highly consistent with those from the S100A6 protein stimulation assay, further confirming the molecular mechanism by which CVB3 promotes the secretion of inflammatory cytokines IL-6, IL-1β, and Ptgs2 in RAW264.7 cells through the S100A6 / RAGE signaling pathway. The RAGE inhibitors FPS-ZM1 and TTP488 can, to some extent, inhibit CVB3-induced inflammatory responses in RAW264.7 cells.

[0230] 8. RAGE inhibitors FPS-ZM1 and TTP488 reduce the changes in ROS levels induced by CVB3 via S100A6

[0231] Next, we investigated the effects of FPS-ZM1 and TTP488 on intracellular ROS levels. The results showed that compared with the S100A6 group, the intracellular green fluorescence intensity of the RAGE inhibitor group decreased, indicating that the ROS level decreased ( Figure 15 ).

[0232] To further verify the role of RAGE in CVB3-induced oxidative stress, RAW264.7 cells were pretreated with RAGE inhibitors 2 h before CVB3 stimulation. The results showed that RAGE inhibitors significantly inhibited the increase in ROS levels induced by CVB3 ( Figure 16 ).

[0233] These results indicate that CVB3 can upregulate ROS levels in RAW264.7 cells through the S100A6 / RAGE signaling pathway, inducing cellular oxidative stress. RAGE-specific inhibitors TTP488 and FPS-ZM1 can effectively block this process, suggesting that they can alleviate cellular oxidative stress.

[0234] 9. RAGE inhibitors FPS-ZM1 and TTP488 alleviate CVB3-induced viral myocarditis in mice

[0235] Based on the results of in vitro experiments, animal experiments were further conducted to evaluate the intervention effects of RAGE inhibitors TTP488 and FPSZM1 on CVB3 viral myocarditis. 7 pfu) were injected intraperitoneally into BALB / c mice. The experimental groups were given FPS-ZM1 (5 mg / kg) or TTP488 (4 mg / kg) by gavage every 2 days. The results showed that mice began to die on the 4th day of CVB3 infection and all mice died on the 6th day of infection. After TTP488 was given, mice began to die on the 5th day and all mice died within 7 days. In the FPS-ZM1 intervention group, mice began to die on the 6th day, and 25% of the mice were still alive on the 8th day of infection ( Figure 17 A). Compared with the CVB3 group, the body weight loss in the TTP488 and FPS-ZM1 groups was significantly less (p<0.05) ( Figure 17 B). These results suggest that RAGE inhibitors can effectively inhibit CVB3 infection.

[0236] Obvious white fiber exudates were observed in the hearts of mice in the CVB3 group (indicated by the yellow arrow); while obvious exudates were observed in both the TTP488 and FPS-ZM1 groups, and the appearance of the hearts was similar to that of the MOCK group ( Figure 17 C). H&E staining results showed that the myocardial cells in the heart tissue of the mice in the MOCK group were neatly arranged and the myocardial fibers were intact. The mice in the CVB3 group showed obvious myocardial tissue rupture, cell necrosis, interstitial edema (indicated by green arrows), and a large number of inflammatory cell infiltration (indicated by blue arrows), forming multiple inflammatory lesions. The degree of myocardial damage and inflammatory infiltration in the mice in the TTP488 group was less than that in the CVB3 group. The myocardial tissue of the mice in the FPS-ZM1 group remained intact, with no obvious rupture or inflammatory cell infiltration ( Figure 17 D).

[0237] The above results indicate that RAGE inhibitors TTP488 and FPS-ZM1 can improve CVB3 viral myocarditis to a certain extent, among which the intervention effect of FPS-ZM1 is better than that of TTP488.

[0238] 10. RAGE inhibitors FPS-ZM1 and TTP488 reduce CVB3-induced expression of IL-1β, IL-6, and Ptgs2 in mouse heart tissue

[0239] To further evaluate the mechanism of action of RAGE inhibitors, the viral load and inflammatory factor expression levels in mouse heart tissue were quantitatively analyzed. RT-qPCR results showed that the expression level of VP1 in the heart tissue of mice in the TTP488 and FPS-ZM1 groups was significantly reduced compared with the CVB3 group (p<0.05) ( Figure 18 A), which indicates that RAGE inhibitors can effectively inhibit the replication of CVB3 in myocardial tissue. CVB3 infection caused the expression levels of IL-6, IL-1β and Ptgs2 in mouse heart tissue to be significantly increased compared with the MOCK group (p<0.05). TTP488 treatment significantly reduced the expression level of IL-6, but had no significant effect on the expression of IL-1β and Ptgs2; while FPS-ZM1 intervention significantly reduced the expression levels of IL-6, IL-1β and Ptgs2 (p<0.05) ( Figure 18 B- Figure 18 D) These results indicate that the RAGE inhibitors TTP488 and FPS-ZM1 inhibit CVB3 replication and downregulate inflammatory cytokine expression in vivo, with FPS-ZM1 showing a stronger effect than TTP488. FPS-ZM1 is a potential drug for the treatment of CVB3 infection.

[0240] 11. RAGE inhibitors FPS-ZM1 and TTP488 inhibit the JAK2 / STAT3 signaling pathway to improve CVB3 viral myocarditis

[0241] Next, the downstream molecular mechanism of RAGE inhibitors in improving CVB3 viral myocarditis was further verified. IHC results showed that the expression of RAGE in mouse heart tissue increased significantly after CVB3 infection; TTP488 and FPS-ZM1 intervention effectively reduced the expression level of RAGE. Subsequently, changes in the expression of JAK2, p-JAK2, STAT3 and p-STAT3 were detected. The results showed that CVB3 infection can activate the JAK2 / STAT3 signaling pathway. TTP488 intervention can partially inhibit the phosphorylation of JAK2 and STAT3; while FPS-ZM1 intervention significantly inhibited the phosphorylation levels of JAK2 and STAT3, making their expression levels basically consistent with those of the MOCK group ( Figure 19In conclusion, TTP488 and FPS-ZM1 can inhibit the JAK2 / STAT3 signaling pathway and improve CVB3 viral myocarditis.

[0242] 12. S100A6 knockout downregulates the expression levels of IL-6 and Ptgs2 to improve CVB3 infection-induced inflammatory response in mouse heart tissue

[0243] To confirm the regulatory role of S100A6 in CVB3 viral myocarditis, a CVB3-infected S100A6- / - mouse model was established. The survival curve results showed that the survival rate of S100A6- / - mice was significantly higher than that of WT mice (p<0.01) ( Figure 20 A), indicating that S100A6 deficiency can effectively reduce the lethality of CVB3 infection. RT-qPCR detection found that CVB3 infection significantly upregulated the expression levels of IL-6, IL-1β, and Ptgs2 in the heart tissue of WT mice; after S100A6 knockout, IL-6 and Ptgs2 in the heart tissue of mice were significantly lower than those in the WT group (p<0.05), but there was no significant change in IL-1β ( Figure 20 B) This in vivo experiment demonstrated that CVB3 promotes the expression of IL-6 and Ptgs2 through S100A6, regulating the occurrence and development of viral myocarditis.

[0244] 13. S100A6 knockout inhibits the RAGE / JAK2 / STAT3 signaling pathway and alleviates CVB3 infection-induced cardiac inflammation in mice

[0245] Next, we further verified the molecular mechanism of S100A6 in regulating CVB3 viral myocarditis in vivo. RT-qPCR results showed that CVB3 infection could upregulate the expression level of RAGE in the heart tissue of WT mice; however, after S100A6 gene knockout, the expression level of RAGE was significantly lower than that of WT mice (p<0.01) ( Figure 21 ). IHC results showed that CVB3 infection could upregulate the expression of RAGE in the heart tissue of WT mice; however, after CVB3 infection of S100A6- / - mice, the expression level of RAGE was lower than that of WT mice ( Figure 21 ). Secondly, the expression changes of key molecules in the JAK2 / STAT3 signaling pathway were detected. The results are as follows Figure 21 As shown in the results, after CVB3 infection, the expression of JAK2, p-JAK2, STAT3, and p-STAT3 in the heart tissues of S100A6- / - mice was downregulated compared with that in the WT group. This result indicates that S100A6 gene deletion inhibits the activation of JAK2 and STAT3, suggesting that S100A6 has a positive regulatory effect on the JAK2 / STAT3 signaling pathway during CVB3 infection.

[0246] The present study demonstrates that CVB3, upon infection of target cells and stimulation of macrophages, promotes the secretion of S100A6 into the cell supernatant. S100A6 subsequently activates its receptor, RAGE, which in turn regulates the expression of IL-6, IL-1β, and Ptgs2, the production of ROS, and the JAK2 / STAT3 signaling pathway, modulating the inflammatory response. Inhibition of RAGE and knockout of S100A6 alleviate CVB3-induced inflammation in cardiac tissue.

[0247] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing a S100A6 protein with low endotoxin content, characterized in that: The following steps are involved: S1 ligated the S100a6 gene into the pET-28a vector to obtain the pET-28a-S100a6 plasmid; S2: introducing the pET-28a-S100a6 plasmid into Escherichia coli, inducing expression, and obtaining bacteria containing the S100A6 protein by centrifugation; S3: lysing and crushing the bacterial cells, centrifuging and filtering to obtain a supernatant; S4 purifies the supernatant through a nickel column to obtain protein solution 1; S5 purifying the protein solution 1 through Q column chromatography to obtain protein solution 2; S6 purifies the protein solution 2 through a desalting column to obtain S100A6 protein.

2. The preparation method according to claim 1, characterized in that In step S5, the protein solution 1 is diluted with Q-Buffer A at a volume ratio of 1:1-4 and then loaded onto the sample, preferably at a volume ratio of 1.5-2.

5.

3. The preparation method according to claim 1, characterized in that The filler in the Q column is Fast Flow.

4. The preparation method according to claim 1, characterized in that In step S5, the sample is loaded at a flow rate of 2.5 to 10 mL / min, and the flow-through is collected after the peak appears; after the sample loading is completed, the Q column is flushed with Q-Buffer A at a flow rate of 2.5 to 10 mL / min until the Cd and UV280 baselines are stable.

5. The preparation method according to any one of claims 1 to 4, characterized in that The vector was linearized with BamHI and NdeⅠ, and the S100a6 gene was ligated to the pET-28a vector by seamless cloning to obtain the pET-28a-S100a6 plasmid.

6. The S100A6 protein with low endotoxin content obtained according to the preparation method according to any one of claims 1 to 5.

7. The S100A6 protein according to claim 6, characterized in that The endotoxin content of the S100A6 protein is: the endotoxin content in each 1 ug of S100A6 protein is less than 0.02 EU.

8. Use of the S100A6 protein with low endotoxin content according to any one of claims 6 or 7 for studying the correlation between the S100A6 gene and a disease in vivo or in vitro cells, preferably, the disease is myocarditis, and more preferably, the disease is CVB3 viral myocarditis.

9. Use of an agent for knocking out the S100A6 gene in the preparation of a drug for treating myocarditis. Preferably, the myocarditis is CVB3 viral myocarditis.

10. Use of an RGAE inhibitor in the preparation of a method for improving or treating CVB3 viral myocarditis, wherein the RGAE inhibitor is preferably TTP488 and / or FPS-ZM1.

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